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Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Objective. Anti–citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA) and have long been regarded as pathogenic. Despite substantial in vitro evidence supporting this claim, reports investigating the proinflammatory effects of ACPAs in animal models of arthritis are rare and include mixed results. Here, we sequenced the plasmablast antibody repertoire of a patient with RA and functionally characterized the encoded ACPAs. Methods. We expressed ACPAs from the antibody repertoire of a patient with RA and characterized their autoantigen specificities on antigen arrays and enzyme-linked immunosorbent assays. Binding affinities were estimated by bio-layer interferometry. Select ACPAs (n = 9) were tested in the collagen antibody–induced arthritis (CAIA) mouse model to evaluate their effects on joint inflammation. Results. Recombinant ACPAs bound preferentially and with high affinity (nanomolar range) to citrullinated (cit) autoantigens (primarily histones and fibrinogen) and to auto-cit peptidylarginine deiminase 4 (PAD4). ACPAs were grouped for in vivo testing based on their predominant cit-antigen specificities. Unexpectedly, injections of recombinant ACPAs significantly reduced paw thickness and arthritis severity in CAIA mice as compared with isotype-matched control antibodies (P ≤ 0.001). Bone erosion, synovitis, and cartilage damage were also significantly reduced (P ≤ 0.01). This amelioration of CAIA was observed for all the ACPAs tested and was independent of cit-PAD4 and cit-fibrinogen specificities. Furthermore, disease amelioration was more prominent when ACPAs were injected at earlier stages of CAIA than at later phases of the model. Conclusion. Recombinant patient-derived ACPAs ameliorated CAIA. Their antiinflammatory effects were more preventive than therapeutic. This study highlights a potential protective role for ACPAs in arthritis. [ABSTRACT FROM AUTHOR]

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens. Periodontal disease and rheumatoid arthritis: The incidence of periodontal disease is high in individuals with rheumatoid arthritis (RA) who also have anti-citrullinated protein antibodies (ACPAs), suggesting a link between these two diseases. Brewer et al. carried out a paired analysis of human and bacterial transcriptomes from blood samples collected longitudinally from RA patients with and without periodontal disease. They identified transcriptional signatures within inflammatory monocyte subsets that correlated with repeated oral bacteremias and clinical arthritis flares in patients with RA and periodontal disease. These oral bacteria were broadly citrullinated, and some of these citrullinated epitopes were the targets of ACPA expressed by RA blood plasmablasts that have undergone affinity maturation. These results confirm that periodontal disease can cause breaches in oral mucosa that release citrullinated bacteria into the blood, which activates inflammatory monocytes and ACPA-specific B cells. —CF [ABSTRACT FROM AUTHOR]

Here, we offer a roadmap for what might be studied next in understanding how EBV triggers MS. We focus on two areas: The first area concerns the molecular mechanisms underlying how clonal antibody in the CSF emanates in widespread molecular mimicry to key antigens in the nervous system including GlialCAM, a protein associated with chloride channels. A second and equally high priority in the roadmap concerns various therapeutic approaches that are related to blocking the mechanisms whereby EBV triggers MS. Therapies deserving of attention include clinical trials with antivirals and the development of 'inverse' vaccines based on nucleic acid technologies to control or to eradicate the consequences of EBV infection. High enthusiasm is given to continuation of ongoing clinical trials of cellular adoptive therapy to attack EBV‐infected cells. Clinical trials of vaccines to EBV are another area deserving attention. These suggested topics involving research on mechanism, and the design, implementation and performance of well‐designed trials are not intended to be an exhaustive list. We have splendid tools available to our community of medical scientists to tackle how EBV triggers MS and then to perhaps change the world with new therapies to potentially eradicate MS, as we have done with nearly complete success for poliomyelitis. [ABSTRACT FROM AUTHOR]

The renin-angiotensin-aldosterone system (RAAS) is a key hormonal system regulating blood pressure. However, expression of RAAS components has recently been detected in immune cells, and the RAAS has been implicated in several mouse models of autoimmune disease. Here, we have identified Ang II as a paracrine mediator, sustaining inflammation in the CNS in the EAE mouse model of MS via TGF-β. Ang II type 1 receptors (AT1Rs) were found to be primarily expressed in CNS-resident cells during EAE. In vitro, astrocytes and microglia responded to Ang II treatment by inducing TGF-β expression via a pathway involving the TGF-β-activating protease thrombospondin-1 (TSP-1). TGF-β upregulation in astrocytes and microglia during EAE was blocked with candesartan (CA), an inhibitor of AT1R. Treatment of EAE with CA ameliorated paralysis and blunted lymphocyte infiltration into the CNS, outcomes that were also seen with genetic ablation of AT1Ra and treatment with an inhibitor of TSP-1. These data suggest that AT1R antagonists, frequently prescribed as antihypertensives, may be useful to interrupt this proinflammatory, CNS-specific pathway in individuals with MS., Introduction The renin-angiotensin-aldosterone system (RAAS) is a major endocrine system, regulating blood pressure and body fluid homeostasis. Angiotensin peptides not only act on the vasculature, heart, kidney, and adrenal gland [...]

Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Further- more, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glyco-protein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow’s milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet. [ABSTRACT FROM AUTHOR]

IgG4-related disease (IgG4-RD) is a fibroinflammatory condition involving loss of B-cell tolerance and production of autoantibodies. However, the relevant targets and role of these aberrant humoral immune responses are not defined. Our aim was to identify novel autoantibodies and autoantigen targets that promote pathogenic responses in IgG4-RD. We sequenced plasmablast antibody repertoires in patients with IgG4-RD. Representative mAbs were expressed and their specificities characterized by using cytokine microarrays. The role of anti–IL-1 receptor antagonist (IL-1RA) autoantibodies was investigated by using in vitro assays. We identified strong reactivity against human IL-1RA by using a clonally expanded plasmablast-derived mAb from a patient with IgG4-RD. Plasma from patients with IgG4-RD exhibited elevated levels of reactivity against IL-1RA compared with plasma from the controls and neutralized IL-1RA activity, resulting in inflammatory and fibrotic mediator production in vitro. IL-1RA was detected in lesional tissues from patients with IgG4-RD. Patients with anti–IL-1RA autoantibodies of the IgG4 subclass had greater numbers of organs affected than did those without anti–IL-1RA autoantibodies. Peptide analyses identified IL-1RA epitopes targeted by anti–IL-1RA antibodies at sites near the IL-1RA/IL-1R interface. Serum from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) also had elevated levels of anti–IL-1RA autoantibodies compared with those of the controls. A subset of patients with IgG4-RD have anti–IL-1RA autoantibodies, which promote proinflammatory and profibrotic meditator production via IL-1RA neutralization. These findings support a novel immunologic mechanism underlying the pathogenesis of IgG4-RD. Anti–IL-1RA autoantibodies are also present in a subset of patients with SLE and RA, suggesting a potential common pathway in multiple autoimmune diseases. [ABSTRACT FROM AUTHOR]

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysregulation and breaks in tolerance that lead to the production of pathogenic autoantibodies. We performed single-cell RNA sequencing of B cells from healthy donors and individuals with SLE which revealed upregulated CD52 expression in SLE patients. We further demonstrate that SLE patients exhibit significantly increased levels of B cell surface CD52 expression and plasma soluble CD52, and levels of soluble CD52 positively correlate with measures of lupus disease activity. Using CD52-deficient JeKo-1 cells, we show that cells lacking surface CD52 expression are hyperresponsive to B cell receptor (BCR) signaling, suggesting an inhibitory role for the surface-bound protein. In healthy donor B cells, antigen-specific BCR-activation initiated CD52 cleavage in a phospholipase C dependent manner, significantly reducing cell surface levels. Experiments with recombinant CD52-Fc showed that soluble CD52 inhibits BCR signaling in a manner partially-dependent on Siglec-10. Moreover, incubation of unstimulated B cells with CD52-Fc resulted in the reduction of surface immunoglobulin and CXCR5. Prolonged incubation of B cells with CD52 resulted in the expansion of IgD+IgMlo anergic B cells. In summary, our findings suggest that CD52 functions as a homeostatic protein on B cells, by inhibiting responses to BCR signaling. Further, our data demonstrate that CD52 is cleaved from the B cell surface upon antigen engagement, and can suppress B cell function in an autocrine and paracrine manner. We propose that increased expression of CD52 by B cells in SLE represents a homeostatic mechanism to suppress B cell hyperactivity. [ABSTRACT FROM AUTHOR]

The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis. Author summary: Viral hepatitis is responsible for more than one million annual deaths worldwide and may progress to liver cirrhosis and hepatocellular carcinoma. The main metabolic cell type of the liver is the hepatocyte. In viral hepatitis, type I interferon (IFN-I) signaling rewires hepatocyte metabolism and serum metabolites to shape disease pathophysiology–an immune-regulatory circuit that might be therapeutically exploited. Here, we show that hepatocyte-intrinsic antiviral IFN-I signaling is both necessary and sufficient to induce wide-spread metabolic changes in hepatocytes. We identify an IFN-I-mediated induction of the hepatic kynurenine pathway via the rate-limiting and liver-specific enzyme TDO2, which controls serum homeostasis of tryptophan by converting it into kynurenine. Loss of TDO2 triggers a so far unknown compensatory mechanism, resulting in a vast increase of circulating kynurenine independent of hepatocyte intrinsic activity of the related IDO-enzymes. This study provides new insights into how inflammation reprograms metabolism of the liver and the kynurenine pathway during viral hepatitis. [ABSTRACT FROM AUTHOR]

High-throughput single-cell technologies have recently emerged as essential tools in biomedical research with great potential for clinical pathology when studying liquid and solid biopsies. We provide an update on current single-cell methods in cerebrospinal fluid research and diagnostics, focusing on high-throughput cell-type specific proteomic and genomic technologies. Proteomic methods comprising flow cytometry and mass cytometry as well as genomic approaches including immune cell repertoire and single-cell transcriptomic studies are critically reviewed and future directions discussed. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSC) display unique suppressive properties on T-cell immunity, thus representing an attractive vehicle for the treatment of conditions associated with harmful T-cell responses such as organ-specific autoimmunity and graft-versus-host disease. Toll-like receptors (TLR) are primarily expressed on antigen-presenting cells and recognize conserved pathogen-derived components. Ligation of TLR activates multiple innate and adaptive immune response pathways to eliminate and protect against invading pathogens. In this work, we show that TLR expressed on human bone marrow-derived MSC enhanced the immunosuppressive phenotype of MSC. Immunosuppression mediated by TLR was dependent on the production of immunosuppressive kynurenines by the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase- 1 (IDO1). Induction of IDO1 by TLR involved an autocrine interferon (IFN)-β signaling loop, which was dependent on protein kinase R (PKR), but independent of IFN-γ. These data define a new role for TLR in MSC immunobiology, which is to augment the immunosuppressive properties of MSC in the absence of IFN-γ rather than inducing proinflammatory immune response pathways. PKR and IFN-β play a central, previously unidentified role in orchestrating the production of immunosuppressive kynurenines by MSC. [ABSTRACT FROM AUTHOR]

The catabolism of tryptophan to immunosuppressive and neuroactive kynurenines is a key metabolic pathway regulating immune responses and neurotoxicity. The rate-limiting step is controlled by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). IDO is expressed in antigen presenting cells during immune reactions, hepatic TDO regulates blood homeostasis of tryptophan and neuronal TDO influences neurogenesis. While the role of IDO has been described in multiple immunological settings, little is known about TDO's effects on the immune system. TDO-deficiency is neuroprotective in C. elegans and Drosophila by increasing tryptophan and specific kynurenines. Here we have determined the role of TDO in autoimmunity and neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We created reporter-TDO mice for in vivo imaging to show that hepatic but not CNS TDO expression is activated during EAE. TDO deficiency did not influence myelin-specific T cells, leukocyte infiltration into the CNS, demyelination and disease activity. TDO-deficiency protected from neuronal loss in the spinal cord but not in the optic nerves. While this protection did not translate to an improved overt clinical outcome, our data suggest that spatially distinct neuroprotection is conserved in mammals and support TDO as a potential target for treatment of diseases associated with neurodegeneration. [ABSTRACT FROM AUTHOR]

Due to their immunosuppressive properties, human mesenchymal stem cells (hMSC) represent a promising tool for cell-based therapies of autoimmune diseases such as multiple sclerosis (MS). Mouse MSC (mMSC) have been used extensively to characterize and optimize route of administration, motility, cellular targets, and immunosuppressive mechanisms in mouse models of autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). Tryptophan (trp) catabolism by indolamine-2,3-dioxygenase 1 (IDO1) is a chief endogenous metabolic pathway that tightly regulates unwanted immune responses through depletion of trp and generation of immunosuppressive kynurenines (kyn). IDO1 activity contributes to the immunosuppressive phenotype of hMSC. Here, we demonstrate that although IDO1 is inducible in bone marrow-derived mMSC by proinflammatory stimuli such as interferon-g (IFN-g) and ligands of toll-like receptors (TLR), it does not lead to catabolism of trp in vitro. This failure to catabolize trp is not due to defective TLR signaling as demonstrated by induction of interleukin 6 (IL-6) by TLR activation. While mMSC suppressed the activation of antigen-specific myelin oligodendrocyte glycoprotein (MOG)-reactive T-cell receptor (TCR) transgenic T-helper (TH) cells in co-culture, neither pharmacologic inhibition nor genetic ablation of IDO1 reversed this suppressive effect. Finally, systemic administration of both, IDO1-proficient and phenotypically identical IDO1-deficient mMSC, equally resulted in amelioration of EAE. mMSC, unlike hMSC, do not display IDO1-mediated suppression of antigen-specific T-cell responses.

Mesenchymal stem cells (MSC) display unique suppressive properties on T-cell immunity, thus representing an attractive vehicle for the treatment of conditions associated with harmful T-cell responses such as organ-specific autoimmunity and graft-versus-host disease. Toll-like receptors (TLR) are primarily expressed on antigen-presenting cells and recognize conserved pathogen-derived components. Ligation of TLR activates multiple innate and adaptive immune response pathways to eliminate and protect against invading pathogens. In this work, we show that TLR expressed on human bone marrow-derived MSC enhanced the immunosuppressive phenotype of MSC. Immunosuppression mediated by TLR was dependent on the production of immunosuppressive kynurenines by the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase-1 (IDO1). Induction of IDO1 by TLR involved an autocrine interferon (IFN)-beta signaling loop, which was dependent on protein kinase R (PKR), but independent of IFN-gamma. These data define a new role for TLR in MSC immunobiology, which is to augment the immunosuppressive properties of MSC in the absence of IFN-gamma rather than inducing proinflammatory immune response pathways. PKR and IFN-beta play a central, previously unidentified role in orchestrating the production of immunosuppressive kynurenines by MSC.