Field of the Invention

The present invention is directed to a method for diagnosing inflammatory diseases based on the marker IRF4 particularly for diagnosing specific stages of inflammatory diseases and/or for determining the effectiveness of specific anti inflammatory drugs

Background of the Invention

Macrophages constitute the first line of defense of the immune system against infections. Since their identification by Metchnikoff as cells capable of ingesting microorganisms, their functional characterization has continuously evolved; yet much is still unknown about key aspects of their biology and how they are regulated. The existence of long-term consequences of the stimulation of macrophages with certain simple (e.g. beta glucans) or complex (e.g. BCG, the mycobacterial vaccine strain) stimuli has been recently recognized and termed ‘innate immune memory’. This concept originally evolved from observations in BCG-vaccinated individuals in which a level of protection against disparate pathogens was identified. Innate immune memory has been defined in terms of the induction of soluble factors (i.e. proinflammatory cytokines). Responses identified as memory have been divided into innate immune training and tolerance; the difference being the nature of the secondary response (heightened versus reduced). Innate immune memory is replicated in vitro by the use of a primary stimulus, a period of resting, and a different secondary stimulus. Although the mechanisms underlying the development of innate immune memory are not completely known, both variations in metabolism (Warburg effect) mediated by the AKT/mTOR/HIF axis, and epigenetic changes are known to occur. The effect of this previous experience on the ability of monocytes/macrophages to internalize/phagocytose microorganisms has been, however, largely unaddressed. This is highly relevant, due to the importance of this process in the elimination of pathogens, and the intimate relationship between phagocytosis and the inflammatory output of macrophages. Moreover, the response to live and killed microorganisms is vastly different, both quantitatively and qualitatively. Therefore, the phenotypic and regulatory mechanisms of innate immune memory cells against pathogens that are able to establish persistent infections are lacking. During a persistent infection, which the host is not able to eradicate, an enhanced secondary response would result in increased inflammatory and therefore, pathological consequences. However, this is not detected in the case of the persistent infection with the causative agent of Lyme borreliosis, Borrelia burgdorferi. B. burgdorferi is one of a few extracellular pathogens that are able to establish persistent infections even after antibiotic treatment, in part because of the need to remain in the mammalian host until ticks acquire the microorganism, which can take several months. In spite of persistently infecting mammalian hosts, including organs in which macrophages are the main responding immune cell such as the heart, the inflammatory response is known to vane over time albeit with bouts of exacerbation in experimentally infected animals while long term cardiac-related anomalies associated with infection with B. burgdorferi in humans are rare.

Transcriptional traits and signalling pathways associated with the short-time stimulation of monocytes/macrophages from both human and murine origin with B. burgdorferi, such as PPAR and the Toll-like receptor (TLR) family member, CD180 has been recently identified (Carreras-Gonzalez A, et ai. (2018) Emerg Microbes Infect 7(1): 19.). However, macrophages are likely exposed to B. burgdorferi during prolonged periods of time. Therefore, the response of these cells may be differentially modulated over time due to transcriptional, epigenetic or metabolic changes, including the integration of primary (to the spirochete) and secondary (to metabolites or inflammatory/anti-inflammatory) factors.

The control of infection by antibodies is probably important during the dissemination phase, when the bacteria are accessible to them, but less relevant once the spirochetes have colonized tissues; in these tissues the control of the spirochete is likely to occur through phagocytosis.

The identification of specific traits that could be therapeutically targeted in ongoing memory responses have not been described. Therefore there is a need in the art of method for diagnosing and the identification of alternative compounds for treating infections.

Summary of the Invention

In a first aspect, the invention relates to an in vitro method for diagnosing an infection in a subject which comprises a) determining the level of expression of IRF4 in a sample from said subject, and b) comparing said level with a reference value, wherein a decreased level of IRF4 compared to the reference value indicates that said subject suffers from an acute infection and wherein an increased level of Irf4 compared to the reference value indicates that said subject suffers from a persistent infection.

In a second aspect, the invention relates to a method for designing a personalized therapy in a subject suffering from an infection, which method comprises a) determining the level of expression of IRF4 in a sample from the subject, and b) comparing the level of expression obtained in a) with a reference value wherein a decreased or increased level of expression of IRF4 compared to the reference value is indicative that said subject is a candidate for receiving a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a Pl- 3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant.

In a third aspect, the invention relates to a combination comprising a compound suitable for treating an infection and a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, an antioxidant and combinations thereof.

In a fourth aspect, the invention relates to the combination according to the invention for use in the prevention and/or treatment of an infection.

In a fifth aspect, the invention relates to a compound for use in the prevention and/or treatment of an infection wherein the compound is selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant.

In a sixth aspect, the invention relates to the use of a kit comprising a reagent suitable for determining the level of expression of IRF4 in a sample for diagnosing an infection in a subject.

Brief description of the Figures

Figure 1. B. burgdorferi induces long-term responses in macrophages that affect phagocytosis and proinflammatory cytokine production. (A) Schematic representation of the working conditions to assess long-term effects of the stimulation of BMMs with B. burgdorferi. The four conditions were defined by the first and secondary stimulations, yielding the conditions UU, UB, BU and BB. (B) TNF production by BMMs acutely (UB) and re-stimulated (BB) with B. burgdorferi, compared to unstimulated (UU) and stimulated and rested (BU) macrophages. (C) TNF production by human peripheral monocytes incubated with B. burgdorferi according to the protocol presented in A. (D) B. burgdorferi binding (upper panel) and internalization (lower panel) by naive (black histograms) and memory (grey histograms) BMMs. The filled grey histogram represents BMMs with no spirochetes added. (E) Increased mean fluorescence intensity (MFI) of memory macrophages over naive cells, incubated for 2 h at 4 °C. The data are presented as percentage increase over acutely stimulated macrophages (UB). (F) Confocal image showing binding of GFP-S. burgdorferi (Bb) to naive (UB) and memory (BB) macrophages. The cells were incubated at 4 °C for 2 hours, fixed and stained with phalloidin and DAPI. (G) Expression of CD11b by naive (black histograms) and memory (grey histograms) macrophages. The figure represents the results of 4 independent mice. (H) Phagocytic index of naive (UB) and memory (BB) macrophages.

Figure 2. B. burgdorferi-induced Irf4 expression regulates proinflammatory cytokine production in memory macrophages. (A) Principal component analysis of the transcriptome of unstimulated (UU), acutely stimulated (UB), stimulated and rested (BU) and re-stimulated (BB) macrophages. (B) Volcano plots representing genes differentially expressed by acutely stimulated (left panel) and re-stimulated (right panel) macrophages versus unstimulated cells. (C) Venn diagram representing genes that are co- and differentially regulated in naive (UB) and re-stimulated (BB) macrophages versus unstimulated cells. The numbers at the top represent genes upregulated, while those at the bottom indicate the number of genes downregulated. (D) Volcano plot showing the differential expression of genes when comparing acute and memory macrophages. The light grey dots represent genes upregulated in BB macrophages and the dark grey dots indicate genes that are upregulated in acutely stimulated macrophages. (E) Motifs associated with the expression of genes specifically upregulated by memory macrophages. (F) Venn diagram showing the genes that are co- or differentially regulated by the transcription factors, SpiB, MAFK and MZF1. The only gene regulated by the 3 transcription factors is Irf4. (G) Open chromatin peaks associated with the genes regulated by SpiB, MAFK and MZF1 in acute and memory macrophages. (H) Phagocytosis by BMMs infected with lentivirus containing shlrf4 (grey histogram) or the control, pLKO (black histogram). The filled grey histogram represents the 4 °C control. The cells were coinfected with lentivirus containing two different shRNA sequences. (I) TNF and IL-6 induction by B. burgdorferi in Irf4-silenced and control BMMs. The cells were stimulated for 20 hours, followed by the determination of the cytokines in the supernatants by ELISA. (J) Lactate production by BMMs with silenced Irf4 and the control vector. Figure 3 mRNA expression levels of pro- and anti-inflammatory cytokines in BMMs differentially exposed to B. burgdorferi. The normalized reads for each condition (UU, UB, BU and BB) as defined in Fig. 1A, are represented. *; p < 0.05

Figure 4. Kinetics of Irf4 gene expression in vitro and in vivo. (A) Irf4 expression in acute and memory macrophages in response to B. burgdorferi, (B) peripheral blood and (C) heart tissue of infected mice.

Figure 5. Irf4-silenced RAW 264.7 cells regulate inflammatory output independently of phagocytosis. (A) Phagocytosis by RAW 264.7 cells containing shlrf4 (light grey and dark gray histograms) compared to control, pLKO-infected cells (black histogram). The filled grey histogram represents the 4°C control. The cells were infected separately with two different shRNA sequences: TRCN0000081548 (48, dark grey histogram) and TRCN0000081549 (49; light grey histogram). (B) TNF induction by B. burgdorferi- stimulation in Irf4-silenced and control cells. (C) Lactate production by RAW 264.7 cells silenced for Irf4 and the control vector. * < 0.05.

Figure 6 B. burgdorferi-induced metabolic changes in memory macrophages. (A) Oxygen consumption rate (OCR) and (B) glycolysis of acute and memory murine macrophages. (C) Lactate production by acute and memory murine macrophages, measured in the supernatants of cells stimulated following the experimental design described in Fig. 1A. (D) Paired LDHA expression in acute and memory human peripheral blood monocytes, as determined by real-time PCR. (E) Intermediate metabolites of the TCA in acute and memory murine macrophages.

Figure 7. Memory murine macrophages switch from and oxidative phosphorylation to a glycolytic metabolism. Normalized Oxygen consumption rate (OCR, A) and extracellular acidification rate (ECAR, B) of acute and memory BMMs. Oligo: oligomycin, FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Rot: rotenone; Anti: antimycin; 2-DG: 2-deoxy glucose. (C) Expression levels of Pfkfb3 in acute and memory macrophages. * < 0.05.

Figure 8 Glycolysis inhibition modulates the response of murine macrophages and modulates inflammation during Lyme borreliosis. (A) Lactate production (B) Phagocytosis and (C) TNF induction by B. burgdorferi in acute and memory macrophages in the presence or absence of the glycolysis inhibitor, 2-deoxyglucose (DG), during the phase of memory generation (48h) or the restimulation period (20h). (D) B. burgdorferi burdens as determined by recA expression over 10 ⁵ Rpl19 copies (E) Macrophage infiltration, (F) Tnf and (G) Irf4 gene expression in infected mouse hearts treated with dichloroacetate starting day 14 of infection (DCA 14). The results shown represent 10 mice per group and are representative of 2 experiments performed with an n = 15 per group. (H) Paired Irf4 gene expression by memory macrophages treated with DG during the re-stimulation with B. burgdorferi. (I) TNF production by memory macrophages treated with 2-DG or left untreated in the presence of B. burgdorferi for 20 h, washed and re-stimulated with the spirochete (20 h) in the absence or presence of 2- DG, following the scheme shown on the right. *, p < 0.05.

Figure 9. Validation of the RNAseq. The upper graphs show normalized reads of a group of selected genes differentially regulated in BMMs from 4 independent mice, as determined by the RNA-seq analysis. The bottom graphs show the fold induction of the same genes determined by qRT-PCR using BMMs from 4 mice. BMMs were stimulated following the schedule from figure 1A.

Figure 10. Glycolysis inhibition modulates the response of murine macrophages to B. burgdorferi. (A) Lactate production (B) Phagocytosis and (C) TNF induction by B. burgdorferi in acute and memory macrophages in the presence or absence of the glycolysis inhibitor, 2-deoxyglucose (2DG), during the restimulation period (20h) with B. burgdorferi. ^* , p < 0.05.

Figure 11. Glycolysis inhibition modulates inflammation during Lyme borreliosis. (A) B. burgdorferi burdens in the heart of infected mice as determined by DNA real-time PCR using primers specific for recA and relative to the house-keeping gene, Rpl19. (B) Macrophage infiltration (Adrgel), and Tnf gene expression in infected mouse hearts treated with 3PO starting day 14 of infection. (C) Irf4 expression in infected hearts in mice treated with 3PO or controls. Uninf., uninfected controls. The results shown represent 5 infected mice per group.

Figure 12. The memory phenotype is reversible and renders macrophages more susceptible to glycolysis inhibition. (A) Irf4 gene expression by memory macrophages treated with 2-DG during the re-stimulation with B. burgdorferi. (B) TNF production by memory and memory-reverted macrophages treated with 2DG in response to B. burgdorferi*, p < 0.05.

Detailed Description of the Invention

Diagnostic method

The authors of the present invention have identified IRF4 as a key regulator of memory macrophage proinflammatory responses and that the levels of IRF4 are different in subjects suffering from an infection compared to a reference value. Therefore, in a first aspect, the invention relates to an in vitro method for diagnosing an infection in a subject which comprises a) determining the level of expression of IRF4 in a sample from said subject, and b) comparing said level with a reference value, wherein a decreased level of IRF4 compared to the reference value indicates that said subject suffers from an acute infection and wherein an increased level of IRF4 compared to the reference value indicates that said subject suffers from a persistent infection.

“Diagnostic method”, as used herein relates to the evaluation of the probability according to which a subject suffers a specific pathology (in this case, an infection). As the skilled in the art will understand, such evaluation may not be correct for 100% of the subjects to be diagnosed, although it preferably is. The term, however, requires being able to identify a statistically significant part of the subjects.

The term “infection”, as used herein, relates to invasion by bacteria, viruses, fungi, protozoa or other microorganisms, referring to the undesired proliferation or presence of invasion of pathogenic microbes in a host organism. It includes the excessive growth of microbes that are normally present in or on the body of a mammal or other organism. More generally, a microbial infection can be any situation in which the presence of a microbial population(s) is damaging to a host mammal. Thus, a microbial infection exists when excessive numbers of a microbial population are present in or on a mammal's body, or when the effects of the presence of a microbial population(s) is damaging the cells or other tissue of a mammal.

In a preferred embodiment, the infection is caused by a bacterium.

The term “bacterium” refers to both gram-negative and gram-positive bacterial cells capable of infecting and causing a disease in a mammalian host, as well as producing infection-related symptoms in the infected host, such as fever or other signs of inflammation, intestinal symptoms, respiratory symptoms, dehydration, and the like.

In one embodiment the bacteria are gram-negative bacteria. In another embodiment the bacteria are gram-positive bacteria. In another further embodiment the bacteria are gram-positive bacteria together with gram-negative bacteria. In another embodiment there is only one bacteria species or different bacteria species; one bacteria genus or different bacteria genus, infecting or causing disease.

In some embodiments, and without limitation, the bacteria is of a genus selected from the group consisting of Acinetobacter, Actinobacillus, Aeromonas, Aggregatibacter, Agrobacterium, Bacillus, Bordetella, Brucella, Burkholderia, Campylobacter, Chromobacterium, Cyanobacteria, Enterobacter, Erwinia, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Micrococcus, Moraxella, Mycobacterium, Neisseria, Nitrosomas, Nocardia, Obesumbacterium, Pantoea, Pasteurella, Pediococcus, Porphyromonas, Prevotella, Proteus, Pseudomonas, Ralstonia, Rhizobium, Rhodobacter, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Tannerella, Treponema, Tsukamurella, Vibrio, Xenorhabdus, Yersinia and mixtures thereof. For example, in some embodiments and without limitation, the bacteria is of a species selected from the group consisting of Aeromonas hydrophila, Aeromonas salmonicida, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Agrobacterium tumefaciens, Bacillus cereus, Bacillus subtilis, Burkholderia cepacia, Campylobacter jejuni, Chromobacterium violaceum, Enterobacter agglomeran, Erwinia carotovora, Erwinia chrysanthemi, Escherichia coli, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pylori, Lactobacillus plantarum, Listeria monocytogenes, Klebsiella pneumoniae, Micrococcus luteus, Mycobacterium tuberculosis, Neisseria meningitidis, Neisseria gonorrhoeae, Nitrosomas europaea, Nocardia carnea, Obesumbacterium proteus, Pantoea stewartii, Pediococcus acidilactici, Prevotella intermedia, Porphyromonas gingivalis, Pseudomonas aureofaciens, Pseudomonas aeruginosa, Pseudomonas phosphoreum, Pseudomonas syringae, Ralstonia solanacearum, Rhiszobium etli, Rhizobium leguminosarum, Rhodobacter sphaeroides, Salmonella typhimurium, Serratia liguefaciens, Serratia marcescens, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus enteritis, Tannerella forsythensis, Treponema denticola, Tsukamurella pulmonis, Vibrio anguillarum, Vibrio fischeri, Vibrio cholerae, Vibrio harveyi,, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, Xenorhabdus nematophilus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia medievalis, Yersinia ruckeri and mixtures thereof.

In a more preferred embodiment, the infection is caused by a microorganism selected from the group consisting of a Lyme disease-causing Borrelia species, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Treponema pallidum, Borrelia hermsii, Borrelia miyamotoii, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Haemophilus influenza, Mycobacterium tuberculosis, Helicobacter pylori, Salmonella typhi, Mycobacterium leprae, Neisseria gonorrhoeae , Chlamydia spp., Bartonella, Legionella pneumophilla and Brucella abortus.

“Lyme disease-causing Borrelia species”, as used herein, relates to Borrelia species capable of inducing Lyme disease. Lyme disease or Lyme borreliosis is an infectious disease caused by the Borrelia bacterium and is transmitted to humans by the bites of infected ticks of the genus Ixodes. In a preferred embodiment, the Lyme disease- causing Borrelia species is selected from the group consisting of Borrelia burgdorferi, Borrelia mayonii, Borrelia afzelii and Borrelia garinii. In a more preferred embodiment, the Lyme disease-causing Borrelia species is Borrelia burgdorferi.

“Acute infection”, as used herein, means the primary stage of an infection during which the immune system elicits an initial response to the recent introduction of the microorganism into a subject.

“Persistent infections" refer to those infections that, in contrast to acute infections, are not effectively cleared by the induction of a host immune response. During such persistent infections, the infectious agent and the immune response reach equilibrium such that the infected subject remains infectious over a long period of time without necessarily expressing symptoms. Persistent infections often involve stages of both silent and productive infection without rapidly killing or even producing excessive damage of the host cells. Persistent infections include for example, latent, chronic and slow infections. The mechanisms by which persistent infections are maintained can involve modulation of the microorganism and cellular gene expression and modification of the host immune response. Reactivation of a latent infection can be triggered by various stimuli, including changes in cell physiology, superinfection by another microorganism, and physical stress or trauma. Host immunosuppression is often associated with reactivation of a number of persistent infections.

The term “subject”, as used herein, refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents. Preferably, the subject is a human of any sex, age or race.

Suitable samples for use in the method of the invention include any bodily fluid selected from the group consisting of blood, serum, plasma, CSF, saliva, gingival crevicular fluid, urine, seminal plasma, tears, and nipple fluid. The term biofluid also includes liquid biopsy. A liquid biopsy, also known as fluid biopsy or fluid phase biopsy is the sampling and analysis of non-solid biological tissue, primarily blood. In a particular embodiment, the biofluid is blood, more particularly a peripheral blood. In another particular embodiment, the sample is a cellular fraction of blood such as for example macrophages, leukocytes, erythrocytes or thrombocytes. In another particular embodiment, the sample is an acellular fraction of blood. Additionally suitable samples are tissue samples. In a preferred embodiment the sample is whole blood sample or blood cell sample.

“IRF4”, also known as Interferon regulatory factor 4 or MUM1 , relates to a transcriptional activator that binds to the interferon-stimulated response element (ISRE) of the MHC class I promoter. In humans the sequence of IRF4 protein corresponds to the accession number Q15306 in the Uniprot database on September 18, 2019.

In a first step, the method for diagnosing an infection according to the invention comprises determining the level of expression of IRF4 in a sample from the subject.

As it is used herein, the term “expression level” refers to the value of a parameter that measures the degree of expression of a specific gene or of the corresponding polypeptide. In a particular embodiment, said value can be determined by measuring the mRNA level of the gene of interest or a variant thereof or by measuring the amount of protein encoded by said gene of interest or a variant thereof. Thus, in the context of the present invention, in a particular embodiment, said expression level comprises determining the level of the mRNA encoded from the IRF4 gene or determining the level of the IRF4 protein or a variant thereof.

Preferably, variants of IRF4 are (i) polypeptides in which one or more amino acid residues are substituted by a preserved or non-preserved amino acid residue (preferably a preserved amino acid residue) and such substituted amino acid may be coded or not by the genetic code, (ii) polypeptides in which there is one or more modified amino acid residues, for example, residues modified by substituent bonding, (iii) polypeptides resulting from alternative processing of a similar mRNA, (iv) polypeptide fragments and/or (v) polypeptides resulting from URI fusion or the polypeptide defined in (i) to (iii) with another polypeptide, such as a secretory leader sequence or a sequence being used for purification (for example, His tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated through proteolytic cut (including multisite proteolysis) of an original sequence. The variants may be post-translationally or chemically modified. Such variants are supposed to be apparent to those skilled in the art.

As known in the art, the “similarity” between two polypeptides is determined by comparing the amino acid sequence and the substituted amino acids preserved from a polypeptide with the sequence of a second polypeptide. The variants are defined to include polypeptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment concerned, more preferably different from the original sequence in less than 25% of residues per segment concerned, more preferably different from the original sequence in less than 10% of residues per segment concerned, more preferably different from the original sequence in only a few residues per segment concerned and, at the same time, sufficiently homologous to the original sequence to preserve functionality of the original sequence. The present invention includes amino acid sequences which are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides may be determined using computer algorithms and methods which are widely known to those skilled in the art. The identity between two amino acid sequences is preferentially determined using BLASTP algorithm [BLASTManual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)]. In a preferred embodiment, the sequence identity is determined throughout the whole length of the polypeptide of IRF4 or throughout the whole length of the variant or of both.

Virtually any conventional method for detecting and quantifying the expression level of a gene can be used within the framework of the present invention for detecting and quantifying the expression level of IRF4. By way of non-limiting illustration, the expression level of a gene can be determined by means of quantifying the mRNA level of said gene or by means of quantifying the level of protein encoded by said gene. Methods for determining the amount of mRNA are well-known in the state of the art. For example, the nucleic acid contained in the sample, such as blood ^' s sample from the subject under study, is extracted according to conventional methods. The extracted mRNA can be detected by hybridization (for example by means of Northern blot analysis or DNA or RNA arrays (microarrays) after converting mRNA into labeled cDNA) and/or amplification by means of a enzymatic chain reaction. In general, quantitative or semi- quantitative enzymatic amplification methods are preferred. The polymerase chain reaction (PCR) or quantitative real-time RT-PCR or semi-quantitative RT-PCR technique is particularly advantageous. Primer pairs are preferably designed for the purpose of superimposing an intron to distinguish cDNA amplification from the contamination from genomic DNA (gDNA). Additional primers or probes, which are preferably labeled, for example with fluorescence, which hybridize specifically in regions located between two exons, are optionally designed for the purpose of distinguishing cDNA amplification from the contamination from gDNA. If desired, said primers can be designed such that approximately the nucleotides comprised from the 5’ end to half the total length of the primer hybridize with one of the exons of interest, and approximately the nucleotides comprised from the 3’ end to half the total length of said primer hybridize with the other exon of interest. Suitable primers can be readily designed by a person skilled in the art. Other amplification methods include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). The amount of mRNA is preferably measured quantitatively or semi-quantitatively. Relevant information about conventional methods for quantifying the expression level of a gene can be found, for example, in Sambrook et al., 2001 [Sambrook, J., et al., “Molecular cloning: a Laboratory Manual”, 3rd ed., Cold Spring Harbor Laboratory Press, N.Y., Vol. 1-3]

To normalize the expression values of one gene among different samples, comparing the mRNA level of the gene of interest (i.e. IRF4) in the samples from the subject object of study with a control RNA level is possible. As it is used herein, a “control RNA” is RNA of a gene the expression level of which does not differ depending on if the subject suffers from an infection or not; a control RNA is preferably an mRNA derived from a housekeeping gene encoding a protein that is constitutively expressed and carrying out essential cell functions. Illustrative, non-limiting examples of housekeeping genes for use in the present invention include GUSB (beta-glucuronidase), PPIA (peptidyl-prolyl isomerase A), b-2-microglobulin, GAPDH, PSMB4 (proteasome subunit beta type-4), ubiquitin, transferrin receptor, 18-S ribosomal RNA, cyclophilin, tubulin, b- actin, 3-monooxygenase/tryptophan 5-monooxygenase tyrosine activation protein (YWHAZ), etc. If the expression level of TLR2 and/or TLR4 is determined by measuring the expression level of transcription product (mRNA) of said gene in a sample from the subject under study, the sample can be treated to physically or mechanically break up the structure of the tissue or cell for the purpose of releasing the intracellular components into an aqueous or organic solution to prepare the nucleic acids for additional analysis. Care is preferably taken to prevent RNA degradation during the extraction process.

In a particular and preferred embodiment of the invention, the expression level of IRF4 is determined by means of determining the expression level of the protein encoded by the IRF4 gene or a variant thereof, because increased expression of a gene is usually accompanied by an increase in the amount of corresponding protein. The term “variant” as used herein, relates to those variant of human IRF4 which appear naturally in other species, i.e. the orthologues of IRF4.

In another particular and preferred embodiment of the invention, the expression level of IRF4 is determined by means of determining the expression level of the protein encoded by the IRF4 gene. The natural variants of IRF4 suitable for their use in the present invention also derive from said sequence by insertion, substitution or deletion of one or more amino acids and include natural alleles, variants resulting from alternative processing and truncate forms which appear naturally. The term “variant” also includes fragments, isoforms and analogues or derivatives of IRF4.

The proteins can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation, protein folding and proteolytic processing, etc. Additionally, the proteins may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation.

The determination of the amount of a protein corresponding to the expression of a specific gene can be performed using any conventional method for protein detection and quantification, for example by means of an immunoassay, etc. By way of non-limiting illustration, said determination can be performed using antibodies with the capability to bind specifically to the protein to be determined (or fragments thereof with the antigenic determinants) and subsequent quantification of the antigen-antibody complex derivatives. The antibodies can be, for example, polyclonal sera, hybridoma supernatants or monoclonal antibodies, fragments of antibodies, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies, humanized antibodies, etc. Said antibodies may (or may not) be labeled with a marker. Illustrative, non-limiting examples of markers that can be used in the present invention include radioactive isotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme cofactors, enzyme substrates, enzyme inhibitors, etc. There is a wide range of well-known assays that can be used in the present invention, such as, for example, assays based on Western-blot or immunoblot techniques, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical or immunohistochemical techniques such as flow cytometry, etc. Other ways of detecting and quantifying the protein include affinity chromatography, ligand binding assay techniques, particle-enhanced turbidimetric immunoassay (PETIA) etc.

The second step of the method of the invention comprises comparing the level of expression of IRF4 obtained in the first step of the method with a reference value.

As it is used herein, the term “reference value” refers to a value obtained in the laboratory and used as a reference for the values or data obtained by means of laboratory examinations of the patients or samples collected from the patients. The reference value or reference level can be an absolute value, a relative value, a value having an upper and/or lower limit, a range of values, a mean value, a median value, or a value compared to a specific control or reference value. The reference value can be based on a value of the individual sample, such as a value obtained from a sample of the subject being tested, for example, but at an earlier time. The reference value can be based on a large number of samples, such as the values of the population of subjects from the same age group, or can be based on a set of samples, including or excluding the sample to be tested. In a preferred embodiment, the reference value corresponds to the level of expression of IRF4 in a sample from the subject not suffering an infection, and more preferably the level of expression of IRF4 in a healthy subject.

Once the reference value has been established, the expression level of IRF4 in the sample from the subject under study is compared with the reference value. As a consequence of this comparison, the expression level of the marker of interest ( IRF4 in the method of the invention) in the sample from the subject can be “greater than”, “less than” or “equal to” said reference value for said gene. In the context of the present invention, it is considered that an expression level of IRF4 in the sample from the subject is “greater than” or “higher than” the reference value for said marker when the expression level of IRF4 in the sample from the subject increases, for example, 5%, 10%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or even more when compared with the reference value for said gene, or when it increases, for example, at least 1.1 -fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or even more when compared with the reference value for said marker. In the context of the present invention, it is also considered that an expression level of the marker of interest ( IRF4 ) in the sample from the subject is “less than” the reference value for said marker when the expression level of IRF4 in the sample from the subject decreases, for example, 5%, 10%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or even 100% when compared with the reference value for said marker.

Once the comparison is made between the expression level of IRF4 in the sample from the subject and the reference value for said marker, the method of the invention allows determining if a subject suffers from an acute infection if the expression level of IRF4 is lower than the reference value or if a subject suffers from a persistent infection if the expression level of IRF4 is higher than said reference value. All the terms and embodiments described elsewhere herein are equally applicable to this aspect of the invention.

Method for designing a personalized therapy

The authors of the present invention have identified the transcriptomic and metabolic changes associated with the memory response of macrophages to microorganisms able to establish long-term infections in mammals. Inhibition of glycolysis reduces the production of TNF by memory macrophages. In vivo, glycolysis inhibition results in decreased cardiac inflammation and reduced expression of IRF4. Therefore the inventors propose a personalized therapy for subjects suffering from an infection. The increased expression of IRF4 results in the reduction of TNF by competition with the pathway that leads to its production; therefore, the increased expression of this molecule will result in decreased inflammation.

Therefore, in another aspect, the invention relates to a method for designing a personalized therapy in a subject suffering from an infection, which method comprises a) determining the level of expression of IRF4 in a sample from the subject, and b) comparing the level of expression obtained in a) with a reference value wherein a decreased or increased level of expression of IRF4 compared to the reference value is indicative that said subject is a candidate for receiving a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a Pl- 3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant.

In a preferred embodiment, the infection is an acute infection. In another preferred embodiment, the infection is a persistent infection.

“Personalized or customized therapy” relates to the match of patients with treatments that are more likely to be effective and cause fewer side effects.

Steps a) and b) have been previously described in detail in relation to the method for diagnosing an infection.

After step b) if a decreased or increased level of expression of IRF4 compared to the reference value is detected, indicates that said subject is a candidate for receiving a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant.

“Inhibitor of an enzyme”, as used herein, in the present case inhibitor of Ras, Pl- 3K, mTOR or STAT3, relates to a molecule capable of inhibiting, blocking or decreasing the activity of the enzyme, including those compounds which prevent expression of the enzyme and which lead to reduced mRNA or protein levels of the enzyme. Thus, suitable inhibitors of a particular enzyme according to the present invention includes dominant negatives of the enzyme, an interference RNA specific for the sequence of the enzyme, an antisense oligonucleotide specific for the sequence of the enzyme, a ribozyme or DNA enzyme specific for the sequence of the enzyme or inhibitory antibodies capable of binding specifically to the enzyme and inhibiting or reducing the enzyme activity. Other compounds capable of inhibiting an enzyme include aptamers and spiegelmers. In a preferred embodiment, the inhibitors of an enzyme, in the present case inhibitor of Ras, PI-3K, mTOR or STAT3 is a small molecule compound.

In a preferred embodiment, the compound is a RAS inhibitor.

“Ras”, as used herein relates to a protein that binds GDP/GTP and possesses intrinsic GTPase activity. In human Ras corresponds to the sequence with accession number P01112 in the Uniprot database September 18, 2019.

Illustrative, non- limitative examples of Ras inhibitors are Manumycin A, Farnesyl thiosalicylic acid, FTI-276 trifluoroacetate salt, L-744,832 Dihydrochloride and MCI-062, Deltarasin, Zn-cyclen, ARS-1620, BIM-46187, Sulindac sulphide, IND12, Kobe2601, 3144, DCAI and ABD7.

In another preferred embodiment, the compound is a PI-3K inhibitor and/or mTOR inhibitor.

“PI-3K”, Phosphoinositide-3-kinase (PI3K), as used herein relates to an enzyme that phosphorylates Ptdlns (Phosphatidylinositol), Ptdlns4P (Phosphatidylinositol 4- phosphate) and Ptdlns(4,5)P2 (Phosphatidylinositol 4,5-bisphosphate) to generate phosphatidylinositol 3,4,5-trisphosphate (PIP3). The catyalytic subunit alpha isoform in human corresponds to the sequence with accession number P42336 in the Uniprot database September 18, 2019.

Illustrative, non-limitative examples of PI-3K inhibitors are Wortmannin, LY294002, hibiscone C, Idelalisib, Copanlisib, Duvelisib, Alpelisib and Umbralisib,

In another preferred embodiment, the compound is a mTOR inhibitor.

“mTOR” as used herein relates to serine/threonine protein kinases and regulate cell proliferation, cell motility, cell growth, cell survival, and transcription. In human mTOR corresponds to the sequence with accession number P42345 in the Uniprot database September 18, 2019.

Illustrative, non-limitative examples of mTOR inhibitors useful in the present invention, are without limitation Rapamycin and its derivatives, sirolimus, temsirolimus, everolimus and ridaforolimus. Alternatively, mTOR/PI3K dual inhibitors can be used according to the invention. Illustrative non-limitative examples are dactolisib, BGT226, SF1126 and PKI-587, LY3023414, PF-05212384, PQR309, P7170, SF-1126, Copanlisib and BEZ235.

In another preferred embodiment, the compound is a STAT3 inhibitor.

“STAT3” as used herein relates to a transcription factor that mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. In human STAT3 corresponds to the sequence with accession number P40763 in the Uniprot database September 18, 2019.

Illustrative, non-limitative examples of STAT3 inhibitors useful in the present invention, are without limitation Galiellalactone, S3295, CPA-1 , CPA-7, STA-21, LLL-3, LLL12, S3I-201, FLLL31, STX-0119, ISS 610, Cpc 3, Cpc 30, Cpc 188, BP-1-102, ISS610, Stattic, OPB-31121, OPB-51602, Curcumin and analogues (FLLL11, FLLL12, FLLL32), AZD1480 and Butein.

In a more preferred embodiment, the compound is a glycolysis inhibitor.

“Glycolysis inhibitor”, “glycolytic inhibitor” or “inhibitor(s) of glycolysis” are intended to refer to compounds or compositions that substantially inhibit or interfere with the activity of one or more enzymes involved in glycolysis. Illustrative non-limitative examples of glycolysis inhibitor are Dichloroacetate, 2-deoxyglucose, 3-bromopyruvate, Lodinamine, 3-(3-pyrididyl)-1-(4-pyridinyl)-2-propene-1-one (3PO), 1-(4-pyridinyl)-3-(2- quinolinyl)-2-Propen-1-one (PFK15), 6-Aminonicotinamide, Oxythiamine Chloride Hydrochloride, Shikonin and FX11. In a preferred embodiment, the glycolysis inhibitor is an inhibitor of pyruvate dehydrogenase kinase, more preferably dichloroacetate (DCA). DCA corresponds to the compound having the CAS number 13425-80-4.

“Pyruvate dehydrogenase kinase”, also pyruvate dehydrogenase complex kinase, PDC kinase, or PDK; EC 2.7.11.2, is a kinase enzyme which acts to inactivate the enzyme pyruvate dehydrogenase by phosphorylating it using ATP. In human, the sequence of PDK1 corresponds to the sequence with accession number Q15118 in the Uniprot dataset September 18, 2019.

Illustrative non-limitative examples of PDK inhibitors are 2-chloropropionate, N- methylnicotinate-dichloroacetate, Nov3r ( (R)-3.3.3-trifluoro-2-hydroxy-2-methyl propionamides), AZD7545 (Aninlide tertiary carbinols, Pfz3 ((N-(2-aminoethyl)-2{3- chloro-4-[(4-isopropylbenzyl)oxi]phenyl}acetamide)), Radicicol, Mitaplatin, Hemoglobin- DCA conjugate, Mito-DCA, Phenulbutyrate, 4,5-diarylisoxazoles, VER-246608 and Betulinic acid. In another preferred embodiment, the inhibitor is an inhibitor of fructose 2, 6 biphosphatase 3, more preferably 3-(3-pyrididyl)-1-(4-pyridinyl)-2-propene-1-one (3PO) ,(1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one -PFK15), 5-triazolo-2-arylpyridazinone, 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP), 5,6,7,8-tetrahydroxy-2-(4- hydroxyphenyl) chrome-4-one (N4A) or 7,8-dihydroxy-3-(4.hydroxyphenyl) chromen-4- one (YN1).

“Fructose 2, 6 bisphosphatase 3” or FBP1, (EC 3.1.3.11), as used herein, relates to an enzyme that converts fructose-1 , 6-bisphosphate to fructose 6-phosphate in gluconeogenesis and the Calvin cycle which are both anabolic pathways. In human, the sequence of FBP1 corresponds to the sequence with accession number P09467 in the Uniprot database September 18, 2019.

3-(3-pyrididyl)-1-(4-pyridinyl)-2-propene-1-one (3PO) corresponds to the compound having the CAS number 55314-16-4. (1-(4-pyridinyl)-3-(2-quinolinyl)-2- propen-1-one) corresponds to the compound having the CAS number 4382-63-2.

In another preferred embodiment, the compound is an IRF4 mimetic. “IRF4 mimetic” or IRF4 peptidomimetic, as used herein relates to various types or classes of molecules, as long as the resulting molecule mimics or resembles the biological activity of Irf4. In an embodiment, the mimetic is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides and peptoids. Examples of some peptidomimetics by the broader definition (e.g., where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below. Whether completely or partially non-peptide in character, peptidomimetics according to this invention may provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in a polypeptide. As a result of this similar active-site geometry, the peptidomimetic may exhibit biological effects that are similar to the biological activity of a polypeptide. In other embodiment, the mimetic include peptidic compounds. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin 10 Pergamon Press (1992).

In other embodiments a peptide mimetic is an oligomer that mimics peptide secondary structure through use of amide bond isosteres and/or modification of the native peptide backbone, including chain extension or heteroatom incorporation; examples of which include azapeptides, oligocarbamates, oligoureas, beta-peptides, gamma-peptides, oligo(phenylene ethynylene)s, vinylogous sulfonopeptides, poly-N- substituted glycines (peptoids) and the like. Methods for designing and synthesizing peptide mimetics are well known to one of skill in the art. In certain embodiments, it is contemplated that a peptide mimetic is used to overcome protease sensitivity, stabilize secondary structure and/or improve bioavailability relative to a naturally occurring IRF4 peptide analogues. In certain embodiments, a peptide mimetic of the invention is a reverse turn mimetic, e.g., an alpha-turn mimetic, a monocyclic beta-turn mimetic, a bicyclic beta-turn mimetic, a gamma-turn mimetic or a monocyclic gamma-turn mimetic.

T echniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure, shape or reactivity. The development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. These techniques aid in the development of novel compositions of higher potency and/or greater bioavailability and/or greater stability than the original polypeptide (Dean (1994), BioEssays, 16: 683-687; Cohen and Shatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich (1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98, all incorporated herein by reference].

By “IRF4 mimetic” is also understood any molecule capable of increasing the level of expression or activity of IRF4, such as an inducer agent or a molecule.

The term “agent inducing expression” or “inducer agent”, as used in this invention, refers to any molecule that is capable of causing an increase in the transcription of a gene. Usually, the gene the transcription of which is induced in response to said inducer agent is under the operative control of a transcription regulatory region which, in turn, has binding sites for a transcription activator the activity of which increases in the presence of said inducer agent. Preferably, the inducer agent is a compound that is easy to administer and easily distributed in the body, and innocuous at the doses used to activate the system. Moreover, it must be capable of penetrating into the desired tissue or organ, and have a suitable half-life (usually of several hours).

In another preferred embodiment the compound is an antioxidant. “Antioxidant”, as used herein relates to a compound that inhibits oxidation. The antioxidant may be natural or synthetic antioxidant. Illustrative, non limitating examples of antioxidants that can be used in the present invention are ascorbic acid, glutathione lipoic acid, carotenes, a- tocopherol, ubiquinol, silibinin, erythropoietin, allopurinol, oxypurinol, N-acetylcysteine (NAC), melatonin or lutein.

In a preferred embodiment, the antioxidant is silibinin.

“Silibinin”, also known as silybin, relates to a mixture of two diastereomers, silybin A and silybin B, in approximately equimolar ratio and corresponds to the compound having the CAS number 22888-70-6.

The compounds to be administered according to the present invention are in a therapeutically effective amount.

The term "effective" amount or a "therapeutically effective amount" of a compound is meant a nontoxic but sufficient amount of the compound to provide the desired effect.

Even though individual needs vary, determination of optimal ranges for effective amounts of the agent of the invention belongs to the common experience of those experts in the art. In general, the dosage needed to provide an effective amount of such compound, which can be adjusted by one expert in the art will vary depending on age, health, fitness, sex, diet, weight, frequency of treatment and the nature and extent of impairment or illness, medical condition of the patient, route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profile of the particular compound used, if using a system drug delivery, and if the compound is administered as part of a combination of drugs.

The compounds to be administered according to the invention can occur at any pharmaceutical form of administration considered appropriate for the selected administration route, for example, by systemic (e.g intravenous, subcutaneous, intramuscular injection), oral, parenteral or topical administration, for which it will include the pharmaceutically acceptable excipients necessary for formulation of the desired method of administration. Additionally, it is also possible to administer the compounds intranasally or sublingually which allows systemic administration by a non-aggressive mode of administration. Also, intraventricular administration may be adequate. A preferred route of delivery is oral.

Those skilled in the art are familiar with the principles and procedures discussed in widely known. Where necessary, the compound is comprised in a composition also including a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

In cases other than intravenous administration, the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, gel, polymer, or sustained release formulation. The composition can be formulated with traditional binders and carriers, as would be known in the art. Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., inert carriers having well established functionality in the manufacture of pharmaceuticals. Various delivery systems are known and can be used to administer a compound according to the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.

Solid dosage forms for oral administration may include conventional capsules, sustained release capsules, conventional tablets, sustained-release tablets, chewable tablets, sublingual tablets, effervescent tablets, pills, suspensions, powders, granules and gels. At these solid dosage forms, the active compounds can be mixed with at least one inert excipient such as sucrose, lactose or starch. Such dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets, effervescent tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can be prepared with enteric coatings.

Liquid dosage forms for oral administration may include emulsions, solutions, suspensions, syrups and elixirs pharmaceutically acceptable containing inert diluents commonly used in the technique, such as water. Those compositions may also comprise adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening agents, flavoring and perfuming agents.

Injectable preparations, for example, aqueous or oleaginous suspensions, sterile injectable may be formulated according with the technique known using suitable dispersing agents, wetting agents and/or suspending agents. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. Sterile oils are also conventionally used as solvents or suspending media.

For topical administration, compounds of the invention can be formulated as creams, gels, lotions, liquids, pomades, spray solutions, dispersions, solid bars, emulsions, microemulsions and similars which may be formulated according to conventional methods that use suitable excipients, such as, for example, emulsifiers, surfactants, thickening agents, coloring agents and combinations of two or more thereof.

Additionally, the compounds may be administered in the form of transdermal patches or iontophoresis devices.

Several drug delivery systems are known and can be used to administer the agents or compositions for use according to the invention, including, for example, encapsulation in liposomes, microbubbles, emulsions, microparticles, microcapsules and similars. The required dosage can be administered as a single unit or in a sustained release form.

Sustainable-release forms and appropriate materials and methods for their preparation are described in, for example, "Modified-Release Drug Delivery Technology", Rathbone, M. J. Hadgraft, J. and Roberts, M. S. (eds.), Marcel Dekker, Inc., New York (2002), "Handbook of Pharmaceutical Controlled Release Technology", Wise, D. L. (ed.), Marcel Dekker, Inc. New York, (2000). In one embodiment of the invention, the orally administrable form of a compound for use according to the invention is in a sustained release form further comprises at least one coating or matrix. The coating or sustained release matrix include, without limitation, natural polymers, semisynthetic or synthetic water-insoluble, modified, waxes, fats, fatty alcohols, fatty acids, natural semisynthetic or synthetic plasticizers, or a combination of two or more of the them. Enteric coatings may be applied using conventional processes known to experts in the art, as described in, for example, Johnson, J. L., "Pharmaceutical tablet coating", Coatings Technology Handbook (Second Edition), Satas, D. and Tracton, A. A. (eds), Marcel Dekker, Inc. New York, (2001), Carstensen, T., "Coating Tablets in Advanced Pharmaceutical Solids", Swarbrick, J. (ed.), Marcel Dekker, Inc. New York (2001), 455-468.

All the terms and embodiments previously described are equally applicable to this aspect of the invention. Combination of the invention

In another aspect, the invention relates to a combination comprising a compound suitable for treating an infection and a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, an antioxidant and combinations thereof.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and a glycolysis inhibitor.

In another preferred embodiment, the combination comprises a compound suitable for treating an infection and an IRF4 mimetic.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and a Ras inhibitor.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and a PI-3K inhibitor.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and a mTOR inhibitor.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and a STAT3 inhibitor.

In a preferred embodiment, the combination comprises a compound suitable for treating an infection and an antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention comprises an IRF4 mimetic and a Ras inhibitor, an IRF4 mimetic and a PI-3K inhibitor, an IRF4 mimetic and a mTOR inhibitor, an IRF4 mimetic and a STAT3 inhibitor, an IRF4 mimetic and an antioxidant, a Ras inhibitor and a PI-3K inhibitor, a Ras inhibitor and a mTOR inhibitor, a Ras inhibitor and a STAT3 inhibitor, a Ras inhibitor and an antioxidant, a PI-3K inhibitor and a mTOR inhibitor, a PI-3K inhibitor and a STAT3 inhibitor, a PI-3K inhibitor and an antioxidant, a mTOR inhibitor and a STAT3 inhibitor, a mTOR inhibitor and an antioxidant, or a STAT3 inhibitor and antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention can comprise an IRF4 mimetic, a Ras inhibitor and a PI-3K inhibitor; an IRF4 mimetic, a Ras inhibitor and a mTOR inhibitor, an IRF4 mimetic, a Ras inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a Ras inhibitor and an antioxidant; an IRF4 mimetic, a PI-3K inhibitor and a mTOR inhibitor; an IRF4 mimetic, a PI-3K inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a PI-3K inhibitor and an antioxidant; an IRF4 mimetic, a mTOR inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a mTOR inhibitor and an antioxidant; an IRF4 mimetic, a STAT3 inhibitor and an antioxidant; a Ras inhibitor, a PI-3K inhibitor and a mTOR inhibitor; a Ras inhibitor, a Pl- 3K inhibitor and a STAT3 inhibitor; a Ras inhibitor, a PI-3K inhibitor and an antioxidant; a Ras inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a Ras inhibitor, a mTOR inhibitor and an antioxidant; a Ras inhibitor, a STAT3 inhibitor and an antioxidant; a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; or a mTOR inhibitor, a STAT3 inhibitor and an antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention comprises an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and a mTOR inhibitor; an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and an antioxidant; an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor and an antioxidant; an IRF4 mimetic, a Ras inhibitor, a STAT3 inhibitor and an antioxidant; an IRF4 mimetic, a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a PI-3K inhibitor ,a mTOR inhibitor and an antioxidant; an IRF4 mimetic, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; an IRF4 mimetic, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; a Ras inhibitor, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; a Ras inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant or a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention comprises a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and a mTOR inhibitor; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and a STAT3 inhibitor; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a glycolysis inhibitor, an IRF4 mimetic, a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, a Ras inhibitor, a Pl- 3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a glycolysis inhibitor, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; a glycolysis inhibitor, a Ras inhibitor, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, a Ras inhibitor, a TOR inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; an IRF4 mimetic, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant, or a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention comprises a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and a STAT3 inhibitor; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, an IRF4 mimetic, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; a glycolysis inhibitor, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant; or an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant.

In another preferred embodiment, in addition to a compound suitable for treating an infection, the combination of the invention comprises a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant.

“Combination" stands for the various combinations of a compound suitable for treating an infection and a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant in a composition, in a combined mixture composed from separate formulations of the single active compounds, such as a "tank-mix", and in a combined use of the single active ingredients when applied in a sequential manner, i.e. one after the other with a reasonably short period, such as a few hours or days or in simulatenous administration.

A combination of a compound suitable for treating an infection and a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant may be formulated for its simultaneous, separate or sequential administration. This has the implication that the combination of the two compounds may be administered:

- as a combination that is being part of the same medicament formulation, the two compounds being then administered always simultaneously.

- as a combination of two units, each with one of the substances giving rise to the possibility of simultaneous, sequential or separate administration.

In a particular embodiment, the compound suitable for treating an infection is independently administered from the compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant (i.e in two units) but at the same time.

In another particular embodiment, the compound suitable for treating an infection is administered first, and then the compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant is separately or sequentially administered.

In yet another particular embodiment, the compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor and an antioxidant is administered first, and then the compound suitable for treating an infection is administered, separately or sequentially, as defined.

In a preferred embodiment, the compound is a glycolysis inhibitor, particularly an inhibitor of pyruvate dehydrogenase kinase, more particularly dichloroacetate, an inhibitor of fructose-2, 6-bisphosphatase 3, more particularly 3-(3-pyridinyl)-1-(4- pyridinyl)-2-propen-1-one or (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one). In another preferred embodiment, the compound is an IRF4 mimetic.

Furthermore, compounds are administered in the same or different dosage form or by the same or different administration route, e.g. one compound can be administered topically and the other compound can be administered orally. Suitably, both compounds are administered orally.

“Compound suitable for treating an infection”, as used herein to any drug capable of killing bacteria, viruses, fungi or parasites or inhibit their growth. Antimicrobial medicines can be grouped according to the microorganisms they act primarily against, antibacterial, antifungal, antiviral and antiparasitic.

In a preferred embodiment, the compound suitable for treating an infections, is suitable for treating a persistent infection. In another preferred embodiment, the compound is suitable for treating an infection caused by a Lyme disease-causing Borrelia species, more preferably doxycycline, amoxicillin, or cefuroxime axetil.

In a preferred embodiment, the Lyme disease-causing Borrelia species is selected from the group consisting of Borrelia burgdorferi, Borrelia mayonii, Borrelia afzelii and Borrelia garinii. In a more preferred embodiment, the Lyme disease-causing Borrelia species is Borrelia burgdorferi.

All the terms and embodiments previously described are equally applicable to this aspect of the invention.

Medical uses

The authors of the present invention have identified the transcriptomic and metabolic changes associated with the memory response of macrophages to microorganisms able to establish long-term infections in mammals. Therefore the inventors proposes novel treatments for infections.

In another aspect, the invention relates to the combination according to the invention for use in the prevention and/or treatment of an infection.

Alternatively, the invention relates to a method for preventing and/or treating an infection comprising administering a combination of the invention to a subject in need thereof.

Alternatively, the invention relates to the use of a combination of the invention for the preparation of a medicament for preventing and/or treating an infection.

In another preferred embodiment, the invention relates to a compound for use in the prevention and/or treatment of an infection wherein the compound is selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant.

Alternatively, the invention relates to a method for preventing and/or treating an infection comprising administering a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant to a subject in need thereof.

Alternatively, the invention relates to the use of a compound selected from the group consisting of a glycolysis inhibitor, an IRF4 mimetic, a Ras inhibitor, a PI-3K inhibitor, a mTOR inhibitor, a STAT3 inhibitor, and an antioxidant for the preparation of a medicament for the prevention and/or treatment of an infection.

As used herein the terms "treat”, "treatment, " or "treatment of refers to reducing the potential for a certain disease or disorder, reducing the occurrence of a certain disease or disorder, and/or a reduction in the severity of a certain disease or disorder, preferably, to an extent that the subject no longer suffers discomfort and/or altered function due to it. For example, “treating” can refer to the ability of a therapy when administered to a subject, to prevent a certain disease or disorder from occurring and/or to cure or to alleviate a certain disease symptoms, signs, or causes. “T reating” also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness. Thus, the terms "treat," "treating" or "treatment of" (or grammatically equivalent terms) refer to both prophylactic and therapeutic treatment regimes. Particularly, “treatment”, as used herein, relates to the administration of a compound suitable for treating an infection to a subject suffering from an infection including the administration in an initial or early stage of a disease, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder.

The term “prevention”, “preventing” or “prevent”, as used herein, relates to the administration of a compound suitable for treating an infection to a subject who has been identified as susceptible to suffering an infection according to the method of the invention. The prevention intends to avoid the appearance of said disease. The prevention can be complete (e.g. the total absence of a disease). The prevention can also be partial, such that for example the occurrence of a disease in a subject is less than that which would have occurred without the administration of the compound suitable for treating an infection. Prevention also refers to reduced susceptibility to a clinical condition.

In a preferred embodiment of the medical uses according to the invention the compound is a RAS inhibitor. Illustrative, non- limitative examples of Ras inhibitors are Manumycin A, Farnesyl thiosalicylic acid, FTI-276 trifluoroacetate salt, L-744,832 Dihydrochloride and MCI-062, Deltarasin, Zn-cyclen, ARS-1620, BIM-46187, Sulindac sulphide, IND12, Kobe2601 , 3144, DCAI and ABD7.

In another preferred embodiment, the compound is a PI-3K inhibitor and/or mTOR inhibitor. Illustrative, non-limitative examples of PI-3K inhibitors are Wortmannin, LY294002, hibiscone C, Idelalisib, Copanlisib, Duvelisib, Alpelisib and Umbralisib. In another preferred embodiment, the compound is a mTOR inhibitor. Illustrative, non-limitative examples of mTOR inhibitors useful in the present invention, are without limitation Rapamycin and its derivatives, sirolimus, temsirolimus, everolimus and ridaforolimus.

Alternatively, mTOR/PI3K dual inhibitors can be used according to the invention. Illustrative non-limitative examples are dactolisib, BGT226, SF1126 and PKI-587, LY3023414, PF-05212384, PQR309, P7170, SF-1126, Copanlisib and BEZ235.

In another preferred embodiment, the compound is a STAT3 inhibitor. Illustrative, non-limitative examples of STAT3 inhibitors useful in the present invention, are without limitation Galiellalactone, S3295, CPA-1 , CPA-7, STA-21, LLL-3, LLL12, S3I-201, FLLL31 , STX-0119, ISS 610, Cpc 3, Cpc 30, Cpc 188, BP-1 -102, ISS610, Stattic, OPB- 31121 , OPB-51602, Curcumin and analogues (FLLL11, FLLL12, FLLL32), AZD1480 and Butein.

In another preferred embodiment, the compound is a glycolysis inhibitor.

In a more preferred embodiment, the glycolysis inhibitor is selected from the group consisting of Dichloroacetate, 2-deoxyglucose, 3-bromopyruvate, Lodinamine, 3- (3-pyrididyl)-1-(4-pyridinyl)-2-propene-1-one (3PO), 1-(4-pyridinyl)-3-(2-quinolinyl)-2- Propen-1-one (PFK15), 6-Aminonicotinamide, Oxythiamine Chloride Hydrochloride, Shikonin and FX11. In a preferred embodiment, the inhibitor is an inhibitor of pyruvate dehydrogenase kinase, more preferably dichloroacetate (DCA). DCA corresponds to the compound having the CAS number 13425-80-4.

In another preferred embodiment, the inhibitor is an inhibitor of fructose 2,6 biphosphatase 3, more preferably 3-(3-pyrididyl)-1-(4-pyridinyl)-2-propene-1-one (3PO) or (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one).

In another preferred embodiment, the compound is an IRF4 mimetic.

In another preferred embodiment the compound is an antioxidant. In a more preferred embodiment the antioxidant is selected from the group consisting of ascorbic acid, glutathione lipoic acid, carotenes, a- tocopherol, ubiquinol or silibinin.

In a preferred embodiment, the medical uses of the invention are for use in the prevention and/or treatment of an infection caused by a microorganism selected from the group consisting of Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii, Borrelia mayonii, Treponema pallidum, Borrelia hermsii, Borrelia miyamotoii, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Haemophilus influenza, Mycobacterium tuberculosis, Helicobacter pylori, Salmonella typhi, Mycobacterium leprae, Neisseria gonorrhoeae, Chlamydia spp., Bartonella, Legionella pneumophilla and Brucella abortus. In a more preferred embodiment, the infection is caused by a Lyme disease-causing Borrelia species, particularly caused by B. burgdorferi.

In another preferred embodiment, the infection is an acute infection. In another preferred embodiment the infection is a persistent infection.

The present invention covers any combination of compounds and diseases.

All the terms and embodiments previously described are equally applicable to this aspect of the invention.

Kit

In another aspect, the invention relates to the use of a kit comprising a reagent suitable for determining the level of expression of IRF4 in a sample for diagnosing an infection in a subject.

In another aspect, the invention relates to the use of a kit comprising a reagent suitable for determining the level of expression of IRF4 in a sample for designing a personalized therapy in a subject suffering from an infection.

Kit, as used herein relates to a product containing the different reagents required for carrying out the methods of the invention packaged so as to allow their transport and storage. Additionally, the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components in the kit. Said instructions can be found in the form of a printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic discs, tapes and the like), optical media (CD-ROM, DVD) and the like.

The term “reagent capable of determining the expression level of IRF4", as used herein, refers to any reagent suitable for detecting the gene product produced by Irf4 gene, wherein the gene product can be a transcriptional product (mRNA) or a translational product (IRF4 protein encoded by the IRF4 gene).

In a particular embodiment, the reagent capable of determining the expression level of IRF4 is a nucleic acid capable of hybridizing specifically with the IRF4 mRNA. Nucleic acids capable of hybridizing specifically with the Irf4 mRNA are, for example, one or more pairs of primer oligonucleotides for the specific amplification of fragments of the mRNA (or their correspondent cDNA) of IRF4 and/or one or more probes for the identification of IRF4 mRNA. As the skilled person understands, the oligonucleotide primers and probes can be used in all techniques of gene expression profiling (RT-PCR, SAGE, TaqMan, Real Time-PCR, FISH, NASBA, etc).

In a preferred embodiment the reagent capable of determining the expression level of IRF4 is a primer oligonucleotide, such as those described in the examples of the invention.

In another particular embodiment, the reagent capable of determining the expression level of IRF4 is an antibody that specifically binds to the I RF4 protein encoded by IRF4 gene. Antibodies, or fragments thereof, capable of specifically binding to IRF4 protein or to variants thereof are, for example, monoclonal and polyclonal antibodies, antibody fragments, Fv, Fab, Fab’ and F(ab’)2, ScFv, diabodies, triabodies, tetrabodies and humanised antibodies. These antibodies can be used in conventional methods for detecting protein expression levels, such as Western-blot or Western transfer, ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzymatic immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips, protein microarrays including specific antibodies or assays based on colloidal precipitation in formats such as dipsticks, etc.

In a preferred embodiment, the reagents adequate for the determination of the expression levels of IRF4 comprise at least 10% of the total amount of reagents forming the kit. In further embodiments, the reagents adequate for the determination of the levels of IRF4 comprise at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount of reagents forming the kit.

According to the present invention, the term “comprises” or “comprising” also includes as one embodiment that no further components or elements are present, that is, it includes as one embodiment the term “consists of” or “consisting of”.

All the terms and embodiments previously described are equally applicable to this aspect of the invention.

The invention will be described by way of the following examples, which are to be considered as merely illustrative and not limitative of the scope of the invention.

Examples

Materials and methods Mice

C57BI/6 (B6) mice were purchased from Charles River Laboratories and bred in the Animal Facility at CIC bioGUNE. All the assays performed were approved by the competent authority (Diputacion de Bizkaia) under European and Spanish directives. CIC bioGUNE ^' s Animal Facility is accredited by AAALAC Inti.

Human cells

Human monocytes were purified from buffy coats of healthy blood donors by positive selection using a human CD14 purification kit (Miltenyi Biotec), as described (Carreras- Gonzalez A, et al. (2018). Emerg Microbes Infect 7(1): 19). The cells were rested overnight before stimulation.

Bacteria

B. burgdorferi s.s. Bb914 (Dunham-Ems SM, et al. (2009) J Clin Invest 119(12):3652- 3665.) and B31 clone 5A15 were used throughout. The spirochetes were grown in BSK- H medium (Sigma Aldrich) in 5-ml tubes at 34 °C and used at a multiplicity of infection of 25.

Murine infections

Six to eight-week old B6 mice were infected with 105 B. burgdorferi B31 clone 5A15 subcutaneously, as described (Hawley KL, et al. (2012) Proc Natl Acad Sci U S A 109(4): 1228- 1232). At the specified times, the mice were treated with dicholoroacetate (DCA; Thermo Fisher Scientific) (2 g/l) in the drinking water (changed twice a week) or intraperitoneally with 50 mg/kg of 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), for a period of 14-28 days. The mice were sacrificed 4-5 weeks post infection. The hearts were cut in half through bisections across the atria and ventricles to isolate DNA and RNA using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) following the manufacturer’s recommendations. RNA was extracted from whole EDTA-containing blood by Trizol/chloroform (80:20) and precipitation with isopropanol, followed by purification with the NucleoSpin® RNA kit (Macherey-Nagel). Bacterial burdens were measured from heart DNA by qPCR targeting recA relative to the murine gene, Rpl19. RNA was reverse-transcribed and used to perform real-time PCR to determine the expression levels of Tnf, Adgrel , Itgam and Irf4 relative to Rpl19, as before. The primers used are listed in Table I.

Cell culture

Bone marrow-derived macrophages (BMMs) were generated from 6-12-week-old B6 mice. Bone marrow cells were obtained from the femoral and tibial shafts and incubated in 100 mm x 15 mm non-treated Petri dishes (Falcon). The cells were incubated for 6 days at 37 °C with 5% C02 in DM EM supplemented with 10% FCS and 10% penicillin-streptomycin plus 30 ng/ml of M-CSF (Miltenyi Biotec, Pozuelo de Alarcon, Madrid, Spain). Non-adherent cells were then discarded and adherent macrophages were scraped, counted and seeded. The macrophage-like cell line, RAW 264.7, was maintained in DMEM supplemented with 10% FCS and 10% penicillin- streptomycin (Thermo Fisher Scientific). Lentiviral particles containing shRNA targeting Irf4 (Mission shRNA library, Sigma Aldrich; TRCN0000081548, TRCN0000081549) were generated as described (34). RAW 264.7 cells were infected with supernatants containing the virus followed by incubation with puromycin at 3 pg/ml (Sigma Aldrich) to produce stable lines. BMMs were infected with lentiviral particles at days 3 and 5 of the differentiation process. The controls used were cells containing the empty vector pLKO.1.

The cells were stimulated with live B. burgdorferi at an m.o.i. of 25 according to the scheme depicted in Fig 1A. In some experiments, the cells were treated either during the first 48 h stimulation (acute) or at the restimulation phase (memory) with the compound 2-deoxyglucose (50 mM). For the reversion of the memory phenotype, the cells were stimulated with B. burgdorferi for 48h, washed and restimulated in the presence of 2-DG for 16-20 h. After washing, the cells were stimulated with the spirochete in the presence or absence of 2-DG for 16-20 h and analyzed.

Phagocytosis assays

Phagocytosis assays were performed as previously described (Hawley KL, et al. (2012) cited supra). BMMs and RAW 264.7 cells were cultured in serum- and antibiotic- free medium for 1 h. GFP expressing B. burgdorferi were then added to the cells at a multiplicity of infection of 25 and incubated at 4 °C for 15 min followed by 37 °C for 2 h. The cells were then washed to eliminate surface bacteria and analyzed by flow cytometry in a BD FACS Canto II cytometer (BD Biosciences, San Agustin de Guadalix, Madrid, Spain). The data were analyzed using FlowJo for Mac, version 10.5.3 (FlowJo, Ashland, OR). The phagocytic index was calculated following the formula: % GFP cells (Test) x MFI (Test) - % GFP cells (4 °C control) x MFI (4 °C control) (Hovius JW, et al. (2009) PLoS Pathog 5(5):e1000447).

Confocal microscopy

Following incubation with B. burgdorferi Bb914 at 4 °C, the cells were washed, fixed with 4% paraformaldehyde for 20 min, permeabilized with PBS containing 0,3% Triton X-100 (VWR, Radnor, PA, USA) and stained with rhodamine-labelled phalloidin and DAPI for 10 min at 37 °C (Thermo Fisher Scientific). After extensive washing with PBS, the cells were mounted using the Prolong Gold Antifade mounting reagent (Thermo Fisher Scientific). The images were obtained employing a Leica TCS SP8 confocal system (Leica Microsystems, Madrid, Spain).

Cytokine ELISA

The levels of murine and human TNF and murine IL-6 in the re-stimulation supernatants were determined by capture ELISA using the Mouse TNF ELISA Set II, the Mouse IL-6 ELISA set (BD Biosciences) and the human TNF ELISA set (Thermo Fisher Scientific), following the manufacturers’ instructions.

RNA isolation

Total RNA was isolated using the NucleoSpin® RNA kit (Macherey-Nagel). The quantity and quality of the RNAs were assessed using the Qubit RNA Assay Kit (Thermo Fisher Scientific) and RNA Nano Chips in a 2100 Bioanalyzer (Agilent Technologies), respectively. RNAseq transcriptomics

Libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (lllumina) following the instructions from the manufacturer. Single-read 50 nt sequencing of pooled libraries was carried out in a HiScanSQ platform (lllumina). The quality control of the sequenced samples was performed using the FASTQC software (www.bioinformatics.babraham.ac.uk/projects/fastq). Reads were mapped against the mouse (mm 10) reference genome using Tophat (Trapnell C, Pachter L, & Salzberg SL (2009) Bioinformatics 25(9): 1105-1111) accounting for spliced junctions. The resulting BAM alignment files for the samples were then used to generate a table of raw counts by Rsubread (Liao Y, Smyth GK, & Shi W (2013) Nucleic Acids Res 41(10):e108). The raw counts table was the input for the Differential Expression (DE) analysis, carried out by DESeq2 (Love Ml, HuberW, & Anders S (2014). Genome Biol 15(12):550.), to identify differentially expressed genes among the different conditions. GO enrichment was tested using the clusterProfiler bioconductor package (Yu G, Wang LG, Han Y, & He QY (2012) OMICS 16(5):284-287), the Panther Database (Thomas PD, et al. (2003) Genome Res 13(9):2129-2141) and DAVID (https://david.ncifcrf.gov) (Huang da W, Sherman BT, & Lempicki RA (2009) Nat Protoc 4(1 ):44-57., Huang da W, Sherman BT, & Lempicki RA (2009) Nucleic Acids Res 37(1): 1-13.). Transcriptomics data were also analyzed using QIAGEN ^' s Ingenuity® Pathway Analysis (I PA®, Qiagen). Motif enrichment analysis was performed using the HOMER software (Heinz S, et al. (2010) Mol Cell 38(4):576-589.) for motif discovery, using the findMotifs.pl script. Motifs of lengths 8, 10, 12 nucleotides were considered in this analysis. Validation of the RNAseq data was performed by real time PCR (Table I, Fig 9).

Real-time PCR

RNA was reverse transcribed using M-MLV reverse transcriptase (Thermo Fisher Scientific). Real-time PCR was then performed using the PerfeCTa SYBR Green SuperMix low ROX (Quantabio) on a QuantStudio™ 6 Real-Time PCR System (Thermo Fisher Scientific). Fold induction of the genes was calculated relative to Rpl19 using the 2- ^DDOI _me hod. The primers used are listed in Table I.

Omni ATAC-seq

The nuclei of stimulated BMMs were isolated in an lodixanol gradient and the DNA was tagmented by a transposition reaction using the tagmentase included in the Nextera DNA Library Prep Kit (lllumina) as previously described (Corces MR, et al. (2017) Nat Methods 14(10):959-962). The transposed DNA was purified using the Qiagen Minielute Kit (Qiagen) and libraries were generated and amplified according to the protocol by Buenostro and cols. (Buenrostro JD, Giresi PG, Zaba LC, Chang HY, & Greenleaf WJ (2013) Nat Methods 10(12):1213-1218.). After amplification, a double-size selection using Agencourt AMPure XP beads (Beckman Coulter) was performed. Library quality was assessed on an Agilent 2100 Bioanalyzer using the Agilent High Sensitivity DNA kit (Agilent Tehcnologies). The libraries were paired-end sequenced to 101 nucleotides, in a HiSeq4000 platform (lllumina). Basecalls were performed using CASAVA version 1.8.2. Individual FASTQ files for each sample were merged prior to the quality control & filtering steps. Quality control of the reads was carried out using FASTQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were filtered from the adapter sequences and their quality score using trim_galore software (http://www.bioinformatics.babraham.ac.uk/projects/trim_galo re/) and only those with at least 20 phred quality score are retained. Reads were mapped to the mm10 reference genome using Bowtie2 with the following parameters (-N 1 and -X 2000). Resulting BAM aligment files were used in as input for peak calling using MACS2 with the following parameters (-f BAMPE -g "mm" -p 0.01 —shift -100 --extsize 200 --broad). Resulting peak calls and BAM alignment files were used in as input for a Differential Binding Analysis pipeline based on DiffBind BioconductoR package (https://bioconductor.org/packages/release/bioc/html/DiffBin d.html).

Metabolic assays

The oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured in differentially stimulated BMMs employing an XF24 extracellular flux analyzer (Agilent). Unstimulated (4 x 10 ⁵ ) and B. burgdo/fer/-stimulated cells (2 x 10 ⁵ ) were seeded per well in a Cell-Tak coated plate (BD Biosciences), and the measurements were normalized to cellular protein amount. For ECAR determination, the cells were previously plated in XF Seahorse medium with 4 mM glutamine and 10 mM pyruvate, while for the mitochondrial stress test the cells were plated in medium containing 4 mM glutamine, 10 mM pyruvate and 25 mM glucose. After 1 hour at 37 °C without C02, three baseline oxidative consumption rate (OCR) and extracellular acidification rate (ECAR) measurements were performed. For glycolysis determination, ECAR was measured at baseline and after sequentially adding glucose (25 mM), Oligomycin (1 mM) and 2-DG (50 mM). In parallel experiments, OCR was determined at baseline and after sequentially adding oligomycin, FCCP, antimycin/rotenone at 1 mM. Lactate production was measured from stimulation supernatants using the Lactate (Trinity Biotech), Liquid L-Lactate Trinder (Biochemical Enterprise) and Lactate-Glo™ Assay kits (Promega).

Determination of metabolic intermediaries of the TCA metabolism

The levels of glutamine, glutamate, malate, citrate and succinate were determined in BMMs by liquid chromatography tandem mass spectrometry (LC-MS/MS). Cellular pellets were homogenized in 500 mI of ice-cold extraction liquid (ice cold methanol/water (50/50 %v/v) and 1 mM stable labelled 13CD3-methionine (methionine- SL) as internal standard) with a tissue homogenizer (FastPrep) in one 30 second cycle at 6,000 rpm. Subsequently, 400 mI of the homogenate were shaken at 1 ,400 rpm for 30 minutes at 4 °C. The samples were then centrifuged for 15 min at 13,000 rpm and 4 °C. One hundred mI of the aqueous phase were placed at -80 °C for 20 min, followed by evaporation in a Speedvac during approximately 3 h. The resulting pellets were resuspended in 100 mI water/acetonitrile (MeCN) (40/60 v/v). Samples were measured with a UPLC system (Acquity, Waters Inc.) coupled to a Time of Flight mass spectrometer (ToF MS, SYNAPT G2, Waters Inc.). A 2.1 x 100 mm, 1.7 pm BEH amide column (Waters Inc.), thermostated at 40 °C, was used to separate the analytes before entering the MS. Mobile phase solvent A (aqueous phase) consisted of 99.5% water, 0.5% FA and 20 mM ammonium formate while solvent B (organic phase) consisted of 29.5% water, 70% MeCN, 0.5% FA and 1 mM ammonium formate. In order to obtain a good separation of the analytes the following gradient was used: from 5% A to 50% A in 2.4 minutes in curved gradient (#8, as defined by Waters), from 50% A to 99.9% A in 0.2 minutes constant at 99.9% A for 1.2 minutes, back to 5% A in 0.2 minutes. The flow rate was 0.250 ml/min and the injection volume 2 pi. After every 6 injections QC low and QC high sample was injected. The MS was operated in positive and negative electrospray ionization, depending on analyte, in full scan mode. The cone voltage was 25 V and capillary voltage was 250 V. Source temperature was set to 120 °C and capillary temperature to 450 °C. The flow of the cone and desolvation gas (both nitrogen) were set to 5 L/h and 600 L/h, respectively. A 2 ng/mL leucine-enkephalin solution in water/acetonitrile/formic acid (49.9/50/0.1 %v/v/v) was infused at 10 mI/min and used for a lock mass which was measured each 36 seconds for 0.5 seconds. Spectral peaks were automatically corrected for deviations in the lock mass. Results

Example 1- B. burgdorferi induces long-term responses in macrophages that affect phagocytosis and proinflammatory cytokine production

The inventors first analyzed the response of macrophages stimulated acutely and those that had been previously exposed to B. burgdorferi. The inventors stimulated murine bone marrow-derived macrophages (BMMs) for 48 h, washed them and re stimulated them with the spirochete for 16 - 20h (condition BB; Fig. 1A). Acutely stimulated macrophages were processed in parallel, except with no stimulation the first 48 h (condition UB). Non-stimulated (condition UU) and stimulated and rested (condition BU) macrophages were also generated (Fig. 1A). The inventors then compared the acutely stimulated and experienced macrophages for their capacity to produce proinflammatory cytokines and their phagocytic activity against B. burgdorferi. Previous exposure to the spirochete resulted in decreased TNF production in response to the bacterium, compared to acutely activated cells (Fig. 1 B). The analysis of purified CD14+ cells from peripheral blood of healthy donors confirmed that in human monocytes, the previous exposure to live B. burgdorferi renders the cells hypo responsive to the spirochete in a subsequent encounter, when using the approach described in Fig. 1A (Fig. 1C).

The capacity to both bind and internalize the spirochete was increased by the previous experience of the phagocytic cells (Fig. 1 D). Thus, the analysis by flow cytometry of macrophages not exposed and previously activated with B. burgdorferi revealed an augmented capacity to bind the spirochete when incubated at 4 °C (Fig. 1 D, E). This resulted in increased internalization when the cells were further incubated at 37 °C (Fig. 1 D). More binding of spirochetes to previously activated macrophages was also observed by confocal microscopy (Fig. 1F). The inventors then analyzed the expression levels of phagocytic receptors under both conditions. The surface expression levels of CD11b were higher in previously stimulated cells, compared to unexposed controls (Fig.

IG), indicating that the enhanced binding capacity of memory macrophages was at least in part, due to the increased expression of the phagocytic receptor. In order to analyze the internalization rate of the spirochete, the inventors compared the phagocytosis index (see Materials and Methods) of macrophages previously unexposed and stimulated with the spirochete. This analysis revealed similar internalization rates for B. burgdorferi (Fig.

I H), indicating that the higher internalization observed in macrophages previously activated was due to the increased ability of these cells to bind the bacterium rather than their increased phagocytic activity. Overall, these data show that macrophages exposed to B. burgdorferi augment the capacity to bind and subsequently internalize the spirochete, albeit with the induction of reduced levels of TNF.

Example 2- B. burgdorferi induces a differential transcriptional profile in acute and memory macrophages.

To identify transcriptional traits specifically induced in naive and previously activated macrophages with B. burgdorferi, the inventors analyzed the transcription profiles of bone marrow-derived macrophages (BMMs) by RNA-seq. The inventors analyzed BMMs that had been stimulated with the spirochete under the 4 conditions shown in Fig. 1A. The four conditions showed distinct transcriptional profiles, as seen in PCA (Fig. 2A) and sample distance matrix analysis. The analysis of the 1 ,000 most regulated genes under the four conditions studied, showed similar patterns of expression for the conditions UU and BU, while UB and BB were also similar. The comparison of genes up- and down-regulated under each condition versus unstimulated (UU) macrophages revealed several differences. For example, in spite of the similarities between the unstimulated (UU) and the previously stimulated (BU) conditions, 1334 genes showed differential regulation when using cut off values of 1 for the absolute log2 Fold Change and p < 0.05 (693 up and 641 down; ). On the other hand, the comparison to unstimulated (UU) macrophages of acutely (UB) and re-stimulated (BB) cells revealed similar number of upregulated and downregulated genes (Fig. 2B). Indeed, among the genes differentially expressed under acute (UB) and restimulated (BB) conditions, a majority (2154) were found to be common (Fig. 2C). Ingenuity Pathway Analysis (IPA) showed that the genes regulated in previously exposed macrophages (BB) were consistent with pathways activated by the acute stimulation of BMMs with B. burgdorferi (UB) (Carreras-Gonzalez A, et al. (2018) Emerg Microbes Infect 7(1 ): 19.), including interferons, TLRs, NOD and cytokines such as IL-1 b . The transcriptional analysis of proinflammatory cytokine production (Tnf, II6, 111 b and 1112b) confirmed the pattern observed for TNF by ELISA, while the levels of 1110 transcripts were highly upregulated in previously activated cells (Fig. 3). However, as observed in acutely stimulated cells (Carreras-Gonzalez A, et al. (2018), cited supra), the IL-10R-dependent signaling pathway was significantly repressed in memory macrophages. A sizeable number of genes appeared differentially regulated in naive (UB) and memory (BB) macrophages (Fig. 2C). In fact, the comparison of both conditions showed that 422 genes were upregulated in memory macrophages, while 277 were downregulated compared to the acute stimulation of the cells (Fig. 2D). Example 3- B. burgdorferi-induced Irf4 expression regulates proinflammatory cytokine production in memory macrophages.

The inventors then performed a comparative transcription factor enrichment analysis of acutely stimulated and previously exposed cells to B. burgdorferi using the HOMER package (Heinz S, et al. (2010) Mol Cell 38(4):576-589.). The inventors identified a set of 3 transcription factors putatively responsible for the transcriptional changes in macrophages previously exposed to the spirochete compared to acutely stimulated cells: SpiB, MAFKand MZF1 (Fig. 2E). The analysis of the genes upregulated in macrophages previously stimulated with B. burgdorferi identified Irf4 as the only gene regulated by these transcription factors (Fig. 2F, Table II).

Table II. Genes upregulated in experienced macrophages stimulated with B. burgdorferi that are putatively regulated by the transcription factors, SpiB, MAFK and MZF1.

An ATAC-seq analysis of UB and BB macrophages showed differences in the chromatin accessibility under both conditions and identified open chromatin peaks in the majority of the genes regulated by SpiB, MAFK and MZF1 that were upregulated in experienced macrophages compared to acutely-stimulated cells. In fact, the analysis of the peaks corresponding to the genes regulated by the three transcription factors showed increased open chromatin in re-stimulated macrophages (Fig. 2G), including Irf4.

The analysis of Irf4 expression in acute and memory cells showed the downregulation of the expression levels of this gene in acute cells, while they increased dramatically upon the generation of memory macrophages (Fig. 4A). Interestingly, the analysis of Irf4 gene expression in peripheral blood monocytes of B. burgdorferi- nfectedi mice showed a similar expression kinetics, with lower levels of expression of Irf4 in the acute phase of the disease, followed by their recovery at later stages (Fig. 4B). In the heart, however, Irf4 gene expression was significantly increased after 3 weeks of infection (Fig. 4C). These results suggest that heart-infiltrating macrophages display a memory phenotype.

The inventors therefore assessed the role of IRF4 on the response of phagocytic cells to the spirochete. First, the inventors generated RAW 264.7 cells with stable expression of silencing RNA for Irf4 (shlrf4). Remarkably, Irf4 silencing did not affect the internalization of B. burgdorferi (Fig. 5A), but resulted in increased TNF production (Fig. 5B). Similarly, Irf4 silencing in BMMs did not affect phagocytosis of B. burgdorferi (Fig. 2H) but resulted in increased levels of both TNF and IL-6 (Fig. 2I). These data show that phagocytosis and the inflammatory output of macrophages can be independently regulated. Example 4- Long-term stimulation with B. burgdorferi induces metabolic changes in macrophages.

IRF4 is a transcriptional regulator associated with metabolic changes in M2 macrophages (Huang SC, et al. (2016) Immunity 45(4):817-830.). The inventors therefore, analyzed the expression levels in our conditions of those genes regulated by IRF4 in M2 macrophages (Huang SC. et al., cited supra), including Pdcd1lg2, Retnlb, Arg1, Chil3, 1110, Lipa, Cd36, Fabp4, Pparg, Ppargdb, and those involved in glycolysis (Slc2a1, Hk1, Hk2, Gpi1, Gapdh, Pfkp and Ldha). Comparing experienced and acutely activated macrophages, the inventors only observed significant increased expression levels of Ldha, Arg1, and 1110 in experienced macrophages, while those of Lipa and Pparg were significantly reduced (Table III).

Table III. Induction of M2-specific //f4-regulated genes in memory macrophages in response to B. burgdorferi, compared to acutely stimulated cells.

Furthermore, silencing of Irf4 in RAW264.7 cells (Fig. 5C) or BMMs (Fig. 2J) did not affect lactate production when stimulated with B. burgdorferi. These data suggested that the increased expression of Irf4 in experienced macrophages does not affect significantly genes associated with metabolic reprogramming of macrophages.

Among the differentially expressed pathways, IPA identified the HIF1a pathway as upregulated in memory macrophages (Table IV).

Table IV. Upstream pathways regulated in memory macrophages compared to acutely stimulated cells. Pathways activated (z-score >2) and repressed (z-score < -2) were identified using the Ingenuity Pathway Analysis tool. The table represents the ten most upregulated and ten most repressed upstream regulator pathways. The full list is provided in Table V.

Table V. Upstream pathways regulated in experienced macrophages compared to acutely stimulated cells. Pathways activated (z-score >2) and repressed (z-score < -2) were identified using the Ingenuity Pathway Analysis tool.

The inventors therefore, proceeded to the analysis of the metabolic status of memory macrophages by Seahorse. Macrophages that had been previously exposed to the spirochete showed decreased oxygen consumption rates (OCR) compared to acutely stimulated BMMs (Fig. 7A, Fig. 6A).The inventors further analyzed the glycolytic capacity of macrophages under the conditions of study (Fig. 7B).The glycolytic capacity of memory macrophages was significantly higher than in acutely stimulated cells (Fig. 6B) and correlated with the presence of increased levels of lactate in the memory supernatants (Fig. 6C) as well as the increased expression of the gene encoding the enzyme lactate dehydrogenase in both murine (Table III) and human cells (Fig. 6D). Moreover, the increased glycolytic capacity correlated with the higher expression levels of Pfkfb3, the gene encoding the positive regulator of glycolysis, fructose-2, 6- bisphosphatase 3 (Fig. 7C). On the other hand, the quantitative analysis by GC-MS of several intermediate metabolites of the tricarboxylic acid cycle (TCA) showed highly increased levels of glutamine, glutamate, succinate, citrate and malate (Fig. 6E). These data confirm previous reports and suggest increased glutaminolysis in re-stimulated macrophages and the conversion of malate to pyruvate to induce higher levels of lactate, while resulting in a deficient tricarboxylic acid cycle and the observed reduced OCR.

Example 5- Glycolysis inhibition modulates the response of murine macrophages and inflammation during Lyme borreiiosis.

The inventors next assessed whether the inhibition of the glycolytic output of macrophages would affect the phagocytic capacity of these cells and/or their inflammatory output. Naive and memory BMMs were stimulated in the presence of the glucose analogue, 2-deoxy glucose (2-DG). In both acute and memory macrophages, the use of 2-DG reduced the production of lactate (Fig. 8A). The inhibition of glycolysis during the secondary stimulation resulted in a significant increased ability of naive macrophages to phagocytose B. burgdorferi (Fig. 8B), accompanied by decreased levels of TNF (Fig. 8C). The inhibition of glycolysis did not, however, affect the phagocytic capacity of memory macrophages (Fig. 8B) although it resulted in lower production of TNF (Fig. 8C). Interestingly, the presence of 2-DG only during the initial stimulation of BMMs (48 h) did not affect the production of TNF by memory macrophages (Fig. 8C). In order to assess the inhibition of glycolysis in vivo during infection with B. burgdorferi or their inflammatory status, the inventors treated the infected animals with the pyruvate dehydrogenase kinase inhibitor, dichloroacetate (DCA) in the drinking water (Caro- Maldonado A, et al. (2014) J Immunol 192(8): 3626-3636.. The mice were provided DCA during the last 2 weeks prior to their analysis, once the peak of infection had commenced. Both macrophage infiltration (as measured by the expression of the Adgrel gene, which encodes for the surface protein F4/80, Fig. 8E) and Tnf expression (Fig. 8F) were reduced in mice treated with DCA compared to the controls. Furthermore, the analysis of Irf4 expression showed a significant reduction of expression in mice treated with DCA (Fig. 8G).

In addition, in order to assess whether the inhibition of glycolysis in vivo during infection with B. burgdorferi would affect the levels of bacteria in the heart (where macrophage infiltration is most evident) or their inflammatory status, the inventors treated the infected animals with the PFKFB3 inhibitor, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-

1-one (3PO). The mice were provided 3PO during the last 3 weeks, once the peak of infection had commenced (at week 2 post infection). The analysis of the bacterial burdens showed that the treatment with 3PO reduced spirochetemia in the heart (Fig. 11 A). Furthermore, both macrophage infiltration (as measured by the expression of the Adgrel gene, which encodes for the surface protein F4/80) and Tnf expression (Fig. 11 B) were reduced in mice treated with 3PO compared to controls. Importantly, the analysis of Irf4 gene expression demonstrated a significant reduction in the hearts of the mice treated with 3PO (Fig. 11C).

These results suggested that in vivo, the inhibition of glycolysis during an ongoing infection with B. burgdorferi results in the reversion of the memory-induced Irf4 expression, while affecting the infiltration of macrophages and the ensuing inflammatory response. Indeed, the inhibition of glycolysis in memory macrophages in vitro resulted in the decreased expression of Irf4 (Fig. 8H) and increased TNF production upon a subsequent stimulation in the absence of 2-DG (Fig. 8I). Interestingly, the presence of

2-DG during the final stimulation dramatically diminished the amount of TNF produced by macrophages (Fig. 8I).

These data suggest that the inhibition of glycolysis results in the reversion of the memory phenotype, leading to increased susceptibility to further treatment with the glycolysis inhibitor. These results also highlight the relevance of Irf4 regulation as a marker of memory generation particularly related to the production of proinflammatory factors.

Because the phagocytic capacity of memory macrophages is not affected by glycolysis inhibition (Fig. 10) our results in vivo suggest the reversion of the memory phenotype upon treatment with 3PO. To determine whether the lower expression levels of Irf4 in the infected heart of the 3PO-treated mice was due to the reversion of the memory phenotype or, alternatively, they just merely reflected the reduced macrophage infiltration, we performed a series of experiments in vitro using BMMs. Our results showed that the inhibition of glycolysis in memory macrophages results in the decreased expression of Irf4 (Fig. 12A) and increased TNF production upon a subsequent stimulation in the absence of 2DG (Fig. 12B). Interestingly, the presence of 2DG during the final stimulation dramatically diminished the amount of TNF produced by macrophages (Fig. 12B). These data suggest that the inhibition of glycolysis results in the reversion of the memory phenotype, leading to increased susceptibility to further treatment with the glycolysis inhibitor. These results highlight the relevance of Irf4 regulation as a marker of memory generation particularly related to the production of proinflammatory factors. Alternatively, these results could be associated with a role played by IRF4 in the maintenance of the macrophage memory phenotype.