Diagnostic and Treatment of Chronic Pathologies Such as Lyme Disease

Abstract

The present invention relates to several biomarkers, including IL-1RA, IFN-? and GM-CSF, whose expressions at the mRNA level and at the protein level, correlate with chronic pathologies, such as Lyme disease, in particular the late chronic form of Lyme disease. More specifically, the invention provides methods and kits for the diagnosis of such chronic pathologies and for the assessment of a patient's response to a treatment, as well as methods and therapeutic agents for the treatment of such chronic pathologies.

Claims

1-15. (canceled)

16. An in vitro method for diagnosing a chronic pathology in a subject, said method comprising steps of: (a) determining, in a biological sample obtained from said subject, the expression level of interleukin-1 receptor antagonist (IL-1RA); and (b) comparing the expression level of IL-1RA determined in step (a) with the expression level of IL-1RA determined in a biological sample obtained from a healthy subject or with a predetermined threshold [IL-1RA].sub.0, wherein an expression level of IL-1RA determined in step (a) that is higher than the expression level of IL-1RA determined for the healthy subject, or that is higher than the predetermined threshold [IL-1RA].sub.0, is indicative of the chronic pathology, and/or comprising steps of: (a) determining, in a biological sample obtained from said subject, the expression level of interferon-gamma (IFN-?); and (b) comparing the expression level of IFN-? determined in step (a) with the expression level of IFN-? determined in a biological sample obtained from a healthy subject or with a predetermined threshold [IFN-?].sub.0, wherein an expression level of IFN-? determiner in step (a) that is lower than the expression level of IFN-? determined for the healthy subject, or that is lower than the predetermined threshold [IFN-?].sub.0, is indicative of the chronic pathology, and/or comprising steps of: (a) determining, in a biological sample obtained from said subject, the expression level of granulocyte-macrophage colony stimulating factor (GM-CSF); and (b) comparing the expression level of GM-CSF determined in step (a) with the expression level of GM-CSF determined in a biological sample obtained from a healthy subject or with a predetermined threshold [GM-CSF].sub.0, wherein an expression level of GM-CSF determiner in step (a) that is lower than the expression level of GM-CSF determined for the healthy subject, or that is lower than the predetermined threshold [GM-CSF].sub.0, is indicative of the chronic pathology.

17. The in vitro method according to claim 16, wherein the biological sample is a blood sample.

18. The in vitro method according to claim 16, wherein determining the expression level of IL-1RA, IFN-? or GM-CSF in a biological sample includes measuring the amount or concentration of IL-1RA mRNA, IFN-? mRNA or GM-CSF mRNA in the biological sample or measuring the amount or concentration of IL-1RA protein, IFN-? protein or GM-CSF protein in the biological sample.

19. The in vitro method according to claim 16, wherein the chronic pathology is Lyme disease.

20. The in vitro method according to claim 16, wherein the chronic pathology is the late chronic form of Lyme disease.

21. The in vitro method according to claim 16, wherein the chronic pathology is selected from the group consisting of autoimmune diseases with an unknown etiology, in particular multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematous, vasculitis, Kawasaki disease, uveitis, Dressler syndrome, myasthenia gravis, scleroderma, Gougerot-Sj?gren syndrome, diabetes mellitus, celiac disease, and autoimmune thyroiditis; inflammatory diseases, in particular psoriasis, inflammatory bowel disease (Crohn's Disease and ulcerative colitis), rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, atherosclerosis, vasculitis, Horton vasculitis, and myasthenia gravis; neurodegenerative diseases, in particular Parkinson's disease, Alzheimer's disease, spinocerebellar ataxia, multisystematic atrophy, Alexander's Disease, progressive supranuclear palsy, and myofasciitis with macrophages; chronic infections caused by slow growing pathogens, in particular tuberculosis, leprosy, mycobacterial infections and Whipple disease; pervasive developmental disorders, in particular autism spectrum disorders; chronic fatigue syndrome; and fibromyalgia.

22. The in vitro method according to claim 16, wherein said method is used for diagnosing the severity of the chronic pathology, or for assessing the evolution of the chronic pathology, or for assessing the response of the subject to a treatment, or for adapting the treatment to the subject, or for monitoring the subject post-treatment, or for the early diagnosis of a relapse of the chronic pathology.

23. An in vitro method for diagnosing a chronic pathology in a subject, said method comprising steps of: (a) determining, in a biological sample obtained from said subject, the expression level of IL-1RA and the expression level of IFN-?; and (b) calculating the ratio [IL-1RA]/[IFN-?] between the expression level of IL-1RA and the expression level of IFN-? determined in step (a); and (c) comparing the ratio [IL-1RA]/[IFN-?] calculated in step (b) with the ratio [IL-1RA]/[IFN-?] determined using a biological sample obtained from a healthy subject or with a predetermined threshold ratio [IL-1RA]/[IFN-?].sub.0, wherein a ratio [IL-1RA]/[IFN-?] calculated in step (b) that is higher than the ratio [IL-1RA]/[IFN-?] determined for the healthy subject, or that is higher than the predetermined threshold ratio [IL-1RA]/[IFN-?].sub.0, is indicative of the chronic pathology; and/or comprising steps of: (a) determining, in a biological sample obtained from said subject, the expression level of IL-1RA and the expression level GM-CSF; (b) calculating the ratio [IL-1RA]/[GM-CSF] between the expression level of IL-1RA and the expression level of GM-CSF determined in step (a); and (c) comparing the ratio [IL-1RA]/[GM-CSF] calculated in step (b) with the ratio [IL-1RA]/[GM-CSF] determined using a biological sample obtained from a healthy subject or with a predetermined threshold [IL-1RA]/[GM-CSF].sub.0, wherein a ratio [IL-1RA]/[GM-CSF] calculated in step (b) that is higher than the ratio [IL-1RA]/[GM-CSF] determined for a healthy subject, or that is higher than the predetermined threshold ratio [IL-1RA]/[GM-CSF].sub.0, is indicative of the chronic pathology.

24. The in vitro method according to claim 23, wherein the biological sample is a blood sample.

25. The in vitro method according to claim 23, wherein determining the expression level of IL-1RA, IFN-? or GM-CSF in a biological sample includes measuring the amount or concentration of IL-1RA mRNA, IFN-? mRNA or GM-CSF mRNA in the biological sample or measuring the amount or concentration of IL-1RA protein, IFN-? protein or GM-CSF protein in the biological sample.

26. The in vitro method according to claim 23, wherein the chronic pathology is Lyme disease.

27. The in vitro method according to claim 23, wherein the chronic pathology is the late chronic form of Lyme disease.

28. The in vitro method according to claim 23, wherein the chronic pathology is selected from the group consisting of autoimmune diseases with an unknown etiology, in particular multiple sclerosis, amyotrophic lateral sclerosis, systemic lupus erythematous, vasculitis, Kawasaki disease, uveitis, Dressler syndrome, myasthenia gravis, scleroderma, Gougerot-Sj?gren syndrome, diabetes mellitus, celiac disease, and autoimmune thyroiditis; inflammatory diseases, in particular psoriasis, inflammatory bowel disease (Crohn's Disease and ulcerative colitis), rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, atherosclerosis, vasculitis, Horton vasculitis, and myasthenia gravis; neurodegenerative diseases, in particular Parkinson's disease, Alzheimer's disease, spinocerebellar ataxia, multisystematic atrophy, Alexander's Disease, progressive supranuclear palsy, and myofasciitis with macrophages; chronic infections caused by slow growing pathogens, in particular tuberculosis, leprosy, mycobacterial infections and Whipple disease; pervasive developmental disorders, in particular autism spectrum disorders; chronic fatigue syndrome; and fibromyalgia.

29. The in vitro method according to claim 23, wherein said method is used for diagnosing the severity of the chronic pathology, or for assessing the evolution of the chronic pathology, or for assessing the response of the subject to a treatment, or for adapting the treatment to the subject, or for monitoring the subject post-treatment, or for the early diagnosis of a relapse of the chronic pathology.

30. A kit for the in vitro diagnosis of a chronic disease, said kit comprising a reagent that specifically reacts with IL-1RA mRNA or protein, or with IFN-? mRNA or protein, or with GM-CSF mRNA or protein.

31. The kit according to claim 30, wherein the reagent is selected from the group consisting of oligonucleotide probes that specifically hybridize to IL-1RA, IFN-? and GM-CSF mRNA transcripts, oligonucleotide primers that specifically amplify IL-1RA, IFN-? and GM-CSF mRNA transcripts, antibodies that specifically recognize and bind the IL-1RA, IFN-? and GM-CSF proteins or a fragment thereof, protein-binding peptides that specifically bind to the IL-1RA, IFN-? and GM-CSF proteins or a fragment thereof.

32. The kit according to claim 30, wherein said reagent is labeled with a detectable moiety.

33. A method for treating a chronic pathology in a subject comprising a step of administering, to the subject, a therapeutically effective amount of a therapeutic agent or a pharmaceutical composition thereof, wherein said therapeutic agent is selected from the group consisting of IL-1RA therapeutic agents, TLR10 therapeutic agents, GM-CSF, INF-? and IL-2.

34. The method according to claim 33, wherein IL-1RA therapeutic agents include IL-1, IL-1, anti-IL-1RA antibodies, and anti-IL-1RA interfering agents, and wherein TLR10 therapeutic agents include anti-TLR10 antibodies.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0158] FIG. 1. Differential expression of (A) IL-1RA, (B) IFN? and (C) GM-CSF in chronic Lyme disease patients, reflecting active immunosuppression. Peripheral blood leukocytes (PBLs) were seeded in tissue culture plates. For T lymphocyte activation. 10.sup.6 PBLs/ml were cultured in RPMI1640+10% fetal calf serum supplemented with 5 ?g/ml of anti-CD28 antibody (clone CD28.2) and 10 ?g/ml of anti-CD3 (clone OKT3) agonist antibodies, in 24-well plates. For plastic monocyte activation, cells were grown similarly. but in the absence of anti-CD3 and CD28 antibodies to avoid T cell activation. After a 24 hour-culture period, culture supernatants were harvested and their IL-1RA. IFN? and GM-CSF contains were determined by using ELISA kits.

[0159] FIG. 2. (A) Screen shot of the very first determination of Cold Disease Index 1. Rough data obtained from IL-1Ra. IFN-? ELISA determination and expressed in pg/ml. The white lines correspond to the values obtained for the healthy controls, and the light and dark grey lines are from to Borrelia seropositive and seronegative patients. respectively. The three black lines corresponding to patients previously excluded because of poly-pathologies. The last column on the right presents the [IL-1RA]/[IFN?] ratio (i.e., Cold Disease Index 1), with a p-Value=0.016. (B) Dot plot representation of Cold Disease Index 1. [IL-1RA]/[IFN?] in healthy persons is compared to [IL-1RA]/[IFN?] in patients suffering from chronic Lyme disease, with a p-Value=0.016.

[0160] FIG. 3. Dot plot representation of Cold Disease Index 2. The ratio [IL-1RA]/[GM-CSF] for by PBLs cultured for 24 hours as described in the caption of FIG. 1 was determined and compared to ratio [IL-1RA]/[GM-CSF] measured in Lyme seronegative patients. Lyme seropositive patients and the group of combined patients. P value between healthy donors and total Lyme patients is p=0.008.

[0161] FIG. 4. Dot plot representation and comparison of Cold Disease Index 1 and Cold Disease Index 2. The values of [IL-1RA]/([IFN?] (Cold Disease Index 1) and of [IL-1RA]/[GM-CSF] (Cold Disease Index 2) measured for Lyme patients and for healthy controls were plotted on vertical and horizontal axes, respectively, and compared.

[0162] FIG. 5. IL-1RA secretion (A), but not IL-1? (B), is specifically over-induced in patients PBLs activated through the Toll-like receptors recognizing the bacteria. Many reports show that Borrelia is recognized the TLR1/TLR2 or TLR2/TLR6 pattern of recognition expressed on surface of monocytes and dendritic cells. Pam3CSK4 and FSL1 molecules can recruit these pathways, respectively, to mimic Borrelia action in these cells. LPS, a TLR4 agonist was used as a sham control. 10.sup.6 PBLs/ml from patients or from healthy donors were cultured in RPMI+10% FCS supplemented, or not, with the TLRs agonists for a 24-hour incubation period. IL-1RA and IL-1? concentrations were determined by using ELISAs kits from R&D Systems. The results obtained show that the recruitment of TLR1/TLR2 or TLR2/TLR6 induced secretion of tremendous amounts of IL-1RA in patient cells. This was not observed in healthy controls. In addition, IL-1?, a closely IL-1RA related gene, both structurally and by its genomic location, displayed a conventional behaviour in response to the used stimuli.

EXAMPLES

[0163] The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data were actually obtained.

Materials and Methods

[0164] . Blood samples were collected from healthy adult donors and late disseminated form Lyme disease suffering adult patients. All samples were obtained after informed consent forms were signed, according to the French rules. Patients were selected based on, at least, the following criteria: polyarthralgias migrating from one joint to another, chronic desocializing tiredness, muscular or osteo-muscular pain, loss of cerebral concentration and anterograde memory problems. Some of the patients also displayed more severe problems such as motor impairments. Half of the patient population was selected based on a positive serology for Borrelia, the other half was seronegative (see Table 1). Peripheral blood leukocytes (PBLs) were isolated by Ficoll-Paque PLUS (GE Healthcare) density centrifugation. Interface cells were harvested, washed twice in RPMI 1640+10% foetal calf serum before being frozen in the presence of a final concentration of 10% DMSO at ?80? C., and then in liquid nitrogen. Serum samples were also preserved, and PBL snap frozen pellets were prepared for DNA analyses. During the time course of the project, the company DiagNucleis Plateforme de diagnostic (http://diagnucleis.fr/) brought the possibility of looking at the blood level for the presence of different forms of Borrelia, bacteria co-infecting the Lyme patients, as well as the parasite Babesia, by using ultra sensitive PCRs. Blood samples sent to this company were analysed and completed the serological results, showing that at least 27 of the 28 studied patients had a pathology associated with Lyme disease (see Table 2). Nevertheless, due to poly-pathologies, three of the patients were excluded from the study.

[0165] Gene Expression and Transcriptome Analysis. Total RNA was extracted from PBLs using an RNeasy Micro kit (Qiagen, Valencia, CA) according to the manufacturer's information. One microgram of total RNA was reversed transcribed into cDNA using random hexamer primers and the SuperScript II reverse transcriptase (Invitrogen). Samples were further analyzed using the PrimeView Human Genome U219 Array technology from Affymetrix/Thermo FisherScientific (Waltham, MA), following the manufacturer protocol. This allowed measurement of gene expression of about 36,000 transcripts and variants in each sample. Plastic activated mononuclear cells and activated T cells were studied both in Lyme patients and healthy donors, and the obtained gene expression profiles were compared by computed analyses.

[0166] Tissue Culture and Cell Activation. For T lymphocyte activation, 10.sup.6 PBLs/ml were cultured in RPMI1640 +10% foetal calf serum supplemented with 5 ?g /ml of anti-CD28 antibody (clone CD28.2) and 10 ?g/ml of anti-CD3 (clone OKT3) agonist antibodies, in 24-well plates.

[0167] For plastic monocyte activation, cells were grown similarly, but in the absence of anti-CD3 and CD28 antibodies avoiding any T cell activation. After a 24 hour-culture period at 37? C., culture supernatants were recovered and stored at ?80? C.up until protein determination. For RNA expression analyses, the tissue culture period of samples was shortened to 6 hours, and cells were snap frozen.

[0168] ELISA determination, and reagents. Concentrations of IL-1Ra, IFN?, GM-CSF and IL-1? present in culture supernatants were determined by using ELISA kits commercialized by R&D Systems (Abingdon, UK), following the manufacturer recommendations. Lipopolysaccharide (LPS) a TLR4 agonist was obtained from Sigma (Saint-Louis, MO). FSL-1, a TLR2/TLR6 agonist and Pam3CSK4, a TLR1/TLR2 agonist, were both obtained from Invivogen (San Diego, CA).

TABLE-US-00001 TABLE 1 Samples from Patients and Healthy Individuals. Date of Sample Birth Date Sample Name Day/month/year Gender C.E. Collection ELISA W. BLOT AUTRES Lym001 9 May 1983 F yes 12 Mar. 2016 ? ? EXCLU poly p. Lym002 27 Sep. 1982 F yes 12 Mar. 2016 ? p41 +/? DNA arrays Lym003 6 Feb. 1962 M yes 12 Mar. 2016 ? + Lym004 15 May 1962 M yes 12 Mar. 2016 + + Lym005 24 Nov. 1974 F yes 12 Mar. 2016 + + Lym006 5 Apr. 1990 M yes 12 Mar. 2016 ? P41 +/? DNA arrays Lym007 30 Apr. 1984 F yes 12 Mar. 2016 ? P41 +/? P39+/? Lym008 7 Apr. 1973 F yes 12 Mar. 2016 + + Lym009 28 Jul. 1982 F yes 2 Apr. 2016 ? P41 +/? EXCLU poly p. Lym010 24 Feb. 1960 F yes 2 Apr. 2016 ? + Lym011 31 May 1967 F yes 2 Apr. 2016 + + Lym012 1 Jan. 1977 F yes 2 Apr. 2016 ? ? Lym013 13 Dec. 1986 F yes 2 Apr. 2016 + + Lym014 1 Mar. 1983 M yes 2 Apr. 2016 + + Lym015 1 Feb. 1971 M yes 2 Apr. 2016 + + Lym016 21 Sep. 1985 F yes 2 Apr. 2016 ? ? EXCLU poly p. Lym017 13 Oct. 1951 F yes 2 Apr. 2016 ? p41+/? DNA arrays Lym018 3 May 1967 F yes 2 Apr. 2016 ? p41 +/? DNA arrays Lym019 7 Nov. 1986 M yes 2 Apr. 2016 ? ? Lym020 25 Oct. 1970 F yes 2 Apr. 2016 + + Lym021 3 Mar. 1984 F yes 2 Apr. 2016 ? ? Lym022 8 Feb. 1972 F yes 2 Apr. 2016 + + Lym023 23 Apr. 1993 F yes 14 May 2016 + + Lym024 24 Mar. 1966 F yes 14 May 2016 + + Lym025 21 Jan. 1981 F yes 14 May 2016 + + Lym026 14 May 1961 F yes 14 May 2016 + + Lym027 3 Oct. 1972 F yes 14 May 2016 + + Lym028 24 Jan. 1980 M yes 14 May 2016 + + T1 18 Sep. 1991 M yes ? ND DNA arrays T2 15 Jun. 1982 F yes ? ND DNA arrays T3 26 May 1991 M yes ? ND DNA arrays T4 10 Apr. 1991 M yes ? ND DNA arrays T5 24 Feb. 1984 F yes ? ND T6 10 Apr. 1992 M yes ? ND T7 26 Mar. 1988 F yes ? ND T8 5 Jul. 1990 M yes ? ND T9 7 Feb. 1989 F yes ? ND T10 24 Oct. 1991 F yes ? ND T11 20 Sep. 1989 F yes ? ND T12 30 Aug. 1990 F yes ? ND N.D.: Not determined. +: positif. ?: negative.

TABLE-US-00002 TABLE 2 qPRC Analysis of Blood Samples in Patients with Chronic Lyme Disease Borrelia Sample senso Borrelia Borrelia name lato Bartonella Anaplasma Babesia ELISA W. B. Lym001 + N.D. N.D. N.D. ? ? Lym002 + N.D. N.D. N.D. ? p41 +/? Lym003 + N.D. N.D. N.D. ? + Lym004 N.D. N.D. N.D. 6.3E+4 + + Lym005 + N.D. N.D. 6.2E+4 + + Lym006 N.D. N.D. N.D. N.D. ? P41 +/? Lym007 + N.D. + 5.4E+4 ? P41+/? P39+/? Lym008 + N.D. N.D. N.D. + + Lym009 N.D. N.D. N.D. 2.5E+4 ? P41 +/? Lym010 N.D. N.D. + 5.1E+4 ? + Lym011 + 10E+5 N.D. N.D. + + Lym012 N.D. N.D. N.D. 6.2E+4 ? ? Lym013 + N.D. N.D. 6.1E+4. + + Lym014 + N.D. N.D. 5.4E+4 + + Lym015 N.D. N.D. N.D. 4.3E+4 + + Lym016 + N.D. + 5.5E+4 ? ? Lym017 + N.D. N.D. N.D. ? p41+/? Lym018 N.D. N.D. N.D. 3.2E+4 ? p41 +/? Lym019 N.D. N.D. N.D. 6.5E+4 ? ? Lym020 N.D. N.D. N.D. 5.3E+4 + + Lym021 N.D. N.D. N.D. N.D. ? ? Lym022 N.D. N.D. N.D. 3.9E+4 + + Lym023 N.D. N.D. N.D. N.D. + + Lym024 N.D. N.D. N.D. N.D. + + Lym025 N.D. N.D. N.D. N.D. + + Lym026 N.D. N.D. N.D. N.D. + + Lym027 N.D. N.D. N.D. 2.9E+5. + + Lym028 N.D. N.D. N.D. 5.8E+4 + + N.D.: Not determined. +: positif. ?: negative.

Results

[0169] The present Inventors have analyzed the functional expression of the genome of circulating leukocytes in patients exhibiting a clinical picture characteristic of the late chronic phase of Lyme disease. This allowed them to demonstrate a major defect in the patients' response to the pathogen, which results in severe immunosuppression in the patients' tissue inflammatory response.

Transcriptome Analysis

[0170] Following isolation, mRNAs were hybridized to DNA chips containing more than 50,000 different cDNA targets, thus covering the entire genome and a large majority of the mRNA splicing. The Inventors have analyzed, on the one hand: the functioning of monocytes/macrophages/dendritic cells, and on the other hand: the functioning of T lymphocytes, after stimulating them by contact with plastic for 6 hours, or by recruitment of the CD3/CD28 pathways, respectively. The goal of the stimulation step is to amplify the level of functioning of genes already active in the patient.

[0171] This allowed the identification of genes that are over- or under-expressed in the patients compared to healthy subjects (data not shown). The genes that were found to be upregulatede are IL-1RA (interleurkin-1 antagonist receptor), SSP1 (secreted phosphoprotein 1 or osteopontin), APOBEC3B (apolipoprotein B mRNA editing enzyme catalytic subunit 3B), TNFAIP6 (tumor necrosis factor-inducible gene 6 product), CCL20 (C-C motif chemokine 20), IL-6 (interleurkin-6), Oncostatin M (OSM), C3 (complement component 3), and RGS16 (regulator of G-protein signaling). The genes that were found to be downregulated are IFN? (interferon gamma), GM-SCF (granulocyte-macrophage colony-stimulating factor), AFF3 (AF4/FMR2 family member 1), 20 alpha (3alpha)-HSD (AKR1C1), CD163 (cluster differentiation 163), IL-13 (interleukin-13), CCL8 (chemokine C-C motif ligand 8), IL-22 (interleukin 22), IL-17F (interleukin 17F), IL-17A (interleukin-17A), and IL-3 (interleukin-3).

[0172] The identified genes are often immunosuppressive genes, in particular in monocytes/macrophages/dentritic cells, and whose expression level displays a 5-fold or 10-fold increase compared to healthy subjects. In the analysis of the results, the present Inventors focused on identified genes whose variation was found to be homogeneous for each group and in all the cell populations studied.

[0173] In chronic Lyme disease patients, the myelo/monocytic/dendritic lineage was found to overexpress, in particular, the interleukin-1 receptor antagonist (IL-1RA), osteopontin (SSP1) or TNF-stimulated gene 6, each of which has different degrees of immunomodulation capabilities. Similarly, the chemokine CCL20/MIP3?, which is involved in immune responses of the inflammatory tissue type, referred to as Th17, was also found to be overexpressed. On the other hand, apart from a rather homogeneous small subexpression of the CD163 and ANFF3 molecules, the Inventors have not noticed a significant down-regulation of genes at the monocytic level in chronic Lyme disease patients compared to healthy individuals.

[0174] As far as T lymphocytes (activated through the CD3/CD28 pathways), the results obtained are practically symmetrical. Namely, the Inventors have not detected genes whose expressions by T cells are homogeneously and specifically increased in chronic Lyme disease patients compared to healthy controls. On the other hand, a strong repression of gene expression was observed in a series of 4 T-response genes, in particular IL-17A, IL-17F and IL-22, which are specific markers of tissue inflammatory lymphocytic immune response (Th17). This means that in chronic Lyme disease patients, unlike healthy subjects, the Th17 response that occurs at the tissue level, particularly in the anti-bacterial response, is tremendously inhibited. Along with IL-17A, IL-17F and IL-22, another T-cell marker, interleukin-3, involved in emergency recruitement and myeloid cell maturation, was also found to be deeply repressed.

[0175] Once identified by DNA chips, some markers, including IL-1RA, osteopontin, CCL20/MIP3?, were reanalyzed using quantitative PCR on all available biological samples (from 28 chronic Lyme disease patients and as many as healthy subjects) in order to confirm the homogeneity of the first experimental results obtaine. The same information was acquired by PCR analysis. Furthermore, there were no differences in the gene expressions measured for the seropositive patients and for the seronegative patients (this was also the case at the protein level). This means that the presence of antibodies is not an essential element of the immune response in the patients suffering from the late chronic form of Lyme disease.

Proteome Analysis

[0176] After having identified a number of transcripts, including cytokines related to the immune response, the present Inventors have measured the concentrations of the corresponding proteins. For this purpose, the leucocytes were activated by contact on plastic and the T lymphocytes were recruited by the CD3/CD28 pathways for 24 hours of culture at 37?C. The supernatants of these cultures were then recovered and the concentration of the different soluble cytokines or molecules was determined by ELISA.

[0177] Preliminary experiments have focused on dosing IL-1RA, osteopontin, CCL20/MIP3? on the one hand, and IL-3 and IL-17A on the other hand. This list was supplementred with a few other cytokines known to be involved in the Th17 (and/or Th1) response, in particular interferon gamma (IFN-?), and GM-CSF, two of the genes that are essential to the Th1 and Th17 responses). In addition, IFN-? is known for its mRNA complex regulation, which can lead to a short half-life of its RNAs under certain circumstances, decoupling the mRNA measurement from the corresponding protein concentrations.

[0178] The present Inventors have also considered GM-CSF (which is structurally similar to IL-3, whose expression was observed to decrease by almost 2-fold in chronic Lyme disease patients compared to healthy subjects), as well as the IL-36 inhibitor (which is structurally or functionally close to IL-1RA). The data obtained at the protein level on all patients corroborate those obtained at the mRNA level, with the exception of IL-17A, where a net decrease was not observed in patients at the protein level (at least in the preliminary experiments presented here). Although the IL-36 receptor assay did not provide any new data: in contrast, the levels of GM-CSF and even more of IFN-? were found to be significantly decreased in chronic Lyme disease patients compared to healthy subjects. In the myelocytic monocyte line, a very high increase of the amount of IL-1RA was observed in patients compared to healthy individuals (see FIG. 1).

Cold Disease Indexes

[0179] The results obtained led the Inventors to define two indexes, which were called Cold Disease Index 1 and Cold Disease Index 2. The term Cold Disease has been used in reference to the French term infection froide (cold infection in English), which encompasses dormant, asymptomatic or latent infections, which are capable of manifesting symptoms under particular circumstances or if activated.

[0180] Cold Disease Index 1 (or CD1) corresponds to [IL-1RA]/[IFN-?], the ratio between the expression level of IL-1RA and the expression level of IFN-?, and Cold Disease Index 2 (or CD2) corresponds to [IL-1RA]/[GM-CSF], the ratio between the expression level of IL-1RA and the expression level of GM-CSF.

[0181] If the CDI is set to 1 in healthy individuals, the ratio [IL-1RA]/[IFN-?] was found to reach values between 100 and 1000 in more than 95% of the chronic Lyme disease patients tested, and the value of CD1 was found to increase as the severity of the disease increases (with a confidence interval of p =0.01) (see FIG. 2).

[0182] If the CD2 is set to 1 in healthy individuals, the ratio [IL-1RA]/[GM-CSF] was found to be vary from 1 to 10 in chronic Lyme disease patients with a confidence of p=0.008 (see FIG. 3).

[0183] A two-dimensional representation, with CD1 presented on the y-axis and CD2 presented on the x-axis, leads to the alignement of the values, which define a straight line (see FIG. 4). This demonstrates that the parameters are indeed related to each other in the immune response in the late chronic form of Lyme disease, further reinforcing the weight of the observations made.

[0184] To the Inventors' knowledge, CD1 and CD2 are the first evidence of a solid, quantifiable proof of a biological defect in the late chronic form of Lyme disease.

IL-1RA Biology in the Context of Lyme Disease

[0185] Among the three cytokines assayed, IL-1RA, IFN-? and GM-CSF, the increase in IL-1RA expression was found to be the most dramatic, and could not have been expected. It should be emphasized that IL-1RA is a potent antagonist of both forms of IL-1, the alpha and beta forms, and contributes to the strong inhibition of the immune response at the B-lymphocyte level (hampering its ability to produce antibodies, and also decreasing part of the synthesis of the so-called acute phase proteins synthesized by the liver during aggression by an organism, which contribute to the innate defense response). This is, in particular, the case of the C-Reaction Protein (CRP), a front-line marker of inflammation in routine analyses, which is very often absent ou present at very low levels in patients with the late chronic form of Lyme disease. Moreover, it has been shown that a synthetic form of IL-1RA (called Anakinra), used as therapeutics in a context that is totally different from Lyme disease, actually leads to a reduction in the synthesis of CRP in man. In humans, IL-1 is also a cytokine that, in combination with IL-23, is essential for polyrization of CD4+ cells toward so-called Th17 cells, which are characteristics of lymphoid tissue inflammation. The role of the Th17, then at the sites, is to eliminate bacteria infiltrating the tissues. The transcriptome analysis has identified several specific markers of the presence of recirculating Th17 in patients. On the other hand, their functionality remains strongly altered, and the expression of their main functional genes (IL-17A, Il-17F and IL-22) is strongly repressed in chronic Lyme disease patients compared to healthy individuals. This probably contributes to explain the inability of said patients to achieve an effective clearance of the pathogen at the tissue level. In addition, IL-1RA neutralized the pyrogenic properties of both IL-1? and IL-1?, which were the main endogenous fever inducers in human: and patients suffering of chronic Lyme disease usually do not have fever, and often even display a small hypothermia.

[0186] In rodent models of inactivation, or overactivation, of IL-1RA in the central nervous system, it has been established that this molecule also plays a major role in the acquisition of short memorya type of memory that is highly impaired in chronic Lyme disease patients and which is, along with desocializing tiredness, and multifocal arthralgias, one of the main symptoms of the late form of the disease.

TLRs and Borrelia

[0187] Borrelia is known to enter the body by sensors called Toll-like Receptors (TLRs) and located on the surface of monocytes, macrophages or dendritic cells (and sometimes on certain populations of T lymphocytes). TLRs are the first line of defense against pathogens, which are identified as exogenous on the basis of their electrical charges, their hydrophobicity, the presence of atypical sugars, or specific traits of the pathogenic nuleic acids (mono-stranded RNAs, bacterial DNA methylation, etc . . . ). If most TLRs are expressed at the surface of the cells, some are also present in the endosome, associated with NOD family molecules, and contribute to clearance of virus and intracellular bacteria (mycobacteria, etc . . . ).

[0188] At the membrane level, TLRs can be paired to increase their recognition capabilities. Thus, the Borrelia bacterium is recognized by the TLR1/TLR2 and TLR2/TLR6 pairs. The bacterium attachment to the TLRs strengthens the pairing, which then transmits an intracellular signal initiating a signaling cascade, which ultimately leads to the expression of the genes that are necessary to induce an efficient immune response. The recruitment of TLR genes by bacteria generally leads to abundant synthesis of IL-1?, IL-6 and IL-8, for example, but also, and sometimes more moderately, of IL-1RA. The latter molecule (as well as IL-10 or TGF-?) is then responsible for lowering the immune response and for a return to homeostasis once the antigen has been eliminated.

[0189] Besides Borrelia, other bacteria, such as mycobacteria, for example, recruit TLR1/TLR2 and TLR2/TLR6. On the other hand, TLR4, another important sensor of bacteria expressing lypopolysaccharides (LPS), absent in Borrelia, is not recognized by Borrelia. The present Inventors have sought to dissect the activation of TLRs by Borrelia, in order to determine if these pathways were indeed responsible for IL-1RA hypersecretion. In order to distinguish the response of the different TLRs, they have taken advantage of the fact that certain chemical molecules are known to specifically bind and activate one of the TLRs. Thus, for example, TLR4 is activated by LPS (used as control), TLR1/TLR2 is activated by the chemical agent: Pam3CSK4, and TLR2/TLR6 is activated by FSL-1.

[0190] These three molecules were added, for 24 hours, to mononuclear blood cell cultures from patients with the late chronic form of Lyme disease (n=6) and from healthy subjects (n=6), and the supernatants were assayed for IL-1RA concentrations. The results obtained are presented in FIG. 5.

[0191] The first observation is that IL-1RA is strongly overexpress by the cells of chronic Lyme disease patients compared to the cells of healthy individuals. Regardless of the nature of the agent stimulating the TLRs of the patients, the overexpression reaches very inhabitual concentrations of 50-70 ng/ml, compared to 10-15 ng/ml for the controls (see FIG. 5(A)).

[0192] The deregulation of the IL-1RA gene was then analyzed with respect to the IL-1? agonist, which often exhibits a joint regulation with IL-1RA. The pathway used as a control, which combines LPS and TLR4, led to an increased expression of IL-1? secretion (about 10 ng/ml), identical for patients and healthy controls. On the other hand, activation of the TLR1/TLR2 and TLR2/TLR6 pathways recruited by Borrelia, did not lead to a significant increase in IL-1?, which does not exceed ?1 ng/ml. This result is important since it means that, in chronic Lyme disease patients, the quantities of antagonist (IL-1RA) produced is 50 to 70 times greater than the quantities of IL-1?. This contributes to better explain the deficiency observed in the immune response of patients, and a fortiori with a shart decline in IFN-? production, one of the pillars of the antibacterial response.