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1.
Nature ; 623(7987): 616-624, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37938773

ABSTRACT

Rheumatoid arthritis is a prototypical autoimmune disease that causes joint inflammation and destruction1. There is currently no cure for rheumatoid arthritis, and the effectiveness of treatments varies across patients, suggesting an undefined pathogenic diversity1,2. Here, to deconstruct the cell states and pathways that characterize this pathogenic heterogeneity, we profiled the full spectrum of cells in inflamed synovium from patients with rheumatoid arthritis. We used multi-modal single-cell RNA-sequencing and surface protein data coupled with histology of synovial tissue from 79 donors to build single-cell atlas of rheumatoid arthritis synovial tissue that includes more than 314,000 cells. We stratified tissues into six groups, referred to as cell-type abundance phenotypes (CTAPs), each characterized by selectively enriched cell states. These CTAPs demonstrate the diversity of synovial inflammation in rheumatoid arthritis, ranging from samples enriched for T and B cells to those largely lacking lymphocytes. Disease-relevant cell states, cytokines, risk genes, histology and serology metrics are associated with particular CTAPs. CTAPs are dynamic and can predict treatment response, highlighting the clinical utility of classifying rheumatoid arthritis synovial phenotypes. This comprehensive atlas and molecular, tissue-based stratification of rheumatoid arthritis synovial tissue reveal new insights into rheumatoid arthritis pathology and heterogeneity that could inform novel targeted treatments.


Subject(s)
Arthritis, Rheumatoid , Humans , Arthritis, Rheumatoid/complications , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/pathology , Cytokines/metabolism , Inflammation/complications , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Synovial Membrane/pathology , T-Lymphocytes/immunology , B-Lymphocytes/immunology , Genetic Predisposition to Disease/genetics , Phenotype , Single-Cell Gene Expression Analysis
2.
Annu Rev Immunol ; 2023 Nov 27.
Article in English | MEDLINE | ID: mdl-38011889

ABSTRACT

Autoimmune diseases with B cell-directed therapeutics approved by the US Food and Drug Administration are surprisingly diverse in clinical manifestations and pathophysiology. In this review, we focus on recent clinical and mechanistic insights into the efficacy of B cell depletion in these diverse autoimmune disorders, the rapidly expanding armamentarium of approved agents, and future approaches. The pathogenic roles for B cells include direct functions such as production of autoantibodies and proinflammatory cytokines and indirect functions via antigen presentation to T cells. The efficacy of B cell-depleting strategies varies across diseases and likely reflects the complexity of disease pathogenesis and relative contribution of B cell roles. Additionally, B cell-depleting therapies do not equally target all B cell subsets in all patients, and this likely explains some of the variability in responses. Recent reports of B cell depletion with novel chimeric antigen receptor (CAR) T cell approaches in an expanding number of autoimmune diseases highlight the potential role of B cell depletion in resetting immune tolerance. The relative importance of eliminating autoreactive B cells and plasma cells and approaches to doing so will also be discussed. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

3.
bioRxiv ; 2023 Mar 21.
Article in English | MEDLINE | ID: mdl-36993527

ABSTRACT

Rheumatoid arthritis (RA) is an autoimmune disease initiated by antigen-specific T cells and B cells, which promote synovial inflammation through a complex set of interactions with innate immune and stromal cells. To better understand the phenotypes and clonal relationships of synovial T and B cells, we performed single-cell RNA and repertoire sequencing on paired synovial tissue and peripheral blood samples from 12 donors with seropositive RA ranging from early to chronic disease. Paired transcriptomic-repertoire analyses highlighted 3 clonally distinct CD4 T cells populations that were enriched in RA synovium: T peripheral helper (Tph) and T follicular helper (Tfh) cells, CCL5+ T cells, and T regulatory cells (Tregs). Among these cells, Tph cells showed a unique transcriptomic signature of recent T cell receptor (TCR) activation, and clonally expanded Tph cells expressed an elevated transcriptomic effector signature compared to non-expanded Tph cells. CD8 T cells showed higher oligoclonality than CD4 T cells, and the largest CD8 T cell clones in synovium were highly enriched in GZMK+ cells. TCR analyses revealed CD8 T cells with likely viral-reactive TCRs distributed across transcriptomic clusters and definitively identified MAIT cells in synovium, which showed transcriptomic features of TCR activation. Among B cells, non-naive B cells including age-associated B cells (ABC), NR4A1+ activated B cells, and plasma cells, were enriched in synovium and had higher somatic hypermutation rates compared to blood B cells. Synovial B cells demonstrated substantial clonal expansion, with ABC, memory, and activated B cells clonally linked to synovial plasma cells. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate RA synovium.

4.
Sci Transl Med ; 14(649): eabo0686, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35704599

ABSTRACT

T cell-derived pro-inflammatory cytokines are a major driver of rheumatoid arthritis (RA) pathogenesis. Although these cytokines have traditionally been attributed to CD4 T cells, we have found that CD8 T cells are notably abundant in synovium and make more interferon (IFN)-γ and nearly as much tumor necrosis factor (TNF) as their CD4 T cell counterparts. Furthermore, using unbiased high-dimensional single-cell RNA-seq and flow cytometric data, we found that the vast majority of synovial tissue and synovial fluid CD8 T cells belong to an effector CD8 T cell population characterized by high expression of granzyme K (GzmK) and low expression of granzyme B (GzmB) and perforin. Functional experiments demonstrate that these GzmK+ GzmB+ CD8 T cells are major cytokine producers with low cytotoxic potential. Using T cell receptor repertoire data, we found that CD8 GzmK+ GzmB+ T cells are clonally expanded in synovial tissues and maintain their granzyme expression and overall cell state in blood, suggesting that they are enriched in tissue but also circulate. Using GzmK and GzmB signatures, we found that GzmK-expressing CD8 T cells were also the major CD8 T cell population in the gut, kidney, and coronavirus disease 2019 (COVID-19) bronchoalveolar lavage fluid, suggesting that they form a core population of tissue-associated T cells across diseases and human tissues. We term this population tissue-enriched expressing GzmK or TteK CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 TteK cells have the potential to drive inflammation.


Subject(s)
COVID-19 , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cytokines/metabolism , Granzymes/metabolism , Humans
5.
Cell Rep ; 39(5): 110766, 2022 05 03.
Article in English | MEDLINE | ID: mdl-35508128

ABSTRACT

Ectopic lymphoid structures (ELS) can develop in rheumatoid arthritis (RA) synovial tissue, but the precise pathways of B cell activation and selection are not well understood. Here, we identify a synovial B cell population characterized by co-expression of a family of orphan nuclear receptors (NR4A1-3), which is highly enriched in RA synovial tissue. A transcriptomic profile of NR4A synovial B cells significantly overlaps with germinal center light zone B cells and an accrual of somatic hypermutation that correlates with loss of naive B cell state. NR4A B cells co-express lymphotoxins α and ß and IL-6, supporting functions in ELS promotion. Expanded and shared clones between synovial NR4A B cells and plasma cells and the rapid upregulation with BCR stimulation point to in situ differentiation. Together, we identify a dynamic progression of B cell activation in RA synovial ELS, with NR4A transcription factors having an important role in local adaptive immune responses.


Subject(s)
Arthritis, Rheumatoid , Synovial Membrane , B-Lymphocytes , Humans , Plasma Cells/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Synovial Membrane/metabolism
6.
Arthritis Rheumatol ; 74(2): 200-211, 2022 02.
Article in English | MEDLINE | ID: mdl-34347945

ABSTRACT

OBJECTIVE: B cells can become activated in germinal center (GC) reactions in secondary lymphoid tissue and in ectopic GCs in rheumatoid arthritis (RA) synovium that may be tumor necrosis factor (TNF) and lymphotoxin (LT) dependent. This study was undertaken to characterize the peripheral B cell compartment longitudinally during anti-TNF therapy in RA. METHODS: Participants were randomized in a 2:1 ratio to receive standard dosing regimens of etanercept (n = 43) or adalimumab (n = 20) for 24 weeks. Eligible participants met the American College of Rheumatology 1987 criteria for RA, had clinically active disease (Disease Activity Score in 28 joints >4.4), and were receiving stable doses of methotrexate. The primary mechanistic end point was the change in switched memory B cell fraction from baseline to week 12 in each treatment group. RESULTS: B cell subsets remained surprisingly stable over the course of the study regardless of treatment group, with no significant change in memory B cells. Blockade of TNF and LT with etanercept compared to blockade of TNF alone with adalimumab did not translate into significant differences in clinical response. The frequencies of multiple activated B cell populations, including CD21- double-negative memory and activated naive B cells, were higher in RA nonresponders at all time points, and CD95+ activated B cell frequencies were increased in patients receiving anti-TNF treatment in the nonresponder group. In contrast, frequencies of transitional B cells-a putative regulatory subset-were lower in the nonresponders. CONCLUSION: Overall, our results support the notion that peripheral blood B cell subsets are remarkably stable in RA and not differentially impacted by dual blockade of TNF and LT with etanercept or single blockade of TNF with adalimumab. Activated B cells do associate with a less robust response.


Subject(s)
Adalimumab/pharmacology , Antirheumatic Agents/pharmacology , Arthritis, Rheumatoid/immunology , B-Lymphocytes/drug effects , B-Lymphocytes/physiology , Etanercept/pharmacology , Tumor Necrosis Factor Inhibitors/pharmacology , Adalimumab/therapeutic use , Adult , Aged , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/drug therapy , Etanercept/therapeutic use , Female , Humans , Male , Middle Aged , Single-Blind Method , Tumor Necrosis Factor Inhibitors/therapeutic use
7.
J Immunol ; 207(11): 2660-2672, 2021 12 01.
Article in English | MEDLINE | ID: mdl-34706932

ABSTRACT

Type I IFN is essential for viral clearance but also contributes to the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE), via aberrant nucleic acid-sensing pathways, leading to autoantibody production. Type III IFN (IFN-λ) is now appreciated to have a nonredundant role in viral infection, but few studies have addressed the effects of IFN-λ on immune cells given the more restricted expression of its receptor primarily to the epithelium. In this study, we demonstrate that B cells display a prominent IFN gene expression profile in patients with lupus. Serum levels of IFN-λ are elevated in SLE and positively correlate with B cell subsets associated with autoimmune plasma cell development, including CD11c+T-bet+CD21- B cells. Although B cell subsets express all IFN receptors, IFNLR1 strongly correlates with the CD11c+CD21- B cell expansion, suggesting that IFN-λ may be an unappreciated driver of the SLE IFN signature and B cell abnormalities. We show that IFN-λ potentiates gene transcription in human B cells typically attributed to type I IFN as well as expansion of T-bet-expressing B cells after BCR and TLR7/8 stimulation. Further, IFN-λ promotes TLR7/8-mediated plasmablast differentiation and increased IgM production. CD11c+ B cells demonstrate IFN-λ hyperresponsive signaling compared with other B cell subsets, suggesting that IFN-λ accelerates plasma cell differentiation through this putative extrafollicular pathway. In summary, our data support type III IFN-λ as a cytokine promoting the Ab-secreting cell pool in human viral and autoimmune disease.


Subject(s)
B-Lymphocytes/immunology , Interferons/immunology , Lupus Erythematosus, Systemic/immunology , Plasma Cells/immunology , Adult , Aged , Cell Differentiation , Female , Humans , Male , Middle Aged , Young Adult
8.
Nat Immunol ; 20(7): 928-942, 2019 07.
Article in English | MEDLINE | ID: mdl-31061532

ABSTRACT

To define the cell populations that drive joint inflammation in rheumatoid arthritis (RA), we applied single-cell RNA sequencing (scRNA-seq), mass cytometry, bulk RNA sequencing (RNA-seq) and flow cytometry to T cells, B cells, monocytes, and fibroblasts from 51 samples of synovial tissue from patients with RA or osteoarthritis (OA). Utilizing an integrated strategy based on canonical correlation analysis of 5,265 scRNA-seq profiles, we identified 18 unique cell populations. Combining mass cytometry and transcriptomics revealed cell states expanded in RA synovia: THY1(CD90)+HLA-DRAhi sublining fibroblasts, IL1B+ pro-inflammatory monocytes, ITGAX+TBX21+ autoimmune-associated B cells and PDCD1+ peripheral helper T (TPH) cells and follicular helper T (TFH) cells. We defined distinct subsets of CD8+ T cells characterized by GZMK+, GZMB+, and GNLY+ phenotypes. We mapped inflammatory mediators to their source cell populations; for example, we attributed IL6 expression to THY1+HLA-DRAhi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.


Subject(s)
Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Gene Expression Profiling , Synovial Membrane/metabolism , Transcriptome , Arthritis, Rheumatoid/pathology , Autoimmunity/genetics , Biomarkers , Computational Biology/methods , Cross-Sectional Studies , Cytokines/metabolism , Fibroblasts/metabolism , Flow Cytometry , Gene Expression , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Humans , Leukocytes/immunology , Leukocytes/metabolism , Monocytes/immunology , Monocytes/metabolism , Signal Transduction , Single-Cell Analysis/methods , Synovial Membrane/pathology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Workflow
9.
Nat Commun ; 9(1): 5127, 2018 12 03.
Article in English | MEDLINE | ID: mdl-30510188

ABSTRACT

The function of B cells in osteoblast (OB) dysfunction in rheumatoid arthritis (RA) has not been well-studied. Here we show that B cells are enriched in the subchondral and endosteal bone marrow (BM) areas adjacent to osteocalcin+ OBs in two murine RA models: collagen-induced arthritis and the TNF-transgenic mice. Subchondral BM B cells in RA mice express high levels of OB inhibitors, CCL3 and TNF, and inhibit OB differentiation by activating ERK and NF-κB signaling pathways. The inhibitory effect of RA B cells on OB differentiation is blocked by CCL3 and TNF neutralization, and deletion of CCL3 and TNF in RA B cells completely rescues OB function in vivo, while B cell depletion attenuates bone erosion and OB inhibition in RA mice. Lastly, B cells from RA patients express CCL3 and TNF and inhibit OB differentiation, with these effects ameliorated by CCL3 and TNF neutralization. Thus, B cells inhibit bone formation in RA by producing multiple OB inhibitors.


Subject(s)
Arthritis, Rheumatoid/immunology , B-Lymphocytes/immunology , Cell Differentiation/immunology , Osteoblasts/immunology , Osteogenesis/immunology , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Arthritis, Rheumatoid/metabolism , B-Lymphocytes/metabolism , Bone Marrow/immunology , Bone Marrow/metabolism , Humans , Male , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Osteoblasts/metabolism , Osteoblasts/pathology , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
10.
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Article in English | MEDLINE | ID: mdl-29996944

ABSTRACT

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.


Subject(s)
Arthritis, Rheumatoid/pathology , Flow Cytometry/methods , High-Throughput Screening Assays/methods , Synovial Membrane/pathology , Cryopreservation , Humans
11.
Cytoskeleton (Hoboken) ; 75(7): 290-306, 2018 07.
Article in English | MEDLINE | ID: mdl-29729126

ABSTRACT

Stu2p is the yeast member of the XMAP215/Dis1/ch-TOG family of microtubule-associated proteins that promote microtubule polymerization. However, the factors that regulate its activity are not clearly understood. Here we report that Stu2p in the budding yeast Saccharomyces cerevisiae interacts with SUMO by covalent and noncovalent mechanisms. Stu2p interacted by two-hybrid analysis with the yeast SUMO Smt3p, its E2 Ubc9p, and the E3 Nfi1p. A region of Stu2p containing the dimerization domain was both necessary and sufficient for interaction with SUMO and Ubc9p. Stu2p was found to be sumoylated both in vitro and in vivo. Stu2p copurified with SUMO in a pull-down assay and vice versa. Stu2p also bound to a nonconjugatable form of SUMO, suggesting that Stu2p can interact noncovalently with SUMO. In addition, Stu2p interacted with the STUbL enzyme Ris1p. Stu2p also copurified with ubiquitin in a pull-down assay, suggesting that it can be modified by both SUMO and ubiquitin. Tubulin, a major binding partner of Stu2p, also interacted noncovalently with SUMO. By two-hybrid analysis, the beta-tubulin Tub2p interacted with SUMO independently of the microtubule stressor, benomyl. Together, these findings raise the possibility that the microtubule polymerization activities mediated by Stu2p are regulated through sumoylation pathways.


Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Tubulin/metabolism , Saccharomyces cerevisiae/metabolism , Sumoylation , Ubiquitin-Protein Ligases
12.
J Immunol ; 199(2): 458-466, 2017 07 15.
Article in English | MEDLINE | ID: mdl-28584005

ABSTRACT

Neutrophils are well characterized as mediators of peripheral tissue damage in lupus, but it remains unclear whether they influence loss of self-tolerance in the adaptive immune compartment. Lupus neutrophils produce elevated levels of factors known to fuel autoantibody production, including IL-6 and B cell survival factors, but also reactive oxygen intermediates, which can suppress lymphocyte proliferation. To assess whether neutrophils directly influence the progression of autoreactivity in secondary lymphoid organs (SLOs), we characterized the localization and cell-cell contacts of splenic neutrophils at several stages in the progression of disease in the NZB/W murine model of lupus. Neutrophils accumulate in SLO over the course of lupus progression, preferentially localizing near T lymphocytes early in disease and B cells with advanced disease. RNA sequencing reveals that the splenic neutrophil transcriptional program changes significantly over the course of disease, with neutrophil expression of anti-inflammatory mediators peaking during early-stage and midstage disease, and evidence of neutrophil activation with advanced disease. To assess whether neutrophils exert predominantly protective or deleterious effects on loss of B cell self-tolerance in vivo, we depleted neutrophils at different stages of disease. Neutrophil depletion early in lupus resulted in a striking acceleration in the onset of renal disease, SLO germinal center formation, and autoreactive plasma cell production. In contrast, neutrophil depletion with more advanced disease did not alter systemic lupus erythematosus progression. These results demonstrate a surprising temporal and context-dependent role for neutrophils in restraining autoreactive B cell activation in lupus.


Subject(s)
Autoimmunity , Disease Progression , Germinal Center/immunology , Lupus Erythematosus, Systemic/immunology , Neutrophils/immunology , Animals , Autoantibodies/biosynthesis , Autoantibodies/immunology , B-Lymphocytes/immunology , Disease Models, Animal , Germinal Center/cytology , Lupus Erythematosus, Systemic/physiopathology , Lymphocyte Activation , Mice , Mice, Inbred NZB , Neutrophils/physiology , Sequence Analysis, RNA , Spleen/cytology , Spleen/immunology , T-Lymphocytes/immunology
13.
Arthritis Rheumatol ; 69(8): 1623-1635, 2017 08.
Article in English | MEDLINE | ID: mdl-28471483

ABSTRACT

OBJECTIVE: Bone marrow-derived mesenchymal stem cells (BM-MSCs) create a special microenvironment for hematopoiesis and immunity and display robust immunomodulatory properties that are impaired in systemic lupus erythematosus (SLE). This study was undertaken to identify the mechanisms of defects in human SLE BM-MSCs. METHODS: Patients fulfilling SLE classification criteria and healthy controls (n = 6 per group) were recruited according to an institutional review board-approved protocol. BM-MSCs were isolated with low-density Ficoll-Hypaque, verified by flow cytometry, and studied using immunocytochemistry, real-time polymerase chain reaction, Western blotting, comet assay, ß-galactosidase assay, and RNA interference. RESULTS: SLE BM-MSCs had a senescent phenotype characterized by a reduced proliferation rate, increased production of reactive oxygen species, increased DNA damage and repair, increased expression of p53 and p16, which block the cell cycle, and altered cytokine production (increased proinflammatory cytokine production and decreased immunomodulatory cytokine production). Moreover, SLE BM-MSCs had a 5-fold increase in interferon-ß (IFNß) levels (P < 0.05 versus healthy controls) and increased IFNß-induced messenger RNAs (mRNAs), including mRNA for the intracellular nucleic acid-sensing adaptor protein mitochondrial antiviral signaling protein (MAVS), whose expression was highly correlated with IFNß levels (r > 0.9, P < 0.01). Since MAVS is known to induce IFNß production, we hypothesized that there is a positive feedback loop between MAVS and IFNß. Notably, silencing of MAVS markedly decreased IFNß, p53, and p16 protein levels and expression of mRNAs for proinflammatory cytokines. CONCLUSION: This study demonstrates a novel pathway for elevated IFNß signaling in SLE that is not dependent on stimulation by immune complexes but rather is cell intrinsic and critically mediated by IFNß and MAVS, implicating new pathways as potential therapeutic targets.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Cellular Senescence/immunology , Interferon-beta/immunology , Lupus Erythematosus, Systemic/immunology , Mesenchymal Stem Cells/immunology , RNA, Messenger/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adult , Blotting, Western , Bone Marrow , Cellular Senescence/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cytokines/genetics , Cytokines/immunology , Feedback , Female , Flow Cytometry , Humans , Interferon-beta/genetics , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/metabolism , Mesenchymal Stem Cells/metabolism , Middle Aged , Phenotype , RNA Interference , Real-Time Polymerase Chain Reaction , Signal Transduction , Tumor Suppressor Protein p53/metabolism
14.
Arthritis Rheumatol ; 68(4): 805-16, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26554541

ABSTRACT

OBJECTIVE: Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage. The mechanisms of bone damage in RA are complex, involving activation of bone-resorbing osteoclasts (OCs) by synoviocytes and Th17 cells. This study was undertaken to investigate whether B cells play a direct role in osteoclastogenesis through the production of RANKL, the essential cytokine for OC development. METHODS: RANKL production by total B cells or sorted B cell subpopulations in the peripheral blood and synovial tissue from healthy donors or anti-cyclic citrullinated peptide-positive patients with RA was examined by flow cytometry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis. To define direct effects on osteoclastogenesis, B cells were cocultured with CD14+ monocytes, and OCs were enumerated by tartrate-resistant acid phosphatase staining. RESULTS: Healthy donor peripheral blood B cells were capable of expressing RANKL upon stimulation, with switched memory B cells (CD27+IgD-) having the highest propensity for RANKL production. Notably, switched memory B cells in the peripheral blood from RA patients expressed significantly more RANKL compared to healthy controls. In RA synovial fluid and tissue, memory B cells were enriched and spontaneously expressed RANKL, with some of these cells visualized adjacent to RANK+ OC precursors. Critically, B cells supported OC differentiation in vitro in a RANKL-dependent manner, and the number of OCs was higher in cultures with RA B cells than in those derived from healthy controls. CONCLUSION: These findings reveal the critical importance of B cells in bone homeostasis and their likely contribution to joint destruction in RA.


Subject(s)
Arthritis, Rheumatoid/metabolism , B-Lymphocytes/metabolism , Bone Resorption/metabolism , Cell Differentiation/immunology , Osteoclasts/metabolism , RANK Ligand/metabolism , RNA, Messenger/metabolism , Acid Phosphatase/metabolism , Arthritis, Rheumatoid/immunology , B-Lymphocytes/immunology , Bone Resorption/immunology , Case-Control Studies , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunohistochemistry , In Vitro Techniques , Isoenzymes/metabolism , Monocytes , Osteoclasts/immunology , RANK Ligand/immunology , Real-Time Polymerase Chain Reaction , Synovial Fluid , Synovial Membrane/cytology , Synovial Membrane/immunology , Synovial Membrane/metabolism , Tartrate-Resistant Acid Phosphatase
15.
Curr Opin Rheumatol ; 27(5): 461-7, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26164595

ABSTRACT

PURPOSE OF REVIEW: Our understanding of the physiological and pathogenic functions of B cells in systemic lupus erythematosus (SLE) and Primary Sjögren's syndrome (pSS) continues to expand. In this review, we discuss novel insights published in the last 18 months into the roles of B cells in systemic autoimmunity. RECENT FINDINGS: Data have continued to expand regarding the diverse mechanisms by which innate immune signals including Toll-like receptors (TLRs) regulate the B cell compartment. Localized B cells and long-lived plasma cells have been identified as playing an important role in target tissue including the development of ectopic lymphoid structures in kidney and salivary gland. In addition to pathogenic roles for B cells, there is mounting evidence for regulatory B cell subsets that play a protective role and new insights into the signals that regulate their development. SUMMARY: The past few years have provided insights into the multiple paths by which innate immune signals can lead to B cell activation in SLE and pSS and the increasingly diverse ways in which B cells contribute to disease expression. Further understanding the imbalance between protective and pathogenic functions for B cells in disease including in understudied target tissue should yield new treatment approaches.


Subject(s)
B-Lymphocytes/immunology , Lupus Erythematosus, Systemic/immunology , Sjogren's Syndrome/immunology , Adaptive Immunity , Autoimmunity , Humans , Immunity, Innate , Immunotherapy
16.
J Immunol ; 192(3): 906-18, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-24379124

ABSTRACT

Inappropriate activation of type I IFN plays a key role in the pathogenesis of autoimmune disease, including systemic lupus erythematosus (SLE). In this study, we report the presence of IFN activation in SLE bone marrow (BM), as measured by an IFN gene signature, increased IFN regulated chemokines, and direct production of IFN by BM-resident cells, associated with profound changes in B cell development. The majority of SLE patients had an IFN signature in the BM that was more pronounced than the paired peripheral blood and correlated with both higher autoantibodies and disease activity. Pronounced alterations in B cell development were noted in SLE in the presence of an IFN signature with a reduction in the fraction of pro/pre-B cells, suggesting an inhibition in early B cell development and an expansion of B cells at the transitional stage. These B cell changes strongly correlated with an increase in BAFF and APRIL expression in the IFN-high BM. Furthermore, we found that BM neutrophils in SLE were prime producers of IFN-α and B cell factors. In NZM lupus-prone mice, similar changes in B cell development were observed and mediated by IFN, given abrogation in NZM mice lacking type-I IFNR. BM neutrophils were abundant, responsive to, and producers of IFN, in close proximity to B cells. These results indicate that the BM is an important but previously unrecognized target organ in SLE with neutrophil-mediated IFN activation and alterations in B cell ontogeny and selection.


Subject(s)
B-Lymphocyte Subsets/immunology , Bone Marrow/immunology , Interferon Type I/physiology , Lupus Erythematosus, Systemic/immunology , Lymphopoiesis/immunology , Neutrophils/immunology , Adult , Animals , B-Cell Activating Factor/biosynthesis , B-Cell Activating Factor/genetics , B-Lymphocyte Subsets/pathology , Bone Marrow/metabolism , Chemokines/biosynthesis , Chemokines/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Disease Models, Animal , Female , Gene Expression Profiling , Gene Expression Regulation/immunology , Humans , Interferon Type I/genetics , Male , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Middle Aged , Neutrophils/metabolism , Transcription Factors/biosynthesis , Transcription Factors/genetics , Up-Regulation/immunology
17.
Mol Biol Cell ; 23(23): 4552-66, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23034179

ABSTRACT

Microtubules and microtubule-associated proteins are fundamental for multiple cellular processes, including mitosis and intracellular motility, but the factors that control microtubule-associated proteins (MAPs) are poorly understood. Here we show that two MAPs-the CLIP-170 homologue Bik1p and the Lis1 homologue Pac1p-interact with several proteins in the sumoylation pathway. Bik1p and Pac1p interact with Smt3p, the yeast SUMO; Ubc9p, an E2; and Nfi1p, an E3. Bik1p interacts directly with SUMO in vitro, and overexpression of Smt3p and Bik1p results in its in vivo sumoylation. Modified Pac1p is observed when the SUMO protease Ulp1p is inactivated. Both ubiquitin and Smt3p copurify with Pac1p. In contrast to ubiquitination, sumoylation does not directly tag the substrate for degradation. However, SUMO-targeted ubiquitin ligases (STUbLs) can recognize a sumoylated substrate and promote its degradation via ubiquitination and the proteasome. Both Pac1p and Bik1p interact with the STUbL Nis1p-Ris1p and the protease Wss1p. Strains deleted for RIS1 or WSS1 accumulate Pac1p conjugates. This suggests a novel model in which the abundance of these MAPs may be regulated via STUbLs. Pac1p modification is also altered by Kar9p and the dynein regulator She1p. This work has implications for the regulation of dynein's interaction with various cargoes, including its off-loading to the cortex.


Subject(s)
Endoribonucleases , Microtubule-Associated Proteins , Mitosis , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Sumoylation , Carrier Proteins/genetics , Carrier Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/genetics , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
18.
Article in English | MEDLINE | ID: mdl-23017325

ABSTRACT

INTRODUCTION: Aspirin's effectiveness in reducing cardiovascular disease events is inadequate in some individuals, a phenomenon termed aspirin "resistance". The hypothesis that combining low dose aspirin with eicosapentaenoic acid and docosahexaenoic acid (EPA+DHA) reduces platelet function in the acute setting has not been investigated. PATIENTS AND METHODS: We conducted a clinical trial of EPA+DHA and aspirin ingestion in healthy adults. Fasting blood samples were drawn at baseline and 4 h after supplementation with EPA/DHA (3.4 g/d), aspirin (81 mg), and both. Platelet function was measured using the Platelet Function Analyzer-100 (PFA-100). Plasma lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), autotaxin, angiogenesis activators, and cytokines were measured. RESULTS: Platelet function decreased with the combination of aspirin+EPA/DHA (p=0.03) but not with either alone (p>0.05). EPA-LPC increased (p=0.002). DISCUSSION AND CONCLUSIONS: Our results demonstrate that a potentially beneficial effect on platelet function occurred within 4h after ingestion of low-dose aspirin and EPA+DHA in healthy adults.


Subject(s)
Aspirin/pharmacology , Blood Platelets/drug effects , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Aspirin/administration & dosage , Docosahexaenoic Acids/administration & dosage , Drug Combinations , Eicosapentaenoic Acid/administration & dosage , Humans , Platelet Function Tests
19.
J Immunol ; 188(8): 3784-90, 2012 Apr 15.
Article in English | MEDLINE | ID: mdl-22427635

ABSTRACT

Negative regulation of innate immune responses is essential to prevent excess inflammation and tissue injury and promote homeostasis. Lysophosphatidic acid (LPA) is a pleiotropic lipid that regulates cell growth, migration, and activation and is constitutively produced at low levels in tissues and in serum. Extracellular LPA binds to specific G protein-coupled receptors, whose function in regulating innate or adaptive immune responses remains poorly understood. Of the classical LPA receptors belonging to the Edg family, lpa2 (edg4) is expressed by dendritic cells (DC) and other innate immune cells. In this article, we show that DC from lpa2(-/-) mice are hyperactive compared with their wild-type counterparts and are less susceptible to inhibition by different LPA species. In transient-transfection assays, we found that lpa2 overexpression inhibits NF-κB-driven gene transcription. Using an adoptive-transfer approach, we found that allergen-pulsed lpa2(-/-) DC induced substantially more lung inflammation than did wild-type DC after inhaled allergen challenge. Finally, lpa2(-/-) mice develop greater allergen-driven lung inflammation than do their wild-type counterparts in models of allergic asthma involving both systemic and mucosal sensitization. Taken together, these findings identify LPA acting via lpa2 as a novel negative regulatory pathway that inhibits DC activation and allergic airway inflammation.


Subject(s)
Asthma/immunology , Dendritic Cells/immunology , Lung/immunology , Lysophospholipids/immunology , NF-kappa B/immunology , Receptors, Lysophosphatidic Acid/immunology , Administration, Inhalation , Adoptive Transfer , Allergens/immunology , Animals , Asthma/pathology , Dendritic Cells/metabolism , Dendritic Cells/pathology , Disease Models, Animal , Female , Gene Deletion , HEK293 Cells , Humans , Inflammation/immunology , Inflammation/pathology , Lung/metabolism , Lung/pathology , Lysophospholipids/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/genetics , Receptors, Lysophosphatidic Acid/deficiency , Receptors, Lysophosphatidic Acid/genetics , Signal Transduction , Transcription, Genetic
20.
J Allergy Clin Immunol ; 128(6): 1216-1224.e11, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21996340

ABSTRACT

BACKGROUND: Disruption of the epithelial barrier might be a risk factor for allergen sensitization and asthma. Viral respiratory tract infections are strongly associated with asthma exacerbation, but the effects of respiratory viruses on airway epithelial barrier function are not well understood. Many viruses generate double-stranded RNA, which can lead to airway inflammation and initiate an antiviral immune response. OBJECTIVES: We investigated the effects of the synthetic double-stranded RNA polyinosinic:polycytidylic acid (polyI:C) on the structure and function of the airway epithelial barrier in vitro. METHODS: 16HBE14o- human bronchial epithelial cells and primary airway epithelial cells at an air-liquid interface were grown to confluence on Transwell inserts and exposed to polyI:C. We studied epithelial barrier function by measuring transepithelial electrical resistance and paracellular flux of fluorescent markers and structure of epithelial apical junctions by means of immunofluorescence microscopy. RESULTS: PolyI:C induced a profound decrease in transepithelial electrical resistance and increase in paracellular permeability. Immunofluorescence microscopy revealed markedly reduced junctional localization of zonula occludens-1, occludin, E-cadherin, ß-catenin, and disorganization of junction-associated actin filaments. PolyI:C induced protein kinase D (PKD) phosphorylation, and a PKD antagonist attenuated polyI:C-induced disassembly of apical junctions and barrier dysfunction. CONCLUSIONS: PolyI:C has a powerful and previously unsuspected disruptive effect on the airway epithelial barrier. PolyI:C-dependent barrier disruption is mediated by disassembly of epithelial apical junctions, which is dependent on PKD signaling. These findings suggest a new mechanism potentially underlying the associations between viral respiratory tract infections, airway inflammation, and allergen sensitization.


Subject(s)
Epithelial Cells/pathology , Poly I-C/immunology , Protein Kinase C/metabolism , Respiratory Mucosa/pathology , Signal Transduction/immunology , Asthma/immunology , Asthma/metabolism , Asthma/pathology , Cell Line , Epithelial Cells/immunology , Epithelial Cells/metabolism , Humans , Immunoblotting , Interferon Inducers/immunology , Interferon Inducers/metabolism , Microscopy, Fluorescence , Permeability , Poly I-C/metabolism , Protein Kinase C/immunology , RNA Interference , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Respiratory Tract Infections/immunology , Respiratory Tract Infections/metabolism , Respiratory Tract Infections/pathology , Tight Junctions/immunology , Tight Junctions/metabolism , Tight Junctions/pathology
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