Subjects at-risk for future development of rheumatoid arthritis demonstrate a PAD4-and TLR-dependent enhanced histone H3 citrullination and proinflammatory cytokine production in CD14(hi) monocytes.

The presence of anti-citrullinated protein/peptide antibodies (ACPA) and epitope spreading across the target autoantigens is a unique feature of rheumatoid arthritis (RA). ACPA are present in the peripheral blood for several years prior to the onset of arthritis and clinical classification of RA. ACPA recognize multiple citrullinated proteins, including histone H3 (H3). Intracellular citrullination of H3 in neutrophils and T cells is known to regulate immune cell function by promoting neutrophil extracellular trap formation and citrullinated autoantigen release as well as regulating the Th2/Th17 T cell phenotypic balance. However, the roles of H3 citrullination in other immune cells are not fully elucidated. We aimed to explore H3 citrullination and cytokine/metabolomic signatures in peripheral blood immune cells from subjects prior to and after the onset of RA, at baseline and in response to ex vivo toll-like receptor (TLR) stimulation. Here, we analyzed 13 ACPA (+) subjects without arthritis but at-risk for future development of RA, 14 early RA patients, and 13 healthy controls. We found significantly elevated H3 citrullination in CD14hi monocytes, as well as CD1c+ dendritic cells and CD66+ granulocytes. Unsupervised analysis identified two distinct subsets in CD14hi monocytes characterized by H3 modification and unique cytokine/metabolomic signatures. CD14hi monocytes with elevated TLR-stimulated H3 citrullination were significantly increased in ACPA (+) at-risk subjects. These cells were skewed to produce TNFα, MIP1β, IFNα, and partially IL-12. Additionally, they demonstrate peptidyl arginine deiminase 4 (PAD4) mediated upregulation of the glycolytic enzyme PFKFB3. These CD14hi monocytes with elevated H3 citrullination morphologically formed monocyte extracellular traps (METs). Taken together, dysregulated PAD4-driven cytokine production as well as MET formation in CD14hi monocytes in ACPA (+) at-risk subjects likely plays an important role in the development of RA via promoting and perpetuating inflammation and generation of citrullinated autoantigens.

Okamato Y ，Ghosh T ，Okamoto T ，Schuyler RP ，Seifert J ，Charry LL ，Visser A ，Feser M ，Fleischer C ，Pedrick C ，August J ，Moss L ，Bemis EA ，Norris JM ，Kuhn KA ，Demoruelle MK ，Deane KD ，Ghosh D ，Holers VM ，Hsieh EWY ... -  《-》

Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis.

Lloyd KA ，Wigerblad G ，Sahlström P ，Garimella MG ，Chemin K ，Steen J ，Titcombe PJ ，Marklein B ，Zhou D ，Stålesen R ，Ossipova E ，Lundqvist C ，Ekwall O ，Rönnelid J ，Mueller DL ，Karlsson MCI ，Kaplan MJ ，Skriner K ，Klareskog L ，Wermeling F ，Malmström V ，Grönwall C ... -  《Frontiers in Immunology》

Extracellular traps and PAD4 released by macrophages induce citrullination and auto-antibody production in autoimmune arthritis.

The mechanisms underlying the transition of rheumatoid arthritis (RA) systemic autoimmunity to the joints remain largely unknown. Here, we demonstrate that macrophages in the secondary lymphoid organs (SLOs) and synovial ectopic lymphoid-like structures (ELSs) express peptidylarginine deiminase 4 (PAD4) in murine collagen induced arthritis (CIA) and synovial biopsies from RA patients. Moreover, peptidyl citrulline colocalized with macrophages in SLOs and ELSs, and depletion of macrophages in CIA decreased lymphoid tissue citrullination and serum anti-citrullinated protein/peptide antibody (ACPA) levels. Furthermore, PAD was released from activated murine and RA synovial tissue and fluid (SF) macrophages which functionally deiminated extracellular proteins/peptides in vitro. Additionally, activated murine and SF macrophages displayed macrophage extracellular trap formation (METosis) and release of intracellular citrullinated histones. Moreover, presentation of citrullinated proteins induced ACPA production in vitro. Thus, lymphoid tissue macrophages contribute to self-antigen citrullination and ACPA production, indicating that their selective targeting would potentially ameliorate citrullination-dependent autoimmune disorders.

El Shikh MEM ，El Sayed R ，Nerviani A ，Goldmann K ，John CR ，Hands R ，Fossati-Jimack L ，Lewis MJ ，Pitzalis C ... -  《-》

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays.

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

Poulsen TBG ，Damgaard D ，Jørgensen MM ，Senolt L ，Blackburn JM ，Nielsen CH ，Stensballe A ... -  《-》

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.

Sur Chowdhury C ，Giaglis S ，Walker UA ，Buser A ，Hahn S ，Hasler P ... -  《-》