Wiley

Inhibitors of p38 demonstrate limited benefit in rheumatoid arthritis (RA), perhaps due to the antiinflammatory functions of p38α. This study was performed to determine if selective deletion of p38α in macrophages affects the severity of arthritis and whether blocking upstream kinases in the p38 pathway, such as MKK‐3 or MKK‐6, avoids some of the limitations of p38 blockade.

Wild‐type (WT) mice and mice with selective deletion of p38α in macrophages (p38α^ΔLysM) were injected with K/BxN sera. Antigen‐induced arthritis was also induced in p38α^ΔLysM mice. Mouse joint extracts were evaluated by enzyme‐linked immunosorbent assay, quantitative polymerase chain reaction (qPCR), and Western blot analysis. Bone marrow–derived macrophages (BMMs) were stimulated with lipopolysaccharide (LPS) and were evaluated by qPCR and Western blotting. Bone marrow chimeras were generated using MKK‐3^−/− and MKK‐6^−/− mice, and K/BxN serum was administered to induce arthritis.

Compared to WT mice, p38α^ΔLysM mice had increased disease severity and delayed resolution of arthritis, which correlated with higher synovial inflammatory mediator expression and ERK phosphorylation. In contrast to WT BMMs cultured in the presence of a p38α/β inhibitor, LPS‐stimulated MKK‐6– and MKK‐3–deficient BMMs had suppressed LPS‐mediated interleukin‐6 (IL‐6) expression but had normal IL‐10 production, dual‐specificity phosphatase 1 expression, and MAPK phosphorylation. WT chimeric mice with MKK‐6– and MKK‐3–deficient bone marrow had markedly decreased passive K/BxN arthritis severity.

Inhibiting p38α in a disease that is dominated by macrophage cytokines, such as RA, could paradoxically suppress antiinflammatory functions and interfere with clinical efficacy. Targeting an upstream kinase that regulates p38 could be more effective by suppressing proinflammatory cytokines while preventing decreased IL‐10 expression and increased MAPK activation.

Neutrophil extracellular traps (NETs) are composed of DNA and antimicrobial proteins, including myeloperoxidase (MPO). Recent studies have demonstrated that impaired regulation of NETs could trigger an autoimmune response. Propylthiouracil (PTU), an antithyroid drug, is associated with a risk of MPO antineutrophil cytoplasmic antibody (ANCA) production and MPO ANCA–associated vasculitis (MPO AAV). This study was undertaken to clarify the mechanism of MPO ANCA production, using the PTU‐induced model of MPO AAV.

NETs were induced by treating human neutrophils with phorbol myristate acetate (PMA) in vitro. We examined whether the addition of PTU influenced the NET formation induced by PMA and the degradation of NETs by DNase I, which is regarded as a regulator of NETs. Furthermore, we examined whether NETs generated by the combination of PMA and PTU induced MPO ANCA and MPO AAV in vivo in rats.

When NETs were induced by PMA with PTU using human neutrophils in vitro, abnormal conformation of NETs was observed. Interestingly, the abnormal NETs were hardly digested by DNase I. Moreover, rats immunized with the abnormal NETs, which had been induced by PMA with PTU using rat neutrophils, produced MPO ANCA and developed pulmonary capillaritis. When rats were given oral PTU with intraperitoneal injection of PMA, pauci‐immune glomerulonephritis and pulmonary capillaritis occurred with MPO ANCA production in the serum.

Our findings indicate that abnormal conformation and impaired degradation of NETs induced by PTU are involved in the pathogenesis of PTU‐induced MPO ANCA production and MPO AAV. These findings suggest that disordered NETs can be critically implicated in the pathogenesis of MPO AAV.

To profile the abundance and diversity of subgingival oral microbiota in patients with never‐treated, new‐onset rheumatoid arthritis (RA).

Periodontal disease (PD) status, clinical activity, and sociodemographic factors were determined in patients with new‐onset RA, patients with chronic RA, and healthy subjects. Multiplexed‐454 pyrosequencing was used to compare the composition of subgingival microbiota and establish correlations between the presence/abundance of bacteria and disease phenotypes. Anti–Porphyromonas gingivalis antibody testing was performed to assess prior exposure to the bacterial pathogen P gingivalis.

The more advanced forms of periodontitis were already present at disease onset in patients with new‐onset RA. The subgingival microbiota observed in patients with new‐onset RA was distinct from that found in healthy controls. In most cases, however, these microbial differences could be attributed to the severity of PD and were not inherent to RA. The presence and abundance of P gingivalis were also directly associated with the severity of PD and were not unique to RA. The presence of P gingivalis was not correlated with anti–citrullinated protein antibody (ACPA) titers. Overall exposure to P gingivalis was similar between patients with new‐onset RA and controls, observed in 78% of patients and 83% of controls. The presence and abundance of Anaeroglobus geminatus correlated with the presence of ACPAs/rheumatoid factor. Prevotella and Leptotrichia species were the only characteristic taxa observed in patients with new‐onset RA irrespective of PD status.

Patients with new‐onset RA exhibited a high prevalence of PD at disease onset, despite their young age and paucity of smoking history. The subgingival microbiota profile in patients with new‐onset RA was similar to that in patients with chronic RA and healthy subjects whose PD was of comparable severity. Although colonization with P gingivalis correlated with the severity of PD, overall exposure to P gingivalis was similar among the groups. The role of A geminatus and Prevotella/Leptotrichia species in this process merits further study.

To examine the relationship of Porphyromonas gingivalis to the presence of autoantibodies in individuals at risk of rheumatoid arthritis (RA).

Study participants included the following: 1) a cohort enriched in subjects with HLA–DR4 and 2) subjects at risk of RA by virtue of having a first‐degree relative with RA. None of the study subjects satisfied the American College of Rheumatology 1987 classification criteria for RA. Autoantibodies measured included anti–citrullinated protein antibody (ACPA; by second‐generation anti–cyclic citrullinated peptide antibody enzyme‐linked immunosorbent assay [ELISA]) and rheumatoid factor (RF; by nephelometry or ELISA for IgA, IgM, or IgG isotype). Individuals were considered autoantibody positive (n = 113) if they had ≥1 RA‐related autoantibody; individuals were further categorized as high risk (n = 38) if they had ACPA or positive findings ≥2 assays for RF. Autoantibody‐negative individuals (n = 171) served as a comparator group. Antibody to P gingivalis, P intermedia, and F nucleatum were measured. Associations of bacterial antibodies with group status were examined using logistic regression.

Anti–P gingivalis concentrations were higher in high‐risk (P = 0.011) and autoantibody positive group (P = 0.010) than in the autoantibody negative group. There were no group differences in anti–P intermedia or anti–F nucleatum concentrations. After multivariable adjustment, anti–P gingivalis concentrations (but not anti–P intermedia or anti–F nucleatum) were significantly associated with autoantibody‐positive and high‐risk status (P < 0.05).

Immunity to P gingivalis, but not P intermedia or F nucleatum, is significantly associated with the presence of RA‐related autoantibodies in individuals at risk of RA. These results support the hypothesis that infection with P gingivalis may play a central role in the early loss of tolerance to self antigens that occurs in the pathogenesis of RA.

To test the hypothesis that autoantigen modifications by peptidylarginine deiminase type 4 (PAD‐4) increase immunoreactivity.

We assembled sera from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Felty's syndrome (FS), and antineutrophil cytoplasmic antibody–associated vasculitides (AAVs), as well as sera from control subjects without autoimmune diseases. The sera were tested for binding to activated neutrophils, deiminated histones, and neutrophil extracellular chromatin traps (NETs). IgG binding to lipopolysaccharide‐activated neutrophils was assessed with confocal microscopy, and binding to in vitro–deiminated histones was measured using enzyme‐linked immunosorbent assay (ELISA) and Western blotting. In addition, we quantitated histone deimination in freshly isolated neutrophils from the blood of patients and control subjects.

Increased IgG reactivity with activated neutrophils, particularly binding to NETs, was paralleled by preferential binding to deiminated histones over nondeiminated histones by ELISA in a majority of sera from FS patients but only in a minority of sera from SLE and RA patients. Immunoblotting revealed autoantibody preference for deiminated histones H3, H4, and H2A in most FS patients and in a subset of SLE and RA patients. In patients with AAVs, serum IgG preferentially bound nondeiminated histones over deiminated histones. Increased levels of deiminated histones were detected in neutrophils from RA patients.

Circulating autoantibodies in FS are preferentially directed against PAD‐4–deiminated histones and bind to activated neutrophils and NETs. Thus, increased reactivity with modified autoantigens in FS implies a direct contribution of neutrophil activation and the production of NET‐associated nuclear autoantigens in the initiation or progression of FS.

To determine whether antibodies against peptidyl arginine deiminase type 4 (PAD‐4) are present in the preclinical phase of rheumatoid arthritis (RA) and to compare the timing and extent of their appearance with those of other preclinical autoantibodies.

Prediagnosis serum samples from 83 patients with RA were evaluated for the presence of anti–PAD‐4 antibody, anti–cyclic citrullinated peptide (anti‐CCP) antibody, and rheumatoid factor. In addition, a control cohort (n = 83) matched by age, sex, race, number of serum samples, and duration of serum storage was tested for the presence of anti–PAD‐4 antibody to determine its sensitivity and specificity for the subsequent development of RA.

Fifteen of 83 patients with RA (18.1%) had at least 1 prediagnosis sample positive for anti–PAD‐4. One of 83 control subjects (1.2%) had at least 1 positive sample, resulting in a sensitivity and specificity of 18.1% and 98.8%, respectively, of anti–PAD‐4 for the future development of RA. The mean duration of anti–PAD‐4 positivity prior to clinical diagnosis was 4.67 years. Anti–PAD‐4 positivity was associated with anti‐CCP positivity (odds ratio 5.13 [95% confidence interval 1.07–24.5]). In subjects with prediagnosis samples that were positive for both antibodies, anti‐CCP positivity predated anti–PAD‐4 positivity in 9 of 13 cases (69%).

Autoantibodies to PAD‐4 are present during the preclinical phase of RA in a subset of patients and are associated with anti‐CCP positivity. Further exploration is needed regarding the timing of appearance and disease‐related effects of PAD‐4 autoimmunity.

Triggering receptor expressed on myeloid cells 1 (TREM‐1) is a cell surface molecule that was recently identified on monocytes and neutrophils. TREM‐1 has been implicated in the early inflammatory responses induced by microbes, but its pathophysiologic role in nonmicrobial inflammation remains unknown. In the present study, we investigated the role of TREM‐1 in acute inflammation induced by monosodium urate monohydrate (MSU) crystals. Induction of TREM‐1 expression by MSU crystal–stimulated murine resident peritoneal macrophages and infiltrating leukocytes in a murine air‐pouch model of crystal‐induced acute inflammation was determined. The biologic role of TREM‐1 in crystal‐induced cytokine production by resident peritoneal macrophages was also investigated.

TREM‐1 expression by resident peritoneal macrophages and infiltrating leukocytes in a murine air‐pouch model was determined by quantitative real‐time polymerase chain reaction, Western blot analysis, and flow cytometry. Cytokine production by resident peritoneal macrophages after incubation with MSU crystals in the presence or absence of an anti–TREM‐1 agonist antibody was determined by enzyme‐linked immunosorbent assay.

TREM‐1 expression by resident peritoneal macrophages was significantly induced after stimulation with the crystals. Maximum expression of TREM‐1 transcripts and protein occurred at 1 and 4 hours after exposure to the crystals, respectively. Costimulation of resident peritoneal macrophages with MSU crystals and an anti–TREM‐1 agonist antibody synergistically increased the production of both interleukin‐1β and monocyte chemotactic protein 1 compared with stimulation with the crystals alone. MSU crystals also induced TREM‐1 expression in infiltrating leukocytes in a murine air‐pouch model of crystal‐induced acute inflammation.

These findings suggest that rapid induction of TREM‐1 expression on resident peritoneal macrophages and neutrophils by MSU crystals may contribute to the development of acute gout through enhancement of inflammatory responses.

MicroRNAs (miRNA) have recently emerged as a new class of modulators of gene expression. In this study we investigated the expression, regulation, and function of miR‐155 and miR‐146a in rheumatoid arthritis (RA) synovial fibroblasts (RASFs) and RA synovial tissue.

Locked nucleic acid microarray was used to screen for differentially expressed miRNA in RASFs treated with tumor necrosis factor α (TNFα). TaqMan‐based real‐time polymerase chain reaction was applied to measure the levels of miR‐155 and miR‐146a. Enforced overexpression of miR‐155 was used to investigate the function of miR‐155 in RASFs.

Microarray analysis of miRNA expressed in RASFs treated with TNFα revealed a prominent up‐regulation of miR‐155. Constitutive expression of both miR‐155 and miR‐146a was higher in RASFs than in those from patients with osteoarthritis (OA), and expression of miR‐155 could be further induced by TNFα, interleukin‐1β, lipopolysaccharide, poly(I‐C), and bacterial lipoprotein. The expression of miR‐155 in RA synovial tissue was higher than in OA synovial tissue. Enforced expression of miR‐155 in RASFs was found to repress the levels of matrix metalloproteinase 3 (MMP‐3) and reduce the induction of MMPs 3 and 1 by Toll‐like receptor ligands and cytokines. Moreover, compared with monocytes from RA peripheral blood, RA synovial fluid monocytes displayed higher levels of miR‐155.

This study provides the first description of increased expression of miRNA miR‐155 and miR‐146a in RA. Based on these findings, we postulate that the inflammatory milieu may alter miRNA expression profiles in resident cells of the rheumatoid joints. Considering the repressive effect of miR‐155 on the expression of MMPs 3 and 1 in RASFs, we hypothesize that miR‐155 may be involved in modulation of the destructive properties of RASFs.