METHODS FOR TREATING AND DIAGNOSING SUSCEPTIBILITY TO CYTOSOLIC PATHOGENS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application Serial No. 60/657,348, filed March 1, 2005, the contents of which are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

[002] Innate immunity is the human body's first line of defense against disease. Each individual responds differently to invading bacterial and viral pathogens, largely due to differences in their innate immue response. For example, it is estimated that 8 million people are infected each year with the pathogen, Mycobacterium tuberculosis (MBT), and over 2 million die annually ¹ . It is estimated that about 10% of the individuals infected actually develop tuberculosis and thus many individuals are carriers.

[003] Genetic variation within host populations is known to play a significant

1 ¹ X role in tuberculosis infection in humans and animals ' . However, the nature of genetic control of host resistance to tuberculosis remains poorly understood, and the individual genes responsible for innate immunity to the pathogen have been elusive. In susceptible individuals, progression of lung tuberculosis often leads to formation of characteristic necrotic 'cavities' that destroy significant portions of the lung. Beyond their life- threatening clinical consequences, these lesions are essential for efficient transmission of MTB via aerosol. In humans, tuberculosis is transmitted primarily via the respiratory route. Therefore, the ability to cause lung disease is considered a key aspect of the pathogen's virulence strategy. Accordingly, developing assays that can be used to look at pathogenic mechanisms that are employed by virulent pathogens, such as MTB during e.g., lung tuberculosis in susceptible individuals, is essential for developing effective prevention and treatment strategies ' .

[004] There is a need in the art to identify ways to modulate innate immunity to intracellular pathogens. It is desirable to develop treatments for a variety of infections and prophylactic therapies, particularly for those who lack adaptive immunity.

SUMMARY OF THE INVENTION

[005] The present invention is based on the identification of a gene and gene product that controls host resistance to cytosolic pathogens in mammals. We have named the murine gene Iprl (intracellular pathogen resistance) because it mediates innate immunity to intracellular pathogens (e.g. Mycobacterium tuberculosis and Listeria monocytogene). The absence or downregulation of Iprl gene expression leads to high susceptibility to infection. In addition, the absence or downregulation of a functional Iprl gene expression leads to high susceptibility to infection. Further, expression of Iprl protein induces apoptosis in infected cells, controls inflammation and thus controls the severity of infection. Accordingly, the present invention is directed to methods for determining a patient's susceptibility to infection by monitoring expression of the human homologue to Iprl.

[006] The present invention is also directed to methods of prophylaxis, as well as methods for treatment of infection by inducing expression of, or administering, the Iprl protein human homologue. Furthermore, it has been shown that Iprl regulates TNF- α and IL-10 expression in macrophages. The presence of Iprl results in an increased TNF-α production, which inhibits pathogen replication and induces cell apoptosis. The absence of Iprl decreases TNF-α production and increases IL-10 production, leading to suppression of apoptosis and more rapid pathogen replication. Thus, methods of prophylaxis and treatment that combine inducing expression of or administering Iprl protein with the upregulation or administration of TNF-α and the downregulation of IL- 10 are contemplated.

[007] In one embodiment, a method for determining a patients susceptibility to infection by a cytosolic pathogen are provided. The methods comprise analyzing gene expression, e.g. gene expression levels, of the human homologue to Iprl (SEQ ID NO:11) in a biological sample, such as, a peripheral blood sample (e.g, in monocytes and macrophages) or a tissue sample (e.g. lung, spleen, heart, liver etc). In one embodiment, a positive gene expression indicates that the individual has innate resistance to infection by cytosolic pathogens. In a preferred embodiment, a negative gene expression indicates that the patient is highly susceptible to infection. As used herein, a negative gene expression includes a lack or decrease in gene expression levels, the presence of a

mutation or polymorphism that results in the expression of a Iprl protein that is nonfunctional (e.g. has imparired antiviral or antibacterial function), or the expression of a protein that has an altered half life. In another embodiment, the method further comprises an analysis of other genetic loci that predict susceptibility to infection by cytosolic pathogens. Other loci may be, for example, loci that predict altered acquired immunity pathways.

[008] Also encompassed in the present methods are an analysis of cytokine levels, such as, for example, TNF-α and IL-10 as predictors of innate immune function. A lowered level of TNF-α and/or an increased level of IL-10 is indicative of susceptibility to infection by cytosolic pathogens. Cytokine levels may be analyzed in conjunction with the levels of Iprl gene expression for the determination of one's susceptibility to infection with a cytosolic pathogen, such as, for example, MBT.

[009] A preferred human homologue of the Iprl protein is SPl 10b ¹⁸ , which is expressed from a gene localized to human chromosome 2. Accordingly, a method for determining a patients susceptibility to infection by a cytosolic pathogen is provided that comprises obtaining a sample from a patient and analyzing gene expression of SPIlOb (SEQ ID NO:1). A positive gene expression indicates that the patient has innate resistance to infection by cytosolic pathogens, while negative gene expression indicates that the individual is highly susceptible to infection.

[0010] As used herein, the term "cytosolic pathogen" refers to any pathogen that enters the cytoplasm upon infection. These include pathogens that directly infect the cell without entering an endosomal pathway, as well as pathogens that initially enter the endosomal pathway but escape from endosomes during the course of infection.

[0011] In one embodiment, the cytosolic pathogen is a bacterial pathogen such as, Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, or Brucella abortus. In another embodiment, the cytosolic pathogen is a viral pathogen, such as Hepatitis C Virus, Epstein Barr virus, or a retrovirus. In one embodiment, the cytosolic pathogen excludes Epstein Barr virus.

[0012] Gene expression (e.g. gene expression levels) can be measured by measuring levels of mRNA (e.g. quantitative RT-PCR and gene expression array analysis). Alternatively, the gene expression levels can be measured by measuring levels of protein. As used herein, an analysis of gene expression may also include an analysis

of protein degradation. Gene expression levels are determined by comparing to levels in a wild type control group. The wild type control group (e.g. sample) should preferably consist of at least 5 individuals, more preferably, at least 20, still more preferably, at least 50 individuals. More preferably, control sets should look at additional criteria such as sex and age to reduce false positives or negatives.

[0013] In one embodiment, one can screen for mutations in a Iprl human homologue gene that affect gene expression and/or protein product function by DNA sequence analysis. One can also detect mutations in a gene product by immunohistochemical methods. For example, one can use an antibody to the carboxy end of the protein to determine if a mutation results in no product or a truncated product. In another embodiment, the mutation in an Iprl human homologue gene results in a nonfunctional or lowered function protein that while expressed at levels similar to a wild type control, functions as if it has decreased expression levels. The mutation or polymorphism may reduce the function so much so as to effectively make the the gene non-functional. Thus, in one embodiment, methods for the detection of mutations and/or polymorphisms that result in either non-functional or lowered function Iprl protein are contemplated.

[0014] Mutations and/or polymorphisms in the Iprl gene may also result in translational modifications that affect protein processing and degradation. As such, methods to screen for mutations and/or polymorphisms that result in modified Iprl proteins are also encompassed. The presence of mutations and/or polymorphisms that result in such modified Iprl protein indicate susceptibility to infection with cytosolic pathogens.

[0015] Methods for treating a patient infected with a cytosolic pathogen are also provided. The method comprises administering to said patient an effective amount of a compound or agent that upregulates expression of a human homologue of Iprl (e.g. SPIlOb (SEQ ID NO:1)).

[0016] Any compound or agent that upregulates expression of a human homologue of Iprl can be used in methods of the invention. In one preferred embodiment, the agent that upregulates gene expression is interferon γ, interferon α, or interferon β. Furthermore, upregulation of a human homologue of Iprl can be combined with upregulation of TNF-α or downregulation of IL-10. Thus, the present invention

further comprises co-administering a compound(s) or agent(s) that upregulates TNF-α or downregulates IL-IO.

[0017] The methods of the invention are useful to treat both patients that innately lack expression of the Iprl human homologue (e.g. SPIlOb) as well as patients that lose expression as the result of having been chronically infected with a pathogen. For example, in tuberculosis, the expression of the Iprl gene is initially upregulated upon infection with the pathogen, however, during chronic infection expression of the Iprl gene is downregulated. Also encompassed in the present invention are mutations and/or polymporphisms that result in lowered or lack of expression of the Iprl human homologue.

[0018] Accordingly, compounds or agents that prevent downregulation of Iprl gene expression are contemplated. In addition, compounds that block inhibitors of the human homologue to Iprl protein, compounds that interefere with post-translational modifications of the human homologue to Iprl protein, and compounds that inhibit degradation of the human homologue to Iprl protein are also contemplated for use in treatment of infection by cytosolic pathogens. Assays to determine such compounds constitute another embodiment of the invention.

[0019] In one embodiment, a method for treating a patient infected with a cytosolic pathogen that comprises administering to said patient an effective amount of Iprl protein human homologue, or a vector containing the corresponding gene, is provided.

[0020] In another embodiment, a method for preventing infection of an individual by a cytosolic pathogen is provided. The method comprises administering to said patient an effective amount of a human protein homologue to Iprl or vector containing said gene encoding such product. Prophylactic treatment is particularly useful for treating individuals at risk of infection.

[0021] Preferably the protein to be administered is a fusion protein, designed such that entry into the cell and/or entry into the nucleus is facilitated. Fragments of the human homologue to Iprl may be administered in leiu of the full length protein. For example, fragments of the human homologue to Iprl that confer antibacterial and/or antiviral activity may be administered. Furthermore, a portion of the human homologue of Iprl that is responsible for TNF-α production may be administered to the patient.

[0022] The proteins of the invention can be administered by administration of a DNA that encodes human protein homologue to Iprl (e.g. contained in a viral expression vector or other vector). Also encompassed is the use of fragments of the DNA that encodes human protein homologue to Iprl . Such fragments should confer antimicrobial and/or antiviral activity.

[0023] The methods of the invention are particularly useful for treating immunocompromised individuals.

BRIEF DESCRIPTION OF FIGURES

[0024] Figures IA to IG show that the sstl locus mediates innate immunity to tuberculosis. Figure IA and Figure IB show the survival of C3H, B6, and the C3H.B6- sstl (sstJ ^R ) mice after i.v. (Figure IA) or aerosol (Figure IB) infection with MTB; Figure 1C shows MTB bacterial loads in the lungs of the sstl congenic mice after the aerosol infection; Figures ID and IE show tuberculosis lung lesions 25 days after i.v. infection (Figure ID) and 12 weeks after aerosol infection, H&E, 4OX original magnification (Figure IE); Figure IF shows FACS analysis of mechanism of cell death of the sstl congenic macrophages infected with MTB (top panels) or BCG (bottom panels) in vitro; Figure IG shows multiplication of MTB (left panel) or M. boy is BCG (right panel) in the sstl congenic macrophages in vitro (*p<0.01, **p<0.001). Error bars represent 95% confidence intervals.

[0025] Figures 2A to 2E show the identification of the sstl candidate gene. Figure 2 A, shows the physical map of the sstl minimal region. (#)- number of recombination events, (M) - polymorphic markers, (C) - chromosome with distances between the markers (kb); (G) - known genes; (RC) - recombinant chromosomes containing the sstl resistant (R) or susceptible (S) alleles, genotypes for each marker are represented by solid (B6) and opened (C3H) boxes; Figure 2B shows analysis of the Ifl75-rs expression in the tuberculosis lung lesions of the sstl congenic mice by RACE. Figure 2C shows the domain structure of the Iprl and its human homolog SPl 10b and location of the PCR primers. Figure 2D shows Iprl and Spl00-rs gene expression in the lungs during MTB infection (Northern blot); Figure 2E shows Iprl gene expression in sstl ^s (S) or sst 1 ^R (R) macrophages infected MTB, BCG or activated with IFN-γ in vitro.

[0026] Figures 3A to 3C show that the lack of the Iprl gene expression in the C3HeB/FeJ substrain correlates with its extreme susceptibility to MTB infection. Figure 3A shows the survival after the intravenous infection with MTB; Figure 3B shows MTB bacterial loads three weeks after the infection (4 mice per strain, ** p<0.001, error bars represent standard deviation); Figure 3C shows the analysis of the sstl -encoded candidate gene expression in the tuberculosis lung lesions by RT-PCR three weeks after the infection.

[0027] Figures 4A to 4F show that the expression of the Iprl transgene in the sstl macrophages confers resistance to intracellular pathogens MTB and L. monocytogenes. Figure 4 A shows RT-PCR of Iprl and Spl00-rs in macrophages isolated from the sstl (S), sstl (R) mice, their Fl hybrids (SxR, RxS) and the Iprl transgenic (Tg) mice, 1 - IFNγ-stimulated, 2 - MTB-infected. Figure 4B shows MTB bacterial loads in the Iprl transgenic (Tg+/-) and control (Tg-/-) mice after infection with MTB (7 mice/strain, * p<0.05); Figures 4C and 4E show the growth of MTB (Figure 4C) and L. monocytogenes (Figure 4E) in the Iprl transgenic and control (sstl ^s ) macrophages (three experiments were performed in triplicates, *p<0.01, **p<0.001, error bars represent 95% confidence interval).

[0028] Figures 5 A to 5B show MTB growth in the organs of the sstl congenic mouse strains and bone marrow chimeras after systemic i.v. infection. Figure 5A shows MTB bacterial loads in the organs from the C3H (sstl ^s ) and the C3H.B6-^ti (sstl ^R ) congenic mice at 2 and 3 weeks after i.v. infection with 10 ⁵ CFU of MTB; Figure 5B shows MTB bacterial loads in the organs of the bone marrow (BM) chimeras 2 and 3 weeks after i.v. infection with 5x10 ⁴ CFU of MTB. Recipient mice were lethally irradiated (650 Gy twice at 4 hour interval) and reconstituted with 2x10 bone marrow cells (S - S, S - R, R - S, R - R = BM Donor - BM Recipient; S = sstl ^s , R = sstl ^R ). Chimeric mice were infected 2 months after the bone marrow transplantation.

[0029] Figures 6 A to 6E show the analysis of the sstl minimal region. Figure 6 A shows the fine mapping of the sstl locus by progeny testing. The sstl allelic composition of each recombinant male (bottom row) was inferred by analysis of co- segregation of the B6-derived segment of chromosome 1 with extended survival times of its progeny after the MTB infection as follows. Parental mice were produced by 4 or 5 generations of backcross on C3HeB/FeJ (sstl ^s ) background. Males that carried

recombination within the B6-derived sstl -containing segment of the mouse chromosome 1 were identified by genotyping. The recombinant males were bred with C3HeB/FeJ females to generate 30-40 backcross progeny for testing. The progeny of each recombinant male were tested for susceptibility to i.v. infection with MTB and genotyped using the microsatellite markers within the sstl region. The survival times of the heterozygous and homozygous backcross progeny for each marker was compared using Student's t test (p=0.001 was used as a threshold). Each column of boxes represents a genotypic class and each row of boxes represents genotypes for an individual microsatellite marker within the sstl candidate region (specified on the left of each row). Solid boxes represent heterozygous (B6 and C3H-derived) and opened boxes homozygous genotype (C3H only). The number of recombinant males, which were tested per each genotypic class, is denoted under each column. The two horizontal lines drawn between DlMit439 and DlMit49 designate the sstl candidate region. The number of recombinants of each genotype that were characterized by progeny testing, is presented at the bottom of each column.

[0030] Figure 6B shows the analysis of expression of the most likely candidate genes in C3HeB/FeJ (S) and C3E.B6-sstl (R) using RT-PCR (left column) and RACE (right column). Total RNA was isolated from lung tuberculosis lesions (Lung) or from bone marrow-derived macrophages (BMph) infected with virulent MTB for 72 hours in vitro. Details of the Ifi75-rs analysis by RACE are presented separately in Fig. 2B. Priority chart of the sstl -encoded candidate genes is presented in Figure 6E (see below).

[0031] Figure 6C shows the number of copies of individual exons of the Ifi 75-rs per genome. Genomic DNA was isolated from the C3HeB/FeJ, C3H.B6-^ti inbred mouse strains and Mus caroli and tested by real time quantitative PCR with 12 pairs of the Iprl exon-specifϊc primers using Amplifluor™ Universal Detection System. A single copy gene encoding brain-derived neurotrophic factor iβdnf) was used as an internal control for normalization. The number of exons per genome was estimated by comparison of normalized values obtained for the C3HeB/FeJ and C3H.B6-^ti genomic DNA with the genomic DNA of Mus caroli, which contains a single copy of the Ifi75 gene (Weichenhan, Mamm Genome 12:590-594, 2001). Each sample was tested in triplicate. The number of exons varied between 40 copies for exon 8 (maximal copy number) and 5 for exon 12 (minimal copy number) indicating that most of the copies of

the Ifi75-rs contained less than a complete set of 12 exons and there are 5 or fewer full length copies of the gene encoded within the B6-derived HSR. The number of the exons 1, 2, and 4 per genome was lower in the sstl mouse.

[0032] Figure 6D shows the heterogeneity of individual exons of the Ifι75-rs demonstrated by Single Strand Conformation Polymorphism (SSCP) analysis. Individual exons of the Ifi75-rs were amplified from the genomic DNA isolated from the C3HeB/FeJ (S) or C3H.B6-^t7 (R) congenic mice and separated on non-denaturing acrylamide gel as described in Methods. Multiple bands were produced with each exon- specific primer pair, suggesting that individual copies of the repeated exons were non identical. There was also a clear difference between the sstl congenic mouse strains in genomic DNA encoding exon 1.

[0033] Figure 6E shows the priority chart of the candidate genes encoded within the sstl region based on their expression pattern. The list of the genes corresponding to the numbers 1-22 is presented in Table 1. The sequences of the Spl00-rs and Ifι75 genes, which are encoded within the repeat region, were described previously (Weichenhan et al, 2001). To prioritize the ^./-encoded genes we used the following criteria: the gene is expressed in critical tissue (lung) and cell type (macrophage), expression of the gene is modulated by the MTB infection, and the gene might be differentially expressed between the sstl disparate animals or cells. Based on priority, all the genes within the sstl region were divided into 5 categories: not expressed in the lungs and macrophages (unlikely candidates, 9 genes); expressed in the lungs, not in macrophages (low priority, 3 genes); expressed in the lungs and macrophages, not induced by MTB infection (priority score 2, medium priority, 10 genes); upregulated by the MTB infection (high priority, 2 genes); differentially expressed between the sstl congenic macrophages (highest priority, 1 gene).

[0034] Figures 7A to 7D show tuberculosis lung lesions in the sstl congenic mice after aerosol infection with MTB. Figure 7A and Figure 7B show tuberculosis lesions in the lungs of the C3HeB/FeJ (C3H) and the sstl resistant congenic mouse strain CSH-Bo-^i at 6 weeks (Figure 7A) and 12 weeks (Figure 7B) after aerosol challenge with MTB. Figure 7C shows tuberculosis granuloma wall adjacent to central necrosis in the lung lesions of the C3H (sstl ^s ) mice (left panel) and lung granuloma of the C3H.B6- sst 1 (sstl ^R ) mice (right panel) 12 weeks after aerosol challenge with MTB. H&E, 200X, original magnification. Figure 7D shows auromine-rodamine staining of the acid fast

bacteria (MTB), 400X, original magnification. Central necrosis with fibrous capsule is seen in lesions of the susceptible C3H mice, with numerous extracellular bacilli seen on fluorescence microscopy within necrotic masses. Lesions of the sstl -resistant congenic mice showed minimal necrosis, abundant lymphocytes and foamy macrophages with scant bacilli upon fluorescence microscopy. Formation of necrotic lesions in the lungs of the sstl -susceptible mice was observed in three independent aerosol experiments. For each mouse strain, 3-4 animals were tested at each time point and necrotic lesions of various sizes were invariably found in the lungs of every sstl -susceptible mouse at 12 weeks after the aerosol challenge with MTB.

[0035] Figures 8 A to 8D shows TUNEL staining of the tuberculosis lung lesions of the sstr ^R and sstl ^s congenic mice. Apoptotic cells in the tuberculosis lung lesions of sstl ^R (Figure 8A and 8C) and sstl ^s (Figure 8B and 8D) congenic mouse strains were identified using a TUNEL assay and counterstained with hematoxylin. In the sstl R mice no necrosis is observed and some focal lesions include cells with TUNEL-positive apoptotic nuclei (Figure 8C, arrow). In the tuberculosis lesions of the sstl S mice the TUNEL-positive staining is associated with cytoplasm and extracellular exudative material (Figure 8D, arrow), which is consistent with necrotic cell death and release of DNA fragments into the exudate. The arrows show a TUNEL-positive nucleus in the resistant mice (Figure 8C) and the TUNEL-positive extracellular exudative material in the susceptible mice (Figure 8D). Figure 8 A and 8B - 10OX; Figure 8C and 8D - 400X original magnification.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention is directed to methods for determining an individual's susceptibility to infection by cytosolic pathogens (e.g. Mycobacterium tuberculosis, Listeria monocytogene, Hepatitis virus and Epstein Barr virus) as well as to methods for prophylaxis treatment and methods for treatment of such pathogenic infections.

[0037] We have identified the Iprl gene as a gene involved in innate immunity to infection by cytosolic pathogens (e.g. Mycobacterium tuberculosis and Listeria monocytogene). The lack of Iprl gene expression correlates with high susceptibility to infection. Further, expression of the Iprl transgene in susceptible cells inhibits

multiplication of the cytosolic pathogen as well as induces apoptosis and controls inflammation. Thus, the present invention is directed to methods for determining a patient's susceptibility to infection by monitoring expression of the human homologue to Iprl protein. The present invention is also directed to methods of prophylaxis, as well as to methods for treatment of infection by inducing expression of, or administering, the Iprl protein human homologue.

[0038] In one embodiment, the term "human homologue to Iprl" refers to a DNA sequence that has at least about 55% homology to SEQ ID NO:11, the full length nucleotide sequence of Iprl. In one embodiment, the term "human homologue to Iprl protein" refers to an amino acid sequence that has 55% homology to SEQ ID NO: 12, the full length amino acid sequence of Iprl protein, more preferably at least about 60% homology, still more preferably, at least about 70% homology, even more preferably, at least about 75% homology, yet more preferably, at least about 80% homology, even more preferably at least about 85% homology, still more preferably, at least about 90% homology, and more preferably, at least about 95% homology. In another embodiment, one looks for a domain involved in causing innate immunity to a cytosolic pathogen. As discussed above, the homology is at least about 50% to 100% and all intervals in between (i.e., 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, etc.).

[0039] The full length nucleotide sequence of Iprl (SEQ ID NO: 11), the full length protein sequence of Iprl protein (SEQ ID NO:12), the SpIOO domain coding sequence of Iprl (SEQ ID NO:13), the SpIOO protein domain of Iprl protein (SEQ ID NO:14), the SAND domain coding sequence of Iprl (SEQ ID NO:15), the SAND protein domain of Iprl protein (SEQ ID NO: 16), the nuclear receptor binding motif coding sequence of Iprl (SEQ ID NO: 17), the nuclear receptor binding protein domain of Iprl protein (SEQ ID NO: 18), and the nuclear localization coding sequence of Iprl (SEQ ID NO: 19), the nuclear localization amino acid sequence of Iprl protein (SEQ ID NO:20) are shown below.

[0040] Iprl full length nucleotide sequence agtgtgctgagatcactttcatttttcttttcttgaagcctgactccccgcgggacttcc aaggcagcataa cttcgggtccagactgggctgtcaggcttttccaggaaggatccaggaaccccttaacta atccaggcagtg acctgggagaactcgggagaacccgtggcagccctcagcatccaggatgttcactctgac caaagccttgga aaaggctcttctccagcatttcatatacatgaaggtgaacatcgcctatgccatcaacaa gccattcccctt cttcgaagcgctccgggacaattccttcatcactgagagaatgtacaaggaatctctgga agcctgtcaaaa tctggtccctctgtccaaagtggtgcacaatattctcaccagtctggagcagactttcca cccgtcagtgct gctgacgttgttcagcaaggtcaacctccgggaataccccagcctggtggcaattttcag aagcttcagaaa

cgttggttatacctacgaagagaaaaacagacccccactgaccctgcttgaagacct ggccaacccagcaga agggtgctcccttcagacactgctgccaccaccccgaccccagatatcgctgccaagtca tctgtcctcagc accgagagtctgtgaccccagagcaaccgcacagccaatcattgagatcctggatgagca gcccagtccttc tccccgagctgtgcctctccttggctgcattcaggaaggaaaaaccactccagtgtcctc cagagatcacca gagaaaagataaggaagactctcgagagatgccccacagtccctcaggacccgagtcagt ggtaaaagatga ctctccagcagcaaatgacctggaaatggccagggaagtaccctgcacacctgcaaacaa gaaagcaagaag aaaaaaacgtccgaactggtcaaattccaaaagaagacggcagaaaaaaaagccccgtca agatgagatgat gggagtggcctcacctggacatggagttcaagagaagctcaaggcagtgagcaggaggac tttgtggaaaga tgactcatctacgaacgtgaaggaggtgaccaagacacagagaacaaggatgaggcgtgc ccagacatccaa ttcacaagagatcagcaaagaggcatcaaaaacaagtggtagaaagaggcccagcacagc acgaagaaccac acaagttccagagaagaccaagaatgacgctgtggatttctctcccacactccctgtgac ctgtggtaaggc caaagggactttgttccaagagaaactgaagcaaggagcctcaaaaaagtgcattcagaa tgaggcaggaga ttggctcactgtaaaggaatttttaaatgaagggggaagggccacatcaaaagactggaa gggcgttatacg ttgtaacggggagacattaagacatctggagcagaaaggacttttgttctttacctccaa gagtaaacctca aaagaagggtgcctagcagatgaactcctgacctgatatgtgctcagctcctgtgctcgc tcgcactctgtc tctgtctctctgtctctctctgtctctctctatctctgtctctctctctctctctctctc tctctctctctc tctgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtctgaactttgcctttgagcct ctgagtgtccca gctctgaggtccttcgtagttgtggtcctcatcacaactgacccagcactgtggtacacg ctcacgatgtcc ttgttcacagctgacttacaatggcaaagtgcatgaatactagmcagtaaaaataaagct gttggtgtytct gtgcgtttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO 11)

[0041] Iprl protein Ml length

MFTLTKALξKαLLQHFIYMKVNIAYAINKPFPFFEALRDNSFITERMYKESLξA CQNLVPLSKVλmNILTSL ξQTFHPSVLLTLFSKVNLREYPSLVAIFRSFRNVGYTYEEKNRPPLTLLξDLANPAEG CSLQTLLPPPRPQI SLPSHLSSAPRVCDPRATAQPIIEILDξQPSPSPRAVPLLGCIQEGKTTPVSSRDHQRK DKξDSREMPHSPS GPESWKDDSPAANDLEMAREVPCTPANKKARRKKRPNWSNSKRRRQKKKPRQDEMMGVAS PGHGVQEKLKA VSRRTLWKDDSSTNVKEVTKTQRTRMRRAQTSNSQEISKEASKTSGRKRPSTARRTTQVP EKTKNDAVDFSP TLPVTCGKAKGTLFQEKLKQGASKKCIQNEAGDWLTVKEFLNEGGRATSKDWKGVIRCNG ETLRHLEQKGLL FFTSKSKPQKKGA (SEQ ID NO 12)

[0042] SpIOO domaincoding sequence ofIprl aaagccttggaaaaggctcttctccagcatttcatatacatgaaggtgaacatcgcctat gccatcaacaag ccattccccttcttcgaagcgctccgggacaattccttcatcactgagagaatgtacaag gaatctctggaa gcctgtcaaaatctggtccctctgtccaaagtggtgcacaatattctcaccagtctggag cagactttccac ccgtcagtgctgctgacgttgttcagcaaggtcaacctccgggaataccccagcctggtg gcaattttcaga agcttcagaaacgttggttata (SEQ ID NO 13)

[0043] SpI00 protein domainofIprl

KALEKALLQHFIYMKVNIAYAINKPFPFFEALRDNSFITERMYKESLEACQNLVPLS KWHNILTSLEQTFH PSVLLTLFSKVNLREYPSLVAIFRSFRNVGY (SEQ ID NO 14)

[0044] SAND domain coding sequence of Iprl cccacactccctgtgacctgtggtaaggccaaagggactttgttccaagagaaactgaag caaggagcctca aaaaagtgcattcagaatgaggcaggagattggctcactgtaaaggaatttttaaatgaa gggggaagggcc acatcaaaagactggaagggcgttatacgttgtaacggggagacattaagacatctggag cagaaa (SEQ ID NO 15)

[0045] SAND proteindomain ofIprl

PTLPVTCGKAKGTLFQEKLKQGASKKCIQNEAGDWLTVKEFLNEGGRATSKDWKGVI RCNGETLRHLEQK (SEQ ID NO 16)

[0046] NRB (nuclear receptor binding) motif coding sequence of Iprl cttcagacactgctg ( SEQ ID NO 17 )

[0047] NRB motif of Iprl

LQTLL ( SEQ ID NO 18 )

[0048] NLS (nuclear localization signal) coding sequence of Iprl aagaaagcaagaagaaaaaaacgtccgaactggtcaaattccaaaagaagacggcagaaa aaaaagσcccgt ( SEQ ID NO 19 )

[0049] NLS motif Iprl

KKARRKKRPNWSNSKRRRQKKKPR ( SEQ ID NO 20 )

[0050] The full length nucleotide sequence of SPIlOb (SEQ ID NO:1), the full length protein sequence of SPl 10b (SEQ ID NO:2), the SpIOO domain coding sequence of SPl 10b (SEQ ID NO:3), the SpIOO protein domain of SPl 10b protein (SEQ ID NO:4), the SAND domain coding sequence of SPIlOb (SEQ ID NO:5), the SAND protein domain of SPl 10b protein (SEQ ID NO:6), the nuclear receptor binding motif coding sequence of SPIlOb (SEQ ID NO: 7), the nuclear receptor binding protein domain of SPl 10b protein (SEQ ID NO: 8), the nuclear localization coding sequence of SPIlOb (SEQ ID NO:9), and the nuclear localization amino acid sequence of SPl 10b protein (SEQ ID NO: 10) are shown below.

[0051] SP 11 Ob full length nucleotide sequence, human gttttgcctgctagcatctccctgtaactctcccaatcttgaggagtgatccctgtccca gcccctggaaag gggcaggaacgacaaactcaaagtccaggatgttcaccatgacaagagccatggaagagg ctctttttcagc acttcatgcaccagaagctggggatcgcctatgccatacacaagccatttcccttctttg aaggcctcctag acaactccatcatcactaagagaatgtacatggaatctctggaagcctgtagaaatttga tccctgtatcca gagtggtgcacaacattctcacccaactggagaggacttttaacctgtctcttctggtga cattgttcagtc aaattaacctgcgtgaatatcccaatctggtgacgatttacagaagcttcaaacgtgttg gtgcttcctatg aacggcagagcagagacacaccaatcctacttgaagccccaactggcctagcagaaggaa gctccctccata ccccactggcgctgcccccaccacaaccccctcaaccaagctgttcaccctgtgcgccaa gagtcagtgagc ctggaacatcctcccagcaaagcgatgagatcctgagtgagtcgcccagcccatctgacc ctgtcctgcctc tccctgcactcatccaggaaggaagaagcacttcagtgaccaatgacaagttaacatcca aaatgaatgcgg aagaagactcagaagagatgcGcagcctcctcactagcactgtgcaagtggccagtgaca acctgatcGccc aaataagagataaagaagaccctcaagagatgccccactctcccttgggctctatgccag agataagagata attctccagaaccaaatgacccagaagagccccaggaggtgtccagcacaccttcagaca agaaaggaaaga aaagaaaaagatgtatctggtcaactccaaaaaggagacataagaaaaaaagcctcccaa gagggacagcct catctagacacggaatccaaaagaagctcaaaagggtggatcaggttcctcaaaagaaag atgactcaactt gtaactccacggtagagacaagggcccaaaaggcgagaactgaatgtgcccgaaagtcga gatcagaggaga tcattgatggcacttcagaaatgaatgaaggaaagaggtcccagaagacgcctagtacac cacgaagggtca cacaaggggcagcctcacctgggcatggcatccaagagaagctccaagtggtggataagg tgactcaaagga aagacgactcaacctggaactcagaggtcatgatgagggtccaaaaggcaagaactaaat gtgcccgaaagt ccagatcgaaagaaaagaaaaaggagaaagatatctgttcaagctcaaaaaggagatttc agaaaaatattc accgaagaggaaaacccaaaagtgacactgtggattttcactgttctaagctccccgtga cctgtggtgagg cgaaagggattttatataagaagaaaatgaaacacggatcctcagtgaagtgcattcgga atgaggatggaa cttggttaacaccaaatgaatttgaagtcgaaggaaaaggaaggaacgcaaagaactgga aacggaatatac gttgtgaaggaatgaccctaggagagctgctgaagagtggacttttgctctgtcctccaa gaataaatctca agagagagttaaatagcaagtgaatttctactaccctctcagtcaccatgttgcagactt tccctgtctgga ggctcaccttagagcttctgagtttccaagctctgagtcacctccacatttgggcatggc atcttcaaaaca

attaatttgcatagttaatttgggatggggaagcaaatgactctaaaataaaaatta aatgaaaaagctcaa aaaaaaaaaaaaaaaaaa (SEQ ID NO 1)

[0052] SP 11 Ob, full length protein, human

MFTMTRAMEEALFQHFMHQKLGIAYAIHKPFPFFEGLLDNSIITKRMYMESLEACRN LIPVSRW HNILTQLERTFNLSLLVTLFSQINLREYPNLVTIYRSFKRVGASYERQSRDTPILLEAPT GLAEG SSLHTPLALPPPQPPQPSCSPCAPRVSEPGTSSQQSDEILSESPSPSDPVLPLPALIQEG RSTSV TNDKLTSKMNAEEDSEEMPSLLTSTVQVASDNLIPQIRDKEDPQEMPHSPLGSMPEIRDN SPEPN DPEEPQEVSSTPSDKKGKKRKRCIWSTPKRRHKKKSLPRGTASSRHGIQKKLKRVDQVPQ KKDDS TCNSTVETRAQKARTECARKSRSEEIIDGTSEMNEGKRSQKTPSTPRRVTQGAASPGHGI QEKLQ WDKVTQRKDDSTWNSEVMMRVQKARTKCARKSRSKEKKKEKDICSSSKRRFQKNIHRRGK PKSD TVDFHCSKLPVTCGEAKGILYKKKMKHGSSVKCIRNEDGTWLTPNEFEVEGKGRNAKNWK RNIRC EGMTLGELLKSGLLLCPPRINLKRELNSK (SEQ ID NO 2)

[0053] SpIOO domain coding sequence ofSPl10b, human

Gaggctctttttcagcacttcatgcaccagaagctggggatcgcctatgccatacac aagccatt tcccttctttgaaggcctcctagacaactccatcatcactaagagaatgtacatggaatc tctgg aagcctgtagaaatttgatccctgtatccagagtggtgcacaacattctcacccaactgg agagg acttttaacctgtctcttctggtgacattgttcagtcaaattaacctgcgtgaatatccc aatct ggtgacgatttacagaagcttcaaacgtgttggtgct (SEQ ID NO 3)

[0054] Sp100 proteindomain ofSP110b, human

EALFQHFMHQKLGIAYAIHKPFPFFEGLLDNSIITKRMYMESLEACRNLIPVSRWHN ILTQLER TFNLSLLVTLFSQINLREYPNLVTIYRSFKRVGA (SEQ ID NO 4)

[0055] SAND domain coding sequence ofSP110b, human

Tgttctaagctccccgtgacctgtggtgaggcgaaagggattttatataagaagaaa atgaaaca cggatcctcagtgaagtgcattcggaatgaggatggaacttggttaacaccaaatgaatt tgaag tcgaaggaaaaggaaggaacgcaaagaactggaaacggaatatacgttgtgaaggaatga cccta ggagagctgctgaagagt (SEQ ID NO 5)

[0056] SAND proteindomain ofSP11Ob, human

CSKLPVTCGEAKGILYKKKMKHGSSVKCIRNEDGTWLTPNEFEVEGKGRNAKNWKRN IRCEGMTL GELLKS (SEQ ID NO 6)

[0057] NRB (nuclear receptor binding) motif coding sequence of SP 110b, human ctaggagagctgctg (SEQ ID NO 7)

[0058] NRB motif of SP 11 Ob, human

LGELL (SEQ ID NO 8 )

[0059] NLS (nuclear localization signal)coding sequence of SPl 10b, human

Aagaaaggaaagaaaagaaaaagatgtatctggtcaactccaaaaaggagacataag aaaaaaag cctcccaagagggacagcctcatctagacacggaatccaaaagaagctcaaaagg (SEQ ID NO 9)

[0060] NLS of SP 11 Ob, human

KKGKKRKRCIWSTPKRRHKKKSLPRGTASSRHGIQKKLKR (SEQ ID NO 10)

[0061] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g. , similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. A sequence which is "unrelated" or "nonhomologous" shares less than 40% identity, though preferably less than 25% identity with a sequence of the present application.

[0062] In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity. The term "homology" describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs. The nucleic acid and protein sequences of the present application may be used as a "query sequence" to perform a search against public databases to, for example, identify other family members, related sequences or homologs. Such searches can be performed using the NBLAST and XBLAST programs (version 2. 0) of Altschul, et al. (1990) J MoI. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the application. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389- 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and BLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0063] As used herein, "identity" means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ea., Oxford University Press, New York, 1988; Biocomputing: Informatics and - 14 Genome Projects, Smith, D. W., ea., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 1 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. I Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. MoI. Biol. 215: 403-410 (1990)). The well known Smith Waterman algorithm may also be used to determine identity.

[0064] As used herein, "high stringency" means the following: hybridization at 42° C in the presence of 50% formamide; a first wash at 65 ⁰ C with 2 x SSC containing 1% SDS; followed by a second wash at 65 ⁰ C with 0.1 x SSC.

[0065] Methods for determining a patients susceptibility to infection by a cytosolic pathogen are provided. The methods comprise determining gene expression, e.g. gene expression levels, of the human homologue to Iprl (SEQ ID NO:11) in a biological sample, such as a peripheral blood sample (e.g, in monocytes and macrophages) or a tissue sample (e.g. lung, spleen, heart, liver etc). A positive gene expression indicates that the individual has innate resistance to infection by cytosolic pathogens, while negative gene expression indicates that the patient is highly susceptible to infection.

[0066] According to the present invention, a "baseline" or "control" can include a normal or negative control and/or a disease or positive control, against which a test level of gene expression can be compared. Therefore, it can be determined, based on the control or baseline level of gene expression, whether a sample to be evaluated for the human homologue of Iprl gene expression has a measurable difference or substantially no difference in gene expression, as compared to the baseline level. In one aspect, the baseline control is a indicative of the level of gene expression as expected in the sample of a normal (e.g., healthy, negative control) patient. Therefore, the term "negative control" used in reference to a baseline level of gene expression typically refers to a baseline level of expression from a population of individuals which is believed to be normal (i.e., not having or developing susceptibility to infection of cytosolic pathogens). In some embodiments of the invention, it may also be useful to compare the gene expression in a test sample to a baseline that has previously been established from a patient or population of patients having decreased or lack of Iprl gene expression. Such a baseline level, also referred to herein as a "positive control", refers to a level of gene expression established from one or preferably a population of individuals who had been positively diagnosed with susceptibility to infection with cytosolic pathogens.

[0067] As used herein, a negative gene expression includes a lack or decrease in gene expression levels, the presence of a mutation or polymorphism that results in the expression of a Iprl protein that is non-functional (e.g. has imparired antiviral or antibacterial function), or the expression of a protein that has an altered half life (e.g. degrades faster than a control group).

[0068] In another embodiment, a preferred human homologue of the Iprl protein is SPl 10b , which is expressed from a gene localized to human chromosome 2. Accordingly, methods for determining a patient's susceptibility to infection by a cytosolic pathogen are provided that comprise determining gene expression levels of SPIlOb (SEQ ID NO:1) in a peripheral blood sample (e.g. monocyte or macrophage) or tissue sample (e.g. lung, spleen, heart, liver etc). A positive gene expression indicates that the individual has innate resistance to infection by cytosolic pathogens, while negative gene expression indicates that the patient is highly susceptible to infection.

[0069] As used herein, "tissue sample" refers to a portion, piece, part, segment, or fraction of a tissue which is obtained or removed from an intact tissue of a patient, preferably a human patient.

[0070] As used herein, a "peripheral blood sample" refers to blood drawn from a patient, preferably a human patient. The term peripheral blood sample also encompasses peripheral blood isolates, such as monocytes or macrophages.

[0071] The present invention also encompasses the use of isolates of a tissue sample in the methods of the invention. As used herein, an "isolate" of a sample refers to a material or composition (e. g., a biological material or composition) which has been separated, derived, extracted, purified or isolated from the sample and preferably is substantially free of undesirable compositions and/or impurities or contaminants associated with the sample.

Measuring levels of gene expression

[0072] Gene expression levels can be measured by any means known to those skilled in the art. In the present invention, it is generally preferred to measure mRNA, The most commonly used methods known in the art for the quantification of mRNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNAse protection assays (Hod, Biotechniques 13:852-854 (1992)); and reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)), U.S. Pat. No. 5,322,770, or (RT-AGLCR) as described by R. L. Marshall, et al., PCR Methods and Applications 4: 80-84 (1994). Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS).

[0073] Other known amplification methods which can be utilized herein include, but are not limited to, the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874-1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et

al., Clin. Chem. 42: 9-13 (1996)) and European Patent Application No. 684315; and target mediated amplification, as described by PCT Publication WO 9322461.

[0074] In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a tissue sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. Samples may be stained with haematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.

[0075] One can also look for mutations such as additions, deletions, substitutions in the amino acid or nucleic acid sequence by standard means, for example, DNA sequence analysis can be used as well as assays to detect single nucleotide polymorphisms (SNPs) or single strand conformation polymorphisms (SSCP). Alternatively, immunohistochemical means can be used. In one embodiment, one can use an antibody to the carboxy portion, most preferably, the carboxy terminus of the protein. This will identify mutations resulting in truncation or lack of expression. Mutation can also include modifications to the nucleic acid in coding and non-coding portions such as methylation or ethylation can prevent expression of the gene.

Reverse Transcriptase PCR (RT-PCR)

[0076] Of the techniques listed above, the most sensitive and most flexible quantitative method is RT-PCR, which can be used to compare mRNA levels in different sample populations, with or without drug treatment, to characterize patterns of gene expression and to discriminate between closely related mRNAs. The first step is the isolation of mRNA from a target sample. The starting material is typically total RNA isolated from human tissue. Thus, RNA can be isolated from a variety of tissues, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, etc. The mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin-fixed) tissue samples.

[0077] General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and

Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995). In particular, RNA isolation can be performed using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions and Paraffin Block RNA Isolation Kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test).

[0078] As RNA cannot serve as a template for PCR, the first step in gene expression profiling by RT-PCR is the reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AMV-RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling. For example, extracted RNA can be reverse-transcribed using a GeneAmp RNA PCR kit (Perkin Elmer, CA, USA), following the manufacturer's instructions. The derived cDNA can then be used as a template in the subsequent PCR reaction.

[0079] Although the PCR step can use a variety of thermostable DNA- dependent DNA polymerases, it typically employs the Taq DNA polymerase, which has a 5 '-3' nuclease activity but lacks a 3 '-5' proofreading endonuclease activity. Thus, TaqMan™ PCR typically utilizes the 5'-nuclease activity of Taq or Tth polymerase to hydrolyze a hybridization probe bound to its target amplicon, but any enzyme with equivalent 5' nuclease activity can be used.

[0080] Two oligonucleotide primers are used to generate an amplicon typical of a PCR reaction. A third oligonucleotide, or probe, is designed to detect nucleotide sequence located between the two PCR primers. The probe is non-extendible by Taq DNA polymerase enzyme, and is labeled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together as they are on the probe. During the amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is free from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule

synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0081] TaqMan™ RT-PCR can be performed using commercially available equipment, such as, for example, ABI PRISM 770O ^Tm Sequence Detection System ⁷ " ¹ (Perkin-Ehner-Applied Biosystems, Foster City, CA, USA), or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In a preferred embodiment, the 5' nuclease procedure is run on a realtime quantitative PCR device such as the ABI PRISM 770O ^Tm Sequence Detection System ^1" " ¹ .

[0082] The system consists of a thermocycler, laser, charge-coupled device (CCD), camera and computer. The system amplifies samples in a 96-well format on a thermocycler. During amplification, laser-induced fluorescent signal is collected in realtime through fiber optics cables for all 96 wells, and detected at the CCD. The system includes software for running the instrument and for analyzing the data.

[0083] 5 '-Nuclease assay data are initially expressed as Ct, or the threshold cycle. As discussed above, fluorescence values are recorded during every cycle and represent the amount of product amplified to that point in the amplification reaction. The point when the fluorescent signal is first recorded as statistically significant is the threshold cycle (Ct).

[0084] To minimize errors and the effect of sample-to-sample variation, RT- PCR is usually performed using an internal standard. The ideal internal standard is expressed at a constant level among different tissues, and is unaffected by the experimental treatment. RNAs most frequently used to normalize patterns of gene expression are mRNAs for the housekeeping genes glyceraldehyde phosphate- dehydrogenase (GAPDH) and P-actin.

[0085] A more recent variation of the RT-PCR technique is the real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (i.e., TaqMan ^Tm probe). Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR. For further details see, e.g. Held et al., Genome Research 6:986-994 (1996).

[0086] The steps of a representative protocol for profiling gene expression using fixed, paraffin-embedded tissues as the RNA source, including mRNA isolation, purification, primer extension and amplification are given in various published journal articles for example: T.E. Godfrey et al,. J. Molec. Diagnostics 2: 84-91, 2000; K. Specht et al., Am. J. Pathol. 158, 419.29, 2001. Briefly, a representative process starts with cutting about 10 um thick sections of paraffin-embedded tumor tissue samples. The RNA is then extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and RNA is reverse transcribed using gene specific promoters followed by RT-PCR.

[0087] According to one aspect of the present invention, PCR primers and probes are designed based upon exon and intron sequences present in the homologous Iprl gene (e.g. SPIlOb) to be amplified. In this embodiment, the first step in the primer/probe design is the delineation of intron sequences within the genes. This can be done by publicly available software, such as the DNA BLAT software developed by Kent, WJ., Genome Res. 12(4):656-64 (2002), or by the BLAST software including its variations. Subsequent steps follow well established methods of PCR primer and probe design.

[0088] In order to avoid non-specific signals, it is important to mask repetitive sequences within the introns when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program available on-line through the Baylor College of Medicine, which screens DNA sequences against a library of repetitive elements and returns a query sequence in which the repetitive elements are masked. The masked intron sequences can then be used to design primer and probe sequences using any commercially or otherwise publicly available primer/probe design packages, such as Primer Express (Applied Biosystems); MGB assay-by-design (Applied Biosystems); Primer3 (Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386). The most important factors considered in PCR primer design include primer length, melting temperature (Tm), and G/C content, specificity, complementary primer sequences, and 3 '-end sequence. In general, optimal PCR primers are generally 17-30

bases in length, and contain about 20-80%, such as, for example, about 50-60% G+C bases. Tm ¹ S between 50 and 80 ⁰ C, e.g. about 50 to 70 ⁰ C are typically preferred.

[0089] For further guidelines for PCR primer and probe design see, e.g. Dieffenbach, CW. et al., "General Concepts for PCR Primer Design" in: PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, pp. 133- 155; Innis and Gelfand, "Optimization of PCRs" in: PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pp. 5-11; and Plasterer, T.N. Primerselect: Primer and probe design, Methods MoI. Biol. 70:520-527 (1997), the entire disclosures of which are hereby expressly incorporated by reference.

Microarrays

[0090] Gene expression can also be identified, or confirmed using the microarray technique. Methods of preparing DNA arrays and their use are well known in the art. (See, for example U.S. Patent Nos: 6,618,6796; 6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. 1995 Science 20:467-470; Gerhold et al. 1999 Trends in Biochem. Sci. 24, 168-173; and Lennon et al. 2000 Drug discovery Today 5: 59-65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858).

[0091] The expression of homologous genes can be measured in either fresh or paraffin-embedded tissue, using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are plated, or arrayed, on a microchip substrate. The arrayed sequences are then hybridized with specific cDNA probes from tissues of interest. Just as in the RT-PCR method, the source of mRNA typically is total RNA isolated from the tissue to be tested, and corresponding control and reference samples. mRNA can be extracted, for example, from frozen or archived paraffin-embedded and fixed (e.g. formalin fixed) tissue samples, which are routinely prepared and preserved in everyday clinical practice.

[0092] In a specific embodiment of the microarray technique, PCR amplified inserts of cDNA clones are applied to a substrate in a dense array. Preferably at least 10,000 nucleotide sequences are applied to the substrate. The microarrayed genes, immobilized on the microchip at 10,000 elements each, are suitable for hybridization under stringent conditions.

[0093] Fluorescently labeled cDNA probes may be generated through incorporation of fluorescent nucleotides by reverse transcription of RJSfA extracted from tissues of interest. Labeled cDNA probes applied to the chip hybridize with specificity to each spot of DNA on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by another detection method, such as a CCD camera. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance. With dual color fluorescence, separately labeled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels (Schena et al, Proc. Natl. Acad. Sci. USA 93(2): 106-149 (1996)).

[0094] Microarray analysis can be performed by using commercially available equipment and/or commercially available microarrays, following manufacturer's protocols, such as by using the Affymetrix GenChip technology, or Incyte's microarray technology.

[0095] Serial Analysis of Gene Expression (SAGE) Serial analysis of gene expression (SAGE) is a method that allows the simultaneous and quantitative analysis of a large number of gene transcripts, without the need of providing an individual hybridization probe for each transcript. First, a short sequence tag (about 10-14 bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is obtained from a unique position within each transcript. Then, many transcripts are linked together to form long serial molecules, that can be sequenced, revealing the identity of the multiple tags simultaneously. The expression pattern of any population of transcripts can be quantitatively evaluated by determining the abundance of individual tags, and identifying the gene corresponding to each tag. For more details see, e.g. Velculescu et al., Science 270:484-487 (1995); and Velculescu et al., Cell 88:243-51 (1997).

MassARRAY Technology

[0096] The MassARRAY (Sequenom, San Diego, California) technology is an automated, high-throughput method of gene expression analysis using mass spectrometry (MS) for detection. According to this method, following the isolation of RNA, reverse transcription and PCR amplification, the cDNAs are subjected to primer extension. The cDNA-derived primer extension products are purified, and dispensed on a chip array that is pre-loaded with the components needed for MALTI-TOF MS sample preparation. The various cDNAs present in the reaction are quantitated by analyzing the peak areas in the mass spectrum obtained.

Analysis of protein levels

[0097] Protein expression can also be measured as a means for determining gene expression levels. In one preferred method, the protein product of the expressed gene to be monitored is detected using an "antibody-based binding moiety" or "antibody" that specifically binds the protein product.

[0098] The term "antibody-based binding moiety" or "antibody" includes immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, e.g., molecules that contain an antigen binding site which specifically binds (immunoreacts with) to the protein to be detected. The term "antibody-based binding moiety" is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with the protein to be detected. Antibodies can be fragmented using conventional techniques. Thus, the term includes segments of proteolytically-cleaved or recombinantly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non limiting examples of such proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, dAbs and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker. The scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites. Thus, "antibody-base binding moiety" includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies. The term "antibody-base binding moiety" is further intended to include humanized antibodies, bispecific antibodies, and chimeric molecules having at least one antigen binding determinant derived from an antibody molecule. In a preferred embodiment, the antibody-based binding moiety detectably labeled.

[0099] "Labeled antibody", as used herein, includes antibodies that are labeled by a detectable means and include, but are not limited to, antibodies that are enzymatically, radioactively, fluorescently, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS.

[00100] In the diagnostic methods of the invention that use antibody based binding moieties for the detection of gene expression levels, the level of protein present in the samples (or gene expression levels) correlates to the intensity of the signal emitted from the detectably labeled antibody.

[00101] In one preferred embodiment, the antibody-based binding moiety is detectably labeled by linking the antibody to an enzyme. The enzyme, in turn, when exposed to it's substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Chemiluminescence is another method that can be used to detect an antibody-based binding moiety.

[OC 102] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays. The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audoradiography. Isotopes which are particularly useful for the purpose of the present invention are ³ H, ¹³¹ 1, ³⁵ S, ¹⁴ C, and preferably ¹²⁵ I.

[00103] It is also possible to label an antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[00104] An antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵² Eu ₅ or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[00105] An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

[00106] In one embodiment, levels of protein are measured by contacting the sample with an antibody-based binding moiety that specifically binds to the protein, or to a fragment of. Formation of the antibody-antigen complex is then detected as a measure of the protein levels.

[00107] Means for detection of protein levels are well known to those skilled in the art. For example, levels of protein can be detected by immunoassays, such as enzyme linked immunoabsorbant assay (ELISA), radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Western blotting, or immunohistochemistry. Immunoassays such as ELISA or RIA, which can be extremely rapid, are more generally preferred.

[00108] There are different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A. Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904. Sandwich ELISA and Competitive ELISA, well known to those in the art, are also useful in methods of the invention.

[00109] Antibody arrays or protein chips can also be employed, see for example U.S. Patent Application Nos: 20030013208A1; 20020155493A1; 20030017515 and U.S. Patent Nos: 6,329,209; 6,365,418, which are herein incorporated by reference in their entirety.

Analysis of protein degradation

[00110] Also encompassed in the present invention is an analysis of protein degradation of the human homologue to Iprl . It has been found that in some instances, the Iprl protein degrades faster in individuals susceptible to infection by cytosolic pathogens. Thus, methods to determine the rate of Iprl protein degradation in an individual are encompassed. At any given time the cellular content of a protein is believed to be regulated by a combination of its synthetic and degradation rates, with each protein having a characteristic pattern of synthesis and degradation to ensure proper cellular function. Methods for analyzing human homologue Iprl protein degradation are thus encompassed and are known to those of skill in the art.

[00111] Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[00112] Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci. 663:48-62 (1992)).

[00113] Any of the above modifications may alter the degradation rate of the human homologue to IprI. Thus, methods to detect such alterations in a test sample, as compared to a control group, are encompassed. In analyzing protein degradation rates, it may be necessary to compare different timepoints in an individual. For example, a test biological sample may be analyzed and compared to a control group at a first time point and a second time point. An increase in the rate of protein degradation in the test sample (e.g. between the first and second time point) as compared to the control group is indicative of susceptibility to infection by cytosolic pathogens.

[00114] As discussed above, individuals having polymorphism or mutation and/or lower levels of expression of the Iprl homolog are at greater risk for infection by a cytosolic pathogen. Identifying such individuals can permit them to take precautions to avoid infection such as staying away from infected individuals, and/or preventative chemotherapy/immunotherapy, etc. Also, identifying changes in such expression can be used to screen individuals that may be infected or at an increased risk for infection. Mutations and/or polymorphisms that reduce the function of the gene so much so as to effectively render the gene non-functional (e.g. unable to render antibacterial or antiviral functions) are encompassed. Thus, methods for analyzing such mutations or polymorphisms are encompassed.

[00115] A polymorphism is typically defined as two or more alternative sequences, or alleles, of a gene in a population. A polymorphic site is the location in the gene at which divergence in sequence occurs. Examples of the ways in which polymorphisms are manifested include restriction fragment length polymorphisms, variable number of tandem repeats, hypervariable regions, minisatellites, di- or multi- nucleotide repeats, insertion elements and nucleotide deletions, additions or substitutions. The first identified allele is usually referred to as the reference allele, or the wild type.

[00116] A patient's biological sample, such as a tissue or peripheral blood sample, may be analyzed for levels of TNF-α and IL-10 in determining susceptibility to infection by cytosolic pathogens. Methods for the analysis of TNF-α and IL-10 are known to those of skill in the art. For example, TNF-α and IL-10 antibodies or antibody fragments may be utilized in immunoassays such as ELISA, immunohisto- or immunocytochemistry or western blot. For a general review of immunoassays, see also Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); and Basic and Clinical Immunology 7th Edition, Stites &

Terr, eds. (1991). It will be appreciated that monitoring of TNF- α and IL-IO levels can also be accomplished through other means well known to those skilled in the art which are not listed herein. For example PCR (polymerase chain reaction) techniques can be used to detect mRNA or DNA expression. Additionally, flow cytometry can be used to detect intracellular TNF- α and IL-IO.

[00117] In a preferred embodiment, an ELISA is utilized. An "ELISA" is an enzyme-linked immunosorbent assay. In typical embodiments herein, an antigen (e.g., TNF-α or IL-10 present in a patient and which is optionally linked or adsorbed onto a surface) is detected through specific interaction with a labeled antibody (or antibodies) specific for the antigen. Those of skill in the art will be familiar with various variations of ELISA which are optionally utilized in embodiments herein.

[00118] Polyclonal murine antibodies to TNF are disclosed by Cerami et al. (EPO Patent Publication 0212489, Mar. 4, 1987). Rubin et al. (EPO Patent Publication 0218868, Apr. 22, 1987) discloses murine monoclonal antibodies to human TNF, the hybridomas secreting such antibodies, methods of producing such murine antibodies, and the use of such murine antibodies in immunoassay of TNF. Yone et al. (EPO Patent Publication 0288088, Oct. 26, 1988) discloses anti-TNF murine antibodies, including mAbs, and their utility in immunoassay diagnosis of pathologies.

[00119] Detection kits for IL-IO determination are available from, e.g., R & D Systems, Inc. (Minneapolis, Minn.), Endogen (Pierce Biotechnology, Inc., Rockford, 111.), and Biosource International (Camarillo, Calif.) among others. For example, R & D Systems' Quantikine[R] HS Human IL-10 assay has found a sample serum/plasma range value of between "non-detectable" and 5.16 pg/mL (n=40, mean of detectable samples=2.00). See, kit package insert.

[00120] A method for treating a patient infected with a cytosolic pathogen is also provided. The method comprises administering to said patient an effective amount of a compound or agent that upregulates expression of a human homologue of Iprl (e.g. Si ³ UOo (SEQ ID NO:!)).

[00121 ] Any compound or agent that upregulates expression of a human homologue of Iprl can be used in methods of the invention. In one preferred embodiment, the agent that upregulates gene expression is interferon γ, interferon α, or

interferon β. The portion of the human homologue of Iprl that upregulates TNF-α production is particularly useful.

[00122] The methods of the invention are useful both for patients that innately lack expression of the Iprl human homologue (e.g. . SPIlOb) and for patients that suffer a decrease in expression, such as those that have been chronically infected with a pathogen. For example, in tuberculosis, the expression of the Iprl gene is initially upregulated upon infection with the pathogen, however, during chronic infection expression of the Iprl gene is downregulated. Accordingly, compounds or agents that prevent downregulation of Iprl gene expression are also useful for treating patients. In addition, compounds that block inhibitors of Iprl human homologue activity are also contemplated for use in treatment of infection by cytosolic pathogens.

[00123] Compounds or agents that induce expression of a human homologue of Iprl (e.g. SPIlOb (SEQ ID NO: I)) can be determined using assays well known to those skilled in the art. For example, cell based assays using reporter gene constructs containing the Iprl promoter (or Iprl human homologue promoter) can be used to identify compounds which modulate expression of the Iprl gene and its human homologue.

[00124] The present invention also provides assays for screening for compounds or agents that prevent downregulation of the Iprl gene (or its human homologue). For example, gene expression can be monitored in cells that have been infected with tuberculosis, in the presence or absence of a test compound.

[00125] The present invention also provides assays for testing of compounds or agents that upregulate Iprl function or activity. These compounds or agents may activate the Iprl mediated pathway. The assays involve the detection of gene expression levels of genes that are modulated by Iprl (or its human homologue). For example, expression of genes that are directly regulated by Iprl or genes whose gene products interact with Iprl (or its human homologue).

[00126] The "compounds" or "agents" of the invention can be DNA, RNA, a small organic molecule, a natural product, protein (e.g., antibody), peptide or peptidomimetic. Compounds that induce gene expression, or that prevent downregulation of expression, can be identified, for example, by screening libraries or collections of

molecules, such as, the Chemical Repository of the National Cancer Institute, as described herein or using other suitable methods.

[00127] Another source of compounds is combinatorial libraries which can comprise many structurally distinct molecular species. Combinatorial libraries can be used to identify lead compounds or to optimize a previously identified lead. Such libraries can be manufactured by well-known methods of combinatorial chemistry and screened by suitable methods, such as the methods described herein.

[00128] The molecular pathway responsible for the effects of Iprl protein on pathogenic infection is presently unknown. However, Iprl and the human SPl 10 proteins contain motifs that are involved in protein-protein interactions (SpIOO domain) ¹⁹ ' ²⁰ , chromatin binding (SAND domain) ²¹ ' ²² , nuclear localization signal (NLS) and the nuclear receptor binding (NRB) motif LXXLL. Recent evidence suggests that human SPl 10 protein, may function as a nuclear hormone receptor transcriptional cofactor ¹⁸ and directly bind the retinoic acid receptor ²³ . Signaling through nuclear receptors, such as the corticosteroid receptor, retinoic acid receptor, PPARs and vitamin D plays an important role in control of various aspects of the macrophage life cycle, including differentiation, activation, response to pathogens and apoptosis ²⁴ .

[00129] The present invention provides in vivo animal models useful for identification of members of the Iprl pathway. In particular, we have generated transgenic mice that expresses a full copy of the Iprl cDNA on a susceptible C3HeB/FeJ background in a macrophage specific manner under control of the human scavenger receptor A promoter (SR-A) (See Example 1). Expression of a single gene of Iprl restored key functions of macrophages related to the pathogenisis of tuberculosis. This mouse model can be used in methods of differential expression analysis. We have also generated congenic mice, C3H.B6-sstl (See Example 1) that are useful for for identification of members of the Iprl pathway. Identification of pathway members may lead to novel targets for "Iprl activation".

[00130] The present invention further provides in vivo animal models for screening of compounds to treat tuberculosis infection. Detailed mechanistic studies of pathogenesis of lung tuberculosis and its genetic control have been limited by the fact that in mouse models of MTB infection, necrotic lesions in the lungs are rarely found unless the mouse is rendered systemically immunodeficient. We have discovered that non-

immunodeficient C3HeB/FeJ mice (See Figure 8) have the characteristic necrotic lesion cavities present in human lung tissue as a result of tuberculosis infection. The sstl susceptible congenic mouse B6.C3H-sstl develops encapsulated necrotic lung lesions that closely resemble tuberculosis cavities in human lungs. Thus, these mice are extremely useful in studying cytosolic infections such as MTB in vivo, for they are the only model currently known that resembles human infection and does not require immunosuppression. These C3H mice can be used to screen for compounds or agents that penetrate the cavity and kill the bacteria inside the cavity. Furthermore, these mice can be used to study the progression of cytosolic infections,

[00131] In addition, the C3H mice can be used to generate transgenic mice that express human Iprl homologs. These mice can be used to screen for and to test compounds that modulate Iprl expression or function.

[00132] A C3H.B6-sstl (sstl resistant) congenic mouse strain has been generated by transferring an approximately 20 cM chromosomal segment containing the sstl resistant allele on C3HeB/FeJ genetic background and as described in Example 1. This strain express the Iprl gene under control of its own promoter and, therefore, the Iprl gene expression is regulated in a physiologically relevant manner. Along with the Iprl transgenic mice, which express the Iprl gene in macrophage-specific manner, the C3H.B6-sstl congenic strain is useful for analysis of the Iprl gene function, characterization of Iprl -mediated effects on immune functions and identification of other proteins and genes, whose function is affected by the Iprl gene.

[00133] We have further generated a congenic strain that does not express the Iprl gene, C57BL/6J.C3H-sstl (susceptible) (See Example II). This strain is particularly useful for studies of Iprl gene function. For example, this strain can be used for generation of transgenic mice that express human homologue of the Iprl gene to study its function, as well as for screening and testing of potential activators and inhibitors of the human Iprl homologue in a whole animal. In addition, these mice are more resistant to infection by cytosolic pathogens (e.g. tuberculosis) than the C3HeB/FeJ parental mice, which also do not express the Iprl gene. Thus, this mouse model can be used to identify compensatory immune mechanisms that function in the absence of the Iprl gene product.

[00134] The C57BL/6J.C3H-sstl strain is particularly useful as an animal model for chronic pulmonary tuberculosis because the mice develop tuberculosis cavities in their

lungs. Accordingly, this mouse model can be used for screening of anti-tuberculosis drugs, for testing of treatment regiments and vaccines, as well as for the development of methods of immunomodulation.

[00135] The invention also provides a method for treating a patient infected with a cytosolic pathogen comprising administering to said patient an effective amount of Iprl protein human homologue (e.g. SPl 10b protein).

[00136] Polypeptides (e.g. SPl 10b protein), fragments and derivatives thereof can be obtained by any suitable method. For example, polypeptides can be produced using conventional recombinant nucleic acid technology such as DNA or RNfA, preferably DNA. Guidance and information concerning methods and materials for production of polypeptides using recombinant DNA technology can be found in numerous treatises and reference manuals. See, e.g., Sambrook et al, 1989, Molecular Cloning - A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press; Ausubel et al. (eds.), 1994, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.; Innis et al. (eds.), 1990 PCR Protocols, Academic Press.

[00137] Alternatively, polypeptides (e.g. SPl 10b) or fragments thereof can be obtained directly by chemical synthesis, e.g., using a commercial peptide synthesizer according to vendor's instructions. Methods and materials for chemical synthesis of polypeptides are well known in the art. See, e.g., Merrifield, 1963, "Solid Phase Synthesis," J. Am. Chem. Soc. 83:2149 -2154.

[00138] A preformed polypeptide (e.g. SPl 10b) can be introduced into a cell using conventional techniques for transporting proteins into intact cells, e.g., by fusing the polypeptide to the internalization peptide sequence derived from Antennapedia (Bonfanti et al., Cancer Res. 57:1442-1446) or to a nuclear localization protein such as HIV tat peptide (U.S. Pat. No. 5,652,122). In some embodiments, when using SPl 10b polypeptide, SP 140 is concurrently introduced or co-expressed in a cell so that SP 140 can recruit SPl 10b into nuclear bodies. See WO 02008383, which is herein incorporated by reference in it's entirety.

[00139] Alternatively, the polypeptide (e.g. SPl 10b) can be expressed in the cell following introduction of a DNA encoding the protein (e.g. SPl 10b -encoding DNA), e.g. in a conventional expression vector or by a catheter or by ex vivo transplants.

[00140] In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Roller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

[00141] In a specific embodiment, viral vectors that contain nucleic acid sequences encoding the Iprl human homologues of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the Iprl human homologue to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,

Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

[00142] Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Another preferred viral vector is a pox virus such as a vaccinia, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91 :225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In one preferred embodiment, adenovirus vectors are used. In another embodiment, lentiviral vectors are used, such as the HIV based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

[00143] Use of Adeno-associated virus (AAV) vectors are also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

[00144] Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

[00145] U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA

into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469 (which are herein incorporated by reference) provide methods for delivering DNA-cationic lipid complexes to mammals. Such cationic lipid complexes or nanoparticles can also be used to deliver protein. The protein will preferably contain a nuclear localization sequence.

[00146] For general reviews of the methods of gene and protein therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu ₅ Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191- 217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

Delivery of Therapeutics (Compound)

[00147] The compounds of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including, but not limited to, improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.

[00148] It should be noted that it can be administered as a compound or as a pharmaceutically acceptable salt and can be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.

[00149] It is also noted that humans are treated generally longer than the mice or other experimental animals exemplified herein, which treatment has a length proportional to the length of the disease process and drug effectiveness. The doses may be single doses or multiple doses over a period of several days, but single doses are preferred.

[00150] When administering the compound of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

[00151] Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Non-aqueous vehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions. Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol and sorbic acid. In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.

[00152] Sterile injectable solutions can be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various of the other ingredients, as desired.

[00153] A pharmacological formulation of the present invention can be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention can be administered parenterally to the patient in the form of slow- release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, vectored delivery, iontophoretic, polymer matrices, liposomes, and microspheres. Examples of delivery systems useful in the present invention include those

presented in U.S. Pat. Nos: 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233; 4,447, 224; 4,439,196 and 4,475,196. Other such implants, delivery systems, and modules are well known to those skilled in the art.

[00154] A pharmacological formulation of the compound utilized in the present invention can be administered orally to the patient. Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable. Known techniques that deliver the compound orally or intravenously and retain the biological activity are preferred.

[00155] In another embodiment, the pharmaceutically acceptable formulations comprise lipid-based formulations. Any of the known lipid-based drug delivery systems can be used in the practice of the invention. For instance, multivesicular liposomes, multilamellar liposomes and unilamellar liposomes can all be used so long as a sustained release rate of the encapsulated active compound can be established. Methods of making controlled release multivesicular liposome drug delivery systems are described in PCT Application Publication Nos: WO 9703652, WO 9513796, and WO 9423697, the contents of which are incorporated herein by reference.

[00156] The composition of the synthetic membrane vesicle is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.

[00157] Examples of lipids useful in synthetic membrane vesicle production include phosphatidylglycerols, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, sphingolipids, cerebrosides, and gangliosides, with preferable embodiments including egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidyleholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

[00158] In preparing lipid-based vesicles containing an active compound such variables as the efficiency of active compound encapsulation, labiality of the active compound, homogeneity and size of the resulting population of vesicles, active compound-to-lipid ratio, permeability, instability of the preparation, and pharmaceutical acceptability of the formulation should be considered.

[00159] Prior to introduction, the formulations can be sterilized, by any of the numerous available techniques of the art, such as with gamma radiation or electron beam sterilization.

[00160] When the agents are delivered to a patient, they can be administered by any suitable route, including, for example, orally (e.g., in capsules, suspensions or tablets) or by parenteral administration. Parenteral administration can include, for example, intramuscular, intravenous, intraarticular, intraarterial, intrathecal, subcutaneous, or intraperitoneal administration. The agent can also be administered orally, transdermally, topically, by inhalation (e.g., intrabronchial, intranasal, oral inhalation or intranasal drops) or rectally. Administration can be local or systemic as indicated. Agents can also be delivered using viral vectors, which are well known to those skilled in the art.

[00161] The pharmaceutically acceptable formulations can be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps.

[00162] Preferred approaches for sustained delivery include use of a polymeric capsule, a minipump to deliver the formulation, a bloerodible implant, or implanted transgenic autologous cells (as described in U.S. Patent No. 6,214,622). Implantable infusion pump systems (such as Infusaid; see such as Zierski, J. et al ,1988; Kanoff, R.B., 1994) and osmotic pumps (sold by Alza Corporation) are available in the art. Another mode of administration is via an implantable, externally programmable infusion pump. Suitable infusion pump systems and reservoir systems are also described in U.S. Patent No. 5,368,562 by Blomquist and U.S. Patent No. 4,731,058 by Doan, developed by Pharmacia Deltec Inc.

[00163] It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the active compound and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

[00164] The amount of agent administered to the individual will depend on the characteristics of the individual, such as general health, age, sex, body weight and

tolerance to drugs as well as the degree, severity and type of rejection. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Typically, an effective amount can range from about 0.1 mg per day to about 100 mg per day for an adult. Preferably, the dosage ranges from about 1 mg per day to about 100 mg per day.

[00165] Antibodies and antigen-binding fragments thereof, particularly human, humanized and chimeric antibodies and antigen-binding fragments can often be administered less frequently than other types of therapeutics. For example, an effective amount of such an antibody can range from about 0.01 mg/kg to about 5 or 10 mg/kg administered daily, weekly, biweekly, monthly or less frequently.

Kits

[00166] The present invention is also directed to commercial kits for the detection of Iprl gene human homologue expression (e.g. SPIlOb) . The kit can be in any configuration well known to those of ordinary skill in the art and is useful for performing one or more of the methods described herein for the detection of gene expression. The kits are convenient in that they supply many if not all of the essential reagents for conducting an assay for the detection of a Iprl homologue (e.g. SPl 10b) in a sample. In addition, the assay is preferably performed simultaneously with a standard or multiple standards that are included in the kit, so that the results of the test can be quantitated or validated.

[00167] The kits include a means for detecting gene expression levels such as antibodies, or antibody fragments, which selectively bind to the Iprl human homologue protein or DNA nucleotide primers suitable for use in methods of detecting niRNA.

[00168] In other embodiments, the assay kits may employ (but are not limited to) the following techniques: competitive and non-competitive assays, radioimmunoassay (RIA), bioluminescence and chemiluminescence assays, fluorometric assays, sandwich assays, immunoradiometric assays, dot blots, enzyme linked assays including ELISA, and immunocytochemistry. For each kit the range, sensitivity, precision, reliability, specificity and reproducibility of the assay are established by means well known to those skilled in the art. The above assay kits would further provide instructions for use.

[00169] It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the

invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those skilled in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications and publications cited herein are incorporated herein by reference.

Definitions

[00170] The following definitions are provided for specific terms which are used in the following written description.

[00171] As used herein, the term "administering" to a patient includes dispensing, delivering or applying an active compound in a pharmaceutical formulation to a subject by any suitable route for delivery of the active compound to the desired location in the subject, including delivery by either the parenteral or oral route, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.

[00172] As used herein, "effective amount" of an agent is an amount sufficient to achieve a desired therapeutic or pharmacological effect, such as an amount sufficient to inhibit the multiplication of a pathogen (e.g. tuberculosis). An effective amount of an agent as defined herein may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the agent to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects of the active compound are outweighed by the therapeutically beneficial effects.

[00173] A therapeutically effective amount or dosage of an agent may range from about 0.001 to 30 mg/kg body weight, with other ranges of the invention including about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, and 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the infection , previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an active compound can include a single treatment or a series of treatments. In one example, a subject is treated with an agent in the range of between about 0.1 to 20 mg/kg body

weight, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of an agent used for treatment may increase or decrease over the course of a particular treatment.

[00174] As used herein, the term "patient" or "subject" or "animal" or "host" refers to any mammal. The patient is preferably a human, but can also be a mammal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, fowl, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

EXAMPLES

[00175] Animals.

[00176] C57BL/6J, C3HeB/FeJ, C3H/HeJ, C3H/HeOuJ, and C3H/HeSnJ inbred mice were obtained from The Jackson Laboratory (Bar Harbor, Maine). The congenic CSRBS-sst 1 (sstl ^R ), B6.C3H-.wtf; and transgenic C3H-TgN(SRA-/jσri) mouse strains were generated in our laboratory. The CSRBβ-sstl congenic mice were obtained by introgression of an approximately 20 cM segment of B6-derived chromosome 1 with a proximal recombination breakpoint between DlMit215 (47 cM) and DlMit334 (49 cM) and a distal limit between DlMitl87 (64 cM) and DlMit200 (75 cM) on the C3HeB/FeJ genetic background using 10 backcrosses. Importantly, the congenic interval transferred from the B6 resistant background did not include the Slcllal gene (formerly known as Nrampl), which is located at 39.2 cM. Thus, the sstl resistant congenic mouse strain CSRBβ-sstl carries the same allele of the Nrampl as the parental sstl susceptible C3HeB/FeJ mice. The B6.C3R-sstl mice were obtained by transferring the sstl susceptible allele on the B6 genetic background using 10 backcrosses. The transgenic C3H~TgN(SRA-ipr7) mice were established by expressing the C57BL/6J-derived Iprl gene under the control of a macrophage specific Scavenger Receptor A (SRA) promoter on the C3HeB/FeJ genetic background.

[00177] Infection of mice with MTB.

[00178] For i.v. infection 1 x 10 ⁵ of live MTB were injected via tail vein in 100 μl of PBS. Aerosol infections were performed using aerosol apparatus manufactured by the College of Engineering Shops at the University of Wisconsin (Madison, WI). Mice were exposed to aerosol for 20 min, which resulted in the deposition of 15 to 30 CFU per

mouse. Mice were sacrificed using halothane anesthesia. Organs were homogenized in PBS containing 0.05% Tween 80 and serial 10-fold dilutions were cultured on 7H10 agar enriched with 10% OADC (Difco, MI) for 3 weeks at 37 ⁰ C. Virulent Mycobacterium tuberculosis (MTB) Erdman strain and avirulent vaccine strain of Mycobacterium bovis BCG (BCG) Pasteur strain were used some studies.

[00179] Analysis ofchemokine mRNA expression in vivo [00180] To isolate total RNA from the organs of MTB-infected mice, organs were snap frozen in liquid nitrogen and stored at -80 ⁰ C. Frozen organs were homogenized in cold TRJZOL reagent (Invitrogen) using tissue homogenizer PRO 200 (Pro Scientific, Inc). Total RNA was isolated according to manufacter's instructions and then treated with RNAase-free DNAse and re-purified using RNeasy Mini Kit (Qiagen). Total RNA was reverse transcribed using Superscript II, and amplified with pairs of chemokine-specific primers, which were designed using Primer 3.0 software (primer sequences are available upon request). For semi-quantitative analysis PCR was performed in the presence of ³² P-labelled dCTP (2 μCi per 40 μl reaction). QuantumRNA 18S Internal Standards kit (Ambion, TX) was used to simultaneously amplify 18S rRNA. Radiactively labelled PCR products were separated on acrylamide gel. The gels were dired and incorporated radioactivity was quantitated by Phospholmager Storm 860 (molecular Dynamics) using ImageQuant software.

[00181] Isolation of murine bone marrow-derived macrophages (BMDM) in vitro.

[00182] BMDM were isolated from femurs and tibias of male C3H, C3η..B6. sstl and C3H.Tg (Iprl) mice (6 to 8 weeks old) and depleted of RBC and neutrophils by gradient centrifugation using Nycodenz. Cells were grown in a complete culture medium 1:1 mix of Dulbecco's modified Eagle's medium (DMEM) and Ham F- 12 (CellGro, MediaTech, Inc., Herndon, VA) supplemented with 10% of Fetal Calf Serum (FCS, Hyclone Logan, UT), rIL-3 at 1 ng/ml (Sigma, St. Louis, MO) and 20% L-929 fibroblast- conditioned medium as a source of macrophage colony stimulating factor in 75 cm ² tissue culture flasks (Costar, OJO) for 2 days to deplete mature adherent cells and enrich for macrophage precursors. To deteπnine MTB growth in macrophages, cells were collected and seeded onto circular 12-mm-diameter glass cover slips placed in a 60-mm-diameter Petri dish and differentiated in macrophages in media containing 20% L929-conditioned

medium for 4 - 6 days, and maintained in complete medium containing 5-10% of L929- conditioned medium. For cell death experiments and intracellular cytokine staining, cells were differentiated into macrophages in 6-wells plates and infected.

[00183] Isolation and infection of murine hone marrow-derived macrophages (BMDM) with MTB and L. monocytogenes (LM) in vitro,

[00184] BMDM were isolated from femurs and tibias of male C3H, C3H.B6-λtf7 and C3H-TgN(SRA-/pri) mice (6 to 8 weeks old) and infected with LM strain 10403 S as previously described ³⁰ . Macrophage monolayers were infected at multiplicity of infection 1 MTB Erdman per 10 macrophages (MOI 1 :10). After 6h the cells were washed with PBS containing 1% FCS (PBS- 1% FCS). The cells were incubated in complete medium containing 10% FCS and three coverslips were removed from the culture at indicated time points and separately lysed with 0.1% Triton X-100. Serial 10-fold dilutions of cell lysates were plated on 7H10 agar containing OADC and incubated for 3 weeks at 37 ⁰ C.

[00185] Differentiation ofapoptotic and necrotic pathway of macrophage cell death.

[00186] Macrophages were infected at MOI 1:10. At indicated time points, cells were stained with 10 nM DiOC ₆ (Molecular Probes) and 0.8 mM Ethidium Bromide (Sigma) for 20 min at 37 ⁰ C, washed three times with PBS, fixed with 1% paraformaldehyde 20 min and washed once again with PBS. Cells were analyzed using BD FACScan flow cytometer (BD Biosciences) to differentiate between live (DiOC ₆ ^high EB ^" ); apoptotic (DiOC ₆ ^low EB ^" ) and necrotic(EB ⁺ ) cells. For Annexin V staining cells were incubated in Annexin binding buffer (10 mM Hepes, 140 mM NaCl and 2.5 mM CaCl ₂ ) and stained with 10 μl Annexin V-Alexa 488 (Molecular Probes) for 20 min, then counterstained with propidium iodide (PI, 1 μg/ml), washed with cold PBS twice, fixed with 1% paraformaldehyde for 30 min, and washed once with PBS. FACS analysis was used to differentiate between early apoptotic (Annexin V+ PI-), late apoptotic (Annexin V+ PI+) and necrotic (Annexin V- PI+) cells.

[00187] Isolation and analysis of the Iprl cDNA.

[00188] The 7/z75-specific oligonucleotide primers (F 1-2 and Rl -3) are presented in Fig. 2c. RACE-PCR was performed using the SMART RACE cDNA Amplification Kit (BD Clontech). The cDNAs were synthesized from the lungs of M.

tuberculosis-infected C3HeB/FeJ and C3H.B6-sstl mice. The RACE amplification products were purified using PCR purification columns (Qiagen), cloned into the plasmid vector pGEM-T (Promega) and sequenced using T7 and SP6 primers. A full length sequence oflprl was confirmed by sequencing the "end-to-end" PCR product obtained using the Fl and R3 primers.

[00189] Statistical analysis,

[00190] Statgraphics Plus, release 4, 1999 (Statgraphics Corp., Rockville, MD) and GraphPad Prizm 3.0 (GraphPad, CA) software were used for the analysis. Comparison of bacterial loads was performed using Student's t-test Results are presented as the mean ± SD. A threshold for statistical significance was p < 0.05. Kaplan Meier Survival curves were generated and compared using the log-rank test (GraphPad Prizm). Intracellular bacterial growth and cell death were analysed by two factors ANOVA (time, genetic backgrounds and experiment). The statistical significance was tested at p<0.05 as critical value using the Student-Newman-Keuls post-test to compare means between both genetic backgrounds. Data are presented as the mean ± 95% confidence interval for mean (95% CI).

[00191] Genotyping.

[00192] DNA was isolated from the tail tips by standard procedure ³² . Genotyping for simple sequence length polymorphism markers polymorphic between C57BL and C3H ³³ was performed by PCR with pairs of oligonucleotide primers (Mouse MapPairs) obtained from Research Genetics (Huntsville, AL). The PCR mix contained IX GeneAmp PCR Buffer II, 2.5 rnM MgCl ₂ , 200 μM of dNTP, 0.4 units of AmpliTaq Gold DNA polymerase (all obtained from Perkin-Elmer), 0.22 μM of forward and reverse primers, and IX Rediload dye (Research Genetics). Five microliters of a 20-fold dilution of the stock mouse tail DNA in sterile deionized water was used per reaction. PCRs were performed in 384-well plates (Robbins Scientific, Mountain View, CA) in a final volume of 11 μl using an Thermocycler PTC-100 (MJ Research, Watertown, MA) with a heated lid as follows: 95°C for 10 min, followed by 35 cycles of 94 ⁰ C for 20 sec, 50°C for 30 sec, 72°C for 40 sec, and a final step at 60°C for 10 min. Four microliters of reaction was loaded directly on 4% Metaphor agarose gel (FMC) and run in IX TBE buffer (90 mM Tris/64.6 mM boric acid/2.5 mM EDTA, pH 8.3) at 10 V/cm with cooling

and recirculation for 30-45 min. The bands were visualized with ethidium bromide staining.

[00193] Gene sequence resources.

[00194] A total of 22 genes in the sstl critical region were defined according to Ensembl (www.ensembl.org/Mus musculus/) and Celera

(www.celeradiscoverysystem.com) databases of mouse genome including those identified by experiments or predicted from the genome sequences (Figure 7). Since the cDNA sequences annotated in both genome databases might have differences due to genetic polymorphisms among different inbred mouse strains and also due to alternative splicing, to design primers for RT-PCR analysis, we retrieved all related cDNA sequences for each gene from the Unigene database (wvvW.ncbi.nlm.nih.gov/entrez/query.fcgiTdb^nigene) at National Center for Biotechnology Information (NCBI) and designed several pairs of oligonucleotide primers for each gene based on the conserved sequence obtained by multiple sequence alignment of all related cDNA sequences.

[00195] RNA Isolation and Northern Analysis.

[00196] RNA was isolated from total lungs with Trizol reagent (Invitrogen) followed by DNase treatment and clean-up with RNeasy columns (Qiagen). Northern blotting was carried out using NorthernMax™ kit (Ambion). Briefly, total RNA from each sample was electrophoresed on a formaldehyde agarose gel and transferred to a nylon membrane. DNA fragments amplified by PCR and purified from agarose gel were labeled with [α- ³² P]dCTP using Prime-a-Gene Labeling System (Promega) to generate DNA probes. ULTRAhyb™ (Ambion) was used for hybridization according to the instructions of the manufacturer.

[00197] RT-PCR and quantitative PCR.

[00198] RNA (2 μg) was reverse transcribed by random decamers and RETROscript™ kit (Ambion), and one twenty-fifth of each product was PCR amplified. Copy number of each individual exon of the Iprl/Ifi75 genes in genomic DNA was quantified by real time PCR with Amplifluor™ Universal Detection System (Intergen). Each sample was set up in triplicate, and a single-copy gene encoding brain-derived neurotrophic factor iBdnj) was used as an internal control.

[00199] Cloning of the Iprl cDNA.

[00200] Oligonucleotide primers were prepared based on a large and most conserved region presented in most i/z75-related cDNA and EST sequences in the GenBank database, The location of primers used for RACE and PCR amplification are shown in Fig. 2C and their sequences are listed below: Fl (5'- ATA ACT TCG GGT CCA GAC TGG GCT GTC AGG -3') (SEQ ID NO:21), Rl (5'- GAT CTC AAT GAT TGG CTG TGC GGT TGC TC -3') (SEQ ID NO:22), F2 (5'- AGA GCA ACC GCA CAG CCA ATC ATT GAG A -3') (SEQ ID NO:23), R2 (5'- CAC AGG TCA CAG GGA GTG TGG GAG AGA AAT C -3') (SEQ ID NO:24), R3 (5'- CAA ACG CAC AGA GAC ACC AAC AGC TTT A -3') (SEQ ID NO:25). RACE-PCR (rapid amplification of cDNA ends) was performed to isolate both 5' and 3' cDNA sequences by using the SMART RACE cDNA Amplification Kit (BD Clontech). Briefly, cDNAs with adaptor ends were synthesized from lungs of M. tuberculosis-infected C3HeB/FeJ and congenic C3R.B6-sstl mice. 5'- and 3'- RACE PCR were performed with Rl and F2 primers, respectively, in combination with the adaptor primer (BD Clontech). The RACE amplification products were purified using PCR purification columns (Qiagen), cloned into the plasmid vector pGEM-T (Promega) and sequenced by using T7 (5'- TAA TAC GAC TCA CTA TAG GG -3') (SEQ ID NO:26) and SP6 (5'- CGC CAA GCT ATT TAG GTG ACA -3') (SEQ ID NO:27) primers. After RACE PCR, a full length cDNA of Iprl from infected lungs of congenic strain C3H.B6-sstl , 1911 bp in length, was then constructed. The full length sequence was further confirmed by sequencing the product of "end-to-end" PCR using Fl and R3 primers.

[00201] Generation of transgenic mouse.

[00202] The XhoI-EcoRI DNA fragment of chimeric human Scavenger Receptor A (SRA) silencer-enhancer-promoter was derived from plasmid pALl which was a generous gift of Dr Christopher Glass ³⁴ . A cDNA fragment containing 140-bp of the 5' untranslated region (UTR) and the complete opening reading frame (ORF) of Iprl was generated by PCR with primer F18 (5'- TTG AAT TCG GGA CTT CCA AGG CAG CAT A -3' (SEQ ID NO:28), EcoRl site underlined) and Rl 8 (5'- GGG CTA CTA GGC ACC CTT CTT TTG AGG TTT A -3' (SEQ ID NO:29), two stop codons underlined). For the macrophage-specific expression of Iprl, the Iprl cDNA was cloned into the EcoRl and Smal (blunt end) sites 3' of the SRA promoter and 5' of the simian virus 40

polyadenylation sites (SV40pA). The 7323-bp SRA-ipri~SV40pA transgene was isolated by digestion with Sail and Notl and injected into fertilized C3HeB/FeJ oocytes. Offspring of the injection was genotyped by two runs of PCR with primer SRFl (5'- GCT AAG ATG CTG GGT TGC TTC TC -3') (SEQ ID NO:30) and SRRl (5'- GAC TTC TGT CTC CTG GTC TCT GTT G -3') (SEQ ID NO:31) or primer F9 (5'- AGA CAT TAA GAC ATC TGG AGC AGA AAG -3') (SEQ ID NO:32) and SV40R2 (5'- GAT GGC ATT TCT TCT GAG CA -3) (SEQ ID NO:33). Transgene-bearing founder mice were backcrossed to the C3HeB/FeJ mice. The transcription of the transgene was confirmed by RT-PCR with primers Fl (5'- ATA ACT TCG GGT CCA GAC TGG GCT GTC AGG -3') (SEQ ID NO:21) and Rl and total RNA prepared from peritoneal macrophages or bone marrow-derived macrophages. Homozygote transgenic mice were generated by intercrossing transgene-positive animals and selection for the transgene homozygotes using quantitative PCR.

[00203] SSCP analysis.

[00204] Genomic DNA was isolated from the tail tips as previously described ³² . SSCP analysis was performed using the primers specific for each of the 12 exons of Iprl gene, labeled with [α- ³² P]dCTP and separated on SequaGel MD (National diagnostics) according to the manufacturer's recommendations. Briefly, 1 μL of PCR product was added per 10 μL of stop solution, the sample was heated to 95 ⁰ C for 5 min, cooled on ice and separated on a 0.6 x gel at a constant power of 7 Watts for 15 hours.

[00205] Infection of mice with MTB.

[00206] For i.v. infection bacteria were diluted by PBS containing 0.05% Tween 80. Mice were infected via tail vein with 1 x 10 ⁵ live bacilli in 100 μl. Aerosol infections were performed using aerosol apparatus developed by Dr. Don Smith and colleagues {Wiegeshaus, 1970 #30} and manufactured by the College of Engineering Shops at the University of Wisconsin (Madison, WI). The bacteria were diluted to 1 x 10 ⁶ CFU in 1 ml. The nebulizer compartment of an airborne-infection apparatus was filled with 75 ml of bacteria suspension and mice were exposed to aerosol for 20 min, followed by 40 min of circulation of clean air. A total of 15 ml of the bacterial suspension was delivered. This resulted in the deposition of 15 to 30 CFU per mouse. The initial deposit was determined in each experiment by plating lung homogenates of 6 mice 24 hours after infection. For determination of the bacterial loads in organs CFU were counted. Mice

were sacrificed using halothane anesthesia. Organs were homogenized in PBS containing 0.05% Tween 80 and serial 10-fold dilutions were plated on 7H10 agar enriched with 10% OADC (Difco, MI). Plates were incubated at 37°C and colonies were enumerated after 21 to 28 days.

[00207] Histopathology.

[00208] Lungs of infected mice were inflated with 10% buffered formaldehyde for more than 24 hours and embedded in paraffin, sectioned and stained with hematoxylin and eosin according to standard procedure at the Harvard rodent histopathology core facility. Histopathological sections were prepared from 3-4 animals per group at each time point. Microphotographs were taken of representative fields. Evaluation of pathologic changes was performed on coded slides by a histopathologist (LK).

[00209] Auramine/Rhodamine staining.

[00210] Identification of acid fast bacilli was performed on formalin-fixed tissue sections using auramine/rhodamine dye (0.1% Auramine O, 0,01% , Rhodamine B (Sigma, MO) in H ₂ O) for 20 min at room temperature in the dark, followed by destaining with 3% HCl in 70% EtOH and counterstained in Mayer's Hematoxylin (VWR).

[00211] Generation of radiation bone marrow chimeras [00212] Bone marrow cells (BMC) were flushed with cold DMEM medium (Gibco BRL) supplemented with 1% of Fetal Bovine Serum (HyClone) and antibiotics (penicillin/streptomycin, 100 U/ml and 100 μg/ml respectively, GibcoBRL) from the femurs and tibias of donor mice. A single-cell suspension was prepared by gently passing the cells by syringe through the needle 22G (Becton Dickinson, Franklin Lakes, NJ). The cells were washed by DMEM/ 1% FBS and nucleated cells counted. Recipient animals were irradiated twice with a dose of 6 Gy at a 4 h interval. Recipient mice were reconstituted with 2 x 10 ⁶ allogeneic BMC 2 h after second irradiation.

[00213] Bacterial strains

[00214] M. tuberculosis (Erdman strain; Trudeau Institute, Saranac Lake, N. Y.) was passed through mice, grown in Dubos oleic albumin complex-enriched Middlebrook 7H9 liquid medium (Difco) containing 0.05% Tween 80 in roller bottles, pelleted by centrifugation (800 g for 10 min), and resuspended in PBS containing 0.05% Tween 80 (PBS-T ween 80). Glycerol was added to a final concentration of 10%, and the bacterial

suspension was aliquoted, frozen and stored at -80°C. Viable bacterial counts were determined by plating serial 10-fold dilutions on Middlebrook 7H10 (Difco) agar plates. An avirulent vaccine strain of Mycobacterium bovis BCG (Pasteur) was used in our studies and was grown and stored as described above for MTB.

[00215] Neutralization of TNFa and IL-10 with specific antibodies.

[00216] To block TNFα and IL-10, anti-TNFα (2.5 μg/ml) Clone MP6-XT3 (Rat IgGl), anti-IL-10 (2.0 μg/ml) Clone JES5-16E3 (Rat IgG2b) were added before and during M. tuberculosis infection. Rat IgGl Clone R3-34 and IgG2b A95-1 were used as isotype controls for Anti-TNFα and anti-IL10 respectively, Blockade antibodies and isotype controls were purchased from BD Pharmingen, CA.

[00217] Measurement of cytokine concentration in the macrophage culture medium.

[00218] Cytokine (IL-10, TNFα, IL- 12, IL-6, IL- lα, IL-I β, IP-10, KC, MCP-I, MIP lα) concentrations were measured in 25 μl of culture supernatant using Linco-plex array (LINCO Research, MO) and Luminex 100 instrument following manufacturer's instructions.

[00219] Caspase 8 activation and blockade

[00220] To detect active Caspase 8, cell cultures were stained for PI as described and washed at room temperature in calcium and Magnesium free PBS, 10 mM HEPES, 2 mM EDTA, pH 7.2, then re suspended in 500 μl of assay buffer and incubated with 5mM solution of Caspase 8 substrate (IETD-Rl 10, Molecular Probes, OR) for 60 minutes at 37 ⁰ C in 5% CO ₂ , protected from light and mixed the twice during incubation to minimize cell settling. Samples were washed once with assay buffer and once with PBS, fixed with 2% PFA and analysed by FACS.

[00221] To block Caspase 8 cells were incubated with a cell-permeable irreversible specific peptide inhibitor of caspase 8 (z-IETD-fmk, Calbiochem, CA) using 5 μM concentration of inhibitor dissolved by sonication in anhydrous DMSO. The cultures were incubated with the inhibitor 2 h before and during infection. Control incubations were performed using the same concentrations of DMSO.

[00222] Iprl gene mediates innate immunity to tuberculosis

[00223] C3HeB/FeJ inbred mice are extremely susceptible to MTB and develop dramatic lung pathology, which leads to their rapid death after infection with virulent

MTB ¹⁰ ' ¹¹ . We generated a congenic mouse strain C3H.B6-,y.stfi (sstl ^R ) that carries the C57BL/6J-derived resistant allele at the sstl locus on the C3HeB/FeJ genetic background. The survival time of the sstl ^R congenic mice infected either intravenously (i.v.) with a high dose of MTB (Fig. IA), or with a low dose of MTB via respiratory route (Fig. IB), relative to their sstl counterparts is significantly lengthened, indicating a profound effect of the locus on anti-tuberculosis immunity. However, the shorter survival of the C3H.B6- sstl (sstl ^R ) mice, as compared to the resistant parental strain C57BL/6J (B6), clearly indicates that the sstl locus is responsible for a significant portion, but not the whole, of the tuberculosis resistance phenotype of the B6 mice.

[00224] The specific effect of the sstl locus on progression of tuberculosis was related to more efficient control of MTB multiplication primarily in the lungs after both respiratory challenge by aerosolized MTB (Fig. 1C), and systemic i.v. infection (Fig. 5A). The development of large necrotic lung lesions within 4 weeks after i.v. infection characteristic of sstl ^s mice was prevented in the presence of the sstl ^R allele (Fig. ID). After a low dose aerosol infection, chronic tuberculosis infection ensued, and the sstl ^s mice developed encapsulated necrotic lung lesions, in some cases reaching approximately one third of the lung lobe (Fig. IE), which resembled tuberculosis cavities in human lungs. Mycobacterium were present both extracellular Iy within necrotic central areas surrounded by the fibrotic capsule as well as within macrophages of the granuloma wall (Fig. 10). In the sstl mice, lung lesions were much smaller and contained fewer macrophages infected with MTB.

[00225] Although the most dramatic effect of the sstl polymorphism on progression of tuberculosis was observed in the lungs; bone marrow transplantation experiments demonstrated that bone marrow-derived, but not lung cells, were responsible for the effect of the sstl locus (Fig. 5B). It is known that T lymphocytes and macrophages play a major role in host resistance to tuberculosis. We have found that, while T lymphocytes are functionally unaffected by the sstl polymorphism (Jobe, in preparation), the sstl disparate macrophages display considerable differences in their ability to control MTB in vitro. The rate of MTB multiplication was significantly higher in the sstl ^s macrophages (Fig. IG, left panel). There was also a clear distinction in mechanism of macrophage cell death after the infection: the sstl macrophages showed characteristic necrosis, while the sstl ^R macrophages underwent apoptosis (Fig. IF, upper

left and upper right panels, respectively). The effect of the sstl locus was much more pronounced upon macrophage infection with virulent MTB, since avirulent vaccine strain of M. bovis BCG failed to multiply in (Fig. IG, right panel) and to induce necrosis of the sstl ^s macrophages (Fig. IF, lower left panel).

[00226] In vivo, many cells within the tuberculosis lung granulomas of the sstl ^R mice contained TUNEL-positive apoptotic nuclei and no necrosis was observed, while the apoptotic nuclei were largely absent from the necrotic lesions of the sstl ^s xmcQ (Figure 8). Thus our studies, both in vivo and in vitro, indicated that the extreme susceptibility to virulent MTB of sstl mice was associated with necrotic death of the susceptible macrophages. Virulent MTB was shown to cause necrosis of the infected epithelial cell lines ⁿ . This necrosis-inducing propensity is specific for virulent MTB and is lost in RDl mutants of MTB that have also dramatically decreased virulence ¹² ' ¹³ . It appears from our studies that, in addition to virulence determinants of the pathogen itself, mechanisms of host cell death depend on the host polymorphic gene(s) encoded within the sstl locus.

[00227] To identify the critical gene(s), we employed a positional cloning strategy (see section under the heading Identification of a candidate gene, IprL within the sstl locus and Fig. 6 for details). First, the sstl minimal region was reduced to an interval between DlMit439 and DlMit49 markers on mouse chromosome 1 (Fig. 6A). This region contains a so-called HSR (for homogeneously stained region) repeat (Fig. 2A). The HSR repeat region is, arguably, the largest repetitive region in the mouse genome ¹⁴ ' ¹⁵ . Its size in inbred mouse strains, is estimated to be between 3.5 and 6 Mb ¹⁵ ' ¹⁶ and it remains unfinished by both mouse genome projects. After identifying and testing progeny of additional recombinants within this interval (Fig. 2A), we concluded that the sstl candidate region encompasses part of the HSR repeat region and a region of mouse chromosome 1 immediately downstream of the repeat, i.e. between the repeat region and the NppC gene. A total of 22 known and predicted genes are encoded within the sstl critical region according to Ensembl and Celera databases of the mouse genome (Figure 7). It was impossible further to reduce the sstl critical region by genetic recombination. Therefore, in the next step we tested expression of each of the sstl -encoded candidate genes in the lungs during tuberculosis infection in vivo and in macrophages infected with MTB in vitro using RT-PCR and RACE. The Ifι75 gene appeared from our studies as the most likely candidate (Fig. 6B and Fig. 6E).

[00228] As shown in Fig. 2B, the 5' RACE products of the Ifi75 in the lungs of tuberculosis-infected mice was strikingly different between the sstl congenic strains: a major single band was amplified from the lungs of the sstl resistant mice, whereas this band was absent from the lungs of the rat/ susceptible strain and, instead, multiple weak products were obtained. Although some aberrant transcripts were present in the lung tissue of the sstl ^R animals as well, the majority of the Ifι75- related transcripts in their tuberculosis lung lesions were represented by a single isoform, which we named as lprl (for intracellular pathogen resistance) to differentiate it from other 7/?75-related sequences (Ifι75~rs) identified by RACE and, perhaps, also encoded within the HSR repeat. The predicted lprl protein is 92% identical to the Mus caroli Ifi75. It contains an SplOO-like domain in its N-terminus, LXXLL-type nuclear receptor binding motif (NRB), bipartite nuclear localization signal (NLS), and a SAND domain in its C-terminus (Fig. 2C),

[00229] Using DNA probes specific for the SpIOO and SAND domains of the lprl, we have analyzed the kinetics of its expression by Northern hybridization in the lungs of the sstl congenic mouse strains during progression of tuberculosis (Fig. 2D). Expression of the lprl gene was detectable in the lungs of the naive and its expression increased significantly 2 weeks after intravenous infection with MTB and remained at elevated levels at later time points. However, expression of the SpIOO and SAND domain-containing Ifi75-rs in the lungs of the sstl susceptible C3HeB/FeJ mice remained below the level of detection by Northern blot hybridization. Instead, the level of transcripts of another gene encoded within the HSR repeat region, Spl00-rs, was elevated in the lungs of the sstl ^s mice (Fig. 2D).

[00230] To investigate expression of the lprl gene in macrophages, we used five overlapping combinations of the lprl -specific PCR primers that cover the full length lprl transcript (Fig. 2E). The lprl gene was expressed in non-activated sstl ^R macrophages and the level of its expression increased after infection with both avirulent BCG and virulent MTB. No expression of the full length transcript of the lprl gene was seen in the sst I ^s macrophages under any stimulation conditions. Macrophages isolated from the tuberculosis lung lesions of the sstl ^R mice also expressed the full length lprl transcript, while those from the sst I ^s mice did not (data not shown). We were unable to detect additional Iprl-velated transcripts induced in macrophages upon infection in vivo and in vitro or stimulation with IFN-γ. Despite the fact that the lprl gene is encoded within the

HSR repeat region, our data suggest that a single major isoform of this gene is expressed in sstl ^R macrophages either before or during tuberculosis infection and this isoform is not expressed in the sstl C3HeB/FeJ mice.

[00231] The C3HeB/FeJ substrain is unique among all other substrains of C3H mice in terms of its extreme susceptibility to tuberculosis ¹⁰ ' ¹¹ . The C3HeB/FeJ mice die abruptly within 3.5-4 weeks after the infection displaying severe lung pathology. In our experiments, the survival time of other substrains of C3H was considerably longer and similar to the sstl ^R congenic strain C3R.B6-sstl (Fig. 3A). The bacterial loads in the lungs of the C3HeB/FeJ mice at 3 weeks post infection were 50 to 100 fold higher as compared to other substrains of C3H and the sstl ^R congenics (Fig. 3B), suggesting a unique allele at the sstl locus. We compared expression of the sstl -encoded candidate genes in the lungs of mice of four C3H substrains and the sstl ^R congenics and found that the lack of expression of the Iprl gene differentiated the C3HeB/FeJ from all other substrains of C3H (Fig. 3C). Since all the C3H substrains originate from a common ancestor ¹⁷ , it is likely that they are genetically identical within the sstl region and a unique de novo mutation has led to the defect of the Iprl gene expression in a C3HeB/FeJ mice and is responsible for a severe defect in their tuberculosis resistance.

[00232] We generated transgenic mice that expressed a full length copy of the Iprl cDNA on the susceptible C3HeB/FeJ background in a macrophage-specific manner under control of the human scavenger receptor A promoter (SR-A). Mature bone marrow-derived macrophages, as well as resident peritoneal macrophages obtained from those mice, expressed the Iprl transgene (Fig. 4A). Despite the fact that the regulation of Iprl gene expression in the transgenic macrophages was clearly less efficient from the SR-A promoter than from the endogenous Iprl promoter (Fig. 4A), when the Iprl transgenic mice were infected with virulent MTB, a statistically significant difference in the bacterial loads between the sstl ^s (Tg -/-) and the Iprl transgenic (Tg +/-) animals was observed in the lungs (Fig. 4B). In vitro, the Iprl transgenic macrophages also controlled multiplication of MTB more effectively (Fig. 4C) and turned on the apoptotic pathway of cell death upon interaction with virulent MTB (Fig. 4D, right panels). The growth of another intracellular pathogen, L. monocytogenes, was dramatically suppressed in the Iprl transgenic macrophages by 50 to 100 fold (Fig. 4E). Similar to the MTB infection, necrotic death accompanied infection of the sstl ^s macrophages with virulent L.

monocytogenes (Fig. 4F, left panel), while the Iprl transgenic macrophages displayed markers of apoptotic death (Fig. 4F, right panel).

[00233] Thus expression of a single gene, Iprl, in the $stl ^s macrophages restored key functions related to pathogenesis of tuberculosis, which are encoded within the sstl locus: greater control of multiplication of virulent MTB in vivo and in vitro as well as an apoptotic mechanism of MTB-induced macrophage cell death. Moreover, the Iprl gene mediated macrophage resistance to another intracellular pathogen, L. monocytogenes, suggesting that the Iprl product controls a common mechanism of innate resistance against several intracellular pathogens.

[00234] The closest homologue of the predicted Iprl protein in humans (41% of identity) is SPl 10b ¹⁸ , which localizes to a region of human chromosome 2 sythenic with the sstl minimal region on mouse chromosome 1. Both the Iprl and the human SPl 10 proteins contain motifs that are involved in protein-protein interactions (SpIOO domain) ' ²⁰ , chromatin binding (SAND domain) ^{2 >22} , nuclear localization signal (NLS) and the nuclear receptor binding (NRB) motif LXXLL. Recent evidence suggests that human SPl 10 protein, may function as a nuclear hormone receptor transcriptional cofactor ¹⁸ and directly bind the retinoic acid receptor ²³ . Signaling through nuclear receptors, such as the corticosteroid receptor, retinoic acid receptor, PPARs and vitamin D plays an important role in control of various aspects of the macrophage life cycle, including differentiation, activation, response to pathogens and apoptosis ²⁴ . Expression of both the Iprl gene and its human homologue SPl 10, is regulated by interferons ²⁵ , additionally implicating a role in immunity in both species. Moreover, polymorphisms in SPIlO gene have been associated with susceptibility to the Hepatitis C virus ²⁶ , and the SPl 10b protein has been shown to physically interact with viral proteins, such as Epstein- Barr virus SM protein and Hepatitis C virus core protein ^{2 >27} . It is an intriguing possibility that the Iprl and SPl 10 proteins mediate cross talk between nuclear receptors, interferon signaling and pathogens. The viruses and, perhaps, intracellular pathogens might have evolved mechanisms to interfere with or exploit the Iprl /SFl 10 function. Taken together, these data show that in mammals, since no Iprl homologues were found in yeasts or insects, the Iprl -related proteins play a novel role in integrating signals generated by intracellular pathogens or viruses with mechanisms regulating activation, gene expression and cell death of host cells .

[00235] Identification of a candidate gene, Iprl, within the sstl locus.

[00236] Fine mapping of the sstl locus.

[00237] Previously we have determined that the B6-derived sstl ^R allele was dominant. Therefore, for fine mapping of the ,Mt/ locus we analyzed progeny of males that carried recombinant chromosome 1 backcrossed to the sstl -susceptible parental strain C3HeB/FeJ. Approximately 30 to 40 backcross progeny of each male were generated and genotyped within the sstl locus. Progeny with no new recombination events within the sstl locus were infected with MTB for phenotyping. The recombinants were used for generating progeny only, but were not characterized phenotypically themselves. The sstl alleles of all recombinants were deduced based on testing of their progeny for tuberculosis susceptibility. Progeny of each recombinant was tested independently. To deduce the sstl alleles of a recombinant, we compared the median survival time (MST) of its progeny that carry the B6-derived segment within the sstl locus with the C3H homozygous littermates using t test. The MST of the progeny that carried the sstl resistant allele varied between 60 - 80 days in independent experiments, while the MST of the C3H homozygous littermates (sstl susceptible) was 27 -30 days. The threshold of significance was established as p=0.001, but typically the p value was lower. Progeny that carried chromosome 1 with new recombination events within the sstl locus, were not infected, but used for the next backcross. Thus, appearance of double crossovers in Figure 6A was a result of two recombination events which occurred sequentially in two subsequent backcrosses: a new recombination event between DlMit415 and DlMit49 occurred in a progeny of a male that already carried a recombinant chromosome with a breakpoint between DlMit49 and DlMitlO. Initially, the candidate region was reduced to an interval between the DlMit439 and DlMit49 (Fig. 6A). In that figure, each column of boxes represents a genotypic class and each row of boxes represents genotypes for an individual microsatellite marker within the sstl candidate region (specified on the left of each row). Solid boxes represent heterozygous (B6 and C3H-derived) and opened boxes homozygous genotype (C3H only). The number of recombinant males, which were tested per each genotypic class, is denoted under each column. The two horizontal lines drawn between DlMit 439 and DlMit49 designate the sstl candidate region. To further reduce the sstl candidate region we have tested an additional 1102 meioses and identified 17 new recombination events between DlMit439 and DlMit80, and 5 new recombination events between DlMit 415 and DlMit49 (Fig. 2A). Each recombinant was used to

generate progeny, which were analyzed for tuberculosis susceptibility, We established that the proximal boundary of the sstl candidate region was delimited by the DlMit438 marker and the distal boundary was delimited by recombinations between DlMMl 5 and the NppC gene (Fig. 2A). Eight recombination events between DlMit438 and WI_WGS_1_86, 182,722 (SNPl, Fig.2A) separated the sstl resistant allele from the D1MM38. The region between D1MM38 and WI_WGS_1_86, 182,722 contains approximately 0.6 Mb of finished sequence and a gap. This gap is due to a presence of a so-called HSR (for homogeneously gained region) repeat. The HSR repeat region is, arguably, the largest repetitive region in the mouse genome and it remains unfinished by both mouse genome projects. Considering the size of the repeat region in inbred mouse strains, which was estimated to be between 3.5 and 6 Mb, it is likely that most of the eight recombination events between DlMit438 and WI_WGS_1_86, 182,722 (SNPl) occurred within the HSR repeat region. No recombination events were found between the sstl resistant allele and polymorphic markers within a 634 kb region between the WI_WGS_1_86, 182,722 (SNPl) and D1MM15 (Fig. 2A). Therefore, we concluded that the minimal candidate region encompasses the distal part of the HSR repeat region and a region of mouse chromosome 1 immediately downstream of the repeat, i.e. between the repeat region and the NppC gene.

[00238] Expression profiling of the sstl -encoded genes during the course of tuberculosis infection.

[00239] Since it was impossible to further reduce the sstl critical region by the genetic recombination, the next step of our positional cloning strategy was based on analysis of expression the sstl -encoded genes during the course of tuberculosis infection. Based on the sstl functional studies, we anticipated that the sstl candidate gene would be expressed in the lungs during tuberculosis infection in vivo and in macrophages infected with MTB in vitro. RNA samples were isolated from the lungs of C3H and C3HB6-sstl congenic mouse strains before and 3 weeks after infection with MTB and from the bone marrow-derived macrophages, which were obtained from the sstl congenic mice and infected with MTB in vitro. A total of 22 genes are encoded within the sstl critical region according to Ensembl and Celera databases of mouse genome (Figure 7). The sequences of the Spl00-rs and Ifi75 genes, which are encoded within the repeat region, were described previously ¹ . Initially the expression analysis was done using RT-PCR (Fig. 6B, left panel) and Affymetrix GeneChip arrays. To prioritize the sstl -encoded

genes we used the following criteria: the gene is expressed in critical tissue (lung) and cell type (macrophage), expression of the gene is modulated by the MTB infection, and the gene might be differentially expressed between the sstl disparate animals or cells. Based on priority, all the genes within the sstl region were divided into 5 categories (Fig. 6E): not expressed in the lungs and macrophages (unlikely candidates, 9 genes); expressed in the lungs, not in macrophages (low priority, 3 genes); expressed in the lungs and macrophages, not induced by MTB infection (medium priority, 10 genes); upregulated by the MTB infection (high priority, 2 genes); differentially expressed between the sstl congenic macrophages (highest priority, 1 gene). To further study the transcripts of the genes that received priority scores of 2 and higher, we used the Rapid Amplification of cDNA Ends (RACE) technique using mRNA isolated from the total lung tissue of the sstl congenic mice at 2, 3 and 4 weeks after infection with MTB. The gene-specific primers for RACE and RT-PCR were designed to anneal to conserved regions of each cDNA, which were identified by sequence alignment of all homologous ' sequences in the NCBI Unigene database. Both the 5' and the 3' ends of mRNA transcripts were amplified using several gene specific and anchor primers and the amplification products were analyzed by gel electrophoresis (Fig. 6B, right panel). The Ifi75 gene-specific transcripts demonstrated differential expression by both RT-PCR and RACE and this gene was considered to be the most likely sstl candidate gene.

[00240] Below is a table of genes within the sstl critical region.

Table 1. Genes within the sstl critical region.

[00241] Isolation and characterization of the Ifi75 -related transcripts from the lung tuberculosis lesions of the sstl ^R mice.

[00242] The Ifι75 gene is encoded within the HSR repeat region, also known as long-range repeat cluster DlLubl. Weichenhan et al isolated two principal component genes encoded within the repeat: Spl00-rs and Ifi75. The Spl00-rs is a chimeric gene that arouse by fusion of the SpIOO and Csprs genes. The mouse Ifl75 gene encodes a putative nuclear hormone receptor co-activator, which is homologous to human "nuclear dot" protein IFI75, also designated SPl 10. Several Mus musculus cDNA and EST sequences in the GenBank database have significant homology to the Mus caroli Ifl75 cDNA (accession number AJ401361).The genome of M. caroli contains single copies of SpIOO, Csprs and Ifι75 genes, while both the Spl00-rs and Ifl75 genes are amplified in M.musculus. It was estimated that a number of the repeat copies ranged from about 50 in the inbred mouse strain C57BL/6, to about 2,000 copies in some isolates of wild mice, which accounts for 0.2 - 6.7% of the haploid mouse genome. Rearranged copies of Iβ75 are homogeneously spread along the repeat region. We considered a possibility that different isoforms of Ifi 75, which we called the //?75-related sequences (Iβ75-rs), might exist within individual repeat elements and might be expressed in different cell types or under different physiological conditions. To isolate the Ifi75-rs, relevant to tuberculosis resistance phenotype, we performed RACE using RNA isolated from tuberculosis lung lesions of the sstl resistant mice. Several oligonucleotide primers were designed based on a large and most conserved region, which is present in known Ifι75-rs sequences, and 5' and 3' cDNA sequences were amplified, cloned and sequenced. As shown in Fig. 2B, the 5' RACE products of the Ifi75-rs in the lungs of tuberculosis-infected mice was strikingly different between the sstl congenic strains: a major single band was amplified from the lungs of the sstl resistant mice, whereas this band was absent from the lungs of the sstl susceptible strain and, instead, multiple weak products were obtained. The sequences of the sstl ^R RACE clones were used to assemble a full length sequence and to design a set of primers based on conserved regions to amplify the full length transcripts of

Iβ75. Location of the primers is presented in Figure 2c and the primer sequences are presented in the Methods. The full length products were possible to obtain only from the lesions of the sstl ^R mice using end-to-end PCR. Although some of the aberrant transcripts were present in the lung tissue of the sstl ^R animals as well, the majority of the Ifl75-rs transcripts in the tuberculosis lung lesions were represented by a single isoform encoding 12 exons, which was 92% identical to the Mus caroli Ifι75. To differentiate between the previously identified Ifι75 transcripts, we named the Ifι75 isoform isolated in our studies from the tuberculosis lung lesions of the C3H.B6-^ti mice as Iprl (for intracellular pathogen resistancel, GenBank accession No. AY845948). The predicted Iprl protein contained a SplOO-like domain in its N-terminus, a LXXLL-type nuclear receptor binding motif (NRB), a bipartite nuclear localization signal (NLS), and a SAND domain in its C-terminus (Fig. 2C). In addition, we have identified another isoform of the Ifi75 in the lungs of the resistant mice that was most likely the product of alternative splicing and encoded a transcript containing a stop ¹ codon after exon 10. In Fig. 2D and e, we demonstrate that a single major isoform of the Ifi75-rs, the Iprl gene, is expressed in sstl macrophages, and is absent from the sstl macrophages obtained from the C3HeB/FeJ mice.

[00243] Using DNA probes specific for the SpIOO and SAND domains of the Iprl, we have analyzed the kinetics of its expression by Northern hybridization in the lungs of the sstl congenic mouse strains during progression of tuberculosis (Fig. 2D). Expression of the Iprl gene was detectable in the lungs of the naive sstl mice, and its expression increased significantly 2 weeks after intravenous infection with MTB and remained at elevated levels at later time points. However, expression of the SpIOO and SAND domain-containing Ifl75-rs in the lungs of the sstl susceptible C3HeB/FeJ mice remained below the level of detection by Northern blot hybridization. Instead, the level of transcripts of another gene encoded within the HSR repeat region, Spl00-rs, was elevated in the lungs of the sstl ^s mice (Fig. 2D). This was detected using a probe that is specific for the Csprs portion of the Spl00-rs, which does not hybridize with a normal transcript of the SpIOO gene. We have also performed RT-PCR of the RNA isolated from the sstl congenic macrophages using primers that specifically amplify the Spl00-rs transcripts, but not the SpIOO gene, because the reverse primer was specific for the Csprs sequence that is not present in the SpIOO cDNA. As shown in Fig. 4 A, Spl00-rs, as well

as the aberrant Ifι75-rs, are expressed in the sstl ^s homozygous (S), heterozygous (SxR and RxS), and the Iprl transgenic macrophages (Tg). However, they are not prominently expressed in the macrophages that are homozygous for the B6-derived resistant allele of the sstl locus (R). The sstl resistant allele in our model is dominant and the tuberculosis resistance of the Fl hybrid mice that are sstl heterozygous is similar to that of the sstl homozygous mice (data not shown). It is, therefore, most likely that the expression of the full length Iprl gene is necessary for tuberculosis resistance and expression of the SpIOO- rs does not confer susceptibility.

[00244] Genetic basis of the Iprl silencing in the sstl mice.

[00245] To investigate the lack of the Iprl gene expression in the sstl C3HeB/FeJ mice, first we used quantitative Real Time PCR of the genomic DNA to compare the number of individual exons of the Iprl gene encoded in the genomes of the sst 1 ^R and the sst I ^s rmcQ. The genomic DNA of Mus caroli, which contains a single copy of the Ifι75 gene ¹ , was used as a reference. We found that both C3H-derived and B6- derived chromosomes contain the HSR repeat. As shown in Fig. 6C, the number copies of individual exons in the sstl resistant mice (C3H.B6-.w77) varied between 40 copies for exon 8 (maximal copy number) and 5 copies for exon 12 (minimal copy number) suggesting that most of the copies of the Ifi75-rs contain less than a complete set of 12 exons. Thus, the HSR repeat, even in the sstl ^R mice, contains mostly aberrant forms of the Ifl75-rs, and very few, perhaps one copy of the full length Iprl gene. The number of exons 1, 2, and 4 was lower in the genome of the sst I ^s mouse strain. Single Strand Conformation Polymorphism (SSCP) analysis of individual exons of the Iprl gene in sstl ^R and sstl ^s congenics demonstrated a clear difference between the congenic mouse strains in genomic DNA encoding exon 1 (Fig. 6D). We hypothesize that a mutation, perhaps a deletion, within the 5' region of a rare full length copy of the Iprl gene resulted in the lack of this gene expression in the sstl -susceptible C3HeB/FeJ mice.

[00246] To summarize, aberrant transcripts of the Ifi75- rs are weakly expressed in macrophages of the sstl susceptible C3HeB/FeJ mice, and the full length Iprl transcript is missing. In contrast, a major Iβ75-rs isoform, which is expressed in macrophages of the sstl resistant mice, is represented by a full length Iprl transcript containing all 12 exons.

EXAMPLE II

[00247] We have generated an additional strain of sstl congenic mice, C57BL/6J.C3H-sstl (susceptible). C57BL/6J.C3H-sstl (susceptible) was generated by transfer of an approximately 20 cM segment of mouse chromosome 1, which contains the susceptible allele of the sstl locus, on the resistant genetic background (C57BL/6J, or B6). Therefore, the B6.C3H-sstl mice, as opposed to the parental B6 mice, do not express the Iprl gene. This is a unique strain, which was generated in our laboratory by 10 backcrosses on B6 genetic background, which is a standard genetic background for testing genetically engineered mice. Since the B6.C3H-sstl mice do not express the Iprl gene, this strain is equivalent of the Iprl knockout mouse. However, generation of the Iprl knockout mouse by standard homologous recombination technique would be currently impossible, because of highly repetitive nature of the chromosomal region encoding the Iprl gene. Thus, the B6.C3H-sstl mouse strain represents a unique resource for studies of the Iprl gene function. For example, it can be used for generation of transgenic mice that express human homologue of the Iprl gene to study its function, as well as for screening and testing of potential activators and inhibitors of the human Iprl homologue in a whole animal.

[00248] In addition these mice are more resistant to tuberculosis infection than the C3HeB/FeJ parental mice, which also do not express the Iprl gene. Therefore, it can be used for identification of compensatory immune mechanisms that function in the absence of the Iprl gene product. Although the B6.C3H-sstl mice survive longer than the C3HeB/FeJ parental mice, a significant proportion of these mice does develop tuberculosis cavities in their lungs. Therefore, this strain represents a unique model of chronic lung tuberculosis, resembling a key element of human pulmonary tuberculosis in adults, such as formation of tuberculosis cavities in the lungs. Although adult pulmonary tuberculosis is the most common and infectious form of tuberculosis infection in humans, until now, no adequate experimental mouse model of this clinical form of tuberculosis existed and progress in developing effective therapeutic and preventive strategies targeting adult pulmonary tuberculosis has been slow. Meanwhile, methods for prevention of formation of the tuberculosis cavities in the lungs and/ or for effective drug delivery to the bacteria within these cavities, could greatly reduce transmission of tuberculosis and would have a significant epidemiological impact.

[00249] Availability of this mouse model of chronic pulmonary tuberculosis with formation of cavities in the infected lungs provides a model for testing of new antituberculosis drugs, treatment regiments, vaccines and methods of immunomodulation. This strain can also be used in studies of pathgenesis of tuberculosis, because the B6.C3H-sstl mice can be bred with genetically engineered mice that carry knockout of other relevant genes on the same C57BL/6J genetic background, thus permitting generation of double and triple knockouts.

EXAMPLE III

[00250] The sstl locus modulates production of inflammatory mediators by the MTB-infected macrophages in vitro.

[00251] To determine whether the sstl locus also modulates production of inflammatory mediators by macrophages, we measured production of a panel of cytokines and chemokines by sstl ^s and sstl ^κ bone marrow-derived macrophages (BMDM) 6 and 30 hours after infection with virulent MTB in vitro. The sstl ^R macrophages produced more TNFα within the first 6 hours post infection. At that time concentrations of IL-10, MCP- 1, IP-10, KC and MIP- lα increased to a similar level on both genetic backgrounds. During the next 24 hours, however, the sstl congenic macrophages demonstrated dichotomous trends: the sstl ^R macrophages (either non-treated or treated with IFNγ) continued to produce higher levels of TNFα, while the susceptible macrophages produced higher levels of IL-10, MCP-I and IP-10. Concentrations of TGF _β in these samples were low and similar for both genotypes. Higher levels of TNFα and lower level of IL-10 production by the sstl ^κ macrophages were also observed at low MOI of 1 bacterium per 10 macrophages.

[00252] Six hours after MTB infection both sstl ^R and sstl ^s macrophages showed approximately 80-fold increases in TNFα mRNA levels, while the increase in IL-10 mRNA was slightly higher in the sstl ^s cells (12-fold as compared to 8-fold in the sstl ^R cells). To test whether the sstl ^s macrophages responded to MTB via an alternative activation pathway, we measured expression of arginase, YmI and FIZZl gene markers of this pathway ³⁵ . MTB infection caused increased expression of arginase, but had no effect on the expression of YmI and FIZZl genes in macrophages of either genetic background. In control experiments macrophages of both strains expressed YmI and FIZZl genes after treatment with IL-4 . When the sstl ^R and sstl ^s macrophages were

stimulated with IFNγ (30 U/ml) and LPS (50 ng/ml) they produced equal amounts of TNFα, but no detectable IL-IO.

[00253] We sought to determine which cytokines are produced by the MTB- infected macrophages themselves, and which are produced by non-infected bystander macrophages upon interaction with the infected cells. We employed a genetically modified strain of virulent MTB that constitutively expresses green fluorescent protein (GFP) or MTB Erdman that was labeled with fluoroclirome CFSE (for short term experiments). This allows identification of MTB -infected macrophages by green fluorescence using flow cytometric analysis. The cytokine production by MTB-infected (GFP-positive) and non-infected (GFP-negative) cells was analyzed using intracellular cytokine staining (ICC) at 24, 48 and 72 hours after infection with MTB in vitro. A major effect of the sstl locus on TNFα and IL-10 production was observed specifically in the cells which harbored the bacteria. At 24 h post infection, all of the MTB-infected sstl ^κ macrophages produced TNFα, but very few of them produced IL-10. In contrast, the sstl macrophages infected by MTB mostly produced IL-10, and very few of them produced TNFα. Importantly, production of both TNFα and IL-10 by the bystander macrophages at that time was similar on both genetic backgrounds. The MTB-infected sstl and sstl macrophages also differed in MCP-I production. However, this difference was observed later, at 48 hours post infection. At 24 hours post infection, the bacterial load per infected macrophage was similar in the sstl and sstl cells, in which the majority of infected macrophages contained a single bacterium. Therefore, the difference in cytokine production by the MTB-infected macrophages cannot be simply accounted for by a difference in bacterial load. It more likely reflects intrinsic differences in regulation of cytokine gene expression in the infected cells.

[00254] In parallel, we used GFP-labelled MTB to examine kinetics of cell death in infected macrophages. MTB-containing (GFP-positive) macrophages were analyzed for membrane damage (PI staining) and apoptosis (Annexin V staining) at 24 h intervals. At 72 hours after a low multiplicity infection, evidence of cell death was primarily limited to MTB-infected cells. The MTB-infected sstl ^κ cells were undergoing apoptosis (PI- negative, Annexin V-positive), while the sstl ^s macrophages typically died by necrosis, i.e. they displayed membrane damage (Pi-positive) in the absence of apoptotic membrane transformation (Annexin V-negative). Of note, in both the sstl and sstl macrophage cultures signs of cell death were observed only 72 hours after the infection and later.

Thus, the earliest macrophage-specific phenotypic expression of the sstl locus in vitro was its effect on cytokine production specifically by MTB-infected cells.

[00255] Production ofTNF-a and IL-JO by macrophages infected with virulent MTB is mediated by the Iprl gene.

[00256] Production of TNFα, IL-10 and IL- 12 by macrophages isolated from the Iprl transgenic C3H.Tg(ipri) mice and from the sstl ^s C3H mice, which lack the Iprl expression, was compared at 12 and 24 hours after the MTB infection in vitro. The MTB-infected Iprl transgenic macrophages produced higher levels of TNF-α at both time points, similar to the sstl ^κ congenic macrophages, which were also tested in parallel. Production of IL-10 was delayed, as compared to TNF-α, but at 24 hours fewer transgenic macrophages produced IL-10 as compared to wild-type C3H, while IL- 12 production increased similarly in MTB-infected macrophages of both genotypes. Thus, the MTB- infected Iprl transgenic macrophages that genetically differ from C3H macrophages only by expression of a single gene (Iprl), demonstrated a pattern of cytokine production similar to that of the sstl ^κ congenic macrophages that differed from C3H by a 20 cM segment of chromosome 1.

[00257] We compared cytokine production by the Iprl disparate macrophages that were infected either with virulent MTB or avirulent vaccine strain of Mycobacterium bovis BCG (BCG). Infection with BCG induced production of TNFα in both Iprl- positive and Iprl -negative macrophages at 12 hours, which declined at 24 hours after the BCG infection. The Iprl transgenic and the sstl ^R macrophages produced higher levels of TNFα when they were infected with MTB then after the BCG infection. On the contrary, production of TNFα by the MTB-infected sstl ^s macrophages was lower as compared to the same cells infected with BCG. Interestingly, at 24 hours post infection with either mycobacteria, the bystander macrophages in these cultures also started to produce TNFα. We have demonstrated that activation of the bystander cells was mediated by TNFα, since neutralization of TNFα using specific antibodies substantially decreased TNFα production by these macrophages.

[00258] The proportion of IL-10 producing cells among the BCG-infected macrophages was low in both genetic backgrounds. Elevated IL-10 production was observed only in macrophages infected with MTB, and proportion of the IL-10 producing cells was significantly higher in the Ipr 1 -negative C3H macrophages. Conversely, IL-12 was rapidly induced by the BCG infection on both genetic backgrounds, less so by MTB,

and we did not observe any impact of the Iprl gene on regulation of this cytokine neither in BCG nor in MTB-infected cells.

[00259] Neutralization of TNFα by specific antibodies significantly enhanced growth rates of MTB in the s$tl ^R and the Iprl transgenic macrophages to the levels similar to the MTB growth in the sstl ^s cells, while having little effect on the bacterial growth in the sstl ^s macrophages. This treatment also blocked apoptosis of the MTB- infected Iprl transgenic macrophages leading to appearance of Annexin V-negative, PI- positive necrotic cells, similar to the cell death phenotype of the sstl ^s macrophages. In both cases, signs of cell death were predominantly associated with the MTB-infected GFP-positive cells.

[00260] TNFα is known to induce apoptosis via caspase 8 activation. Therefore, we compared caspase 8 activity in the sstl ^R congenic, the Iprl transgenic and the sstl ^s C3H macrophages 72 hours after infection with MTB. Activation of caspase 8 was observed in macrophages that expressed the Iprl gene. The caspase 8-positive cells were also Pi-negative, i.e. at this point they had not undergone membrane damage. Conversely, most of the s$tl ^s macrophages were Pi-positive and caspase 8-negative consistent with a necrotic mechanism for their cell death. Neutralization of TNFα using specific antibodies blocked caspase 8 activation, as did the specific peptide inhibitor of caspase 8 zIETD-fmk. Both of these treatments also inhibited apoptotic cell death of the MTB-infected Iprl transgenic macrophages, which resulted in appearance of Annexin V- negative, Pi-positive necrotic cells.

[00261 ] Thus, blockade of TNFα switched the phenotype of the Iprl -transgenic and the sstl macrophages to the phenotype characteristic for Ipr 1 -negative sstl C3HeB/FeJ macrophages, when control of bacterial multiplication and mechanism of cell death were assessed. However, neutralization of TNFα failed to upregulate production of IL-10 by the sstl macrophages to the levels observed in the MTB-infected sstl cells suggesting that production of IL-10 is regulated by the Iprl gene via a TNFα-independent mechanism.

[00262] We combined the sstl ^s macrophages with the sstl ^κ macrophages, which are capable of TNFα production after MTB infection, infected the mixed population of the sstl ^R and sstl ^s macrophages with MTB and studied mechanism of their cell death. To differentiate between the sstl ^R and the sstl ^s macrophages in a mixed culture, we stained

macrophages of one genotype with CFSE. Reciprocal staining was performed to control for potential adverse effect of CFSE on macrophages, but this switch did not affect the outcome of the experiment. The susceptible phenotype was dominant in mixed cultures: apoptosis was blocked and necrotic death was observed in macrophages of both sstl and sstl ^R genotypes. Necrosis was abolished by IL-10-specific neutralizing antibodies. Importantly, we observed appearance of equal numbers of Annexin V-positive cells apoptotic cells among macrophages of both sstl ^R and sstl ^s genotypes. These experiments demonstrated that, in principle, the sstl ^s macrophages were capable of entering apoptotic pathway, but this was blocked by IL-IO secreted by the susceptible cells.

[00263] Blockade of IL-IO also initiated the apoptotic cell death pathway in isolated cultures of MTB-infected sstl ^s macrophages. This effect was TNFα-mediated since apoptosis was blocked again, when the sstl ^s macrophages, were also treated with anti-TNFα antibodies in addition to neutralizing anti-IL-10. This suggested that either the low amount of TNFα, which was initially produced by the MTB-infected sstl macrophages, was sufficient, upon ILlO neutralization, to induce apoptosis, or that the inhibition of IL-10 restored production of TNFα in the MTB-infected sstl ^s cells.

[00264] The later possibility was tested using intracellular cytokine staining. Initially, neutralization of IL-10 resulted in increased TNFα production by both the MTB- infected cells and the non-infected sstl ^s macrophages at 12 hours after the infection. However, by 24 hours, the majority of the infected cells ceased TNFα production, while the by-stander macrophages still produced some. Thus, TNFα production by the MTB- infected sst I ^s cells was not sustainable over longer period even in the presence of IL-10- neutralizing antibodies. This is clearly different from the kinetics of TNFα production by the MTB-infected sstl ^R macrophages, in which high levels of this cytokine were observed within 24 - 48 hours after the MTB infection.

[00265] Anti-IL-10 treatment improved control of MTB replication by the sstl macrophages, although this effect was only partial. No effect of IL-10 blockade on MTB control by the sstl ^R and the Iprl transgenic macrophages was registered in this experiment, which is consistent with low levels of IL-10 production by the resistant cells.

[00266] Cell autonomous control of TNFa production by the sstl locus.

[00267] To avoid possible artifacts related to incomplete inhibition of specific cytokines with antibodies in the above experiments, we studied production of cytokines by the MTB-infected macrophages in co-cultures of the sstl ^κ and sstl ^s cells. In these experiments macrophages of one genotype were labeled with red fluorescent dye (SNARFl), mixed with non-labelled macrophages of the opposite sstl genotype and the mixed population was infected with CFSE-labeled MTB. Production of TNFα, IL-IO and IL- 12 by cells of each genotype in mixed population was determined by intracellular cytokine staining and plotted separately for the MTB-infected and non-infected macrophages of each genotype. This approach eliminates environmental variables and highlights cell specific phenotypes. Two major observations were made regarding TNFα production in these settings. First, 24 hours after infection the MTB-infected sstl ^R macrophages as well as the non-infected macrophages of both sstl genotypes produced similar amounts of TNFα. Meanwhile TNFα production by the sstl ^s MTB-infected cells was profoundly depressed. This effect of the sstl locus on TNFα production was restricted to MTB -infected sstl ^s macrophages. Interestingly, IL-10 production by the sstl and the sstl cells in co-cultures was similar and intermediate between the two genotypes, indicating that production of this by the sstl ^κ macrophages could be upregulated by soluble mediators or cell-cell contact with the sstl ^s cells. Production of IL- 12 by the sstl and the sstl macrophages in these settings was also similar. Thus, production of TNFα by the MTB-infected macrophages is strictly controlled by the Iprl gene in a cell autonomous manner, and this effect perhaps constitutes mechanistic basis of an important role that this genes plays in innate immunity to infection with virulent MTB.

[00268] We have shown that the Iprl gene plays a role in cytokine production by macrophages infected with virulent cytosolic pathogens, such as, Mycobacterium tuberculosis. We found that the Iprl gene controlled cytokine gene expression specifically in macrophages that contained intracellular MTB. Iprl gene expression was necessary for sustainable production of TNFα, while the MTB-infected macrophages that lack Iprl produced IL-10. Our findings that production of TNFα and IL-10 production is reciprocally controlled in the MTB-infected macrophages by the Iprl gene demonstrates that the balance of these cytokines is also influenced by the genetic makeup of the host.

[00269] The references cited herein are incorporated by reference in their entirety.

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