Persistent virus-specific and clonally expanded antibody-secreting cells respond to induced self-antigen in the CNS.

B cells contribute to the pathogenesis of both cellular- and humoral-mediated central nervous system (CNS) inflammatory diseases through a variety of mechanisms. In such conditions, B cells may enter the CNS parenchyma and contribute to local tissue destruction. It remains unexplored, however, how infection and autoimmunity drive transcriptional phenotypes, repertoire features, and antibody functionality. Here, we profiled B cells from the CNS of murine models of intracranial (i.c.) viral infections and autoimmunity. We identified a population of clonally expanded, antibody-secreting cells (ASCs) that had undergone class-switch recombination and extensive somatic hypermutation following i.c. infection with attenuated lymphocytic choriomeningitis virus (rLCMV). Recombinant expression and characterisation of these antibodies revealed specificity to viral antigens (LCMV glycoprotein GP), correlating with ASC persistence in the brain weeks after resolved infection. Furthermore, these virus-specific ASCs upregulated proliferation and expansion programs in response to the conditional and transient induction of the LCMV GP as a neo-self antigen by astrocytes. This class-switched, clonally expanded, and mutated population persisted and was even more pronounced when peripheral B cells were depleted prior to autoantigen induction in the CNS. In contrast, the most expanded B cell clones in mice with persistent expression of LCMV GP in the CNS did not exhibit neo-self antigen specificity, potentially a consequence of local tolerance induction. Finally, a comparable population of clonally expanded, class-switched, and proliferating ASCs was detected in the cerebrospinal fluid of relapsing multiple sclerosis (RMS) patients. Taken together, our findings support the existence of B cells that populate the CNS and are capable of responding to locally encountered autoantigens.

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity.

Adaptive immune repertoires are composed by the ensemble of B and T-cell receptors within an individual, reflecting both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Here, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following lymphocytic choriomeningitis virus (LCMV) infection, CD8+ T cells with binding specificity restricted to two distinct LCMV peptides, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T-cell inflation, and regulation. Together, this dataset provides a resource for immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

Tissue-resident memory CD8+ T cells cooperate with CD4+ T cells to drive compartmentalized immunopathology in the CNS.

In chronic inflammatory diseases of the central nervous system (CNS), immune cells persisting behind the blood-brain barrier are supposed to promulgate local tissue destruction. The drivers of such compartmentalized inflammation remain unclear, but tissue-resident memory T cells (TRM) represent a potentially important cellular player in this process. Here, we investigated whether resting CD8+ TRM persisting after cleared infection with attenuated lymphocytic choriomeningitis virus (LCMV) can initiate immune responses directed against cognate self-antigen in the CNS. We demonstrated that time-delayed conditional expression of the LCMV glycoprotein as neo-self-antigen by glia cells reactivated CD8+ TRM. Subsequently, CD8+ TRM expanded and initiated CNS inflammation and immunopathology in an organ-autonomous manner independently of circulating CD8+ T cells. However, in the absence of CD4+ T cells, TCF-1+ CD8+ TRM failed to expand and differentiate into terminal effectors. Similarly, in human demyelinating CNS autoimmune lesions, we found CD8+ T cells expressing TCF-1 that predominantly exhibited a TRM-like phenotype. Together, our study provides evidence for CD8+ TRM-driven CNS immunopathology and sheds light on why inflammatory processes may evade current immunomodulatory treatments in chronic autoimmune CNS conditions.

Contrasting role of NLRP12 in autoinflammation: evidence from a case report and mouse models.

OBJECTIVE: To explore at the molecular level the phenotype of a patient suffering an autoinflammatory syndrome which was diagnosed as familial cold autoinflammatory syndrome type 2 (FCAS-2). To explore the functions of Nlrp12 in inflammation using mouse models. METHODS: Whole exome sequencing and Nlrp12 targeted resequencing were performed on DNA isolated from the patient and her family members. In vivo and ex vivo models of inflammation (urate crystals-dependent acute joint inflammation and urate crystals-induced peritonitis) were analysed in Nlrp12-deficient and Nlrp12-competent mice. RESULTS: A rare missense NLRP12 variant (c.857C>T, p.P286L) was identified in the patient and her healthy relatives. Nlrp12-deficient mice exhibit reduced systemic inflammation and neutrophilic infiltration. CONCLUSION: Nlrp12 mediates proinflammatory functions in mice. In humans, the identification of Nlrp12 variants must be cautiously interpreted depending on clinical and paraclinical data to diagnose FCAS-2.

Persistence of self-reactive CD8+ T cells in the CNS requires TOX-dependent chromatin remodeling.

Self-reactive CD8+ T cells are important mediators of progressive tissue damage in autoimmune diseases, but the molecular program underlying these cells' functional adaptation is unclear. Here we characterize the transcriptional and epigenetic landscape of self-reactive CD8+ T cells in a mouse model of protracted central nervous system (CNS) autoimmunity and compare it to populations of CNS-resident memory CD8+ T cells emerging from acute viral infection. We find that autoimmune CD8+ T cells persisting at sites of self-antigen exhibit characteristic transcriptional regulation together with distinct epigenetic remodeling. This self-reactive CD8+ T cell fate depends on the transcriptional regulation by the DNA-binding HMG-box protein TOX which remodels more than 400 genomic regions including loci such as Tcf7, which is central to stemness of CD8+ T cells. Continuous exposure to CNS self-antigen sustains TOX levels in self-reactive CD8+ T cells, whereas genetic ablation of TOX in CD8+ T cells results in shortened persistence of self-reactive CD8+ T cells in the inflamed CNS. Our study establishes and characterizes the genetic differentiation program enabling chronic T cell-driven immunopathology in CNS autoimmunity.

NKG2D ligands in inflammatory joint diseases: analysis in human samples and mouse models.

OBJECTIVES: NKG2D ligands (NKG2DLs) are stress-inducible molecules involved in multiple inflammatory settings. In this work, we quantified MICA, an NKG2DL, in the synovial fluid of patients suffering various arthritides and measured Nkg2dLs gene expression in murine models of acute joint inflammation. METHODS: Soluble MICA (sMICA) was quantified by ELISA is synovial fluids harvested from patients suffering osteoarthritis, rheumatoid arthritis, psoriatic arthritis, calcium pyrophosphate crystal arthritis, urate crystal arthritis and reactive arthritis. Transcripts encoding murine NKG2DLs were quantified by RT-qPCR in the joints of mouse models of rheumatoid arthritis, urate crystal arthritis and osteoarthritis. RESULTS: Marked overproduction of sMICA was observed in the synovial fluid of RA patients. Mouse studies highlighted the complex transcriptional regulation of Nkg2d ligands encoding genes depending on the inflammatory setting and microenvironment CONCLUSIONS: sMICA quantification could be an interesting biomarker to identify acute inflammation in RA patients in whom classical markers (i.e. anti-citrullinated protein antibodies, ACPA) are undetectable.

JAK-STAT Targeting Offers Novel Therapeutic Opportunities in Sepsis.

Sepsis is a life-threatening condition caused by exaggerated host responses to infections taking place in two phases: (i) a systemic (hyper)inflammatory response syndrome (SIRS), participating in multiple organ failure (MOF), a major complication of septic shock, followed by (ii) a compensatory anti-inflammatory response syndrome (CARS), leading to sepsis-induced immunosuppression and resulting in late infections and long-term mortality. The Janus kinase-signal transducer and activator of transcription (JAK-STAT)-dependent signaling pathway is involved in both manifestations, hence playing a key role during sepsis. It is also involved in emergency myelopoiesis, which participates in host defense. The aim of this review is to highlight and refine the recent implications of this signaling pathway in sepsis and illustrate why its central position makes it a potential biomarker and therapeutic target.

A mouse model of MSU-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1ß as relevant therapy for gout patients.

Rationale: The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1ß in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1ß secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Methods: Acute urate crystal inflammation was obtained by subcutaneous injections of MSU crystals in mice. Symptoms were followed by scoring, cytokine quantification by ELISA and western blot, gene expression by RT-qPCR and RNAseq; Magnetic Resonance Imaging was also used to assess inflammation. Results: We provide an extensive clinical, biological and molecular characterization of an acute uratic inflammation mouse model which accurately mimics human gout. We report the efficacy of topical imiquimod treatment and its impact on Interferon-dependent down modulation of Il-1ß gene expression in this experimental model. Conclusion: Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities for gout patients.

Temporomandibular joint damage in K/BxN arthritic mice.

Rheumatoid arthritis (RA) is an autoimmune disease affecting 1% of the world population and is characterized by chronic inflammation of the joints sometimes accompanied by extra-articular manifestations. K/BxN mice, originally described in 1996 as a model of polyarthritis, exhibit knee joint alterations. The aim of this study was to describe temporomandibular joint (TMJ) inflammation and damage in these mice. We used relevant imaging modalities, such as micro-magnetic resonance imaging (µMRI) and micro-computed tomography (µCT), as well as histology and immunofluorescence techniques to detect TMJ alterations in this mouse model. Histology and immunofluorescence for Col-I, Col-II, and aggrecan showed cartilage damage in the TMJ of K/BxN animals, which was also evidenced by µCT but was less pronounced than that seen in the knee joints. µMRI observations suggested an increased volume of the upper articular cavity, an indicator of an inflammatory process. Fibroblast-like synoviocytes (FLSs) isolated from the TMJ of K/BxN mice secreted inflammatory cytokines (IL-6 and IL-1ß) and expressed degradative mediators such as matrix metalloproteinases (MMPs). K/BxN mice represent an attractive model for describing and investigating spontaneous damage to the TMJ, a painful disorder in humans with an etiology that is still poorly understood.

DICER1: A Key Player in Rheumatoid Arthritis, at the Crossroads of Cellular Stress, Innate Immunity, and Chronic Inflammation in Aging.

Loss-of-function or knockout mouse models have established a fundamental role for the RNAse III enzyme DICER1 in development and tissue morphogenesis and/or homeostasis. These functions are currently assumed to result mainly from the DICER1-dependent biogenesis of microRNAs which exhibit important gene expression regulatory properties. However, non-canonical DICER1 functions have recently emerged. These include interaction with the DNA damage response (DDR) pathway and the processing of cytotoxic non-coding RNAs, suggesting that DICER1 might also participate in the regulation of major cellular processes through miRNA-independent mechanisms. Recent findings indicated that reduced Dicer1 expression, which correlates with worsened symptoms in mouse models of joint inflammation, is also noted in fibroblast-like synoviocytes (FLS) harvested from rheumatoid arthritis (RA) patients, as opposed to FLS cultured from biopsies of osteoarthritic patients. In addition, low DICER1 levels are associated with the establishment of cellular stress and its associated responses, such as cellular senescence. Senescent and/or stressed cells are associated with an inflammatory secretome (cytokines and chemokines), as well as with "find-me" and "eat-me" signals which will attract and activate the innate immune compartment (NK cells, macrophages, and neutrophils) to be eliminated. Failure of this immunosurveillance mechanism and improper restauration of homeostasis could lead to the establishment of a systemic and chronic inflammatory state. In this review, we suggest that reduced DICER1 expression contributes to a vicious cycle during which accumulating inflammation and premature senescence, combined to inadequate innate immunity responses, creates the appropriate conditions for the initiation and/or progression of autoimmune-autoinflammatory diseases, such as RA.

Therapeutic Perspectives for Interferons and Plasmacytoid Dendritic Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a multifactorial immune disease exhibiting diverse clinical responses to specific therapeutic agents. Such heterogeneity reflects variable activation of signaling pathways. Consequently, RA physiopathology has been linked to many immune cells and factors, with controversial observations for interferons (IFNs). In this opinion article, we review the roles of these cytokines and the cells that produce them in light of recent data: clinical observations showing that expression of IFN-dependent genes does not reflect RA activity and RA mouse models in which the stimulation of IFN-dependent pathways provided disease protection. We suggest that epicutaneous stimulation of the IFN network is an attractive possibility to limit neutrophil infiltration or activation, thus providing therapeutic benefits to RA patients refractory to current therapies.

Anti-inflammatory effect of active nanofibrous polymeric membrane bearing nanocontainers of atorvastatin complexes.

AIM: We developed polymeric membranes for local administration of nonsoluble anti-inflammatory statin, as potential wound patch in rheumatic joint or periodontal lesions. METHODS: Electrospun polycaprolactone membranes were fitted with polysaccharide-atorvastatin nanoreservoirs by using complexes with poly-aminocyclodextrin. Characterization methods are UV-Visible and X-ray photoelectron spectroscopy, molecular dynamics, scanning and transmission electron microscopy. In vitro, membranes were seeded with macrophages, and inflammatory cytokine expression were monitored. RESULTS & CONCLUSION: Stable inclusion complexes were formed in solution (1:1 stability constant 368 M-1, -117.40 kJ mol-1), with supramolecular globular organization (100 nm, substructure 30 nm). Nanoreservoir technology leads to homogeneous distribution of atorvastatin calcium trihydrate complexes in the membrane. Quantity embedded was estimated (70-90 µg in 30 µm × 6 mm membrane). Anti-inflammatory effect by cell contact-dependent release reached 60% inhibition for TNF-α and 80% for IL-6. The novelty resides in the double protection offered by the cyclodextrins as drug molecular chaperones, with further embedding into biodegradable nanoreservoirs. The strategy is versatile and can target other diseases.

Therapeutic Modulation of Plasmacytoid Dendritic Cells in Experimental Arthritis.

OBJECTIVE: The role of plasmacytoid dendritic cells (PDCs) and type I interferons (IFNs) in rheumatoid arthritis (RA) remains a subject of controversy. This study was undertaken to explore the contribution of PDCs and type I IFNs to RA pathogenesis using various animal models of PDC depletion and to monitor the effect of localized PDC recruitment and activation on joint inflammation and bone damage. METHODS: Mice with K/BxN serum-induced arthritis, collagen-induced arthritis, and human tumor necrosis factor transgene insertion were studied. Symptoms were evaluated by visual scoring, quantification of paw swelling, determination of cytokine levels by enzyme-linked immunosorbent assay, and histologic analysis. Imiquimod-dependent therapeutic effects were monitored by transcriptome analysis (using quantitative reverse transcriptase-polymerase chain reaction) and flow cytometric analysis of the periarticular tissue. RESULTS: PDC-deficient mice showed exacerbation of inflammatory and arthritis symptoms after arthritogenic serum transfer. In contrast, enhancing PDC recruitment and activation to arthritic joints by topical application of the Toll-like receptor 7 (TLR-7) agonist imiquimod significantly ameliorated arthritis in various mouse models. Imiquimod induced an IFN signature and led to reduced infiltration of inflammatory cells. CONCLUSION: The therapeutic effects of imiquimod on joint inflammation and bone destruction are dependent on TLR-7 sensing by PDCs and type I IFN signaling. Our findings indicate that local recruitment and activation of PDCs represents an attractive therapeutic opportunity for RA patients.