Commensal microbiota influence systemic autoimmune responses
2015; Springer Nature; Volume: 34; Issue: 4 Linguagem: Inglês
10.15252/embj.201489966
ISSN1460-2075
AutoresJens Van Praet, Erin Donovan, Inge Vanassche, Michael Drennan, Fien Windels, Amélie Dendooven, Liesbeth Allais, Claude Cuvelier, Fons A. J. van de Loo, Paula S. Norris, Andrey Kruglov, Sergei A. Nedospasov, Sylvie Rabot, Raúl Y. Tito, Jeroen Raes, Valérie Gaboriau‐Routhiau, Nadine Cerf–Bensussan, Tom Van de Wiele, Gérard Eberl, Carl F. Ware, Dirk Elewaut,
Tópico(s)T-cell and B-cell Immunology
ResumoArticle19 January 2015free access Commensal microbiota influence systemic autoimmune responses Jens T Van Praet Jens T Van Praet Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Erin Donovan Erin Donovan Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Inge Vanassche Inge Vanassche Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Michael B Drennan Michael B Drennan Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Fien Windels Fien Windels Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Amélie Dendooven Amélie Dendooven Department of Pathology, University Medical Center, Utrecht, the Netherlands Search for more papers by this author Liesbeth Allais Liesbeth Allais Department of Pathology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Claude A Cuvelier Claude A Cuvelier Department of Pathology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Fons van de Loo Fons van de Loo Department of Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands Search for more papers by this author Paula S Norris Paula S Norris Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Andrey A Kruglov Andrey A Kruglov German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany Belozersky Institute of Physico-Chemical Biology and Biological Faculty, Lomonosov Moscow State University, Moscow, Russia Search for more papers by this author Sergei A Nedospasov Sergei A Nedospasov Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, and Lomonosov Moscow State University, Moscow, Russia Search for more papers by this author Sylvie Rabot Sylvie Rabot INRA, UMR1319 Micalis, Jouy-en-Josas, France AgroParisTech, Micalis, Jouy-en-Josas, France Search for more papers by this author Raul Tito Raul Tito Bioinformatics and (eco-)systems Biology Laboratory, Department of Microbiology and Immunology, Rega Institute, VIB Center for the Biology of Disease, KU Leuven, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Bioinformatics and (eco-)systems Biology Laboratory, Department of Microbiology and Immunology, Rega Institute, VIB Center for the Biology of Disease, KU Leuven, Belgium Search for more papers by this author Valerie Gaboriau-Routhiau Valerie Gaboriau-Routhiau INRA, UMR1319 Micalis, Jouy-en-Josas, France INSERM UMR1163, Laboratory of Intestinal Immunity, Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France Search for more papers by this author Nadine Cerf-Bensussan Nadine Cerf-Bensussan INSERM UMR1163, Laboratory of Intestinal Immunity, Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France Search for more papers by this author Tom Van de Wiele Tom Van de Wiele Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium Search for more papers by this author Gérard Eberl Gérard Eberl Lymphoid Tissue Development Group, Institut Pasteur, Paris, France Search for more papers by this author Carl F Ware Carl F Ware Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Dirk Elewaut Corresponding Author Dirk Elewaut Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium VIB Inflammation Research Center, Ghent University, Ghent, Belgium Search for more papers by this author Jens T Van Praet Jens T Van Praet Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Erin Donovan Erin Donovan Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Inge Vanassche Inge Vanassche Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Michael B Drennan Michael B Drennan Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Fien Windels Fien Windels Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Amélie Dendooven Amélie Dendooven Department of Pathology, University Medical Center, Utrecht, the Netherlands Search for more papers by this author Liesbeth Allais Liesbeth Allais Department of Pathology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Claude A Cuvelier Claude A Cuvelier Department of Pathology, Ghent University Hospital, Ghent, Belgium Search for more papers by this author Fons van de Loo Fons van de Loo Department of Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands Search for more papers by this author Paula S Norris Paula S Norris Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Andrey A Kruglov Andrey A Kruglov German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany Belozersky Institute of Physico-Chemical Biology and Biological Faculty, Lomonosov Moscow State University, Moscow, Russia Search for more papers by this author Sergei A Nedospasov Sergei A Nedospasov Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, and Lomonosov Moscow State University, Moscow, Russia Search for more papers by this author Sylvie Rabot Sylvie Rabot INRA, UMR1319 Micalis, Jouy-en-Josas, France AgroParisTech, Micalis, Jouy-en-Josas, France Search for more papers by this author Raul Tito Raul Tito Bioinformatics and (eco-)systems Biology Laboratory, Department of Microbiology and Immunology, Rega Institute, VIB Center for the Biology of Disease, KU Leuven, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Bioinformatics and (eco-)systems Biology Laboratory, Department of Microbiology and Immunology, Rega Institute, VIB Center for the Biology of Disease, KU Leuven, Belgium Search for more papers by this author Valerie Gaboriau-Routhiau Valerie Gaboriau-Routhiau INRA, UMR1319 Micalis, Jouy-en-Josas, France INSERM UMR1163, Laboratory of Intestinal Immunity, Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France Search for more papers by this author Nadine Cerf-Bensussan Nadine Cerf-Bensussan INSERM UMR1163, Laboratory of Intestinal Immunity, Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France Search for more papers by this author Tom Van de Wiele Tom Van de Wiele Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium Search for more papers by this author Gérard Eberl Gérard Eberl Lymphoid Tissue Development Group, Institut Pasteur, Paris, France Search for more papers by this author Carl F Ware Carl F Ware Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA Search for more papers by this author Dirk Elewaut Corresponding Author Dirk Elewaut Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium VIB Inflammation Research Center, Ghent University, Ghent, Belgium Search for more papers by this author Author Information Jens T Van Praet1,‡, Erin Donovan1,‡, Inge Vanassche1, Michael B Drennan1, Fien Windels1, Amélie Dendooven2, Liesbeth Allais3, Claude A Cuvelier3, Fons Loo4, Paula S Norris5, Andrey A Kruglov6,7, Sergei A Nedospasov8, Sylvie Rabot9,10, Raul Tito11, Jeroen Raes11, Valerie Gaboriau-Routhiau9,12, Nadine Cerf-Bensussan12, Tom Van de Wiele13, Gérard Eberl14, Carl F Ware5 and Dirk Elewaut 1,15 1Laboratory for Molecular Immunology and Inflammation, Department of Rheumatology, Ghent University Hospital, Ghent, Belgium 2Department of Pathology, University Medical Center, Utrecht, the Netherlands 3Department of Pathology, Ghent University Hospital, Ghent, Belgium 4Department of Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands 5Infectious and Inflammatory Disease Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA 6German Rheumatism Research Center (DRFZ), A Leibniz Institute, Berlin, Germany 7Belozersky Institute of Physico-Chemical Biology and Biological Faculty, Lomonosov Moscow State University, Moscow, Russia 8Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, and Lomonosov Moscow State University, Moscow, Russia 9INRA, UMR1319 Micalis, Jouy-en-Josas, France 10AgroParisTech, Micalis, Jouy-en-Josas, France 11Bioinformatics and (eco-)systems Biology Laboratory, Department of Microbiology and Immunology, Rega Institute, VIB Center for the Biology of Disease, KU Leuven, Belgium 12INSERM UMR1163, Laboratory of Intestinal Immunity, Université Paris Descartes-Sorbonne Paris Cité and Institut Imagine, Paris, France 13Laboratory of Microbial Ecology and Technology, Ghent University, Ghent, Belgium 14Lymphoid Tissue Development Group, Institut Pasteur, Paris, France 15VIB Inflammation Research Center, Ghent University, Ghent, Belgium ‡These authors contributed equally to this work *Corresponding author. Tel: +329 332 2240; E-mail: [email protected] The EMBO Journal (2015)34:466-474https://doi.org/10.15252/embj.201489966 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life. Synopsis Antinuclear antibodies (ANA) are features of generalized autoimmune diseases. Here, we show that induction of ANA spontaneously occurs in mice lacking gut-associated lymphoid tissues. This was caused by colonization of the intestine by segmented filamentous bacteria, which are potent inducers of Th17 responses. Microbiota and IL-17R-dependent mechanism of ANA induction embark in the neonatal phase of life, leading to systemic autoimmunity in adult. ANA induction may occur in the absence of all secondary lymphoid organs, particularly gut-associated lymphoid tissues Spontaneous occurrence of ANA in the absence of lymphotoxin is RORγt and IL-17R dependent. Neonatal colonization of the intestine by segmented filamentous bacteria in the absence of gut-associated lymphoid tissues predisposes for ANA induction in adult life Introduction Antinuclear antibodies (ANA) are a hallmark feature of generalized autoimmune diseases (von Muhlen & Tan, 1995). These clinically heterogeneous conditions, such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), are characterized by immune-mediated tissue damage in multiple organs, caused by aberrant responses of the adaptive immune system. Immunodominant autoantigens recognized by systemic autoantibodies are often DNA- or RNA-associated protein complexes. Underlying mechanisms that mediate the breach of tolerance against these nuclear autoantigens are only partially understood. In autoimmune-prone strains of mice, antigen-producing cells have been located in secondary lymphoid tissue, and both extrafollicular and germinal center responses have been implicated in the production of such autoantibodies (William et al, 2002). The lymphotoxin-β receptor (LTβR) functions as receptor for both membrane-bound lymphotoxin (LTα1β2) and LIGHT (TNF superfamily member 14). Lymphotoxin-β receptor signaling controls the development of secondary lymphoid organs and is continuously required in adults for homeostasis and structural architecture of the thymus and secondary lymphoid organs (Futterer et al, 1998). LT-deficient mice thus serve as a prototypic model for studying the influence of secondary lymphoid organs in immune processes. Although antibody responses are impaired in LT-deficient animals due to the absence of follicular dendritic cell networks, germinal center formation and somatic hypermutation can still occur. Based on the presence of perivascular lymphocytic infiltrates in multiple organs as well as organ-specific autoantibodies, an autoimmune phenotype has been defined for the LT-deficient animals (Boehm et al, 2003). However, the role of the disturbed thymic medulla in this autoimmune phenotype is a matter of controversy (Chin et al, 2003; Martins et al, 2008). Whether systemic autoimmune responses do occur in the absence of secondary lymphoid tissues is another area of uncertainty (Boehm et al, 2003; Chin et al, 2003). Given conflicting reports on systemic autoimmune responses in Ltbr−/− mice, we sought to investigate whether autoantibodies directed against nuclear antigens can appear in the absence of secondary lymphoid tissues utilizing Ltbr−/− mice. Results and Discussion We found that ~25% of LTβR-deficient mice developed systemic autoimmune responses by three months of age (Fig 1A) using a validated immunodetection system for a broad range of nuclear antigens (Supplementary Fig S1). The immunoassay system identified diverse ANA, including anti-U1RNP, anti-Sm, anti-Scl70/topoisomerase-I, anti-centromere protein B, anti-SSA/Ro52, and anti-Jo1 (Fig 1B and C). Antibodies to these nuclear autoantigens are strongly associated with SLE, SSc, and polymyositis (von Muhlen & Tan, 1995). In contrast, no anti-dsDNA was found (Supplementary Fig S2). By six months of age, the prevalence of autoantibodies remained the same, but more mice developed multiple reactivities (Fig 1B and C). We semiquantitatively determined the titers of these mice (Fig 1B). We could not detect any autoimmune reactivity at six weeks of age despite immune maturation, suggesting a delayed stochastic penetrance of the autoimmune phenotype characteristic in most autoimmune diseases. As LT-deficient animals have a spleen, we sought to determine whether ANA can be generated in asplenic mice by intercrossing Hox-11−/− and Ltbr−/− mice. These double knockout mice still developed the pathological autoantibody responses at the same prevalence, demonstrating that aberrant systemic autoimmune responses can develop in the complete absence of secondary lymphoid organs (Fig 1A). Figure 1. Systemic autoimmune responses in mice lacking secondary lymphoid organs 3-month-old wild-type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA. 6-month-old Ltbr−/− mice (n = 15); titers of individual mice. LIA of 6-month-old Ltbr−/− mice, wild-type mice, and 3-month-old Rorγt-Ltb−/− mice. LTβR-Fc or control immunoglobulin (Ig)-treated wild-type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment. 3-month-old cell-specific LT knockout mice; percentage of mice with at least one ANA. Bone marrow cells from wild-type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild-type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months. Download figure Download PowerPoint Histological examination of Ltbr−/− mice confirmed the presence of lymphocytic infiltrates in multiple organs. Given the association of systemic autoimmune responses with generalized autoimmune disease, we specifically looked for characteristic pathological features. However, compared to wild-type mice, no difference was observed in kidney damage, and skin and esophageal sclerosis (Supplementary Fig S3). Furthermore, renal histology and proteinuria were not different between antibody-positive and antibody-negative Ltbr −/− mice (data not shown). We then evaluated whether structural defects in LT-deficient mice lead to ANA production. To this end, we used an LTβR-Fc fusion protein, which acts as a soluble decoy receptor blocking LTαβ and LIGHT (Rennert et al, 1996). Blocking LTβR signaling at various phases of ontogeny and early postnatally results in the temporally patterned absence of secondary lymphoid organs (lymph nodes, Peyers' patches (PP), and cryptopatches (CP)) (Rennert et al, 1996; Bouskra et al, 2008). We observed that blocking LTβR signaling during late ontogeny through six weeks of age resulted in the appearance of ANA at the age of three months with a comparable spectrum and titers as LTβR-deficient mice (Fig 1D and data not shown), and as demonstrated previously, these mice lacked CP, PP, and isolated lymphoid follicles (ILF) (data not shown). In contrast, mice lacking peripheral lymph nodes and PP by blocking LTβR signaling during early ontogeny did not develop autoantibodies. In addition, blocking LTβR signaling during adulthood, which disrupts splenic architecture, also did not result in ANA formation (Fig 1D). To rule out a role of LTβR signaling in the thymus during the perinatal window, we performed thymus transplant experiments. Fetal thymi from Ltbr−/− or wild-type mice were depleted of hemato-poietic cells and then grafted under the kidney capsules of nude mice, creating Ltbr−/−→nude mice and wild-type→nude mice. Three months after engraftment, mice were sacrificed and T-cell repopulation was verified in the liver and spleen by flow cytometry (Fig 2A and B). Ltbr−/− and wild-type thymi contained approximately equal number of thymocytes, with similar distribution among the different T-cell subsets (Fig 2A and B). Levels of total IgG were also not different between the two groups (Fig 2C). Importantly, no ANA could be detected in the serum samples of nude mice engrafted with LTβR-deficient thymic lobes (Fig 2D). We thus concluded that systemic autoimmune responses can develop in the absence of CP and ILF. Figure 2. Central tolerance against nuclear antigens is not impaired in lymphotoxin-deficient mice Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages. Numbers represent the percentages within the indicated regions (left panel). No significant differences were found. Data represent means ± s.e.m. T-cell reconstitution of spleen was assessed by flow cytometry. Numbers represent the percentages within the indicated regions. Total IgG from nude mice engrafted with Ltbr−/− or wild-type thymi was determined by ELISA. No significant differences were found. Data represent means ± s.e.m. Sera were collected 3 months after transplantation and tested for ANA; percentage of mice with at least one ANA reactivity. Download figure Download PowerPoint We next wanted to resolve which membrane LT-expressing cell type in the lamina propria of the gut was involved in the maintenance of tolerance against nuclear antigens. To this end, we generated mice deficient in LTβ in T cells (T-Ltb−/−), B cells (B-Ltb−/−) or RORγt+ cells (Rorγt-Ltb−/−), and littermate controls. We could only detect autoantibodies with a similar spectrum as Ltbr−/− in mice lacking membrane LT in RORγt-positive cells (Fig 1E). As we were able to test only small sample sizes of cell-specific Ltb−/− mice, we also collected sera from cell-specific Lta−/− mice. Alike cell-specific Ltb−/− mice, only Rorγt-Lta−/− but neither T-Lta−/− nor B-Lta−/− developed ANA at the age of three months (data not shown). To resolve which LTβR-expressing cells in the lamina propria are involved in the maintenance of the tolerance, we performed reciprocal bone marrow transfer experiments between wild-type and Ltbr−/− mice. As shown in Fig 1F, both wild-type→Ltbr−/− and Ltbr−/−→wild-type chimeras developed autoantibodies. We thus conclude that communication via the LT-LTβR axis between RORγt+ innate lymphoid cells (ILC) and both radio-resistant and bone marrow-derived cells is essential to maintain tolerance. RORγt+ ILC have been shown to be essential in the defense of epithelial surfaces and play an important role in the intestinal homeostasis with symbiotic microbiota by the production of IgA, IL-17, and IL-22 production (Vivier et al, 2009). This regulatory control in the gut prompted us to examine the relationship between the gut microbiota and ANA production. Moreover, early postnatal blocking of LTβR signaling leads to a 10-fold expansion of the normal ileal microbiota, including bacteria belonging to the Clostridiales, Bacteroides, and Enterobacteriaceae groups (Bouskra et al, 2008). We first assessed whether elimination of the gut microbiota influenced ANA production. Pregnant mice and their offspring were treated with broad-spectrum antibiotics until the age of 3 months. As previously observed in germfree mice, this caused a significant enlargement of the cecum (data not shown). Ltbr−/− mice receiving antibiotics had a reduced prevalence of ANA compared to a large series of 369 untreated consecutively born three-month-old animals (Fig 3A, upper panel). In this series, we observed some variation in the ANA positivity between litters, but no significant differences in prevalence between cages weaned from the same breading cage (data not shown). This points to a maternal but not a cage effect on the phenotype. Therefore, we opted to analyze offspring of at least 2 litters in further experiments. To further substantiate a role for the gut microbiota, we treated germfree C57BL/6 mice with the LTβR-Fc fusion protein from gestational day 18 until six weeks after birth as described above. These experiments were performed in the INRA Anaxem germfree animal facilities. Similarly, germfree animals had a reduced prevalence of ANA (Fig 3A, lower panel). Furthermore, we observed a higher frequency of ANA-positive animals compared to the experiments with LTβR-Fc fusion protein performed in the Ghent University vivarium. Figure 3. Development of antinuclear antibodies in lymphotoxin-deficient mice is influenced by gut microbiota Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild-type mice were treated with the LTβR-Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months. 16S rRNA gene analysis of luminal samples of wild-type, ANA-positive, and ANA-negative Ltbr−/− mice. Left panel: Beta diversity plot using multidimensional scaling on Bray–Curtis dissimilarities distances. Right panel: Hierarchical cluster analysis using Pearson's correlation. Bacteria community compositions differ in the different groups. Please note that Candidatus arthromitus, according to Thompson et al (2012), should be renamed Candidatus savagella. Real-time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA-positive and ANA-negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m. C3H/HeN germfree pregnant mice were treated with the LTβR-Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day. LTβR-Fc-treated wild-type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months Download figure Download PowerPoint We next assessed whether gut microbiota composition differed between wild-type, antibody-positive, and antibody-negative LTβR-deficient animals existed. In a first screening, we performed a community profiling on the luminal, mucosal, and fecal microbiome by DGGE (data not shown) and 16S rRNA sequencing, both of which showed the same results. As shown on Fig 3B, we observed genotype and antibody-specific clustering of the three animal groups. The most striking differences between groups revealed a species belonging to the segmented filamentous bacteria (SFB), characterizing animals with multiple reactivities as a separate group compared to controls that are in a cluster dominated by Methylobacterium (Fig 3B). These data were confirmed by quantitative PCR with primers specific for SFB genes (Fig 3C). The presence of the Methylobacterium genus has been associated with contamination in samples with low biomass (Barton et al, 2006; Salter et al, 2014). However, we feel that this is not the case here—the fecal material used in this study has large biomass that should dilute the contaminant rather than allowing for its dominance in the profile. Furthermore, a neighbor-joining tree shows that the observed Methylobacterium forms a separate cluster than the one produced by the reported contaminants (Supplementary Fig S5). Histological analyses of different gut parts comparing ANA-positive and ANA-negative LTβR-deficient animals, however, revealed no inflammatory differences (Supplementary Fig S6). We also directly compared the impact of monocolonization of LTβR-Fc-treated mice with SFB versus the nonadherent and nonvirulent strain of E. coli MG1655 on autoantibody induction in the INRA facilities. We found an increased prevalence of ANA in SFB as opposed to E. coli monocolonized mice (Fig 3D). In SFB monocolonized mice, ANA frequency was markedly higher in mice lacking all gut-associated lymphoid tissue compared to mice lacking PP only (Supplementary Fig S4B). ILF and PP contain the stromal microenvironment for IgA production, a critical antibody that helps maintain gut homeostasis (Eberl, 2007). Furthermore, it was shown that SFB can also induce IgA responses via de novo induction of tertiary lymphoid tissue in contrast to E. coli in mice lacking PP and cryptopatch-derived ILF (Lecuyer et al, 2014). Because an aberrant expansion of SFB was reported in IgA-deficient mice, it could be anticipated that IgA was protective against ANA induction. Yet ANA are present at comparable levels in mice with gut tertiary follicles, which have substantial concentrations of IgA in feces, as in mice lacking all gut lymphoid tissues, which have hardly any IgA detectable in the feces (data not shown). This argues against a critical role for IgA in LT-dependent ANA induction. SFB are important for inducing a robust T-helper cell type 17 population in the small intestinal lamina propria of the mouse gut (Wu et al, 2010) and has the ability to induce such responses inside and outside gut organized lymphoid tissue (Lecuyer et al, 2014). To assess the potential role of IL-17 or IL-25 in the model as an effector cytokine, we treated IL-17R-deficient mice with the LTβR-Fc fusion protein from gestational day 18 until six weeks after birth as described above. In contrast to wild-type mice, this treatment did not induce significant systemic autoimmune responses (Fig 3E). In BXD2, a mouse model for lupus, IL-17 was also identified as an important effector cytokine for systemic autoimmune responses (Hsu et al, 2008). It was reported that intestinal IgA and IgG production plasma cells, while mostly antigen specific, include a relatively high frequency of cells secreting autoreactive and polyspecific antibodies (Benckert et al, 2011; Scheid et al, 2011). This is in contrast to the bone marrow IgG plasma cells where autoreactivity is relatively rare. Because we were unable to find immune-mediated pathology despite the presence of multiple ANA reactivity, this could point to production to polyreactive intestinal B cells due to impaired architecture rather than active regulation of autoantibody responses. In such a scenario, SFB could alter the threshold for induction of ANA given its marked ability to generate potent Th17 responses. In summary, we report that in the absence of LT IL-17R-dependent systemic autoimmune responses are associated with increased SFB colonization. Overall, our findings enforce a new paradigm that neonatal co
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