Innate immune pattern recognition receptors sense nucleic acids from microbes and orchestrate cytokine production to resolve infection. Inappropriate recognition of host nucleic acids also results in autoimmune disease. In this study, we use a model of inflammation resulting from accrual of self DNA (DNase II(-/-) type I IFN receptor [Ifnar](-/-)) to understand the role of pattern recognition receptor-sensing pathways in arthritis and autoantibody production. Using triple knockout (TKO) mice deficient in DNase II/IFNaR together with deficiency in either stimulator of IFN genes (STING) or absent in melanoma 2 (AIM2), we reveal central roles for the STING and AIM2 pathways in arthritis. AIM2 TKO mice show limited inflammasome activation and, similar to STING TKO mice, have reduced inflammation in joints. Surprisingly, autoantibody production is maintained in AIM2 and STING TKO mice, whereas DNase II(-/-) Ifnar(-/-) mice also deficient in Unc93b, a chaperone required for TLR7/9 endosomal localization, fail to produce autoantibodies to nucleic acids. Collectively, these data support distinct roles for cytosolic and endosomal nucleic acid-sensing pathways in disease manifestations.

Our aim was to examine the impact of TLR5 ligation in rheumatoid arthritis (RA) and experimental arthritis pathology. Studies were conducted to investigate the role of TLR5 ligation on RA and mouse myeloid cell chemotaxis or osteoclast formation, and in addition, to uncover the significance of TNF-α function in TLR5-mediated pathogenesis. Next, the in vivo mechanism of action was determined in collagen-induced arthritis (CIA) and local joint TLR5 ligation models. Last, to evaluate the importance of TLR5 function in RA, we used anti-TLR5 Ab therapy in CIA mice. We show that TLR5 agonist, flagellin, can promote monocyte infiltration and osteoclast maturation directly through myeloid TLR5 ligation and indirectly via TNF-α production from RA and mouse cells. These two identified TLR5 functions are potentiated by TNF-α, because inhibition of both pathways can more strongly impair RA synovial fluid-driven monocyte migration and osteoclast differentiation compared with each factor alone. In preclinical studies, flagellin postonset treatment in CIA and local TLR5 ligation in vivo provoke homing and osteoclastic development of myeloid cells, which are associated with the TNF-α cascade. Conversely, CIA joint inflammation and bone erosion are alleviated when TLR5 function is blocked. We found that TLR5 and TNF-α pathways are interconnected, because TNF-α is produced by TLR5 ligation in RA myeloid cells, and anti-TNF-α therapy can markedly suppress TLR5 expression in RA monocytes. Our novel findings demonstrate that a direct and an indirect mechanism are involved in TLR5-driven RA inflammation and bone destruction.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1β that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

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.

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^hi sublining fibroblasts, IL1B⁺ pro-inflammatory monocytes, ITGAX⁺TBX21⁺ autoimmune-associated B cells and PDCD1⁺ peripheral helper T (T_PH) cells and follicular helper T (T_FH) 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^hi fibroblasts and IL1B production to pro-inflammatory monocytes. These populations are potentially key mediators of RA pathogenesis.

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.

Objective

Delayed detection of LN associates with worse outcomes. There are conflicting recommendations regarding a threshold level of proteinuria at which biopsy will likely yield actionable management. This study addressed the association of urine protein:creatinine ratios (UPCR) with clinical characteristics and investigated the incidence of proliferative and membranous histology in patients with a UPCR between 0.5 and 1.

Methods

A total of 275 SLE patients (113 first biopsy, 162 repeat) were enrolled in the multicentre multi-ethnic/racial Accelerating Medicines Partnership across 15 US sites at the time of a clinically indicated renal biopsy. Patients were followed for 1 year.

Results

At biopsy, 54 patients had UPCR <1 and 221 had UPCR ≥1. Independent of UPCR or biopsy number, a majority (92%) of patients had class III, IV, V or mixed histology. Moreover, patients with UPCR <1 and class III, IV, V, or mixed had a median activity index of 4.5 and chronicity index of 3, yet 39% of these patients had an inactive sediment. Neither anti-dsDNA nor low complement distinguished class I or II from III, IV, V or mixed in patients with UPCR <1. Of 29 patients with baseline UPCR <1 and class III, IV, V or mixed, 23 (79%) had a UPCR <0.5 at 1 year.

Conclusion

In this prospective study, three-quarters of patients with UPCR <1 had histology showing class III, IV, V or mixed with accompanying activity and chronicity despite an inactive sediment or normal serologies. These data support renal biopsy at thresholds lower than a UPCR of 1.

In lupus nephritis the pathological diagnosis from tissue retrieved during kidney biopsy drives treatment and management. Despite recent approval of new drugs, complete remission rates remain well under aspirational levels, necessitating identification of new therapeutic targets by greater dissection of the pathways to tissue inflammation and injury. This study assessed the safety of kidney biopsies in patients with SLE enrolled in the Accelerating Medicines Partnership, a consortium formed to molecularly deconstruct nephritis. 475 patients with SLE across 15 clinical sites in the USA consented to obtain tissue for research purposes during a clinically indicated kidney biopsy. Adverse events (AEs) were documented for 30 days following the procedure and were determined to be related or unrelated by all site investigators. Serious AEs were defined according to the National Institutes of Health reporting guidelines. 34 patients (7.2%) experienced a procedure-related AE: 30 with haematoma, 2 with jets, 1 with pain and 1 with an arteriovenous fistula. Eighteen (3.8%) experienced a serious AE requiring hospitalisation; four patients (0.8%) required a blood transfusion related to the kidney biopsy. At one site where the number of cores retrieved during the biopsy was recorded, the mean was 3.4 for those who experienced a related AE (n=9) and 3.07 for those who did not experience any AE (n=140). All related AEs resolved. Procurement of research tissue should be considered feasible, accompanied by a complication risk likely no greater than that incurred for standard clinical purposes. In the quest for targeted treatments personalised based on molecular findings, enhanced diagnostics beyond histology will likely be required.