Inflammasome activation and formation of ASC specks in patients with juvenile idiopathic arthritis.

Objective: The formation of large intracellular protein aggregates of the inflammasome adaptor ASC is a hallmark of inflammasome activation and characteristic of autoinflammation. Inflammasome activated cells release the highly proinflammatory cytokine IL-1ß in addition to ASC specks into the extracellular space. Autoinflammatory activity has been demonstrated in systemic JIA, however minimal data exist on the role of inflammasomes in other JIA subtypes. We therefore investigated, if pyroptotic cells are present in the circulation of oligo- and poly-articular JIA. Methods: Peripheral blood of JIA patients (n = 46) was investigated for ASC speck formation, a key step in inflammasome activation, by flow cytometry and immunofluorescence. Free ASC and proinflammatory cytokine levels were determined by ELISA and multiplex assay. Results: Oligo-articular JIA patients showed a significantly increased proportion of ASC speck+ monocytes compared to poly-articular JIA patients. In serum free ASC alone is not sufficient to assess inflammasome activity and does not correlate with ASC speck+ monocytes. Compared to control several cytokines were significantly elevated in samples of JIA patients. JIA serum containing antinuclear antibodies, incubated with ASC specks boosts a secondary inflammation by IL-1ß production in macrophages. Conclusion: For the first time, we detect ex vivo inflammasome activation by ASC speck formation in oligo- and poly-articular JIA patients. Most notably, inflammasome activation was significantly higher in oligo- compared to poly-articular JIA patients. This data suggests that inflammasome derived autoinflammation may have a greater influence in the previously thought autoimmune oligo-articular JIA patients.

Decreased IgG4 ACPA levels in responders and increased CD1c+ classical dendritic cells in non-responders of patients with rheumatoid arthritis under therapy.

The treatment options for patients suffering from rheumatoid arthritis expanded over the last years. However, reliable biomarkers to guide therapy decisions are still warranted. Therefore, we here evaluated the value of antibodies against citrullinated peptide antigens (ACPA) IgG subclasses and peripheral blood antigen presenting cells as biomarkers to monitor and predict therapy response of patients with rheumatoid arthritis. Thirty-four ACPA-positive RA patients were enrolled and monitored for 3 months after therapy begin. ACPA IgG1 and IgG4 serum levels were quantified by ELISA. Phenotyping of the B cell, monocytic, and dendritic cell lineages was performed via flow cytometry. Three months after therapy begin, the responders showed a significant decrease in IgG4 ACPA levels, and this was independent of the individual treatment regimen. The non-responders showed a significant increase in CD1c+ classical dendritic cells (cDC). Furthermore, the baseline disease activity score 28 and the baseline percentage of cDC allowed for some prediction of future therapy responses. We here suggest IgG4 ACPA levels as biomarkers to monitor therapy response in RA. The increase in CD1c+ cDC among non-responders to therapy remains enigmatic and requires future elucidation of the underlying mechanisms.

The Prerequisites for Central Tolerance Induction against Citrullinated Proteins in the Mouse.

OBJECTIVES: To assess the prerequisites for negative selection of peptidylcitrulline-specific T cells in the thymus. In detail, we here analyzed murine medullary thymic epithelial cells for the expression of peptidylarginine deiminases (PAD) and subsequent citrullination. METHODS: Medullary thymic epithelial cells were sorted, their mRNA was isolated and the expression of Pad genes was analyzed by quantitative PCR. Citrullination was detected by Western Blot in lysates of sorted medullary thymic epithelial cells and histologically by immunofluorescence of thymic thin sections. RESULTS: Pad2 and Pad4 are the main Pad isoforms expressed in mature medullary thymic epithelial cells of the mouse and their levels of expression are comparable to that of insulin (Ins2), another highly and promiscuously expressed protein in the thymus. Citrullination was detected in medullary thymic epithelial cells as shown by Western Blot and immunofluorescence. CONCLUSIONS: Even though we here show that the murine thymus harbors the prerequisites for central tolerance to PAD and citrullinated peptides, it remains an open question whether the emergence of peptidylcitrulline-specific T cells and of autoantibodies recognizing citrullinated epitopes is caused by a failure of central or peripheral tolerance mechanisms.