A microbial symbiosis factor prevents intestinal inflammatory disease

Humans are colonized by multitudes of commensal organisms representing members of five of the six kingdoms of life; however, our gastrointestinal tract provides residence to both beneficial and potentially pathogenic microorganisms. Imbalances in the composition of the bacterial microbiota, known as dysbiosis, are postulated to be a major factor in human disorders such as inflammatory bowel disease. We report here that the prominent human symbiont Bacteroides fragilis protects animals from experimental colitis induced by Helicobacter hepaticus, a commensal bacterium with pathogenic potential. This beneficial activity requires a single microbial molecule (polysaccharide A, PSA). In animals harbouring B. fragilis not expressing PSA, H. hepaticus colonization leads to disease and pro-inflammatory cytokine production in colonic tissues. Purified PSA administered to animals is required to suppress pro-inflammatory interleukin-17 production by intestinal immune cells and also inhibits in vitro reactions in cell cultures. Furthermore, PSA protects from inflammatory disease through a functional requirement for interleukin-10-producing CD4+ T cells. These results show that molecules of the bacterial microbiota can mediate the critical balance between health and disease. Harnessing the immunomodulatory capacity of symbiosis factors such as PSA might potentially provide therapeutics for human inflammatory disorders on the basis of entirely novel biological principles.     

Peripheral education of the immune system by colonic commensal microbiota

The instruction of the immune system to be tolerant of self, thereby preventing autoimmunity, is facilitated by the education of T cells in a specialized organ, the thymus, in which self-reactive cells are either eliminated or differentiated into tolerogenic Foxp3(+) regulatory T (T(reg)) cells. However, it is unknown whether T cells are also educated to be tolerant of foreign antigens, such as those from commensal bacteria, to prevent immunopathology such as inflammatory bowel disease. Here we show that encounter with commensal microbiota results in the peripheral generation of T(reg) cells rather than pathogenic effectors. We observed that colonic T(reg) cells used T-cell antigen receptors (TCRs) different from those used by T(reg) cells in other locations, implying an important role for local antigens in shaping the colonic T(reg)-cell population. Many of the local antigens seemed to be derived from commensal bacteria, on the basis of the in vitro reactivity of common colon T(reg) TCRs. These TCRs did not facilitate thymic T(reg)-cell development, implying that many colonic T(reg) cells arise instead by means of antigen-driven peripheral T(reg)-cell development. Further analysis of two of these TCRs by the creation of retroviral bone marrow chimaeras and a TCR transgenic line revealed that microbiota indigenous to our mouse colony was required for the generation of colonic T(reg) cells from otherwise naive T cells. If T cells expressing these TCRs fail to undergo T(reg)-cell development and instead become effector cells, they have the potential to induce colitis, as evidenced by adoptive transfer studies. These results suggest that the efficient peripheral generation of antigen-specific populations of T(reg) cells in response to an individual's microbiota provides important post-thymic education of the immune system to foreign antigens, thereby providing tolerance to commensal microbiota.     

ICOS co-stimulatory receptor is essential for T-cell activation and function

T-lymphocyte activation and immune function are regulated by co-stimulatory molecules. CD28, a receptor for B7 gene products, has a chief role in initiating T-cell immune responses. CTLA4, which binds B7 with a higher affinity, is induced after T-cell activation and is involved in downregulating T-cell responses. The inducible co-stimulatory molecule (ICOS), a third member of the CD28/CTLA4 family, is expressed on activated T cells. Its ligand B7H/B7RP-1 is expressed on B cells and in non-immune tissues after injection of lipopolysaccharide into animals. To understand the role of ICOS in T-cell activation and function, we generated and analysed ICOS-deficient mice. Here we show that T-cell activation and proliferation are defective in the absence of ICOS. In addition, ICOS -/- T cells fail to produce interleukin-4 when differentiated in vitro or when primed in vivo. ICOS is required for humoral immune responses after immunization with several antigens. ICOS-/- mice showed greatly enhanced susceptibility to experimental autoimmune encephalomyelitis, indicating that ICOS has a protective role in inflammatory autoimmune diseases.     

Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. Here we show that a spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA.     

TACI and BCMA are receptors for a TNF homologue implicated in B-cell autoimmune disease

B cells are important in the development of autoimmune disorders by mechanisms involving dysregulated polyclonal B-cell activation, production of pathogenic antibodies, and co-stimulation of autoreactive T cells. zTNF4 (BLyS, BAFF, TALL-1, THANK) is a member of the tumour necrosis factor (TNF) ligand family that is a potent co-activator of B cells in vitro and in vivo. Here we identify two receptors for zTNF4 and demonstrate a relationship between zTNF4 and autoimmune disease. Transgenic animals overexpressing zTNF4 in lymphoid cells develop symptoms characteristic of systemic lupus erythaematosus (SLE) and expand a rare population of splenic B-Ia lymphocytes. In addition, circulating zTNF4 is more abundant in NZBWF1 and MRL-lpr/lpr mice during the onset and progression of SLE. We have identified two TNF receptor family members, TACI and BCMA, that bind zTNF4. Treatment of NZBWF1 mice with soluble TACI-Ig fusion protein inhibits the development of proteinuria and prolongs survival of the animals. These findings demonstrate the involvement of zTNF4 and its receptors in the development of SLE and identify TACI-Ig as a promising treatment of autoimmune disease in humans.     

Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitope

In autoimmune type 1 diabetes, pathogenic T lymphocytes are associated with the specific destruction of insulin-producing beta-islet cells. Identification of the autoantigens involved in triggering this process is a central question. Here we examined T cells from pancreatic draining lymph nodes, the site of islet-cell-specific self-antigen presentation. We cloned single T cells in a non-biased manner from pancreatic draining lymph nodes of subjects with type 1 diabetes and from non-diabetic controls. A high degree of T-cell clonal expansion was observed in pancreatic lymph nodes from long-term diabetic patients but not from control subjects. The oligoclonally expanded T cells from diabetic subjects with DR4, a susceptibility allele for type 1 diabetes, recognized the insulin A 1-15 epitope restricted by DR4. These results identify insulin-reactive, clonally expanded T cells from the site of autoinflammatory drainage in long-term type 1 diabetics, indicating that insulin may indeed be the target antigen causing autoimmune diabetes.     

Antigen-induced apoptotic death of Ly-1 B cells responsible for autoimmune disease in transgenic mice.

Studies on transgenic mice expressing immunoglobulins against self-antigens have shown that self-tolerance is maintained by active elimination (clonal deletion), functional inactivation (clonal anergy) of self-reactive B cells, or a combination of both. We have established and characterized a transgenic mouse line expressing an anti-erythrocyte autoantibody. In contrast to other autoantibody transgenic lines, about 50% of the animals of this transgenic line suffer from autoimmune disease, indicating a loss of self-tolerance. Here we show that peritoneal Ly-1 B cells (also known as B-1 cells) are responsible for this autoimmune disease in our transgenic mice. A few self-reactive Ly-1 B cells that have somehow escaped the deletion mechanism expand in the peritoneum because of the absence of self-antigen. These Ly-1 B cells are eliminated in vivo by apoptosis once exposed to self-antigen. On the basis of these results we propose a novel autoantibody production mechanism whereby self-reactive B cells sequestered in compartments free of self-antigens may survive, proliferate and be activated for generation of pathogenic autoantibodies in autoimmune diseases.     

Can B cells turn on virgin T cells?

The first event in the initiation of an immune response is the capture and presentation of antigen to T cells. Such presentation involves two distinct steps: (1) display of the antigen, which requires uptake, processing and re-expression of the antigen in association with MHC molecules on the presenting cell surface; and (2) triggering, in which the presenting cell provides signals leading to the activation of the responding T cell. Two sorts of cells can capture antigens, the 'professional' antigen-presenting cells (APCs) such as dendritic cells and macrophages, and the B cells. Both types of cells can display antigens and the APCs are known to be able to trigger resting T cells. But despite in vitro evidence that certain B-cell types can reactivate previously-activated T cells, it is not yet clear whether a B cell can initiate an immune response by providing the signals necessary to activate a resting T cell. We reasoned that resting B cells should not have this capacity because of the problems this would present with tolerance to self idiotypes. By exploiting the unique properties of the avian haematopoietic system, we have examined the presenting capacity of B cells in vivo and found that resting B cells are indeed unable to activate resting T cells.     

Response to self antigen imprints regulatory memory in tissues

Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (T(reg) cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated T(reg) cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, T(reg) cells function to confer 'regulatory memory' to the target tissue. These findings provide a framework for understanding how T(reg) cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.     

Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors

Autoreactive B cells are present in the lymphoid tissues of healthy individuals, but typically remain quiescent. When this homeostasis is perturbed, the formation of self-reactive antibodies can have serious pathological consequences. B cells expressing an antigen receptor specific for self-immunoglobulin-gamma (IgG) make a class of autoantibodies known as rheumatoid factor (RF). Here we show that effective activation of RF+ B cells is mediated by IgG2a-chromatin immune complexes and requires the synergistic engagement of the antigen receptor and a member of the MyD88-dependent Toll-like receptor (TLR) family. Inhibitor studies implicate TLR9. These data establish a critical link between the innate and adaptive immune systems in the development of systemic autoimmune disease and explain the preponderance of autoantibodies reactive with nucleic acid-protein particles. The unique features of this dual-engagement pathway should facilitate the development of therapies that specifically target autoreactive B cells.     

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.     

Innate immunity and intestinal microbiota in the development of Type 1 diabetes

Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.     

Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells

On activation, T cells undergo distinct developmental pathways, attaining specialized properties and effector functions. T-helper (T(H)) cells are traditionally thought to differentiate into T(H)1 and T(H)2 cell subsets. T(H)1 cells are necessary to clear intracellular pathogens and T(H)2 cells are important for clearing extracellular organisms. Recently, a subset of interleukin (IL)-17-producing T (T(H)17) cells distinct from T(H)1 or T(H)2 cells has been described and shown to have a crucial role in the induction of autoimmune tissue injury. In contrast, CD4+CD25+Foxp3+ regulatory T (T(reg)) cells inhibit autoimmunity and protect against tissue injury. Transforming growth factor-beta (TGF-beta) is a critical differentiation factor for the generation of T(reg) cells. Here we show, using mice with a reporter introduced into the endogenous Foxp3 locus, that IL-6, an acute phase protein induced during inflammation, completely inhibits the generation of Foxp3+ T(reg) cells induced by TGF-beta. We also demonstrate that IL-23 is not the differentiation factor for the generation of T(H)17 cells. Instead, IL-6 and TGF-beta together induce the differentiation of pathogenic T(H)17 cells from naive T cells. Our data demonstrate a dichotomy in the generation of pathogenic (T(H)17) T cells that induce autoimmunity and regulatory (Foxp3+) T cells that inhibit autoimmune tissue injury.     

T cells become licensed in the lung to enter the central nervous system

The blood–brain barrier (BBB) and the environment of the central nervous system (CNS) guard the nervous tissue from peripheral immune cells. In the autoimmune disease multiple sclerosis, myelin-reactive T-cell blasts are thought to transgress the BBB and create a pro-inflammatory environment in the CNS, thereby making possible a second autoimmune attack that starts from the leptomeningeal vessels and progresses into the parenchyma. Using a Lewis rat model of experimental autoimmune encephalomyelitis, we show here that contrary to the expectations of this concept, T-cell blasts do not efficiently enter the CNS and are not required to prepare the BBB for immune-cell recruitment. Instead, intravenously transferred T-cell blasts gain the capacity to enter the CNS after residing transiently within the lung tissues. Inside the lung tissues, they move along and within the airways to bronchus-associated lymphoid tissues and lung-draining mediastinal lymph nodes before they enter the blood circulation from where they reach the CNS. Effector T cells transferred directly into the airways showed a similar migratory pattern and retained their full pathogenicity. On their way the T cells fundamentally reprogrammed their gene-expression profile, characterized by downregulation of their activation program and upregulation of cellular locomotion molecules together with chemokine and adhesion receptors. The adhesion receptors include ninjurin 1, which participates in T-cell intravascular crawling on cerebral blood vessels. We detected that the lung constitutes a niche not only for activated T cells but also for resting myelin-reactive memory T cells. After local stimulation in the lung, these cells strongly proliferate and, after assuming migratory properties, enter the CNS and induce paralytic disease. The lung could therefore contribute to the activation of potentially autoaggressive T cells and their transition to a migratory mode as a prerequisite to entering their target tissues and inducing autoimmune disease.     

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

In humans, up to 75% of newly generated B cells and about 30% of mature B cells show some degree of autoreactivity. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model B-cell antigen receptor (BCR) transgenic systems have highlighted the critical role of functional unresponsiveness or ‘anergy’. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B-cell tolerance. However, it remains unclear whether the mature diverse B-cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which BCR signalling rapidly and robustly induces green fluorescent protein expression under the control of the Nur77 regulatory region, antigen-dependent and antigen-independent BCR signalling events in vivo during B-cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure, in turn, tunes the responsiveness of BCR signalling in B cells at least partly by downmodulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or anergy exists in the mature B-cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.     

ATP drives lamina propria T(H)17 cell differentiation

Interleukin (IL)-17-producing CD4(+) T lymphocytes (T(H)17 cells) constitute a subset of T-helper cells involved in host defence and several immune disorders. An intriguing feature of T(H)17 cells is their selective and constitutive presence in the intestinal lamina propria. Here we show that adenosine 5'-triphosphate (ATP) that can be derived from commensal bacteria activates a unique subset of lamina propria cells, CD70(high)CD11c(low) cells, leading to the differentiation of T(H)17 cells. Germ-free mice exhibit much lower concentrations of luminal ATP, accompanied by fewer lamina propria T(H)17 cells, compared to specific-pathogen-free mice. Systemic or rectal administration of ATP into these germ-free mice results in a marked increase in the number of lamina propria T(H)17 cells. A CD70(high)CD11c(low) subset of the lamina propria cells expresses T(H)17-prone molecules, such as IL-6, IL-23p19 and transforming-growth-factor-beta-activating integrin-alphaV and -beta8, in response to ATP stimulation, and preferentially induces T(H)17 differentiation of co-cultured naive CD4(+) T cells. The critical role of ATP is further underscored by the observation that administration of ATP exacerbates a T-cell-mediated colitis model with enhanced T(H)17 differentiation. These observations highlight the importance of commensal bacteria and ATP for T(H)17 differentiation in health and disease, and offer an explanation of why T(H)17 cells specifically present in the intestinal lamina propria.     

Class IV semaphorin Sema4A enhances T-cell activation and interacts with Tim-2

Semaphorins are a family of phylogenetically conserved soluble and transmembrane proteins. Although many soluble semaphorins deliver guidance cues to migrating axons during neuronal development, some members are involved in immune responses. For example, CD100 (also known as Sema4D), a class IV transmembrane semaphorin, signals through CD72 to effect nonredundant roles in immune responses in a ligand-receptor system that is distinct from any seen previously in the nervous system. Here we report that the class IV semaphorin Sema4A, which is expressed in dendritic cells and B cells, enhances the in vitro activation and differentiation of T cells and the in vivo generation of antigen-specific T cells. Treating mice with monoclonal antibodies against Sema4A blocks the development of an experimental autoimmune encephalomyelitis that is induced by an antigenic peptide derived from myelin oligodendrocyte glycoprotein. In addition, expression cloning shows that the Sema4A receptor is Tim-2, a member of the family of T-cell immunoglobulin domain and mucin domain (Tim) proteins that is expressed on activated T cells.     

The T-cell repertoire is heavily influenced by tolerance to polymorphic self-antigens

T cells with V beta 3+ alpha beta receptors are deleted by self-tolerance in mice with particular major histocompatibility complex/self-antigen combinations. This also occurs for other V beta elements. Polymorphism in the major histocompatibility complex and/or the self-antigens that cause massive deletion of T cells using particular V beta elements may be maintained by the need to balance the advantage of a diverse T-cell repertoire against the potential involvement of those elements in autoimmune disease.     

Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells (TH1 and TH2) at the site of inflammation. The diversity of TH1 and TH2 function is not predetermined but depends on signals that drive the cells towards either subset. Histamine, released from effector cells (mast cells and basophils) during inflammatory reactions can influence immune response. Here we report that histamine enhances TH1-type responses by triggering the histamine receptor type 1 (H1R), whereas both TH1- and TH2-type responses are negatively regulated by H2R through the activation of different biochemical intracellular signals. In mice, deletion of H1R results in suppression of interferon (IFN)-gamma and dominant secretion of TH2 cytokines (interleukin (IL)-4 and IL-13). Mutant mice lacking H2R showed upregulation of both TH1 and TH2 cytokines. Relevant to T-cell cytokine profiles, mice lacking H1R displayed increased specific antibody response with increased immunoglobulin-epsilon (IgE) and IgG1, IgG2b and IgG3 compared with mice lacking H2R. These findings account for an important regulatory mechanism in the control of inflammatory functions through effector-cell-derived histamine.     

Self-tolerance alters T-cell receptor expression in an antigen-specific MHC restricted immune response

The influence of major histocompatibility complex (MHC) gene products on the T-lymphocyte alpha beta receptor (TCR) repertoire is well documented, but how specificity is also generated for a diverse array of foreign peptide antigens is unknown. One proposed mechanism is that the TCR repertoire is selected by the recognition of processed self-antigens bound to MHC molecules. Here, we examine the influence of non-MHC-encoded self-antigens on the TCR repertoire expressed in an antigen-specific immune response. Most pigeon cytochrome c-specific, Ek alpha Ek beta (Ek) Ia-restricted T cells from B10.A mice express a product of the V alpha 11 gene family in association with a V beta 3 gene-encoded protein. We therefore examined V alpha 11 and V beta 3 gene expression in cytochrome c-specific T-cell lines derived from various mouse strains with different non-MHC genetic backgrounds. T cells from several strains failed to express any V beta 3 due to tolerance induced by Mlsc-encoded self-antigens. Variable levels of V alpha 11 messenger RNA (mRNA) were expressed by antigen-specific T cells from all the strains. In one strain V beta 3 was expressed in the relative absence of V alpha 11. These results directly demonstrate that self-tolerance alters TCR gene usage in the immune response to a foreign antigen, and indicate that TCR V alpha and V beta proteins may, in part, be independently selected.