Multiple sclerosis and the intestine: Chasing the microbial offender.

Multiple sclerosis (MS) affects more than 2.8 million people worldwide but the distribution is not even. Although over 200 gene variants have been associated with susceptibility, studies of genetically identical monozygotic twin pairs suggest that the genetic make-up is responsible for only about 20%-30% of the risk to develop disease, while the rest is contributed by milieu factors. Recently, a new, unexpected player has entered the ranks of MS-triggering or facilitating elements: the human gut microbiota. In this review, we summarize the present knowledge of microbial effects on formation of a pathogenic autoreactive immune response targeting the distant central nervous system and delineate the approaches, both in people with MS and in MS animal models, which have led to this concept. Finally, we propose that a tight combination of investigations of human patients with studies of suitable animal models is the best strategy to functionally characterize disease-associated microbiota and thereby contribute to deciphering pathogenesis of a complex human disease.

A B cell-driven EAE mouse model reveals the impact of B cell-derived cytokines on CNS autoimmunity.

In multiple sclerosis (MS), pathogenic T cell responses are known to be important drivers of autoimmune inflammation. However, increasing evidence suggests an additional role for B cells, which may contribute to pathogenesis via antigen presentation and production of proinflammatory cytokines. However, these B cell effector functions are not featured well in classical experimental autoimmune encephalomyelitis (EAE) mouse models. Here, we compared properties of myelin oligodendrocyte glycoprotein (MOG)-specific and polyclonal B cells and developed an adjuvant-free cotransfer EAE mouse model, where highly activated, MOG-specific induced germinal center B cells provide the critical stimulus for disease development. We could show that high levels of MOG-specific immunoglobulin G (IgGs) are not required for EAE development, suggesting that antigen presentation and activation of cognate T cells by B cells may be important for pathogenesis. As our model allows for B cell manipulation prior to transfer, we found that overexpression of the proinflammatory cytokine interleukin (IL)-6 by MOG-specific B cells leads to an accelerated EAE onset accompanied by activation/expansion of the myeloid compartment rather than a changed T cell response. Accordingly, knocking out IL-6 or tumor necrosis factor α in MOG-specific B cells via CRISPR-Cas9 did not affect activation of pathogenic T cells. In summary, we generated a tool to dissect pathogenic B cell effector function in EAE development, which should improve our understanding of pathogenic processes in MS.

Visualizing the activation of encephalitogenic T cells in the ileal lamina propria by in vivo two-photon imaging.

Autoreactive encephalitogenic T cells exist in the healthy immune repertoire but need a trigger to induce CNS inflammation. The underlying mechanisms remain elusive, whereby microbiota were shown to be involved in the manifestation of CNS autoimmunity. Here, we used intravital imaging to explore how microbiota affect the T cells as trigger of CNS inflammation. Encephalitogenic CD4+ T cells transduced with the calcium-sensing protein Twitch-2B showed calcium signaling with higher frequency than polyclonal T cells in the small intestinal lamina propria (LP) but not in Peyer's patches. Interestingly, nonencephalitogenic T cells specific for OVA and LCMV also showed calcium signaling in the LP, indicating a general stimulating effect of microbiota. The observed calcium signaling was microbiota and MHC class II dependent as it was significantly reduced in germfree animals and after administration of anti-MHC class II antibody, respectively. As a consequence of T cell stimulation in the small intestine, the encephalitogenic T cells start expressing Th17-axis genes. Finally, we show the migration of CD4+ T cells from the small intestine into the CNS. In summary, our direct in vivo visualization revealed that microbiota induced T cell activation in the LP, which directed T cells to adopt a Th17-like phenotype as a trigger of CNS inflammation.

Brain autoimmunity: the CD8 question(s).

In this issue of Immunity, Na et al. (2012) show that the purging of central nervous system (CNS)-specific CD8(+) T cell repertoire requires direct contact with antigen expressing oligodendrocytes and inflammation tips the balance toward autoimmunity.

Brain Autoimmunity and Intestinal Microbiota: 100 Trillion Game Changers.

T cells play a critical role in autoimmune diseases in the brain, particularly in multiple sclerosis (MS). Since T cells are normally prevented from crossing the blood-brain barrier (BBB), autoimmunity requires prior activation of naturally occurring autoreactive T cells in peripheral tissue. Recently, a critical role for the microbiota in this activation process has emerged. Here, we review the role of gut-associated lymphoid tissues (GALT) as a major site for the phenotypic changes that allow the migration of autoreactive T cells to the brain. Additionally, we examine the involvement of the microbiota in clinical MS as well as other brain disorders such as Parkinson's disease (PD), stroke, and psychiatric disorders.

Gut microbiota from multiple sclerosis patients enables spontaneous autoimmune encephalomyelitis in mice.

There is emerging evidence that the commensal microbiota has a role in the pathogenesis of multiple sclerosis (MS), a putative autoimmune disease of the CNS. Here, we compared the gut microbial composition of 34 monozygotic twin pairs discordant for MS. While there were no major differences in the overall microbial profiles, we found a significant increase in some taxa such as Akkermansia in untreated MS twins. Furthermore, most notably, when transplanted to a transgenic mouse model of spontaneous brain autoimmunity, MS twin-derived microbiota induced a significantly higher incidence of autoimmunity than the healthy twin-derived microbiota. The microbial profiles of the colonized mice showed a high intraindividual and remarkable temporal stability with several differences, including Sutterella, an organism shown to induce a protective immunoregulatory profile in vitro. Immune cells from mouse recipients of MS-twin samples produced less IL-10 than immune cells from mice colonized with healthy-twin samples. IL-10 may have a regulatory role in spontaneous CNS autoimmunity, as neutralization of the cytokine in mice colonized with healthy-twin fecal samples increased disease incidence. These findings provide evidence that MS-derived microbiota contain factors that precipitate an MS-like autoimmune disease in a transgenic mouse model. They hence encourage the detailed search for protective and pathogenic microbial components in human MS.

Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.

In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.

Distinct oligoclonal band antibodies in multiple sclerosis recognize ubiquitous self-proteins.

Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.

The Genetic Background of Mice Influences the Effects of Cigarette Smoke on Onset and Severity of Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS.

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

The blood­brain 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.

The immunoregulator soluble TACI is released by ADAM10 and reflects B cell activation in autoimmunity.

BAFF and a proliferation-inducing ligand (APRIL), which control B cell homeostasis, are therapeutic targets in autoimmune diseases. TACI-Fc (atacicept), a soluble fusion protein containing the extracellular domain of the BAFF-APRIL receptor TACI, was applied in clinical trials. However, disease activity in multiple sclerosis unexpectedly increased, whereas in systemic lupus erythematosus, atacicept was beneficial. In this study, we show that an endogenous soluble TACI (sTACI) exists in vivo. TACI proteolysis involved shedding by a disintegrin and metalloproteinase 10 releasing sTACI from activated B cells. The membrane-bound stub was subsequently cleaved by γ-secretase reducing ligand-independent signaling of the remaining C-terminal fragment. The shed ectodomain assembled ligand independently in a homotypic way. It functioned as a decoy receptor inhibiting BAFF- and APRIL-mediated B cell survival and NF-κB activation. We determined sTACI levels in autoimmune diseases with established hyperactivation of the BAFF-APRIL system. sTACI levels were elevated both in the cerebrospinal fluid of the brain-restricted autoimmune disease multiple sclerosis correlating with intrathecal IgG production, as well as in the serum of the systemic autoimmune disease systemic lupus erythematosus correlating with disease activity. Together, we show that TACI is sequentially processed by a disintegrin and metalloproteinase 10 and γ-secretase. The released sTACI is an immunoregulator that shares decoy functions with atacicept. It reflects systemic and compartmentalized B cell accumulation and activation.

Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination.

Active multiple sclerosis lesions show inflammatory changes suggestive of a combined attack by autoreactive T and B lymphocytes against brain white matter. These pathogenic immune cells derive from progenitors that are normal, innocuous components of the healthy immune repertoire but become autoaggressive upon pathological activation. The stimuli triggering this autoimmune conversion have been commonly attributed to environmental factors, in particular microbial infection. However, using the relapsing-remitting mouse model of spontaneously developing experimental autoimmune encephalomyelitis, here we show that the commensal gut flora-in the absence of pathogenic agents-is essential in triggering immune processes, leading to a relapsing-remitting autoimmune disease driven by myelin-specific CD4(+) T cells. We show further that recruitment and activation of autoantibody-producing B cells from the endogenous immune repertoire depends on availability of the target autoantigen, myelin oligodendrocyte glycoprotein (MOG), and commensal microbiota. Our observations identify a sequence of events triggering organ-specific autoimmune disease and these processes may offer novel therapeutic targets.

The gut-brain connection: triggering of brain autoimmune disease by commensal gut bacteria.

In a transgenic model of spontaneous experimental autoimmune encephalomyelitis, autoimmune attack against the CNS requires the presence of an intact commensal gut flora. Extending this observation to human autoimmune disease, such as multiple sclerosis, we postulate that the pathogenic reaction requires the coincidence of at least three factors: a permissive genetic disposition, a pro-inflammatory intestinal microbial profile, and the accumulation of autoreactive T cells in the gut-associated lymphatic tissue. This concept may offer new approaches to diagnostic markers and non-invasive therapies.

Distinction and temporal stability of conformational epitopes on myelin oligodendrocyte glycoprotein recognized by patients with different inflammatory central nervous system diseases.

Autoantibodies targeting conformationally intact myelin oligodendrocyte glycoprotein (MOG) are found in different inflammatory diseases of the CNS, but their antigenic epitopes have not been mapped. We expressed mutants of MOG on human HeLa cells and analyzed sera from 111 patients (104 children, 7 adults) who recognized cell-bound human MOG, but had different diseases, including acute disseminated encephalomyelitis (ADEM), one episode of transverse myelitis or optic neuritis, multiple sclerosis (MS), anti-aquaporin-4 (AQP4)-negative neuromyelitis optica (NMO), and chronic relapsing inflammatory optic neuritis (CRION). We obtained insight into the recognition of epitopes in 98 patients. All epitopes identified were located at loops connecting the ß strands of MOG. The most frequently recognized MOG epitope was revealed by the P42S mutation positioned in the CC'-loop. Overall, we distinguished seven epitope patterns, including the one mainly recognized by mouse mAbs. In half of the patients, the anti-MOG response was directed to a single epitope. The epitope specificity was not linked to certain disease entities. Longitudinal analysis of 11 patients for up to 5 y indicated constant epitope recognition without evidence for intramolecular epitope spreading. Patients who rapidly lost their anti-MOG IgG still generated a long-lasting IgG response to vaccines, indicating that their loss of anti-MOG reactivity did not reflect a general lack of capacity for long-standing IgG responses. The majority of human anti-MOG Abs did not recognize rodent MOG, which has implications for animal studies. Our findings might assist in future detection of potential mimotopes and pave the way to Ag-specific depletion.

Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions.

The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.