In addition to their pivotal role in thrombosis and wound repair, platelets participate in inflammatory responses. We investigated the role of platelets in the autoimmune disease rheumatoid ...arthritis. We identified platelet microparticles--submicrometer vesicles elaborated by activated platelets--in joint fluid from patients with rheumatoid arthritis and other forms of inflammatory arthritis, but not in joint fluid from patients with osteoarthritis. Platelet microparticles were proinflammatory, eliciting cytokine responses from synovial fibroblasts via interleukin-1. Consistent with these findings, depletion of platelets attenuated murine inflammatory arthritis. Using both pharmacologic and genetic approaches, we identified the collagen receptor glycoprotein VI as a key trigger for platelet microparticle generation in arthritis pathophysiology. Thus, these findings demonstrate a previously unappreciated role for platelets and their activation-induced microparticles in inflammatory joint diseases.
The normal synovium forms a membrane at the edges of joints and provides lubrication and nutrients for the cartilage. In rheumatoid arthritis, the synovium is the site of inflammation, and it ...participates in an organized tissue response that damages cartilage and bone. We identified cadherin-11 as essential for the development of the synovium. Cadherin-11-deficient mice have a hypoplastic synovial lining, display a disorganized synovial reaction to inflammation, and are resistant to inflammatory arthritis. Cadherin-11 therapeutics prevent and reduce arthritis in mouse models. Thus, synovial cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis.
How innate T cells (ITC), including invariant natural killer T (iNKT) cells, mucosal-associated invariant T (MAIT) cells, and γδ T cells, maintain a poised effector state has been unclear. Here we ...address this question using low-input and single-cell RNA-seq of human lymphocyte populations. Unbiased transcriptomic analyses uncover a continuous 'innateness gradient', with adaptive T cells at one end, followed by MAIT, iNKT, γδ T and natural killer cells at the other end. Single-cell RNA-seq reveals four broad states of innateness, and heterogeneity within canonical innate and adaptive populations. Transcriptional and functional data show that innateness is characterized by pre-formed mRNA encoding effector functions, but impaired proliferation marked by decreased baseline expression of ribosomal genes. Together, our data shed new light on the poised state of ITC, in which innateness is defined by a transcriptionally-orchestrated trade-off between rapid cell growth and rapid effector function.
Fibroblasts are major contributors to and regulators of inflammation and dominant producers of interleukin-6 (IL-6) in inflammatory diseases like rheumatoid arthritis. Yet, compared to leukocytes, ...the regulation of inflammatory pathways in fibroblasts is largely unknown. Here, we report that analyses of genes coordinately upregulated with IL-6 pointed to STAT4 and leukemia inhibitory factor (LIF) as potentially linked. Gene silencing revealed that STAT4 was required for IL-6 transcription. STAT4 was recruited to the IL-6 promoter after fibroblast activation, and LIF receptor (LIFR) and STAT4 formed a molecular complex that, together with JAK1 and TYK2 kinases, controlled STAT4 activation. Importantly, a positive feedback loop involving autocrine LIF, LIFR, and STAT4 drove sustained IL-6 transcription. Besides IL-6, this autorine loop also drove the production of other key inflammatory factors including IL-8, granulocyte-colony stimulating factor (G-CSF), IL-33, IL-11, IL-1α, and IL-1β. These findings define the transcriptional regulation of fibroblast-mediated inflammation as distinct from leukocytes.
Display omitted
•During inflammation, human fibroblasts upregulate LIF and STAT4•LIF acts in an autocrine manner via LIF receptor to promote STAT4 activation•Activated STAT4 together with NF-κB/p65-p52 and C/EBPβ enhances IL-6 transcription•LIFR/STAT4 circuit also regulates IL-8, G-CSF, IL-33, IL-11, IL-1α, and IL-1β
Growing evidence implicates fibroblasts as inflammatory cells in sites of peripheral inflammation. Nguyen and colleagues demonstrate that regulation of IL-6 along with a set of other inflammatory cytokines and chemokines is regulated by a positive feedback loop involving LIF, LIF receptor, and STAT4 that selectively operates in fibroblasts.
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-DRA
sublining fibroblasts, IL1B
pro-inflammatory monocytes, ITGAX
TBX21
autoimmune-associated B cells and PDCD1
peripheral helper T (T
) cells and follicular helper T (T
) 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-DRA
fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.
Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ ...localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.
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 T
CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 T
cells have the potential to drive inflammation.
The role of stromal fibroblasts in chronic inflammation is unfolding. In rheumatoid arthritis, leukocyte-derived cytokines TNF and IL-17A work together, activating fibroblasts to become a dominant ...source of the hallmark cytokine IL-6. However, IL-17A alone has minimal effect on fibroblasts. To identify key mediators of the synergistic response to TNF and IL-17A in human synovial fibroblasts, we performed time series, dose–response, and gene-silencing transcriptomics experiments. Here we show that in combination with TNF, IL-17A selectively induces a specific set of genes mediated by factors including cut-like homeobox 1 (CUX1) and IκBζ (NFKBIZ). In the promoters of CXCL1, CXCL2, and CXCL3, we found a putative CUX1–NF-κB binding motif not found elsewhere in the genome. CUX1 and NF-κB p65 mediate transcription of these genes independent of LIFR, STAT3, STAT4, and ELF3. Transcription of NFKBIZ, encoding the atypical IκB factor IκBζ, is IL-17A dose-dependent, and IκBζ only mediates the transcriptional response to TNF and IL-17A, but not to TNF alone. In fibroblasts, IL-17A response depends on CUX1 and IκBζ to engage the NF-κB complex to produce chemoattractants for neutrophil and monocyte recruitment.
The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, ...becomes inflamed and invasive, and destroys the joint
. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity
; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.
CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18 ⁺ invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide ...antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.
PDF
