Marlies Meisel

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

A nonpathogenic virus can promote inflammatory immunity to dietary antigens and may be linked to the development of celiac disease. Viruses compound dietary pathology Reoviruses commonly infect humans and mice asymptomatically. Bouziat et al. found that immune responses to two gut-infecting reoviruses take different paths in mice (see the Perspective by Verdu and Caminero). Both reoviruses invoked protective immune responses, but for one reovirus, when infection happened in the presence of a dietary antigen (such as gluten or ovalbumin), tolerance to the dietary antigen was lost. This was because this strain prevented the formation of tolerogenic T cells. Instead, it promoted T helper 1 immunity to the dietary antigen through interferon regulatory factor 1 signaling. Celiac disease patients also exhibited elevated levels of antibodies against reovirus. Science, this issue p. 44; see also p. 29 Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

Natural polyreactive IgA antibodies coat the intestinal microbiota

Programmed recognition of microbiota Increasingly, we recognize that the gut is a specialized organ for maintaining microbial symbioses alongside nutritional functions. The gut produces large quantities of immunoglobulin A (IgA), which adheres to the surface of gut microbes. Bunker et al. discovered that antibodies produced by naïve small intestinal plasma cells are recirculated and enriched within Peyers patches, independently of exogenous antigen and T cell help. The resulting polyreactive IgAs are released into the gut lumen and bind to microbial surface glycans, thus innately recognizing the gut microbiota. Polyreactive IgAs appear to be a product of the coevolution of host and microbiota to maintain symbiotic homeostasis. Science, this issue p. eaan6619 Inherently polyreactive antibodies fuel homeostatic intestinal immunoglobulin A responses to the normal gut microbiota. INTRODUCTION Immunoglobulin A (IgA) is the most abundant mammalian antibody isotype, constituting more than 80% of all antibody-secreting plasma cells at steady state. IgA is particularly prevalent at barrier surfaces such as the intestinal mucosa, where it forms a first line of defense in conjunction with innate mediators, including mucus and antimicrobial peptides. IgA is thought to coat and contain the resident commensal microbiota and provide protection against enteric pathogens. IgA responses occur under normal homeostatic conditions and involve both T cell–dependent and T cell–independent pathways of differentiation in mucosa-associated lymphoid tissues such as Peyer’s patches. However, despite its abundance, the specificity of homeostatic IgA has long remained elusive. RATIONALE To elucidate the specificity and origins of homeostatic IgA, we performed unbiased, large-scale cloning and characterization of monoclonal antibodies (mAbs) from single murine IgA plasma cells and other B cell populations of different origins. All antibodies were expressed recombinantly with an IgG1 isotype to compare their reactivity independent of their monomeric or multimeric nature. RESULTS Panels of single cell–derived mAbs were cloned from various B cell and IgA plasma cell populations, and their microbiota-reactivity was characterized by using a combination of bacterial flow cytometry and 16S ribosomal RNA (rRNA) sequencing. Additionally, mAbs were assayed by enzyme-linked immunosorbent assay (ELISA) for polyreactivity—a peculiar property of certain antibodies that facilitates binding to a variety of structurally diverse antigens. Several insights emerged from this characterization: (i) Microbiota-reactive and polyreactive antibodies arose naturally in all naïve B cell populations but were significantly enriched among IgA-secreting plasma cells. (ii) Microbiota-reactive and polyreactive antibodies from naïve B cells and IgA plasma cells showed similar patterns of binding to a broad, but defined, subset of microbiota. This binding included many members of Proteobacteria but largely excluded those of Bacteroidetes and Firmicutes, the predominant phyla in the colon. Interestingly, broadly neutralizing antibodies against influenza virus, which had previously been shown to be frequently polyreactive, were also commonly microbiota-reactive and displayed binding patterns that resembled IgAs. These patterns of microbiota-reactivity thus appear to be a general property of polyreactive antibodies. (iii) The microbiota-reactive and polyreactive IgA repertoire emerged via a mechanism that was largely independent of T cell help or somatic hypermutation. Instead, naturally microbiota-reactive and polyreactive recirculating naïve B cells were selected to become IgA plasma cells in Peyer’s patches. Although some antibodies subsequently acquired somatic mutations, these did not substantially alter their reactivity. (iv) Differentiation of microbiota-reactive and polyreactive IgAs occurred independent of microbiota or exogenous dietary antigen. Analysis of germ-free mice and germ-free mice fed an antigen-free diet demonstrated that microbiota-reactive and polyreactive IgA plasma cells arose naturally, even in the absence of exogenous antigens. CONCLUSION We conclude that homeostatic intestinal IgAs are natural polyreactive antibodies with innate specificity to microbiota. These data suggest that IgA antibodies, though derived from the adaptive immune system, possess innate-like recognition properties that may facilitate adaptation to the vast and dynamic array of exogenous microbiota and dietary antigens encountered at mucosal surfaces. Large-scale analysis of mAbs reveals the specificity and origins of homeostatic intestinal IgA. Panels of single cell–derived mAbs were cloned from murine IgA plasma cells and other B cell populations and characterized for microbiota-reactivity by bacterial flow cytometry and 16S rRNA sequencing or for polyreactivity against structurally diverse antigens, including DNA, insulin, lipopolysaccharide (LPS), flagellin, albumin, cardiolipin, and keyhole-limpet hemocyanin (KLH) by ELISA. This approach revealed that intestinal IgAs are natural polyreactive antibodies with innate specificity to microbiota. FSC, forward scatter. Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. Here we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer’s patches. This selection process occurred independent of microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. These findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.

Intestinal Microbiota Modulates Gluten-Induced Immunopathology in Humanized Mice

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk.

Coronin 1A is an essential regulator of the TGFβ receptor/SMAD3 signaling pathway in Th17 CD4(+) T cells.

Transforming growth factor β (TGFβ) plays a central role in maintaining immune homeostasis by regulating the initiation and termination of immune responses and thus preventing the development of autoimmune diseases. In this study, we describe an essential mechanism by which the actin regulatory protein Coronin 1A (Coro1A) ensures the proper response of Th17 CD4(+) T cells to TGFβ. Coro1A has been established as a key player in T cell survival, migration, activation, and Ca(2+) regulation in naive T cells. We show that mice lacking Coro1a developed less severe experimental autoimmune encephalomyelitis (EAE). Unexpectedly, upon the re-induction of EAE, Coro1a(-/-) mice exhibited enhanced EAE signs that correlated with increased numbers of IL-17 producing CD4(+) cells in the central nervous system (CNS) compared to wild-type mice. In vitro differentiated Coro1a(-/-) Th17 CD4(+) T cells consistently produced more IL-17 than wild-type cells and displayed a Th17/Th1-like phenotype in regard to the expression of the Th1 markers T-bet and IFNγ. Mechanistically, the Coro1a(-/-) Th17 cell phenotype correlated with a severe defect in TGFβR-mediated SMAD3 activation. Taken together, these data provide experimental evidence of a non-redundant role of Coro1A in the regulation of Th17 CD4(+) cell effector functions and, subsequently, in the development of autoimmunity.

Antibiotic-induced perturbations in microbial diversity during post-natal development alters amyloid pathology in an aged APP SWE /PS1 ΔE9 murine model of Alzheimer’s disease

Recent evidence suggests the commensal microbiome regulates host immunity and influences brain function; findings that have ramifications for neurodegenerative diseases. In the context of Alzheimer’s disease (AD), we previously reported that perturbations in microbial diversity induced by life-long combinatorial antibiotic (ABX) selection pressure in the APPSWE/PS1ΔE9 mouse model of amyloidosis is commensurate with reductions in amyloid-β (Aβ) plaque pathology and plaque-localised gliosis. Considering microbiota-host interactions, specifically during early post-natal development, are critical for immune- and neuro-development we now examine the impact of microbial community perturbations induced by acute ABX exposure exclusively during this period in APPSWE/PS1ΔE9 mice. We show that early post-natal (P) ABX treatment (P14-P21) results in long-term alterations of gut microbial genera (predominantly Lachnospiraceae and S24-7) and reduction in brain Aβ deposition in aged APPSWE/PS1ΔE9 mice. These mice exhibit elevated levels of blood- and brain-resident Foxp3+ T-regulatory cells and display an alteration in the inflammatory milieu of the serum and cerebrospinal fluid. Finally, we confirm that plaque-localised microglia and astrocytes are reduced in ABX-exposed mice. These findings suggest that ABX-induced microbial diversity perturbations during post-natal stages of development coincide with altered host immunity mechanisms and amyloidosis in a murine model of AD.

Cbl-b mediates TGFβ sensitivity by downregulating inhibitory SMAD7 in primary T cells

T cell-intrinsic transforming growth factor β (TGFβ) receptor signaling plays an essential role in controlling immune responses. The RING-type E3 ligase Cbl-b has been shown to mediate the sensitivity of T cells to TGFβ; however, the mechanism underlying this process is unknown. This study shows that SMAD7, an established negative regulator of TGFβ receptor (TGFβR) signaling, is a key downstream effector target of Cbl-b. SMAD7 protein levels, but not SMAD7 mRNA levels, are upregulated in cblb(-/-) T cells. Cbl-b directly interacts with and ubiquitinates SMAD7, suggesting that Cbl-b posttranscriptionally regulates SMAD7. In support of this notion, concomitant genetic loss of SMAD7 in cblb(-/-) mice restored TGFβ sensitivity on T cell cytokine responses and abrogated the tumor rejection phenotype of cblb(-/-) mice. These results demonstrate an essential and non-redundant role for Cbl-b in controlling TGFβR signaling by directly targeting SMAD7 for degradation during T cell responses in vitro and in vivo.

The kinase PKCα selectively upregulates interleukin-17A during Th17 cell immune responses.

Summary Transforming growth-factor β (TGFβ) has been implicated in T helper 17 (Th17) cell biology and in triggering expression of interleukin-17A (IL-17A), which is a key Th17 cell cytokine. Deregulated TGFβ receptor (TGFβR) signaling has been implicated in Th17-cell-mediated autoimmune pathogenesis. Nevertheless, the full molecular mechanisms involved in the activation of the TGFβR pathway in driving IL-17A expression remain unknown. Here, we identified protein kinase C α (PKCα) as a signaling intermediate specific to the Th17 cell subset in the activation of TGFβRI. We have shown that PKCα physically interacts and functionally cooperates with TGFβRI to promote robust SMAD2-3 activation. Furthermore, PKCα-deficient (Prkca−/−) cells demonstrated a defect in SMAD-dependent IL-2 suppression, as well as decreased STAT3 DNA binding within the Il17a promoter. Consistently, Prkca−/− cells failed to mount appropriate IL-17A, but not IL-17F, responses in vitro and were resistant to induction of Th17-cell-dependent experimental autoimmune encephalomyelitis in vivo.

Nuclear orphan receptor NR2F6 directly antagonizes NFAT and RORγt binding to the Il17a promoter

Interleukin-17A (IL-17A) is the signature cytokine produced by Th17 CD4+ T cells and has been tightly linked to autoimmune pathogenesis. In particular, the transcription factors NFAT and RORγt are known to activate Il17a transcription, although the detailed mechanism of action remains incompletely understood. Here, we show that the nuclear orphan receptor NR2F6 can attenuate the capacity of NFAT to bind to critical regions of the Il17a gene promoter. In addition, because NR2F6 binds to defined hormone response elements (HREs) within the Il17a locus, it interferes with the ability of RORγt to access the DNA. Consistently, NFAT and RORγt binding within the Il17a locus were enhanced in Nr2f6-deficient CD4+ Th17 cells but decreased in Nr2f6-overexpressing transgenic CD4+ Th17 cells. Taken together, our findings uncover an example of antagonistic regulation of Il17a transcription through the direct reciprocal actions of NR2F6 versus NFAT and RORγt.

Interleukin-15 promotes intestinal dysbiosis with butyrate deficiency associated with increased susceptibility to colitis

Dysbiosis resulting in gut-microbiome alterations with reduced butyrate production are thought to disrupt intestinal immune homeostasis and promote complex immune disorders. However, whether and how dysbiosis develops before the onset of overt pathology remains poorly defined. Interleukin-15 (IL-15) is upregulated in distressed tissue and its overexpression is thought to predispose susceptible individuals to and have a role in the pathogenesis of celiac disease and inflammatory bowel disease (IBD). Although the immunological roles of IL-15 have been largely studied, its potential impact on the microbiota remains unexplored. Analysis of 16S ribosomal RNA-based inventories of bacterial communities in mice overexpressing IL-15 in the intestinal epithelium (villin-IL-15 transgenic (v-IL-15tg) mice) shows distinct changes in the composition of the intestinal bacteria. Although some alterations are specific to individual intestinal compartments, others are found across the ileum, cecum and feces. In particular, IL-15 overexpression restructures the composition of the microbiota with a decrease in butyrate-producing bacteria that is associated with a reduction in luminal butyrate levels across all intestinal compartments. Fecal microbiota transplant experiments of wild-type and v-IL-15tg microbiota into germ-free mice further indicate that diminishing butyrate concentration observed in the intestinal lumen of v-IL-15tg mice is the result of intrinsic alterations in the microbiota induced by IL-15. This reconfiguration of the microbiota is associated with increased susceptibility to dextran sodium sulfate-induced colitis. Altogether, this study reveals that IL-15 impacts butyrate-producing bacteria and lowers butyrate levels in the absence of overt pathology, which represent events that precede and promote intestinal inflammatory diseases.

Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

Protein kinase C θ (PKCθ) is involved in signaling downstream of the T cell antigen receptor (TCR) and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures) and in vivo (experimental autoimmune encephalomyelitis model, EAE) techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt), accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ) and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ−/− CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.