FIELD OF THE INVENTION

[0001] This invention relates to the use of a NOD2 agonist for the treatment of monocytopenia and for triggering conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^low monocytes.

BACKGROUND OF THE INVENTION

Monocytes

[0002] Although blood monocytes are composed of three subsets in human, only two main subsets have been described in mice. Based on expression levels of Ly6C, mice monocyte subsets can be categorized as inflammatory (Ly6C ^hlgh ) monocytes, which massively extravasate in inflamed tissues in response to chemokines and patrolling (Ly6C ^low ) monocytes, which act as vascular sentinels and are involved in tissue repair.

[0003] The molecular mechanisms that regulate the generation and development of the two main subsets of blood monocytes are still being defined, but it has become broadly accepted that Ly6C ^hlgh cells are obligatory precursors of their Ly6C ^low counterparts. Monocytes emerge from the bone marrow as Ly6C ^hlgh cells, in which they represent more than 90% of monocytes. Microenvironment modifications are thought to trigger the transition from Ly6C ^hlgh cells to Ly6C ^low monocytes. In this process, the critical transcription factors, C/ΕΒΡ-β, KLF2 and NR4A1 appear to regulate monocytes maturation. Indeed, NR4A1, a member of the orphan nuclear receptor family, has emerged as a potential master transcriptional regulator for the differentiation from Ly6C ^hlgh cells to Ly6C ^low monocytes. First, NR4A1 deficient mice were found to carry Ly6C ^hlgh monocytes that have lost their capacity to differentiate into Ly6C ^low cells. This is illustrated well by the almost complete absence of Ly6C ^low cells in peripheral blood. Also, it was found that the generation of Ly6C ^l0W cells could be dependent on C/ΕΒΡ-β and KLF2, both of which bind the NR4A1 promoter and control its expression. C/ΕΒΡ-β -deficient mice, similarly to NR4A1 knockout mice, exhibited an absence of Ly6C ^low monocytes. The transcription factor C/ΕΒΡ-β is also found highly expressed in Ly6C ^low monocytes. It also regulates myelopoiesis by controlling cell development and survival of Ly6C ^low monocytes. Depletion of KLF2 in conditional knockout experiments systems had similar effects on the Ly6C ^ow subset. KLF factors are highly expressed in Ly6C ^low monocytes and seem to regulate Ly6C ^low monocytes development by interacting with the E2 enhancer region of NR4A1 promoter.

[0004] Nonclassical patrolling monocytes are characterized by their unique ability to actively patrol the vascular endothelium under homeostatic. Patrolling monocyte subsets (CX3CRl ^high Ly6C in mouse and CXSCRl^CD^CD^ in humans) are distinct from the classical monocyte subsets (CCR2 ^high Ly6C ⁺ in mouse and CCR2 ^high CD14 ⁺⁺ CD16 in humans) and exhibit unique functions in the vasculature. Patrolling monocytes function in several disease settings to remove damaged cells and debris from the vasculature and have been associated with wound healing in damaged tissues.

[0005] Inflammatory responses trigger recruitment of effector leukocytes to the site of infection. The recruited effector cells are activated to eliminate infectious agents and participate further to the immune cell recruitment by secreting proinflammatory cytokines and chemokines. The first wave of recruitment consists mostly of neutrophils. These polymorphonuclear cells are the most abundant leukocyte population in the peripheral blood. They mediate the early phase of inflammation by ingesting microbes by phagocytosis and by producing microbial molecules. Monocytes/macrophages act as the other major player in the early and late phases of inflammation. Human macrophage populations are derived from monocytes, which display remarkable plasticity and can be divided in three subsets. The "classical monocytes", which are characterized by expression CD14 ⁺ 7CD16 ^" , are also known as inflammatory monocytes, represent between 80-90% of blood monocytes. These cells are believed to differentiate in Ml macrophages in inflammatory conditions. Resident, non- classical or "patrolling" monocytes are characterized by expression CD 147 CD16 ⁺⁺ . These cells patrol the blood cell vessel in a crawling behavior and act as a peripheral watchdog. The third monocyte subset is characterized by expression of CD14 ⁺ 7CD16 ⁺ , their exact role is still under debate but are most likely involved in inflammatory processes.

[0006] There can be numerous causes for the reduction in number of blood monocytes causing monocytopenia, such as acute infections, stress, treatment with glucocorticoids, aplastic anemia, hairy cell leukemia, acute myeloid leukemia, treatment with myelotoxic drugs and genetic syndromes, as for example MonoMAC syndrome. A patient suffering from monocytopenia is generally more prone to infections. It is thus desirable to have a method for restoring the level of monocyte counts and especially the number of patrolling monocytes, to alleviate or correct a monocytopenia of such patient.

[0007] In other cases, there are treatments administered to a patient for a specific disease or condition that will cause or aggravate a monocytopenia. Most often in these cases, the treatment is discontinued to allow the patient to recover from the monocytopenia. However interruption of the treatment of the disease or condition is generally not optimal for treating or eradicating the primary cause of the disease or condition. Having a method for treating monocytopenia and increasing the number of blood monocytes, which is mostly due to an increase of patrolling subset, may allow for the treatment to be maintained.

SUMMARY OF THE INVENTION

[0008] In accordance with a first aspect of the invention, there is provided with the use of a NOD2 agonist for the treatment of a patient afflicted with monocytopenia.

[0009] In accordance with a second aspect of the invention, there is provided with the use of a NOD2 agonist for the conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^l0W monocytes.

[0010] Still, in a further aspect, there is provided with the use of a NOD2 agonist for increasing the number of patrolling monocytes in a subject in need thereof.

[0011] In accordance with another aspect, there is provided a method for treating a patient afflicted with monocytopenia, said method comprising the step of administering to said patient a therapeutically effective dose of aNOD2 agonist.

[0012] Yet, in a further aspect, there is provided a method for the conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^low monocytes, said method comprising the step of administering to a patient in need thereof a therapeutically effective dose of a NOD2 agonist.

[0013] In accordance with a further aspect, there is provided a method for increasing the number of patrolling monocytes in a patient in need thereof, said method comprising the step of administering to said patient a therapeutically effective dose of a NOD2 agonist. [0014] In another aspect, there is provided a NOD2 agonist for use in a method for treating a patient afflicted with monocytopenia, said method comprising the step of administering to said patient a therapeutically effective dose of a NOD2 agonist.

[0015] Still, in another aspect, there is provided a NOD2 agonist for use in a method for the conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^low monocytes, said method comprising the step of administering to a patient in need thereof a therapeutically effective dose of aNOD2 agonist.

[0016] In a yet another aspect, there is provided a NOD2 agonist for use in a method for increasing the number of patrolling monocytes in a patient in need thereof, said NOD2 agonist being administered in a sufficient dose for increasing the number of patrolling monocytes in said patient.

[0017] In a further aspect, there is provided a composition comprising a NOD2 agonist and a pharmaceutically acceptable carrier or excipient, for use in methods according to the various aspects described herein.

[0018] Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Embodiments of the present invention will now be described, by way of example only, with reference to the attached Figures, wherein:

[0020] Fig. 1 illustrates the effect of MDP on peripheral blood monocyte frequencies in wild-type mice;

[0021] Figs. 2A-2E illustrate the effect of MDP on the differentiation of inflammatory CDl lb/CD115/Ly6C ^high monocytes to patrolling CDl lb/CD115/Ly6C ^low monocytes in wild- type mice.

[0022] Figs. 3 illustrates the effect of N-Acetyl-MDP, N-glycolyl-MDP and L18-MDP and Murabutide on the in vivo distribution of monocytes. [0023] Figs. 4A-4D illustrate that MDP effects are N0D2-dependent.

[0024] Figs. 5A-5D illustrate the effect of MDP on the restoration of CDl lb/CD115/Ly6C ^l0W monocyte populations in mice lacking patrolling monocytes (NR4A1 ^{" "} mice background).

[0025] Figs. 6A-6D illustrate the polarizing effect of MDP on human blood monocytes in vitro.

[0026] Figs. 7A and 7B illustrate the effect of MDP treatment on monocytopenic mice and illustrate the effect of the absence of chemokine receptors CCR2 and CX3CR1 on monocyte subsets.

[0027] Fig. 8A illustrates a schematic representation of the kinetics of blood monocyte repopulation following treatment with MDP, where monocytes in the circulation were first depleted using liposomes loaded with clodronate.

[0028] Fig. 8B illustrates the results of the kinetics assays schematically represented in Fig. 8A.

[0029] Fig. 8C illustrates the flow cytometry detection at 48, 72, and 96 hours, of monocytes in clodronate-treated mice vs clodronate and MDP-treated mice.

[0030] Fig. 8D illustrates bar charts showing the evolution of total monocytes, Ly6C ^hlgh and Ly6C ^low monocytes at 24, 48, 72, and 96 hours on monocytes depleted mice treated with MDP.

[0031] Fig. 9A illustrates a schematic representation of a test to evaluate the effect of NOD2 triggering on Ly6C ^hlgh monocyte conversion, where mice were injected with fluorochrome DiO-labeled liposomes (DiO-lipo) after clodronate-liposome induced depletion of monocytes.

[0032] Fig. 9B illustrates the flow cytometry detection of monocytes at 72, 96, 120, and 144 hours post treatment with fluorochrome DiO-labeled liposomes (DiO-lipo) or fluorochrome DiO-labeled liposomes (DiO-lipo) and MDP. [0033] Figs 1 OA- 1 OF illustrate the effect of MDP treatment on the frequencies of Ly 6C and Ly6C ^high in blood, bone marrow and spleen of WT mice.

[0034] Figs. 11A and 11B illustrate the flow cytometry effect of an MDP treatment on the phenotype of circulating pDC, cDC and neutrophils.

[0035] Figs. 12A and 12B illustrate a flow cytometry approach to discriminate between different progenitors associated with the monocyte maturation pathway, including LSK, MDP cells and cMOP.

[0036] Figs. 13A and 13B are images taken from intravital microscopy data illustrating crawling cells expressing GFP proteins in MDP-treated cells and control cells.

[0037] Figs. 13C and 13D illustrate flow cytometry results confirming that these monocytes (in Figs. 13A and 13B) display a patrolling phenotype.

[0038] Fig. 14A illustrates the gene profile in both subsets of monocytes following MDP treatment.

[0039] Fig. 14B is a bar graph illustrating an increase of C/ΕΒΡ-β expression in purified inflammatory Ly6C ^hlgh monocytes and a suppression of C/ΕΒΡ-β expression in patrolling Ly6C ^low monocytes following in vivo activation of NOD2, as measured by RNA-seq.

[0040] Fig. 14C is a bar graph illustrating the increase of C/ΕΒΡ-β expression in blood monocytes as measured by RT-qPCR analysis upon NOD2 activation.

DETAILED DESCRIPTION

[0041] As used herein, the expression "therapeutically effective amount" refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter alia an increase in the number of patrolling monocytes in the blood of a subject, and alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the pharmaceutical formulation as taught herein. [0042] The invention relates to the converting potential of the NOD2 signaling pathway upon activation by NOD2 agonists, which drive the differentiation of inflammatory monocytes into patrolling monocytes

[0043] In the present application, it is demonstrated that triggering of NOD2 receptor by an agonist such as muramyl dipeptide converts blood inflammatory Ly6C ^hlgh monocytes into patrolling Ly6C ^low monocytes. Administration of muramyl dipeptide to NR4A1 ^{" "} mice, which lack Ly6C ^low monocytes, and to Ly6C ^low -depleted mice (such as when treated with clodronate), leads to the emergence of blood patrolling monocytes expressing phenotypic profile of typical Ly6C ^low monocytes, including high expression of CX3CR1 and LFA1. Furthermore, by direct examination of blood monocytes of muramyl dipeptide-treated mice using intravital microscopy, it was observed that converted Ly6C ^low monocytes are capable to patrol endothelium of blood vessels and that their presence in inflamed tissues contributes to reduce inflammatory response. Together, the results demonstrate that NOD2 is involved in the regulation of the phenotype and functions of blood monocytes and highlight a new method for increasing blood levels of anti-inflammatory Ly6C ^low monocytes.

[0044] Indeed, following triggering of NOD2 with MDP, a large proportion of inflammatory monocytes acquire the expression of surface markers that typically characterize patrolling monocytes. This monocyte subset is recognized to patrol blood vessels and play potential roles as scavengers. Crawling of patrolling monocytes on the luminal side of the endothelium mediated by the integrin LFA-1 is a typical function of patrolling monocytes. Intravital microscopy observations made by the inventors have clearly shown that treatment of CX3CRl ^gfp/+ mice with MDP increased the number of patrolling Ly6C ^low monocytes compared to naive mice under steady-state conditions. The most convincing results came from the use of NR4A1 ^{" "} mice which lack patrolling Ly6C ^low monocytes. Following administration of MDP, a significant proportion of blood monocytes which were found to express typical patrolling markers were detected by flow cytometry. In addition, since Ly6C ^l0W monocytes are absent in NR4A1 ^{" "} mice, these results indicate that Ly6C ^lg monocytes give rise to patrolling Ly6C ^low monocytes following treatment with MDP, and therefore suggest that switching of inflammatory monocytes into patrolling phenotype is not due to a massive exit of Ly6C ^low monocytes or a signal from the bone marrow, but is to a cellular differentiation from Ly6C ^hlgh monocytes. This was also supported in experiments using mice pretreated with clodronate before MDP administration in order to deplete all monocytes in the circulation. The presence of circulating Ly6C ^ow was detected as soon as 2 days post-MDP treatment despite that this monocyte subset begins to repopulate the blood stream only on the 6 ^th day after clodronate injection. In addition, it was observed by intravital microscopy that significant proportions of these monocytes had the capacity of crawling within blood vessels. These results demonstrated that "transformed" Ly6C ^low monocytes can acquire not only the phenotype of patrolling monocytes but also their capacity to patrol endothelium of blood vessels. The effects of NOD2 triggering were also observed with primary human monocytes since the inventors found that CD14 ⁺⁺ CD 16 ^" monocytes give rise to a significant increase of patrolling CD14 ^± CD16 ⁺⁺ monocytes following treatment with MDP, supporting the potential of NOD2 in promoting the differentiation of inflammatory monocytes into patrolling phenotype.

[0045] The differentiation of inflammatory monocytes into patrolling phenotype following NOD2 triggering also occurred in bone marrow and in spleen of mice. In contrast, MDP treatment had no effect on phenotype of circulating dendritic cells and neutrophils. The explanation for these results lies in the relationship between NOD2 and NR4A1 since NR4A1 seems to act as an important differentiation factor for Ly6C ^low monocytes. The expression of NR4A1 mRNA and protein are much more lower in Ly6C ^hlgh monocytes than in Ly6C ^low monocytes. After treatment with MDP, Ly6C ^hlgh monocytes were found to strongly express (at least 40-fold) more NR4A1 than the unstimulated control. Thus, activation of NR4A1 gene via NOD2 contribute to initiate the switch of Ly6C ^hlgh into Ly6C ^low monocytes at both phenotype and function levels. However, even if NR4A1 is expressed in dendritic cells and in neutrophils, NOD2 triggering did not affect NR4A1 expression or their phenotype suggesting that NR4A1 is not linked with NOD2 activation in these cell types.

[0046] As used herein, "Ly6C ^hlgh monocytes" is used interchangeably with "Inflammatory monocytes".

[0047] In the development of the present invention, MDP, NAcMDP, N-glycolyl-MDP, L18-MDP and murabutide were tested. Surprisingly, only MDP, NAcMDP, N-glycolyl- MDP, and L18-MDP were found to induce the switching from inflammatory monocytes to patrolling, monocytes. Hence, as used herein , MDP is meant to refer to interchangeably to any one of MDP, NAcMDP, N-glycolyl-MDP, and L18-MDP. [0048] As used herein, "patrolling monocytes" is used interchangeably with "Ly6C ^ow monocytes" and "non-classical monocytes".

[0049] Many NOD2 agonists are known in the art, see for example Ogawa et al, (Curr. Bioact. Compd, 7(3), 180-197, 2011), Fritz et al. (Nature immunology, 7(12), 1250-1257, 2006) and www.invivogen.com. For example, without limitations, preferred NOD2 agonists useful herein are muramyl dipeptide (MDP) and derivatives thereof, such as N-acetyl muramyl dipeptide (NAcMDP), N-Glycolyl-MDP, and L-18 MDP . NOD-like receptor ligand also refers to any modified molecules from the aforementioned NOD2 agonists that can bind to NOD2 and lead to the conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^low monocytes. In fact, as used herein, the MDP derivatives does not include those that the D-isoglutamine residue of the MDP was replaced with L-glutamic acid or L- glutamine. Indeed, in the case of Murabutide, this compound does not induce the conversion of inflammatory Ly6C ^hlgh monocytes to patrolling Ly6C ^low monocytes. That could be explained by the presence in the molecular structure of an amide and an ester residue instead of the carboxyl group. The latter seems very important for the switching activity observed with N-glycolyl-MDP, N-acetyl-MDP or L-18-MDP (1).

Mice

[0050] C57B1/6 (wild-type) mice were purchased from Charles River Laboratories and NOD2 deficient mice (Nod2 ^~ ) from Jackson Laboratory. Mice deficient for NR4A1 (Nr4&r' ^~ ) were kindly provided by Dr. Claude Rouillard (CHU de Quebec Research Center, Universite Laval, Quebec, Canada) and CCR2 (Ccr2 ^/_ ), Cx3cr 1 ^/ , GFP transgenic mice (Gfp ⁺ ) were kindly provided by Dr. Serge Rivest (CHU de Quebec Research Center, Universite Laval, Quebec, Canada). All colonies were maintained at the animal facilities of CHU de Quebec Research Center. Mice were 4 to 10 weeks old unless otherwise indicated. Experiments were conducted in accordance with the guidelines of animal research ethics boards of Laval University.

Mice treatments

[0051] N-acetyl-Muramyl dipeptide (MDP; Invivogen) was diluted in 0.9% saline and intravenously injected at 10 mg/kg unless otherwise indicated. Treatment was administrated daily and mice were sacrificed 48 hours following treatments, unless otherwise indicated. N- Glycolyl-MDP, and L-18 MDP (Invivogen) were diluted in 0.9% saline and intravenously injected at 1.25, 2.5, 5 and 10 mg/kg. Mice were sacrificed 48 hours after drug administration. Control mice were injected with 0.9% saline for all experiments.

In vivo depletion of murine blood monocyte subsets

[0052] Blood mononuclear phagocyte depletion was achieved by intravenous administration of 0.1 ml of dichloromethylene-bisphononate (clodronate)-loaded liposomes (Clo-lipo; Clodronate Liposomes). PBS-loaded liposomes (PBS-lipo) were used as a negative control. Monocyte depletion was monitored by flow cytometry. 3,3'- dioctadecyloxacarbocyanine perchlorate (DiO; Molecular Probes) lipophilic tracer was incorporated into PBS-liposomes at a concentration of 25 μg/ml for in vivo labelling of mononuclear phagocytes following manufacturer's instructions.

Isolation of human primary monocytes and treatment with MDP

[0053] Plasma from healthy donors was isolated from peripheral blood by centrifugation. Mononuclear cells were isolated using Ficoll density gradient (Wisent) as reported in Lacerte, P. et al. (Overexpression of TLR2 and TLR9 on monocyte subsets of active rheumatoid arthritis patients contributes to enhance responsiveness to TLR agonists. Arthritis Res Ther 18, 10 (2016)) and treated with MDP (10 μg/ml) overnight at 37°C. Cells were stained for flow cytometry as described in flow cytometry section. Classical, intermediate and non- classical monocyte subsets were identified by flow cytometry as follows: CD14 ⁺⁺ CD16 ^" (classical) (PI), CD14 ⁺ CD16 ⁺ (intermediate) (P2) and CD14 ^± CD 16 ⁺⁺ (non-classical) (P3).

Flow cytometry analysis

[0054] Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes. Bone marrow cells were collected from both femur and tibia of mice by flushing the bone marrow with DPBS (Wisent) 10% fetal bovine serum (FBS; Wisent). Harvested cells were filtered through a 40 μηι cell strainer and washed with DPBS. Spleen cells were collected by mechanically disruption and digestion with 180 U/ml DNAse I (Sigma Aldrich) and 1 mg/ml collagenase (Sigma Aldrich). Cells were passed through a 40 μηι cell strainer and washed with HBSS-EDTA. Erythrocytes from all samples were lysed with red blood cell (RBC) lysis buffer. Peritoneal exudate cells (PEC) were isolated by centrifugation of peritoneal lavages obtained by injection (2x2ml) of DPBS supplemented with 3% FBS into the peritoneal cavity. All cells were incubated with anti-CD16/32 (clone 93; BioLegend) to block Fc receptors. Mice monocytes were identified using CD45 (clone 30F11; BD Biosciences), Ly6G (clone 1A8; BD Biosciences), CDl lb (clone Ml/70; BD Biosciences), Ly6C (clone HK1.4; BioLegend) and CD115 (clone AFS98; BioLegend) antibodies. For the characterization of surface marker antigens of blood monocytes, F4/80 (clone BM8; BioLegend), CD43 (clone S7; BD Biosciences), CD62L (clone MEL-14, BioLegend), LFA-1 (clone H155-78; BioLegend), CD49b (clone DX5; BioLegend), MHC-II (clone M5/114.15.2; BD Biosciences), CDl lc (clone HL-3; BD Biosciences), 7/4 (AbD Serotec), CCR2 (clone 475301, R&D system) and CX3CR1 (polyclonal, R&D system) antibodies were added to the previous panel. Spleen monocytes were identified using Ly6G, NKl. l (clone PK136; BD Biosciences), CD3 (clone 17A2; eBiosciences), CD19 (clone eBiolD3; eBiosciences), CDl lb, CDl lc and Ly6C antibodies. Spleen dendritic cells were identified using CD45, CDl lc, MHC-II, CDl lb, B220 (clone RA3-6B2; BioLegend), and BST2 (clone eBiol29c(129c); eBioscience) antibodies. Monocytes and precursors from bone marrow were identified using CD117 (clone 2B8; BD Biosciences), CDl lb, Sca-1 (clone D7; BD Biosciences), Ly6G, NKl.l, CD115, Ly6C, CD135 (clone A2F10; eBiosciences), CD3 and CD 19 antibodies. A viability dye (LIVE/DEAD Fixable; Molecular Probes) was added to the previous panels to discriminate live cells. Human blood monocytes were analyzed by flow cytometry based on their expression of CD 14 and CD 16 using CD14-PE-Cy7 (clone M5E2) and CD16-A647 (clone 3G8) antibodies (BD Biosciences). Classical, intermediate and non- classical monocytes were specifically identified by selective gating strategy as follows: CD14 ⁺⁺ CD16 ^" (classical), CD14 ⁺ CD16 ⁺ (intermediate) and CD14 ^± CD16 ⁺⁺ (non-classical). All analyses were performed on BD LSR II or sorted on BD FACS Aria II. Data were analyzed with FACS Diva software (BD Biosciences).

Two-photon intravital microscopy imaging

[0055] Blood vessels were labeled with Qdot™ 750 (Qtracker 705, 5% in PBS (Invitrogen) administered through the tail vein. Animals were anesthetized with isoflurane and placed under the two-photon microscope using a special support. For intravital microscopy of the ear, skin was fixed on slide and covered with 0.9% saline. For peritoneal intravital microscopy, a small midline abdominal incision was made to expose the parietal peritoneum and tissue was covered with a slide covered with 0.9% saline. Intravital microscopy was carried out using MPE two-photon microscope. The two-photon Mai Tai DeepSee™ laser (Spectra-Physics) was tuned at 900 nm for all the experiments. Tissues were imaged using an Olympus™ Ultra 25* MPE water immersion objective (1.05 NA), with filter set bandwidths optimized for GFP (520-560 nm) and Qdot 705 (669-800 nm) imaging. Detector sensitivity and gain were set to achieve the optimal dynamic range of detection. Using the Olympus Fluoview™ software (version 3.0a), images with resolution 512x512 pixels were acquired for 15 minutes at 1.5* zoom factor and 2.5 frames per second. No Kalman filter was used to avoid slowing down the acquisition speed.

Library preparation and sequencing by RNA-seq

[0056] Blood Ly6C ^high and Ly6C ^low monocytes, from vehicle or MDP-treated (18 hours) wild-type mice, were enriched by cell sorting. Total RNA from sorted Ly6C ^hlgh and Ly6C ^low monocytes was isolated using EZ-10 DNAaway™ RNA miniprep (Bio Basic) according to manufacturer's protocol. RNA quality was checked using a TapeStation™ 2200 (Agilent Technologies). RNA integrity number (RIN) for all samples was > 7.7. The Illumina TruSeq™ RNA sample preparation V2 kit (Illumina Inc.) was used to prepare mRNA sequencing libraries, according to manufacturer's protocol. Three libraries were independently prepared for each condition. Briefly, 50 ng of total RNA was used for poly A mRNA selection using oligo-dT attached magnetic beads. Fragmented mRNA was used as template for cDNA synthesis by reverse transcriptase with random primers. The cDNA was further converted into double stranded DNA that was end-repaired to incorporate the specific index adaptor for multiplexing, followed by a purification step with Agencourt AMPure XP beads™ (Beckman Coulter) and an amplification for 15 cycles. The quality of final libraries were examined with a DNA screentape D1000™ on a TapeStation™ 2200 and the quantification was done on the QBit 3.0™ fluorometer (ThermoFisher Scientific) as well as by (q)PCR using KAPA library quantification (KAPABiosystems). Subsequently, RNA-seq libraries with unique index were pooled in equimolar ratio (14 samples/pool) and sequenced using both lanes of a rapid run flowcell on an HiSeq 2500™ system at the Next-Generation Sequencing Platform (Genomics Center from CHU de Quebec Research Center) for paired- ends 100 pb sequencing. The average insert size for the paired-end librairies was 400 bp.

Bioinformatic analysis of RNAseq

[0057] Reads from HiSeq2500 Illumina™ were first processed with Trimmomatic v0.33 software to remove low quality with a ILLUMINACLIP value of 2:30:10, a TRAILING value of 30 and a MINLEN value of 36. Reads were then aligned of the mmlO reference genome using Tophat v2.0.14 and Bowtie v2.2.5 with default values for the parameters. The raw reads were splitted into two sequencing lanes, the aligned reads were thus merged and sorted using samtools vl .2 with default values. Duplicated reads were marked with Picard's MarkDuplicate software vl .130 with the ASSUME SORTED and CREATE INDEX values set at true. Gene abundance and differential expression were calculated using the Cuffquant and Cuffdiff softwares from the Cufflinks suite™ v2.2. Gene counts were obtained with the htseq-count software v0.6.1pl -s no and -m intersection-noempty parameters values. The annotation used for the quantification, differential expression analysis and gene count was downloaded from USCS on May 23, 2014. Gene counts were analyzed with PCA using the PCA function from the FactoMineR package.

Statistical analysis

[0058] Statistical analyses were carried out with Prism software (GraphPad 6.0) and were performed using unpaired f-test or otherwise indicated. The level of significance was set at p < 0.05, as identified in the Figures and Tables with the sign "*".

[0059] The ensuing description provides exemplary embodiment(s) only, and is not intended to limit the scope, applicability or configuration of the disclosure. Rather, the ensuing description of the exemplary embodiment(s) will provide those skilled in the art with an enabling description for implementing an exemplary embodiment. It being understood that various changes may be made in the function and arrangement of elements without departing from the spirit and scope as set forth in the appended claims.

EXAMPLE I

Effect of MDP on Monocytes Frequencies in Mice

[0060] The monocyte mobilizing effect of Muramyl dipeptide (MDP) was evaluated in an in vivo model using wild-type C57B1/6 mice (4-6 week old). MDP was diluted in 0,9% saline and intravenously injected at lOmg/kg. Control mice were injected with 0,9% saline. Treatment was administrated daily for 4 days and mice were sacrificed 24 hours following the last injection, unless otherwise indicated. Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and then transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2 (composed of 150 mM NH ₄ C1, 10μΜ HEPES, O. lmM Na ₂ EDTA pH 8.0). Cell staining for flow cytometry was performed as follows: samples were first incubated with anti-CD 16/32 to block nonspecific antibodies interaction with Fc receptors and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb, Ly6C, and CD115 for 30 minutes. Samples were washed twice and analysis was performed on a BD SORP LSR II™. Monocytes were identified as CD457CD1 lb ⁺ /CDl 15 ⁺ /Ly6G ^" cells.

[0061] Two groups of wild-type C57B1/6 mice were constituted as follows:

• Group 1: treated with 0,9% saline (40 mice) (10 sacrificed/day) (placebo); and

• Group 2: treated with muramyl dipeptide diluted in 0,9% saline (20 mice)(5 sacrificed/day) (lOmg/kg).

[0062] The results obtained, as reported in Fig. 1, indicate that MDP induces a marked increase in peripheral blood monocytes by 48h with a twofold increase (> 8%). Monocytes levels gradually increase throughout the protocol until final mice sacrifice. Mice treated with placebo displayed a steady level of monocytes with frequencies averaging 4% of total white blood cells (Mono/CD45).

EXAMPLE II

Effect of MDP on the Differentiation of Inflammatory CD 1 lb/CD 115/Ly6C ^{hi h} Monocytes into Patrolling CDllb/CD115/Ly6C ^low Monocytes in Mice Peripheral Blood

[0063] The monocyte differentiating effect of MDP was evaluated in an in vivo model using wild-type C57B1/6 mice (4-6 week old). Muramyl dipeptide was diluted in 0,9% saline and intravenously injected at lOmg/kg. Control mice were injected with 0,9% saline. Treatment was administrated daily for 4 days and mice were sacrificed 24 hours following the last injection, unless otherwise indicated. Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and then transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2. Cell staining for flow cytometry was performed as follows: samples were first incubated with anti- CD 16/32 to block nonspecific antibodies interaction with Fc receptors and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb, Ly6C, and CD115 for 30 minutes. Samples were washed twice and analysis was performed on a BD SORP LSR II™. Inflammatory and patrolling monocytes were respectively identified as CD45 ⁺ /CDl lb ⁺ /CD115 ⁺ /Ly6C ^high /Ly6G ^" and

CD45 ⁺ /CDl lb ⁺ /CD115 ⁺ /Ly6C ^l0W /Ly6G ^" cells. A monocyte maturation intermediate is also observed and is characterised by expression of CD45 ⁺ /CDl lb ⁺ /CD115 ⁺ /Ly6C ^int /Ly6G ^" . [0064] Two groups of wild-type C57B1/6 mice were constituted as follows:

• Group 1 : treated with 0,9% saline (placebo); and

• Group 2: treated with muramyl dipeptide diluted in 0,9% saline (lOmg/kg).

[0065] The results obtained indicate that MDP induced a marked decrease in Ly6C ^hlgh monocyte by 24 hours and remains low for the remainder of the protocol (Fig. 2A). More importantly, Ly6C ^low monocytes begin to increase by 24 hours and appear to reach a maximum by 48 hours post-treatment (Fig. 2C). Interestingly, the intermediate population appears to peak mostly in the beginning of the protocol, as expected for this transitory cell population (Fig. 2B). In Figs. 2D and 2E, FACS analysis is represented to illustrate the shift from CDl lb/CD115/Ly6C ^high monocytes (red) into patrolling CDl lb/CD115/Ly6C ^low monocytes (green), in either the placebo (Fig. 2D) or in the treated mice (Fig. 2E).

EXAMPLE III

In vivo treatment with various NOD2 agonists

[0066] As illustrated in Fig. 3, N-Acetyl-MDP, N-Glycolyl-MDP and L18-MDP, but not Murabutide, drive polarization of circulating Ly6C ^hlgh monocytes into Ly6C ^low monocytes. Wild type mice were treated with vehicle (white histogram) or increasing doses (1.25, 2.5, 5 and 10 mg/kg, black histograms) of NOD2 agonists. Animals were sacrificed 48 hours following treatment and percentages of Ly6C ^hlgh , Ly6C ^inter and Ly6C ^low blood monocytes were analyzed by flow cytometry. Data are presented as mean ± SEM of two independent experiments (n=4 mice per dose). * p < 0.05, ** p < 0.01 and *** p < 0.001 (Two-way Anova followed by Tuckey test).

[0067] The results show that comparable polarizing effects were obtained with two other NOD2 agonists, N-Glycolyl-MDP and L18-MDP (Fig.3). No effect was detected when using murabutide. Although all the evaluated NOD2 agonists are known to bind and activate the NOD2 receptor, only three had the capacity to induce conversion of Ly6C ^high to Ly6C ^l0W monocytes. This result indicates that a specific triggering mechanism is required to induce the monocytes "switching" effect, which results in an increase in the Ly6C ^low monocytes subset.

EXAMPLE IV Effect of NOD2 Receptor Knockdown on MDP Activity

[0068] The effect of NOD2 inactivation on MDP activity was evaluated in an in vivo model using NOD2 knock-out (KO) mice as compared to wild-type C57B1/6 mice (4-6 week old). Muramyl dipeptide was diluted in 0,9% saline and intravenously injected at lOmg/kg. Control mice were injected with 0,9% saline. Treatment was administrated daily for 4 days and mice were sacrificed 24 hours following the last injection, unless otherwise indicated. Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and then transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2. Cell staining for flow cytometry was performed as follows: samples were first incubated with anti-CD 16/32 to block nonspecific antibodies interaction with Fc receptors and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb, Ly6C, and CD115 for 30 minutes. Samples were washed twice and analysis was performed on a BD SORP LSR II™. Monocytes were identified as CD45 ⁺ /CDl lb ⁺ /CD115 ⁺ /Ly6G ^" cells. Inflammatory and patrolling monocytes were respectively identified as CD45 ⁺ /CDl lb7CD115 ⁺ /Ly6C ^high /Ly6G ^" and CD457CDl lb7CD1157Ly6C ^low /Ly6G ^" cells. A monocyte maturation intermediate is also observed and is characterised by expression of CD457CDl lb7CD1157Ly6C ^int /Ly6G ^" .

[0069] Two mice groups were constituted as follows:

• Group 1: NOD2 ^{" "} mice treated with 0,9% saline (placebo); and

• Group 2: NOD2 ^" mice treated with muramyl dipeptide diluted in 0,9% saline (lOmg/kg).

[0070] The results obtained indicate that there are no MDP-dependent increases of the monocyte population in the NOD2 ^{" "} mice background (Fig. 4A). More importantly, the monocyte differentiation observed in MDP-treated naive mice is absent, and monocyte subpopulations appear unaffected throughout the protocol in the NOD2 mice background (Fig. 4B-4D). EXAMPLE V

Effect of MDP on the Restoration of CDllb/CD115/ly6C ^low Monocyte Populations in

Mice Lacking Patrolling Monocytes (NR4Al ^"/_ Mice Background)

[0071] The monocyte differentiating effect of MDP was evaluated in an in vivo model using NR4A1 ^{" "} mice (4-6 week old). NR4A1 ^{" "} mice are characterized by a maturation block, which results in a Ly6C ^low patrolling monocytes deficiency. This experiment is performed to evaluate whether MDP can bypass the NR4A1 -dependent maturation block and restore Ly6C ^low patrolling monocytes population. Muramyl dipeptide was diluted in 0,9% saline and intravenously injected at lOmg/kg. Control mice were injected with 0,9% saline. Treatment was administrated daily for 4 days and mice were sacrificed 24 hours following the last injection, unless otherwise indicated. Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and then transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2. Cell staining for flow cytometry was performed as follows: samples were first incubated with anti-CD 16/32 to block nonspecific antibodies interaction with Fc receptors and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb, Ly6C, and CD115 for 30 minutes. Samples were washed twice and analysis was performed on a BD SORP LSR II™. Inflammatory and patrolling monocytes were respectively identified as CD457CDl lb7CD1157Ly6C ^high /Ly6G ^" and CD457CDl lb7CD1157Ly6C ^low /Ly6G ^" cells. A monocyte maturation intermediate is also observed and is characterised by expression of CD457CD1 lb ⁺ /CDl 15 ⁺ /Ly6C ^int /Ly6G ^" .

[0072] Two groups of wild-type NR4A1 ^{" "} mice were constituted as follows:

• Group 1: treated with 0,9% saline (placebo); and

• Group 2: treated with muramyl dipeptide diluted in 0,9% saline (lOmg/kg).

[0073] The results obtained indicate that there are no MDP-dependent increases of the monocyte population in the NR4A1 ^{" "} mice background (Fig. 5A). This result suggested that Ly6C ^hlgh monocytes remained in the bone marrow in absence of NR4A1. In contrast, it was found that levels of Ly6C ^hlgh monocytes, which represent approximately 85% of total blood monocytes in NR4A1 ^{" "} mice (See Table 1), were reduced by 40% to 45% following MDP treatment as compared to the controls (Fig. 5B). More importantly, the percentage of Ly6C ^low monocytes strongly increased to reach an average of 16% after 48 hours compared to naive NR4A1 mice which have « 2-3% of circulating Ly6C monocytes (Fig. 5D). Although fluctuations of Ly6C ^int monocytes occur, general tendency shows an increase of Ly6C ^int after MDP treatment that reflects a transient status of this monocyte subset (Fig. 5C). The results obtained indicate that MDP can restore Ly6C ^low patrolling monocytes population in the NR4A1 ^{" "} mice background.

[0074] Table 1 shows the frequencies (%) and absolute counts of Ly6C ^high , Ly6C ^inter and Ly6C ^low blood monocytes in naive wild-type and NR4A1 ^{" "} mice (n=12) as determined by flow cytometry analysis. Results are presented as mean ± SEM of two independent experiments.

Table 1

Distribution of monocytes between the 3 types

Wild-Type NR4A1 ^"

Frequency (%) 4.6 ± 0.4 1.7 ± 0.1

Total

Monocytes

Absolute Count

146.0 ± 19 46.0 ± 6 (x 10 ³ cells/ml)

Frequency (%) 62.9 ± 1.7 84.6 ± 1.9 high

Ly6C

Absolute Count

90.0 ± 14 39.0 ± 5 (x 10 ³ cells/ml)

Frequency (%) 14.5 ± 0.9 12.3 ± 1.4

Ly6C"

Absolute Count

21.0 ± 3 5.0 ± 1 (x 10 ³ cells/ml)

Frequency (%) 22.5 ± 1.5 3.3 ± 0.6 low

Ly6C

Absolute Count

33.5 ± 5 1.0 ± 0.3 (x 10 ³ cells/ml) EXAMPLE VI

Effect of MDP on the Polarization of Primary Human Blood Monocytes In Vitro

[0075] The results presented herein in mice clearly show that MDP induced a differentiation of inflammatory CDl lb/CD115/Ly6C ^hlgh monocytes into patrolling CDl lb/CD115/Ly6C ^l0W monocytes in mice peripheral blood. To assess its polarization potential in human, the monocyte differentiating effect of MDP was evaluated in an ex vivo model using PBMC isolated from blood of healthy human donor. Mononuclear cells were isolated using Ficoll density gradient (Wisent, Quebec, Canada), and were first separated from lymphocyte population by cell adherence on Petri dishes. After Ficoll isolation, cell were stimulated overnight with Muramyl dipeptide at lC^g/ml. Enriched monocyte subsets were washed twice in Hanks Balanced Salt Sodium (HBSS; Wisent) and cells were surface stained with LIVE/DEAD Blue (Invitrogen), CD45-PerCP-Cy5.5 (2D1) (eBioscience, San Diego, CA, USA), CD91-FITC (A2-MR-a2), CD14-PE-Cy7 (M5E2) and CD16-A700 (3G8) (BD Biosciences), as described in a standardized flow cytometry assay (Ferrer DG et al. Cytometry A. 2014 Jul;85(7):601-10). Classical, intermediate and non-classical monocytes were specifically identified by selective gating strategy as followed: CD14 ⁺⁺ CD 16 ^" (classical), CD14 ⁺ CD16 ⁺ (intermediate) and CD14 ^± CD16 ⁺⁺ (non-classical).

[0076] Classical, intermediate and non-classical monocytes were respectively identified as

• Classical: Live/CD45/SSC ^mid /CD9l7CD14 ⁺ 7CD16 ^" cells (Blue);

• Intermediate: Live/CD45/SSC ^mid /CD91 ⁺ /CD14 ⁺ /CD16 ⁺ cells (Red); and

• Non-classical: Live/CD45/SSC ^mid /CD9l7CD147CD16 ⁺⁺ cells (Green).

[0077] Fig. 6 shows the flow cytometry analysis of CD14 ⁺⁺ CD 16 ^" (PI, blue), CD14 ⁺ CD16 ⁺ (P2, red) and CD14 ^± CD16 ⁺⁺ (P3, green) monocyte subsets in freshly isolated (Fig.6A), placebo-treated (cell cultures) (Fig.6B) or MDP-treated (Fig.6C) human mononuclear cells for 18 hours. Fig. 6D shows the frequencies (%) of blood CD14 ^± CD16 ⁺⁺ (P3) monocyte subsets from freshly isolated, placebo-treated, or MDP-treated human mononuclear cells for 18 hours. Dot plots show the distribution and mean (± SEM, horizontal lines) of monocytes subset frequencies of three independent experiments. Each symbol represents an independent individual. * p < 0.05 and **** p < 0.0001 (unpaired t-test). [0078] Freshly isolated monocytes are mainly constituted of more than 80% of classical monocytes, 2-3% of intermediate monocytes and 5-7% of non-classical monocytes (Fig 6A). While in vitro cultures of monocytes seem to promote the frequencies of intermediate monocytes (Fig.6B), the percentages of non-classical monocytes were not affected. On the other hand, after 24 hours of in vitro treatment with MDP, it was found that percentages of non-classical monocytes (P3) increased to approximately 15% (Fig. 6C and 6D), indicating that CD14 ⁺⁺ CD 16 ^" monocytes switch into CD14 ^± CD16 ⁺⁺ monocytes when stimulated with MDP. The increase of intermediate monocytes following in vitro treatment is suggesting a reflection of the transition into the CD14 ^± CD16 ⁺⁺ phenotype.

Example VII

NOD2 Triggering Restores Monocyte Frequency in Monocytopenic Mice

[0079] The effect of NOD2 activation on blood monocyte levels was evaluated in CCR2- deficient mice, which are monocytopenic. The Ly6C ^hlgh monocytes in these KO mice also have a reduced mobilization potential during inflammatory responses. Additionally, we studied the NOD2 triggering effect in CX3CR1 -deficient mice, which are characterized by Ly6C ^low monocytes with reduced crawling potential.

[0080] In Fig. 7, flow cytometry analysis of blood monocytes from Ccr2 ^{" "} and Cx3crl ^{" "} mice (n=6 mice/group) following daily treatment with vehicle (solid lines) or MDP (dashed lines) are illustrated. Results are presented as in Fig. 7A as percentages of total monocytes and in Fig. 7B as percentages of Ly6C ^hlgh , Ly6C ^inter and Ly6C ^low monocyte subsets. The inset in Fig. 7A shows the wild-type mice treated with vehicule (solid line) or MDP (48 hours, dashed line) to illustrate the increase in blood monocytes following MDP treatment. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 (unpaired t-test). Results show that MDP treatment restores monocyte frequency to normal levels by 48h (Fig 7A). In Fig 7B, it was further evaluated if the absence of chemokine receptors CCR2 and CX3CR1 on monocyte subsets may affect MDP-induced differentiation of Ly6C ^hlgh into Ly6C ^low monocytes. As expected, deficiency of CCR2 reduced the percentage of total recruited blood monocytes as compared to WT controls (Fig. 7A) since Ly6C ^hlgh monocyte recruitment is strongly mediated through CCR2 receptor. Deletion of CX3CR1 did not affect the effects of MDP treatment on total blood monocytes. However, it was observed that in both CCR2 ^{" "} and CX3CR1 mice, MDP treatment strongly increased the percentage of Ly6C ^low monocytes (Fig. 7B), supporting the intrinsic capacity of NOD2 to polarize monocytes from an inflammatory to a patrolling phenotype.

Example VIII

In vivo treatment with MDP converts Ly6C ^{hl h} monocytes into Ly6C ^low patrolling monocytes

[0081] In order to mimic monocytopenia in a murine model and to further confirm the capacity of MDP to convert inflammatory Ly6C ^hlgh into patrolling Ly6C ^low monocytes in the circulation, monocytes were depleted by administration of liposomes loaded with clodronate and the kinetics of blood monocyte repopulation was monitored following treatment with the NOD2 agonist MDP. Fig. 8A shows the experimental design of clodronate liposomes (Clo- lipo) and MDP administration. Wild-type mice (n=6 mice/group) were injected with PBS- liposomes (PBS-lipo) as control or Clo-lipo in order to deplete blood monocytes. MDP was injected daily 24 hours following clodronate administration and animals were sacrificed at indicated times. Fig. 8B shows the schematic representation of reemerging Ly6C ^high (red) and Ly6C ^low (green) blood monocytes following clodronate liposomes (Clo-lipo) administration. Wild type mice (n=4 mice/group) were injected (i.v.) with Clo-lipo and sacrificed at indicated days. Absolute counts of reemerging monocyte subsets are presented as mean ± SEM of two independent experiments. Monocytes are completely eliminated by day 1 post-liposome injection («1% remaining) (Fig. 8B) but patrolling monocytes remain absent and only re-emerge by day 6 post-clodronate injection, which facilitates the visualization of Ly6C ^low monocyte emergence by flow cytometry. Fig. 8C shows the flow cytometry analysis of blood Ly6C ^hlgh and Ly6C ^low monocyte subsets following vehicle (saline) or MDP treatment of Clo-lipo-injected mice. Animals were sacrificed at indicated times post treatment. In clodronate-treated mice, it was found that exclusively Ly6C ^hlgh monocytes reached normal levels in 48 hours (-88%) and Ly6C ^low monocytes remained absent (Figs. 8C). In contrast, when mice are treated with MDP, Ly6C ^low monocytes had started to repopulate the blood stream as soon as 48 hours and gradually increased up to 96 hours to reach 24.6% (Fig. 8C and Table 2). In addition, it was also observed that the number of Ly6C ^int monocytes rapidly increased following treatment with MDP, suggesting that Ly6C ^hlgh monocytes give rise to Ly6C ^low monocytes via a cellular transition in Ly6C ^int phenotype. Fig. 8D shows the percentage of total, Ly6C ^hlgh and Ly6C ^low blood monocytes in control (PBS-lipo) or clodronate (Clo-lipo) injected mice daily treated with vehicle or MDP. Animals were sacrificed at indicated times following Clo-lipo administration.

Table 2

Time distribution of monocytes between the 3 subsets after depletion

Where: * denotes p value < 0.05

*** denotes p value < 0.001

**** denotes p value < 0.0001

[0082] To confirm more directly the consequences of NOD2 triggering on Ly6C ^lg monocyte conversion, mice were injected with fluorochrome DiO-labeled liposomes (DiO- lipo) after clodronate-liposome induced depletion of monocytes (Fig. 9). Fig. 9A shows the experimental design of DiO-labelled liposomes and MDP treatment. DiO-labelled liposomes (DiO-lipo) or PBS-liposomes (control) were injected intravenously in wild-type mice 48 hours following Clo-lipo administration in order to label newly synthesized Ly6C ^hlgh monocytes. Thereafter, mice were daily treated with vehicle or MDP and sacrificed at indicated times. Fig. 9B shows the flow cytometry analysis of Ly6C ^hlgh and Ly6C ^low DiO- labelled monocyte subsets at indicated times following vehicle (upper panels) or MDP (lower panels) treatment. Data are presented as mean ± SEM of three independent experiments. * p < 0.05 ** p < 0.01 *** p < 0.001 (Two-Way ANOVA) compared to indicated groups.

[0083] As expected, the large majority of re-emerged DiO-labeled monocytes have the Ly6C ^hlgh phenotype (-81%) at 96 hours while the percentage of Ly6C ^low gradually increased with time to reach «6% at 120 hours (Fig. 9B). In mice treated with MDP, Ly6C ^low monocytes started to emerge at 96 hours and rapidly increased to reach «32% after 120 hours and even 60% at 144 hours post-treatment (Fig. 9B). Again, these results confirm that Ly6C ^hlgh give rise to Ly6C ^low monocytes in the circulation and show that triggering of NOD2 receptor accelerates such conversion of Ly6C ^hlgh monocytes into Ly6C ^low phenotype.

Example IX

NOD2 triggering gives rise to Ly6C ^low patrolling monocytes

in bone marrow and in spleen of mouse

[0084] Figs. 10A, IOC, and 10E, show the flow cytometry gating strategies of total, Ly6C ^high and Ly6C ^low monocytes in blood (10A), bone marrow (IOC) and spleen (10E) of wild-type mice. Blood monocytes are gated as CD45 ⁺ CDl lb ⁺ CD115 ⁺ after exclusion of neutrophils, bone marrow monocytes as Live ⁺ Lin ^" CD117 ^" CD115 ⁺ CDl lb ⁺ and spleen monocytes as Live ⁺ Lin ^" CDl lb ⁺ CDl lc ^" . Monocyte subsets are analyzed according to their Ly6C expression for blood, bone marrow and spleen cells from vehicle (control) or MDP (48 hours) treated mice. Frequencies of Ly6C ^low and Ly6C ^hlgh monocytes are shown in Fig. 10B for blood cells, Fig. 10D for bone marrow cells and Fig. 10F for spleen cells of mice treated with vehicle or MDP at indicated times. Percentage of Ly6C ^low monocytes in bone marrow of mice is very low, but significantly higher in spleen of mice. It was then evaluated whether MDP treatment of mice could also induce differentiation of Ly6C ^hlgh into Ly6C ^low monocytes in these two compartments. In bone marrow, monocytes were identified as Lin ^" CD 117 ^" CD115 ⁺ CDl lb ⁺ Ly6C ⁺ and in spleen as Lin ^" CD117 ^" CDl lb ⁺ CDl lc ^" Ly6C ⁺ . As observed in blood, treatment with MDP significantly increases frequencies of Ly6C ^low in bone marrow after 48 hours, but percentages of Ly6C ^hlgh remain stable (Figs. 10A to 10D). In spleen of MDP-treated mice, percentages of Ly6C ^hlgh monocytes rapidly decreased after 24 hours but as observed in the circulation, percentages of Ly6C ^low monocytes were found to increase following 48 hours of treatment (Figs. 10E and 10F).

[0085] Fig. 11A shows the flow cytometry analysis of plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC) in spleen of wild-type mice (n=5 mice/group) treated with vehicle or MDP (24 hours). Fig. 11B shows the flow cytometry analysis of blood neutrophils of wild-type mice (n=5 mice/group) treated with vehicle or MDP (24 hours). Data are representative of two independent experiments. In vivo triggering of NOD2 did not affect phenotype of circulating pDC, cDC and neutrophils (See Figs. 11 A and 1 IB).

[0086] The inventors have also used a flow cytometry approach to discriminate between different progenitors associated with the monocyte maturation pathway, including LSK, MDP cells and cMOP (See Figs 12A and 12B). Fig. 12A shows the flow cytometry gating strategies of hematopoietic monocyte precursors (LSK, cMoP and MDP cells) in bone marrow of wild-type mice (n=4 mice/group). Fig. 12B shows the frequencies (%) of LSK, MDP cells and cMOP in wildtype mice (n=4 mice/group) treated with vehicle or MDP at indicated times. Data are presented as percentage of monocyte precursors. Dot plots show the distribution and mean (± SEM, horizontal lines) of monocyte precursor frequencies of three independent experiments. Each symbol represents an independent individual. The results indicate that triggering of NOD2 by MDP does not affect monocyte precursors, thus supporting the intrinsic activity of MDP on monocyte differentiation.

Example X

Characterization of surface antigen expression and biological functions

of differentiated Ly6C ^low monocytes following MDP treatment

[0087] In addition to Ly6C and the chemokine receptor CX3CR1, patrolling monocyte subset can be distinguished from inflammatory monocytes by expression of various antigens and by specific biological functions. Firstly, the phenotypic features of isolated blood Ly6C ^low monocytes was determined from MDP-treated NR4A1 ^{" "} mice, and of converted monocytes from naive WT mice pretreated with clodronate before MDP administration. In that case, only Ly6C ^hlgh monocytes were restored in the circulation at 48 hours post- administration of clodronate. Flow cytometry analysis revealed that MDP-induced Ly6C ^low monocytes in clodronate-treated mice as well as NR4A1 ^{" "} mice express high levels of LFA1 and display typical marker expression characterizing patrolling monocytes such as a negative expression of CD62L, 7/4 and CD49b and a positive expression of CDl lc (See Table 3). Table 3 shows the comparison of the surface marker expression of blood Ly6C ^low monocytes (grey) isolated from naive, Clo-lipo and NR4A1 ^{" "} mice, to those of Ly6C ^hlgh monocytes from WT mice following treatment with MDP (n=6 mice/group). Expression levels of surface antigens have been assigned arbitrary symbols that represent (-) no expression, (±) marginal expression, (+) positive expression and (++) strong expression. *: CX3CRI expression on monocyte subsets was determined using Cx3crl ^+/gfp mice except for NR4A1 ^{" "} mice where CX3CR1 antibody was used.

Table 3

Characterization of monocytes

[0088] Surprisingly, expression of CX3CR1 and CCR2 was not modulated by MDP treatment in NR4A1 ^{" "} mice as it was observed in mice pretreated with clodronate prior MDP administration.

[0089] In contrast to Ly6C ^hlgh inflammatory monocytes, one major feature of Ly6C ^low monocytes is their capacity of crawling on the lumina side of the blood vessels in steady-state conditions. To determine whether MDP-induced Ly6C ^low monocytes can patrol the endothelium of the vessels, the inventors have undertaken intravital confocal microscopy imaging within capillaries of the ear in CXSCRl ⁸ *^ mice. This mouse strain was used to facilitate tracking of patrolling monocytes. When indicated, mice were injected with clodronate to completely eliminate monocytes prior treatment with NOD2 agonist, MDP. As mentioned above, patrolling monocytes start to re-emerge in circulation only by day 6 post- clodronate injection, while Ly6C ^hlgh monocytes appear by day 2 (Fig. 8B). It was thus assumed that the presence of Ly6C ^low patrolling monocytes detected in blood by days 2-3 should arise from Ly6C ^hlgh . Indeed, intravital observation revealed that following treatment with MDP, a significant number of crawling cells expressing the GFP proteins is detected as compared to the control groups treated with a placebo (Figs. 13A and 13B). Figs 13A and 13B are intravital imaging of monocytes in the dermis (ear) of Cx3crl ^+/gfp mice treated with vehicle or MDP (72 hours post-treatment) (Fig. 13 A) and Cx3cr ^1+gfp mice injected with clodronate-liposomes (clo-lipo) 24 hours prior to vehicle or MDP treatment (Fig. 13B). Images were recorded at indicated time following clo-lipo administration. Scale bar 20 mm. Figs. 13C and 13D show gating strategies of Ly6C ^high , Ly6C ^inter and Ly6C ^low blood monocytes from corresponding Cx3cr ^1+/gfp in vivo imaged mice. Cytometry analysis also confirmed that these monocytes display a patrolling phenotype (Figs. 13C and 13D)

Example XI

Gene encoding profile by NOD2-activated Ly6C ^low and Ly6C ^{hl h} monocytes

[0090] Next, the gene profile in both subsets of monocytes following MDP treatment was evaluated. To evaluate gene expression profiles in both monocytes subset following NOD2 activation by MDP treatment, the inventors performed differential RNA sequence analysis. Briefly, muramyl dipeptide was diluted in 0.9% saline and intravenously injected at lOmg/kg in wild type mice. Control mice were injected with vehicle.

[0091] Two mice groups were constituted as follow:

• Group 1: wild type mice were treated with 0.9% saline.

• Group 2: wild type mice were treated with muramyl dipeptide diluted in 0.9% saline (lOmg/kg).

[0092] Mice were sacrificed 18 hours after treatment and blood Ly6C ^high and Ly6C ^low monocytes from vehicle or MDP-treated wild type mice were purified by high speed cell sorting. Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and the transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2. Cell staining for flow cytometry was performed as follows: samples were first incubated with anti-CD16/32 to block non-specific antibody interaction with FC receptor and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb, Ly6C and CD115 for 30 minutes. Samples were washed twice and cell sorting was performed on a BD FACSAria™ II cell sorter. Inflammatory and patrolling monocytes were respectively identified as CD457CD1 lb7CDl 15 ⁺ /Ly6C ^high /Ly6G ^" and CD457CD1 lb ⁺ /CDl 15 ⁺ /Ly6C ^low /Ly6G ^" cells.

[0093] Total RNA from sorted Ly6C ^high and Ly6C ^low monocytes was isolated using EZ- 10 DNAaway RNA miniprep (Bio Basic) according to manufacturer's protocol. RNA sequencing (RNAseq) experiments were performed as described hereinabove.

[0094] Fig. 14A shows the fold change in gene expression as measured by RNA sequencing in sorted Ly6C ^hlgh and Ly6C ^low monocytes from wild type mice treated with MDP (18 hours). Genes were ordered based on a significative fold change in gene expression measured in the Ly6C ^hlgh inflammatory and Ly6C ^low patrolling monocyte subtypes. Data are presented as mean of three independent experiments and are significantly different (p < 0.05) after Benjamini-Hochberg correction for multiple testing as compared to the corresponding monocyte subtype of vehicle treated animals. *: Infinite fold change.

[0095] Comparative analysis show that NOD2 activation by MDP leads to substantial gene regulation. As previously published, 584 and 2058 genes were differentially expressed in Ly6C ^high and Ly6C ^low monocytes after NOD2 activation, respectively. CXCR3 and its ligand CXCL10 are strongly suppressed in Ly6C ^hlgh monocytes after MDP treatment. Axl is also suppressed in Ly6C ^hlgh monocytes. M2 chemokines like CCL17 and CCL22 and the suppressor SOCS2 were found to be significantly activated in both subsets of monocytes. The gene coding for SiglecE, a negative regulator of acute inflammation, was activated in Ly6C ^low monocytes following administration of MDP, while M2 signature genes such as Argl, Aloxl5, Tgm2 and MIF are found to be upregulated in Ly6C ^hlgh monocytes, suggesting that these cells are engaged in a differentiation pathway favoring a M2-like functions.

[0096] Most importantly, NOD2 activation by MDP leads to an increase of the transcription factor CCAATT/enhancer-binding protein b (C/ΕΒΡβ) in inflammatory Ly6C ^hlgh monocytes as compared to vehicle treated control cells (Fig 14A-14B). This transcription factor is highly expressed in monocytes, specifically in Ly6C ^low monocytes. It functions as a critical regulatory factor for the differentiation and survival of Ly6C ^low cells. These results indicate that Ly6C ^hlgh monocytes modify expression in critical regulatory genes upon NOD2 activation that promote and control differentiation into Ly6C ^low cells.

[0097] The progression from Ly6C ^hlgh monocytes to Ly6C ^low cells is characterized by substantial gene expression changes, which reflect modification in cellular function. These gene expression profiles are also greatly affected when NOD2 is activated by MDP to induce a transition from Ly6C ^high to a Ly6C ^low cells.

[0098] C/ΕΒΡβ is a critical regulator in monopoiesis and controls the differentiation from Ly6C ^hlgh to Ly6C ^low monocytes. To confirm the upregulation of C/ΕΒΡβ in monocytes after NOD2 activation, RT-qPCR analyses were performed on monocytes isolated from vehicle and MDP treated mice 1 hour after treatment. Briefly, monocytes isolated from vehicle and MDP treated mice were purified by high speed cell sorting.

[0099] Two mice groups were constituted as follow:

• Group 1 : wild type mice were treated with 0.9% saline.

• Group 2: wild type mice were treated with muramyl dipeptide diluted in 0.9% saline (lOmg/kg).

[00100] Blood samples were collected by cardiac puncture using HBSS-EDTA coated syringes and the transferred into K3-EDTA coated tubes. Erythrocytes from all samples were lysed with red blood cell lysis buffer pH 7.2. Cell staining for flow cytometry was performed as follows: samples were first incubated with anti-CD16/32 to block non-specific antibody interaction with FC receptor and cell staining was performed by incubating white blood cells with antibodies specific to CD45, Ly6G, CDl lb and CD115 for 30 minutes. Samples were washed twice and cell sorting was performed on a BD FACSAria™ II cell sorter. Monocytes were identified as CD45 ⁺ /CDl lb ⁺ /CD115 ⁺ /Ly6G ^" cells.

[00101] DNA was extracted from blood monocytes and amplified using SYBR® GreenER™ qPCR SuperMix Universal (Life Technologies) with the following primers to detect C/ΕΒΡβ gene (forward: 5' - GTTCCCTCGA AGCCAAACCT - 3' (SEQ ID NO: l) and reverse: 5' - TATAAACCTC CCGCTCGGC - 3')(SEQ ID NO:2). [00102] Results indicate and confirm that N0D2 activation by MDP treatment increases expression of C/ΕΒΡβ gene in blood monocytes (Fig 14C). More importantly, these results indicate that the NOD2-induced conversion of monocytes is probably regulated, in part, by the increase of C/ΕΒΡβ, a critical factor in the differentiation to Ly6C ^low cells. This result is also consistent with Ο/ΕΒΡβ'β role in NR4A1 regulation. Indeed, both C/ΕΒΡβ and NR4A1- deficient mice showed an almost complete reduction of Ly6C ^low monocytes, which is a "hallmark" phenotype of NR4A1 -deficient mice.

[00103] The foregoing disclosure of the exemplary embodiments of the present invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many variations and modifications of the embodiments described herein will be apparent to one of ordinary skill in the art in light of the above disclosure. The scope of the invention is to be defined only by the claims appended hereto, and by their equivalents.