Crystal structure of Porphyromonas gingivalis peptidylarginine deiminase: implications for autoimmunity in rheumatoid arthritis.

BACKGROUND: Periodontitis (PD) is a known risk factor for rheumatoid arthritis (RA) and there is increasing evidence that the link between the two diseases is due to citrullination by the unique bacterial peptidylarginine deiminase (PAD) enzyme expressed by periodontal pathogen Pophyromonas gingivalis (PPAD). However, the precise mechanism by which PPAD could generate potentially immunogenic peptides has remained controversial due to lack of information about the structural and catalytic mechanisms of the enzyme. OBJECTIVES: By solving the 3D structure of PPAD we aim to characterise activity and elucidate potential mechanisms involved in breach of tolerance to citrullinated proteins in RA. METHODS: PPAD and a catalytically inactive mutant PPAD(C351A) were crystallised and their 3D structures solved. Key residues identified from 3D structures were examined by mutations. Fibrinogen and α-enolase were incubated with PPAD and P. gingivalis arginine gingipain (RgpB) and citrullinated peptides formed were sequenced and quantified by mass spectrometry. RESULTS: Here, we solve the crystal structure of a truncated, highly active form of PPAD. We confirm catalysis is mediated by the following residues: Asp130, His236, Asp238, Asn297 and Cys351 and show Arg152 and Arg154 may determine the substrate specificity of PPAD for C-terminal arginines. We demonstrate the formation of 37 C-terminally citrullinated peptides from fibrinogen and 11 from α-enolase following incubation with tPPAD and RgpB. CONCLUSIONS: PPAD displays an unequivocal specificity for C-terminal arginine residues and readily citrullinates peptides from key RA autoantigens. The formation of these novel citrullinated peptides may be involved in breach of tolerance to citrullinated proteins in RA.

Tissue-resident, extravascular Ly6c- monocytes are critical for inflammation in the synovium.

Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue. Here, we identify and characterize an intravascular synovial monocyte population resembling circulating non-classical monocytes and an extravascular tissue-resident monocyte-lineage cell (TR-MC) population distinct in surface marker and transcriptional profile from circulating monocytes, dendritic cells, and tissue macrophages that are conserved in rheumatoid arthritis (RA) patients. TR-MCs are independent of NR4A1 and CCR2, long lived, and embryonically derived. TR-MCs undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. Moreover, pathways that are activated in TR-MCs at the peak of arthritis overlap with those that are downregulated in LFA1-/- TR-MCs. These findings show a facet of mononuclear cell biology that could be imperative to understanding tissue-resident myeloid cell function in RA.

Transcriptional profiling of pediatric cholestatic livers identifies three distinct macrophage populations.

BACKGROUND & AIMS: Limited understanding of the role for specific macrophage subsets in the pathogenesis of cholestatic liver injury is a barrier to advancing medical therapy. Macrophages have previously been implicated in both the mal-adaptive and protective responses in obstructive cholestasis. Recently two macrophage subsets were identified in non-diseased human liver; however, no studies to date fully define the heterogeneous macrophage subsets during the pathogenesis of cholestasis. Here, we aim to further characterize the transcriptional profile of macrophages in pediatric cholestatic liver disease. METHODS: We isolated live hepatic immune cells from patients with biliary atresia (BA), Alagille syndrome (ALGS), and non-cholestatic pediatric liver by fluorescence activated cell sorting. Through single-cell RNA sequencing analysis and immunofluorescence, we characterized cholestatic macrophages. We next compared the transcriptional profile of pediatric cholestatic and non-cholestatic macrophage populations to previously published data on normal adult hepatic macrophages. RESULTS: We identified 3 distinct macrophage populations across cholestatic liver samples and annotated them as lipid-associated macrophages, monocyte-like macrophages, and adaptive macrophages based on their transcriptional profile. Immunofluorescence of liver tissue using markers for each subset confirmed their presence across BA (n = 6) and ALGS (n = 6) patients. Cholestatic macrophages demonstrated reduced expression of immune regulatory genes as compared to normal hepatic macrophages and were distinct from macrophage populations defined in either healthy adult or pediatric non-cholestatic liver. CONCLUSIONS: We are the first to perform single-cell RNA sequencing on human pediatric cholestatic liver and identified three macrophage subsets with distinct transcriptional signatures from healthy liver macrophages. Further analyses will identify similarities and differences in these macrophage sub-populations across etiologies of cholestatic liver disease. Taken together, these findings may allow for future development of targeted therapeutic strategies to reprogram macrophages to an immune regulatory phenotype and reduce cholestatic liver injury.

Phosphatase PTPN22 Regulates Dendritic Cell Homeostasis and cDC2 Dependent T Cell Responses.

Dendritic cells (DCs) are specialized antigen presenting cells that instruct T cell responses through sensing environmental and inflammatory danger signals. Maintaining the homeostasis of the multiple functionally distinct conventional dendritic cells (cDC) subsets that exist in vivo is crucial for regulating immune responses, with changes in numbers sufficient to break immune tolerance. Using Ptpn22-/- mice we demonstrate that the phosphatase PTPN22 is a highly selective, negative regulator of cDC2 homeostasis, preventing excessive population expansion from as early as 3 weeks of age. Mechanistically, PTPN22 mediates cDC2 homeostasis in a cell intrinsic manner by restricting cDC2 proliferation. A single nucleotide polymorphism, PTPN22R620W, is one of the strongest genetic risk factors for multiple autoantibody associated human autoimmune diseases. We demonstrate that cDC2 are also expanded in mice carrying the orthologous PTPN22619W mutation. As a consequence, cDC2 dependent CD4+ T cell proliferation and T follicular helper cell responses are increased. Collectively, our data demonstrate that PTPN22 controls cDC2 homeostasis, which in turn ensures appropriate cDC2-dependent T cell responses under antigenic challenge. Our findings provide a link between perturbations in DC development and susceptibility to a broad spectrum of PTPN22R620W associated human autoimmune diseases.

Transcriptional Profiling of Synovial Macrophages Using Minimally Invasive Ultrasound-Guided Synovial Biopsies in Rheumatoid Arthritis.

OBJECTIVE: Currently, there are no reliable biomarkers for predicting therapeutic response in patients with rheumatoid arthritis (RA). The synovium may unlock critical information for determining efficacy, since a reduction in the numbers of sublining synovial macrophages remains the most reproducible biomarker. Thus, a clinically actionable method for the collection of synovial tissue, which can be analyzed using high-throughput strategies, must become a reality. This study was undertaken to assess the feasibility of utilizing synovial biopsies as a precision medicine-based approach for patients with RA. METHODS: Rheumatologists at 6 US academic sites were trained in minimally invasive ultrasound-guided synovial tissue biopsy. Biopsy specimens obtained from patients with RA and synovial tissue from patients with osteoarthritis (OA) were subjected to histologic analysis, fluorescence-activated cell sorting, and RNA sequencing (RNA-seq). An optimized protocol for digesting synovial tissue was developed to generate high-quality RNA-seq libraries from isolated macrophage populations. Associations were determined between macrophage transcriptional profiles and clinical parameters in RA patients. RESULTS: Patients with RA reported minimal adverse effects in response to synovial biopsy. Comparable RNA quality was observed from synovial tissue and isolated macrophages between patients with RA and patients with OA. Whole tissue samples from patients with RA demonstrated a high degree of transcriptional heterogeneity. In contrast, the transcriptional profile of isolated RA synovial macrophages highlighted different subpopulations of patients and identified 6 novel transcriptional modules that were associated with disease activity and therapy. CONCLUSION: Performance of synovial tissue biopsies by rheumatologists in the US is feasible and generates high-quality samples for research. Through the use of cutting-edge technologies to analyze synovial biopsy specimens in conjunction with corresponding clinical information, a precision medicine-based approach for patients with RA is attainable.

The caspase-8/RIPK3 signaling axis in antigen presenting cells controls the inflammatory arthritic response.

BACKGROUND: Caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, but maintains functions beyond cell death that involve suppression of receptor-interacting serine-threonine kinases (RIPKs). A genome-wide association study meta-analysis revealed an SNP associated with risk of rheumatoid arthritis (RA) development within the locus containing the gene encoding for caspase-8. Innate immune cells, like macrophages and dendritic cells, are gaining momentum as facilitators of autoimmune disease pathogenesis, and, in particular, RA. Therefore, we examined the involvement of caspase-8 within these antigen-presenting cell populations in the pathogenesis of an arthritis model that resembles the RA effector phase. METHODS: Cre LysM Casp8 flox/flox and Cre CD11c Casp8 flox/flox mice were bred via a cross between Casp8 flox/flox and Cre LysM or Cre CD11c mice. RIPK3 -/- Cre LysM Casp8 flox/flox and RIPK3 -/- Cre CD11c Casp8 flox/flox mice were generated to assess RIPK3 contribution. Mice were subjected to K/BxN serum-transfer-induced arthritis. Luminex-based assays were used to measure cytokines/chemokines. Histological analyses were utilized to examine joint damage. Mixed bone marrow chimeras were generated to assess synovial cell survival. Flow cytometric analysis was employed to characterize cellular distribution. For arthritis, differences between the groups were assessed using two-way analysis of variance (ANOVA) for repeated measurements. All other data were compared by the Mann-Whitney test. RESULTS: We show that intact caspase-8 signaling maintains opposing roles in lysozyme-M- and CD11c-expressing cells in the joint; namely, caspase-8 is crucial in CD11c-expressing cells to delay arthritis induction, while caspase-8 in lysozyme M-expressing cells hinders arthritis resolution. Caspase-8 is also implicated in the maintenance of synovial tissue-resident macrophages that can limit arthritis. Global loss of RIPK3 in both caspase-8 deletion constructs causes the response to arthritis to revert back to control levels via a mechanism potentially independent of cell death. Mixed bone marrow chimeric mice demonstrate that caspase-8 deficiency does not confer preferential expansion of synovial macrophage and dendritic cell populations, nor do caspase-8-deficient synovial populations succumb to RIPK3-mediated necroptotic death. CONCLUSIONS: These data demonstrate that caspase-8 functions in synovial antigen-presenting cells to regulate the response to inflammatory stimuli by controlling RIPK3 action, and this delicate balance maintains homeostasis within the joint.

Association of Distinct Fine Specificities of Anti-Citrullinated Peptide Antibodies With Elevated Immune Responses to Prevotella intermedia in a Subgroup of Patients With Rheumatoid Arthritis and Periodontitis.

OBJECTIVE: In addition to the long-established link with smoking, periodontitis (PD) is a risk factor for rheumatoid arthritis (RA). This study was undertaken to elucidate the mechanism by which PD could induce antibodies to citrullinated peptides (ACPAs), by examining the antibody response to a novel citrullinated peptide of cytokeratin 13 (CK-13) identified in gingival crevicular fluid (GCF), and comparing the response to 4 other citrullinated peptides in patients with RA who were well-characterized for PD and smoking. METHODS: The citrullinomes of GCF and periodontal tissue from patients with PD were mapped by mass spectrometry. ACPAs of CK13 (cCK13), tenascin-C (cTNC5), vimentin (cVIM), α-enolase (CEP-1), and fibrinogen ß (cFIBß) were examined by enzyme-linked immunosorbent assay in patients with RA (n = 287) and patients with osteoarthritis (n = 330), and cross-reactivity was assessed by inhibition assays. RESULTS: A novel citrullinated peptide cCK13-1 (444 TSNASGR-Cit-TSDV-Cit-RP458 ) identified in GCF exhibited elevated antibody responses in RA patients (24%). Anti-cCK13-1 antibody levels correlated with anti-cTNC5 antibody levels, and absorption experiments confirmed this was not due to cross-reactivity. Only anti-cCK13-1 and anti-cTNC5 were associated with antibodies to the periodontal pathogen Prevotella intermedia (P = 0.05 and P = 0.001, respectively), but not with antibodies to Porphyromonas gingivalis arginine gingipains. Levels of antibodies to CEP-1, cFIBß, and cVIM correlated with each other, and with smoking and shared epitope risk factors in RA. CONCLUSION: This study identifies 2 groups of ACPA fine specificities associated with different RA risk factors. One is predominantly linked to smoking and shared epitope, and the other links anti-cTNC5 and cCK13-1 to infection with the periodontal pathogen P intermedia.

Expression of citrulline and homocitrulline residues in the lungs of non-smokers and smokers: implications for autoimmunity in rheumatoid arthritis.

INTRODUCTION: Smoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4). METHODS: Lung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test. RESULTS: Within the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase. CONCLUSIONS: We have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.

The case for measuring antibodies to specific citrullinated antigens.

Anti-citrullinated protein/peptide antibodies (ACPA) are the principal autoantibody system associated with rheumatoid arthritis (RA), with diagnostic sensitivity of 70% and specificity of 95%. Current testing for ACPA uses the anti-cyclic citrullinated peptide assay (anti-CCP) which measures a generalized reactivity with citrulline-containing peptides, thus giving no insight into reactivity to specific RA antigens. Of these, the best characterized are, α-enolase, fibrinogen/fibrin, vimentin, Type 2 collagen and filaggrin, antibodies to each of which are found in approximately 30-60% of RA cases. Given reports of cross-reactivity between citrullinated antigens, we discuss whether or not measuring these specific antibodies could aid: clinical diagnosis, identification of clinical subsets and drug responses, or provide insight into pathogenic mechanisms or etiology of RA.