NKT Cell-Dependent Amelioration of a Mouse Model of Multiple Sclerosis by Altering Gut Flora
2008; Elsevier BV; Volume: 173; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.080622
ISSN1525-2191
AutoresHiroaki Yokote, Sachiko Miyake, J. Ludovic Croxford, Shinji Oki, Hidehiro Mizusawa, Takashi Yamamura,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoImproved hygiene has been suggested to influence certain autoimmune disorders, such as multiple sclerosis. In this study, we addressed whether altering the composition of gut flora may affect susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We administered a mixture of non-absorbing antibiotics, kanamycin, colistin, and vancomycin (KCV), orally to mice induced to develop EAE. The antibiotic treatment, beginning 1 week prior to sensitization, altered the composition of gut flora and, intriguingly, also ameliorated the development of EAE. While this result was associated with a reduced production of pro-inflammatory cytokines from the draining lymph node cells, a reduction of mesenteric Th17 cells was found to correlate with disease suppression. In addition, we found that Vα14 invariant NKT (iNKT) cells were necessary for maintaining the mesenteric Th17 cells. The homologous effects of KCV treatment and iNKT cell depletion led us to speculate that KCV treatment may suppress EAE by altering the function of iNKT cells. Consistent with this hypothesis, KCV treatment did not suppress EAE that was induced in iNKT cell-deficient mice, although it was efficacious in mice that lacked Vα19 mucosal-associated invariant T cells. Thus, gut flora may influence the development of EAE in a way that is dependent on iNKT cells, which has significant implications for the prevention and treatment of autoimmune diseases. Improved hygiene has been suggested to influence certain autoimmune disorders, such as multiple sclerosis. In this study, we addressed whether altering the composition of gut flora may affect susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We administered a mixture of non-absorbing antibiotics, kanamycin, colistin, and vancomycin (KCV), orally to mice induced to develop EAE. The antibiotic treatment, beginning 1 week prior to sensitization, altered the composition of gut flora and, intriguingly, also ameliorated the development of EAE. While this result was associated with a reduced production of pro-inflammatory cytokines from the draining lymph node cells, a reduction of mesenteric Th17 cells was found to correlate with disease suppression. In addition, we found that Vα14 invariant NKT (iNKT) cells were necessary for maintaining the mesenteric Th17 cells. The homologous effects of KCV treatment and iNKT cell depletion led us to speculate that KCV treatment may suppress EAE by altering the function of iNKT cells. Consistent with this hypothesis, KCV treatment did not suppress EAE that was induced in iNKT cell-deficient mice, although it was efficacious in mice that lacked Vα19 mucosal-associated invariant T cells. Thus, gut flora may influence the development of EAE in a way that is dependent on iNKT cells, which has significant implications for the prevention and treatment of autoimmune diseases. The immunopathology of autoimmune diseases is still poorly understood, although comprehensive and multidisciplinary approaches continue to give us new insight into the mechanisms of disease. Previous studies have generally supported a pathogenic role of interferon (IFN)γ-producing Th1 cells in autoimmune diseases such as multiple sclerosis (MS) that affect the central nervous system (CNS).1Steinman L Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system.Cell. 1996; 85: 299-302Abstract Full Text Full Text PDF PubMed Scopus (796) Google Scholar As Th1 cells are cross-regulated by Th2 cells producing interleukin (IL)-4, IL-5, and IL-13, the counterbalance between Th1 and Th2 cells has been posed as a key issue in understanding the pathogenesis of MS.2Coffman RL Origins of the TH1-TH2 model: a personal perspective.Nat Immunol. 2006; 7: 539-541Crossref PubMed Scopus (156) Google Scholar However, the traditional “Th1/Th2” paradigm is now facing a fundamental challenge since a third class of helper CD4+ T cells, named Th17 cells, have been found to cause autoimmune inflammation.3Langrish CL Chen Y Blumenschein WM Mattson J Basham B Sedgwick JD McClanahan T Kastelein RA Cua DJ IL-23 drives a pathogenic T cell population that induces autoimmune inflammation.J Exp Med. 2005; 201: 233-240Crossref PubMed Scopus (3280) Google Scholar, 4Harrington LE Hatton RD Mangan PR Turner H Murphy TL Murphy KM Weaver CT Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages.Nat Immunol. 2005; 6: 1123-1132Crossref PubMed Scopus (3850) Google Scholar, 5Park H Li Z Yang XO Chang SH Nurieva R Wang YH Wang Y Hood L Zhu Z Tian Q Dong C A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17.Nat Immunol. 2005; 6: 1133-1141Crossref PubMed Scopus (3494) Google Scholar Th17 cells are IL-23-dependent cells that are distinct from Th1 and Th2 cells in their ability to produce IL-176Steinman L A brief history of TH17, the first major revision in the TH1/TH2 hypothesis of T cell-mediated tissue damage.Nat Med. 2007; 13: 139-145Crossref PubMed Scopus (1123) Google Scholar, 7Bettelli E Oukka M Kuchroo VK TH-17 cells in the circle of immunity and autoimmunity.Nat Immunol. 2007; 8: 345-350Crossref PubMed Scopus (1279) Google Scholar, 8Yamamura T Interleukin 17-producing T-helper cells and autoimmune diseases: time for a paradigm shift?.Curr Rheumatol Rep. 2007; 9: 93-95Crossref PubMed Scopus (3) Google Scholar and their use of the RORγt transcription factor.9Ivanov II McKenzie BS Zhou L Tadokoro CE Lepelley A Lafaille JJ Cua DJ Littman DR The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (4022) Google Scholar Although the relationship between Th17 cells and Th1 or Th2 cells remains to be fully characterized, Th17 cells are likely to exert a predominant pathogenic activity in various inflammatory conditions associated with autoimmunity or allergy either independently or collaboratively with Th1 cells.10Kebir H Kreymborg K Ifergan I Dodelet-Devillers A Cayrol R Bernard M Giuliani F Arbour N Becher B Prat A Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation.Nat Med. 2007; 13: 1173-1175Crossref PubMed Scopus (1242) Google Scholar It is widely accepted that development of autoimmune disease is under control of both genetic and environmental factors. For example, recent whole genome analysis has revealed that several genes including human leukocyte antigen-DR are positively linked with the susceptibility to MS.11Hafler DA Compston A Sawcer S Lander ES Daly MJ De Jager PL de Bakker PI Gabriel SB Mirel DB Ivinson AJ Pericak-Vance MA Gregory SG Rioux JD McCauley JL Haines JL Barcellos LF Cree B Oksenberg JR Hauser SL Risk alleles for multiple sclerosis identified by a genomewide study.N Engl J Med. 2007; 357: 851-862Crossref PubMed Scopus (1422) Google Scholar In contrast, most of our knowledge about environmental factors relies on epidemiological data. Results of migration studies, as well as the reported presence of clusters or outbreaks of MS, have illustrated potential environmental influences on MS, including infection, stress, sunlight exposure, and sex hormone.12Marrie RA Environmental risk factors in multiple sclerosis aetiology.Lancet Neurol. 2004; 3: 709-718Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar, 13Ascherio A Munger KL Environmental risk factors for multiple sclerosis. Part I: the role of infection.Ann Neurol. 2007; 61: 288-299Crossref PubMed Scopus (787) Google Scholar, 14Ascherio A Munger KL Environmental risk factors for multiple sclerosis. Part II: noninfectious factors.Ann Neurol. 2007; 61: 504-513Crossref PubMed Scopus (548) Google Scholar While an altered intestinal microflora has been suggested to be an environmental risk factor for rheumatoid arthritis,15Nieuwenhuis EE Visser MR Kavelaars A Cobelens PM Fleer A Harmsen W Verhoef J Akkermans LM Heijnen CJ Oral antibiotics as a novel therapy for arthritis: evidence for a beneficial effect of intestinal Escherichia coli.Arthritis Rheum. 2000; 43: 2583-2589Crossref PubMed Scopus (25) Google Scholar inflammatory bowel disease,16Danese S Sans M Fiocchi C Inflammatory bowel disease: the role of environmental factors.Autoimmun Rev. 2004; 3: 394-400Crossref PubMed Scopus (310) Google Scholar and human allergy and asthma,17Umetsu DT McIntire JJ Akbari O Macaubas C DeKruyff RH Asthma: an epidemic of dysregulated immunity.Nat Immunol. 2002; 3: 715-720Crossref PubMed Scopus (539) Google Scholar the status of gut flora has rarely been evaluated as a potential risk factor for MS. Recent studies have shown that animals bred in a germfree environment are characterized by having low densities of lymphoid cells in the gut mucosa, a reduced size of specialized follicle structures, and low concentrations of immunoglobulins in the peripheral blood.18Falk PG Hooper LV Midtvedt T Gordon JI Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.Microbiol Mol Biol Rev. 1998; 62: 1157-1170Crossref PubMed Google Scholar, 19Butler JE Sun J Weber P Navarro P Francis D Antibody repertoire development in fetal and newborn piglets. III Colonization of the gastrointestinal tract selectively diversifies the preimmune repertoire in mucosal lymphoid tissues.Immunology. 2000; 100: 119-130Crossref PubMed Scopus (103) Google Scholar, 20Tannock GW Molecular assessment of intestinal microflora.Am J Clin Nutr. 2001; 73: 410S-414SPubMed Scopus (159) Google Scholar, 21Mazmanian SK Liu CH Tzianabos AO Kasper DL An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system.Cell. 2005; 122: 107-118Abstract Full Text Full Text PDF PubMed Scopus (2064) Google Scholar It is also of note that the intestinal lamina propria (LP) has been identified as a site that is constitutively inhabited by Th17 cells.9Ivanov II McKenzie BS Zhou L Tadokoro CE Lepelley A Lafaille JJ Cua DJ Littman DR The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (4022) Google Scholar Thus the dialogue between host and bacteria at the mucosal interface seems to be critical in the development of the competent immune system. To explore a possible role of intestinal microflora in the development of autoimmune disease, we tested if oral administration of the mixture of non-absorbing antibiotics kanamycin, colistin, and vancomycin (KCV) could modify the development of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 (B6) mice sensitized against a myelin oligodendrocyte glycoprotein (MOG) peptide of amino acids 35 to 55 [MOG (35–55)]. Here we report that continuous oral KCV treatment, starting one week before immunization, significantly suppressed the development of EAE along with altering gut flora. Suppression of EAE was accompanied by a reduced production of pro-inflammatory cytokines from the draining lymph nodes (dLNs) in response to MOG (35–55). While the antibiotic treatment suppressed MOG (35–55) reactive Th17 cells within the mesenteric lymph nodes (MLNs), it also reduced the total number of mesenteric Th17 cells in naïve mice. Furthermore, unexpectedly we found that the Th17 cells in the MLNs are greatly reduced in Cd1−/− mice or Jα281−/− mice, which lack invariant Vα14 natural killer T (iNKT) cells,22Yamamura T Sakuishi K Illes Z Miyake S Understanding the behavior of invariant NKT cells in autoimmune diseases.J Neuroimmunol. 2007; 191: 8-15Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar and that the KCV-induced reduction of the mesenteric Th17 cells was only marginal in the iNKT cell-deficient mice. As such, KCV treatment and iNKT cell deletion showed homologous effects on the mesenteric Th17 cells, which led us to speculate that gut flora may influence the development of CNS autoimmune disease in a way dependent of iNKT cells. Consistently, oral KCV treatment did not alter the development of EAE in iNKT cell-deficient mice. These results indicate that iNKT cells play a critical role in the dialogue between host and commensal flora. Six-week-old female B6 mice were purchased from CLEA Laboratory Animal Corporation (Tokyo, Japan). Mr1−/− mice were provided by Dr. Susan Gilfillan, (Washington University School of Medicine, St. Louis)23Treiner E Duban L Bahram S Radosavljevic M Wanner V Tilloy F Affaticati P Gilfillan S Lantz O Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1.Nature. 2003; 422: 164-169Crossref PubMed Scopus (792) Google Scholar and were backcrossed to B6 mice for ten generations. β2-microglobulin−/− mice were purchased from Jackson Laboratories. Cd1 −/−24Sonoda KH, Exley M, Snapper S, Balk SP, Stein-Streilein J: CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site. J Exp Med 190: 1215–1226Google Scholar and Jα281−/−25Cui J Shin T Kawano T Sato H Kondo E Toura I Kaneko Y Koseki H Kanno M Taniguchi M Requirement for Vα14 NKT cells in IL-12-mediated rejection of tumors.Science. 1997; 278: 1623-1626Crossref PubMed Scopus (1160) Google Scholar mice were provided by Dr. Steve B. Balk (Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA) and Dr. Masaru Taniguchi (Riken Research Center for allergy and Immunology, Yokohama, Japan) respectively. These mice were also back-crossed to B6 mice for ten generations. Animals were maintained in specific pathogen-free conditions in accordance with the institutional guidelines. For induction of EAE, B6 mice were injected subcutaneously with 100 μg MOG (35–55) (MEVGWYRSPFSRVVHLYRNGK) (TORAY Laboratory, Tokyo, Japan) and 1 mg heat-killed Mycobacterium tuberculosis H37RA (Difco) emulsified in incomplete Freund's adjuvant. 200 ng of pertussis toxin (List Biological Laboratories) in 200 μl PBS was injected i.p. on days 0 and 2 after immunization. Clinical symptoms of EAE were daily evaluated and scored as follows: 0, no clinical signs; 1, loss of tail tonicity; 2, impaired righting reflex; 3, partial hindlimb paralysis; 4, total hindlimb paralysis; 5, moribund or dead. To treat mice with a mixture of non-absorbing antibiotics, we used a previously described protocol after adding minor modifications.26Bashir ME Louie S Shi HN Nagler-Anderson C Toll-like receptor 4 signaling by intestinal microbes influences susceptibility to food allergy.J Immunol. 2004; 172: 6978-6987PubMed Google Scholar Briefly, to examine the effects of altering gut flora, a group of mice were given ad libitum access to drinking water supplemented with kanamycin (1 mg/ml), colistin (2000 U/ml), and vancomycin (0.1 mg/ml). Normal drinking water was given to another group of mice serving as control. For immunological studies of MLNs, LPLs, and splenocytes, the antibiotic-containing water was continuously given for 1 week until individual experiments were conducted. To evaluate the effect of antibiotics on EAE and recall responses, the treatment was started 1 week before immunization, and continued during the entire observation period. To measure cell proliferation and cytokine production, we stimulated lymph node cells (1 × 106/well) with anti-CD3 antibody (2C11) at 5 μg/ml for 72 hours in 96-well round-bottomed plates. For evaluating MOG (35–55)-specific recall responses, we stimulated lymph node cells (1 × 106/well) with MOG (35–55) peptide at 1 to 100 μmol/L for 72 hours. The cells were suspended in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum, 2 mmol/L l-glutamine, 100 U/ml of penicillin-streptomycin, 2 mmol/L sodium pyruvate and 50 mmol/L β-mercaptoethanol. T cell proliferation to MOG (35–55) was determined by measuring the incorporation of [3H] thymidine (1 μCi/well) during the last 24 hours of culture in a β-1205 counter (Pharmacia, Uppsala, Sweden). Assays were conducted in triplicate wells and data were expressed as counts per minute (c.p.m.). Culture supernatant was collected 72 hours after stimulation, and cytokines in the supernatant were measured by using cytometric bead array kits for mouse inflammatory cytokines (BD Biosciences) and IL-17 enzyme-linked immunosorbent assay (ELISA) kit (R&D systems). Cells were stained with fluorescence-labeled specific antibodies after incubation with anti-CD16/32 to avoid nonspecific staining and were analyzed with a FACSCalibur (BD). Except for Foxp3-APC from eBioscience, all of the other antibodies were obtained from BD Pharmingen. For flow cytometric analysis of CNS-infiltrated cells, spinal cords were homogenized, passed through 70-μm nylon mesh and separated by Percoll density-gradient centrifugation to obtain single-cell suspensions. In some experiments, paraffin-embedded spinal cords were stained with either luxol fast blue or H&E for conventional histological analysis. Cells collected from MLN were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and Ionomycin (750 ng/ml) for 5 hours in the presence of GolgiPlug (BD Biosciences). Cells were first stained extracellularly with PerCP-conjugated anti-CD4, APC-conjugated anti-T cell receptor-β and α-GalCer-loaded Dimer X recombinant soluble dimeric mouse CD1 days (BD Pharmingen), and then stained with fluorescein isothiocyanate-conjugated mAb A85-1 specific for mouse IgG1 (BD Pharmingen), and fixed and permeabilized with Fixation/Permiabilization solution (BD Biosciences). Finally, cells were stained intracellulary with phycoerythrin-conjugated anti-IL-17 (BD Biosciences). Samples were acquired on a FACSCalibur (BD Biosciences), and data were analyzed with CELLQuest software (BD Biosciences). Intestines were removed from euthanized mice and placed in ice-cold PBS containing 25 mmol/L HEPES. After removal of residual mesenteric fat tissue, Peyer's patches were carefully excised, and the intestine was opened longitudinally. The intestine was then thoroughly washed in ice-cold PBS and cut into 1.5-cm pieces. The pieces were incubated four times in 5 ml of 5 mmol/L EDTA, in 10% fetal calf serum/25 mmol/L HEPES/PBS for 15 minutes at 37°C with fast rotation (200 rpm). After each round of incubation, the epithelial cell layer, containing the intraepithelial lymphocytes, was removed. After the fourth EDTA incubation, the pieces were washed in PBS, and placed in 25 ml of RPMI containing 20% fetal calf serum, 25 mmol/L HEPES, and 300 U/ml of Collagenase H (Roche). Digestion was performed three times by incubating the pieces at 37°C for 40 minutes with slow rotation (100 rpm). The solution was then vortexed intensely and passed through a 70-mm cell strainer. The pieces were collected and placed into fresh digestion solution. The procedure was repeated three times. Supernatants from all three digestions from a single small intestine were combined, washed once in cold PBS, resuspended in 5 ml of the 40% fraction of a 40:80 Percoll gradient, and overlaid on 2 ml of the 80% fraction in a 15 ml Falcon tube. Percoll gradient separation was performed by centrifugation for 20 minutes at 2800 rpm at room temperature. LPLs were collected at the interphase of the Percoll gradient, washed once, and resuspended in FACS buffer or T cell medium. The cells were used immediately for experiments. The SV Total RNA isolation kit (Promega) was used for isolation of total RNA from mesenteric lymphocytes or splenocytes according to the manufacturer's instruction. First-strand cDNA was generated with the Advantage-RT kit (Clontech). The Light Cycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics) was used for quantitative PCR analysis. Gene expression values were normalized to expression of the hypoxanthine guanine phosphoribosyl transferase (Hprt) as ‘housekeeping’ gene. QuantiTect Primer Assay (Qiagen) was used for amplification of IL-21 and IL-23. The other primers used were as follows: HPRT forward, 5′-GTTGGATACAGGCCAGACTTTGTTG-3′; HPRT reverse, 5 ′-GAGGGTAGGCTGGCCTATAGGCT-3′; RORγtforward, 5′-TGTCCTGGGCTACC CTACTG-3′; RORγt reverse, 5′-GTGCAGGAGTAGGCCACATT-3′; TGF-β1 forward, 5′-TGCGCTTGCAGAGATTAAAA-3′; TGF-β1 reverse, 5′-GCTGAATCGAAA GCCCTGTA-3′; IL-6 forward, 5′-TTCCATCCAGTTGCCTTCTT-3′; IL-6 reverse, 5′-CAGAATTGCCQATTGCCATTGCACAAC-3′. EAE clinical severity was daily scored as mean ± SEM for each group, and analyzed by the Mann-Whitney U nonparametric ranking test. Differences in cumulative scores of each group of mice were evaluated by Student's t-test. Cytokine secretion data were analyzed with Student's t-test. With an attempt to modulate the composition of intestinal flora, we treated wild-type B6 mice orally with a combination of antibiotics KCV as described in Materials and Methods. Because these antibiotics are not absorbed through gut mucosa,27Chambers HF General Consideration of Antimicrobial Therapy.in: Brunton LL Lazo JS Parker KL Goodman LS Gilman AG Goodman & Gilman's The Pharmacological Basis of Therapeutics eleventh edition. McGraw-Hill, New York2005: 1095-1110Google Scholar any effect caused by this treatment is thought to arise from within the gut lumen. To examine whether our treatment protocol would change the composition of intestinal flora, we applied the DNA microarray system referred to as ‘FloraArray’28Yokoi T Kaku Y Suzuki H Ohta M Ikuta H Isaka K Sumino T Wagatsuma M ‘FloraArray’ for screening of specific DNA probes representing the characteristics of a certain microbial community.FEMS Microbiol Lett. 2007; 273: 166-171Crossref PubMed Scopus (7) Google Scholar and made a comprehensive analysis for intestinal flora derived from KCV-treated mice and control mice. To compare the signal intensities of intestinal flora from the two groups of mice, MA plots were illustrated from the fluorescent images. Although each spot on the FloraArray is derived from a number of different strains in the commensal microflora, this analysis gives us useful information regarding the composition of gut flora. The MA-plot analysis revealed that 722 out of 1536 spots showed more than twofold increase in the fecal DNA sample from KCV-treated mice as compared with those from control mice. By contrast, 894 spots showed more than twofold increase in fecal DNA from control mice as compared with the mice treated with antibiotics (Figure 1). We additionally performed quantitative PCR analysis and revealed that the antibiotic treatment caused differential and reciprocal changes in the quantity of each bacterium species. For example, a great reduction of Lactobacillus murinus and Bacteroides fragilis was seen in the feces from KCV-treated mice, whereas Bacteroides thetaiotaomicron was significantly increased in the same samples of feces (data not shown). These results demonstrate that the protocol of the antibiotic treatment significantly affects the content of intestinal flora. We next addressed whether the change of intestinal flora could also modulate the progression of EAE, an animal model of MS. When we continuously treated the mice with KCV-containing drinking water from 1 week before immunization, clinical signs of MOG (35–55)-induced EAE were significantly suppressed in comparison with control mice (Figure 2A). Accordingly, histological examination showed a reduced infiltration of mononuclear cells and less noticeable demyelination at the lumbar region of the spinal cord of the treated mice (Figure 2B). Moreover, we observed a lower number of total CNS infiltrating cells at an active stage of EAE (day 18) in KCV-treated mice than in control mice when we isolated mononuclear cells from CNS of those mice (data not shown). In parallel, we examined the recall responses of the dLNs to MOG (35–55) on day 11 after immunization. Although proliferation rates of the dLNs in response to MOG (35–55) were comparable between KCV-treated mice and control mice (Figure 3A), the dLN cells from the treated mice produced significantly lower amounts of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-17 in response to MOG (35–55) (Figure 3B), consistent with the suppressed signs of EAE. We also measured the recall response of the MLNs to MOG (35–55). The MLN cells from control mice immunized with MOG (35–55) showed significant responses to the MOG peptide in the proliferative responses as well as IL-17 production (Figure 3C). However, those from KCV-treated mice showed only marginal responses, indicating that induction of MOG (35–55) specific encephalitogenic Th17 cells in both dLNs and MLNs is impaired by an alteration of intestinal contents caused by the antibiotic treatment.Figure 3Reduced MOG-specific responses in dLN and MLN cells from KCV-treated mice. A: Effect of KCV on the lymphocyte proliferative responses. Draining lymph nodes (dLNs) were removed from control or KCV-treated mice 11 days after immunization with MOG (35–55) and the total lymphoid cells (1 × 106) were stimulated with varying doses of MOG (35–55) peptide for 72 hours. Proliferative responses were assessed by [3H] thymidine incorporation. Data are from one of three independent experiments, showing the mean of triplicate samples. B: Effect of KCV treatment on MOG (35–55)-reactive T cells in the dLN. Supernatants were collected after stimulating the dLN cells of day 11 with 100 μmol/L MOG (35–55) peptide in vitro for 72 hours. Cytokine concentration was measured by cytometric bead array or ELISA as described in Materials and Methods. Data represent the mean ± SEM of duplicated samples from one of three separate experiments (n = 2 mice). *P < 0.05 (two-tailed Student's t-test.) C: Effect of KCV treatment on MOG (35–55)-reactive T cells in the MLN. Whole MLN cells were isolated from control or KCV-treated mice (n = 2) 11 days after EAE induction. The cells were stimulated with MOG (35–55) as conducted for dLN cells and proliferative responses (upper panel) and IL-17 production (lower panel) were measured. IL-17 was measured by using ELISA. Data represent the mean ± SEM of triplicate samples from one of two independent experiments (n = 2 mice). *P < 0.05 (two-tailed Student's t-test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) MLNs are thought to offer an important site for the functional cross talk between intestinal microflora and gut immunity.29Ochi H Abraham M Ishikawa H Frenkel D Yang K Basso AS Wu H Chen ML Gandhi R Miller A Maron R Weiner HL Oral CD3-specific antibody suppresses autoimmune encephalomyelitis by inducing CD4+ CD25− LAP+ T cells.Nat Med. 2006; 12: 627-635Crossref PubMed Scopus (214) Google Scholar, 30Worbs T Bode U Yan S Hoffmann MW Hintzen G Bernhardt G Forster R Pabst O Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells.J Exp Med. 2006; 203: 519-527Crossref PubMed Scopus (546) Google Scholar Next we investigated whether the antibiotic treatment induced an alteration of the MLN cell functions in naïve wild-type mice. First we compared the ability of the MLN cells to produce pro-inflammatory cytokines on stimulation with plate-bound anti-CD3 antibody. Proliferative responses of the MLN cells were not affected or slightly suppressed at most by KCV treatment. Interestingly, MLN cells from KCV-treated mice secreted significantly lower amounts of IL-6 and IL-17 compared with those from control mice, whereas production of TNF-α and IFN-γ was not significantly suppressed (Figure 4, A and B). In contrast, splenocytes from both groups of mice showed essentially similar result following stimulation with anti-CD3 (Figure 4, A and B). Recently, Ivanov et al showed that an orphan nuclear receptor RORγt is the key transcription factor that orchestrates the differentiation of the Th17 cell lineage.9Ivanov II McKenzie BS Zhou L Tadokoro CE Lepelley A Lafaille JJ Cua DJ Littman DR The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells.Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (4022) Google Scholar They also showed that Th17 cells tend to accumulate in the mucosa of the small intestine. Quantitative RT-PCR analysis revealed a lower expression of RORγt in the MLN cells from KCV-treated mice as compared with control mice (Figure 4C). We also found that the MLN cells from KCV-treated mice secreted significantly greater amounts of IL-10 than those from control mice (Figure 4A), suggesting that the mesenteric T cells would acquire less inflammatory properties after the antibiotic treatment. Next we examined whether this treatment may alter the composition of lymphocytes in the MLN. We found that the total number of MLN cells was almost equal in KCV-treated and control mice (data not shown). Furthermore, flow cytometric analysis demonstrated that the proportion of dendritic cells, macrophage/monocytes, B cells, conventional CD4+ and CD8+ T cells, NK cells, and NKT cells in the MLN did not change after treatment with KCV (data not shown). These data indicate that the antibiotic treatment protocol does not exhibit any cytotoxic effect on the mesenteric lymphocyte populations, although it remarkably alters the cytokine profile of T cells. We also examined the frequency of Foxp3+ regulatory CD4+ T cells in the MLN. Although recent studies have revealed the presence of reciprocal developmental pathways between Th17 cells and Foxp3+ regulatory T cells,31Bettelli E Carrier Y Gao W Korn T Strom TB Oukka M Weiner HL Kuchroo VK Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.Nature. 2006; 441: 235-238Crossref PubMed Scopus (5
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