Inflammatory Cytokine Neutralizing Antibody Treatment Could Recover Increased Follicular Helper and Follicular Regulatory T Cells in Murine Models of Arthritis

Objective. We aimed to illustrate the changes of follicular helper (TFH) and follicular regulatory (TFR) cells in rheumatoid arthritis using the collagen-induced arthritis (CIA) model and clarity the impact of anti-inflammatory treatment on TFH and TFR cells.Methods. We established 10-week-old CIA model and used flow cytometry to analyze the changes of TFH and TFR cells in peripheral blood and spleen at different time points (5w, 7w, 10w,13w). The expression of TIGIT, CD226, ICOS and PD-1 characterizing the functions of TFH and TFR were also analyzed. The function of spleen TFH and TFR cells from CIA was further analyzed. The effects of anti-inflammatory antibody treatments on the subpopulation and function changes of TFH and TFR were also analyzed in CIA mice.Results. The levels of TFH and TFR were significantly increased in spleen and peripheral blood of CIA mice. After treatment, TFH and TFR cells decreased significantly. TIGIT+ and TIGIT+CD226-TFH cells in CIA mouse spleen were elevated and PD-1 and ICOS expression on TFH and TFR cells in the spleen were also significantly increased. The ability of TFH to secrete IL-21 and help B cells, and TFR to secrete IL-10 and inhibit TFH were both enhanced in CIA mouse spleen. After antibody treatment, cell subsets and functions were significantly recovered.Conclusion. In the pathogenesis of rheumatoid arthritis, TFH and TFR cells in the germinal center increases and their functions are enhanced. After the treatment by inflammatory factor antibodies, TFH and TFR subsets and their functions can be significantly recovered.

Effect of (−)-Epigallocatechin Gallate on Activation of JAK/STAT Signaling Pathway by Staphylococcal Enterotoxin A

Staphylococcal enterotoxin A (SEA), which is a superantigen toxin protein, binds to cytokine receptor gp130. Gp130 activates intracellular signaling pathways, including the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. The effects of SEA on the JAK/STAT signaling pathway in mouse spleen cells were examined. After treatment with SEA, mRNA expression levels of interferon gamma (IFN-γ) and suppressor of cytokine-signaling 1 (SOCS1) increased. SEA-induced IFN-γ and SOCS1 expression were decreased by treatment with (−)-epigallocatechin gallate (EGCG). The phosphorylated STAT3, Tyr705, increased significantly in a SEA concentration-dependent manner in mouse spleen cells. Although (−)-3″-Me-EGCG did not inhibit SEA-induced phosphorylated STAT3, EGCG and (−)-4″-Me-EGCG significantly inhibited SEA-induced phosphorylated STAT3. It was thought that the hydroxyl group at position 3 of the galloyl group in the EGCG was responsible for binding to SEA and suppressing SEA-induced phosphorylation of STAT3. Through protein thermal shift assay in vitro, the binding of the gp130 receptor to SEA and the phosphorylation of STAT3 were inhibited by the interaction between EGCG and SEA. As far as we know, this is the first report to document that EGCG inhibits the binding of the gp130 receptor to SEA and the associated phosphorylation of STAT3.

NO-stressed Y. pseudotuberculosis have decreased cell division rates in the mouse spleen

Fluorescence dilution approaches can detect bacterial cell division events, and can detect if there are differential rates of cell division across individual cells within a population. This approach typically involves inducing expression of a fluorescent protein, and then tracking partitioning of fluorescence into daughter cells. However, fluorescence can be diluted very quickly within a rapidly replicating population, such as pathogenic bacterial populations replicating within host tissues. To overcome this limitation, we have generated a revTetR reporter construct, where mCherry is constitutively expressed, and repressed by addition of tetracyclines, resulting in fluorescence dilution within defined timeframes. We show that mCherry signal is diluted in replicating populations, and that mCherry signal accumulates in growth-inhibited populations, including during exposure to inhibitory concentrations of antibiotics and during nitric oxide exposure. Furthermore, we show that tetracyclines can be delivered to the mouse spleen during Yersinia pseudotuberculosis infection. We defined a drug concentration that results in even exposure of cells to tetracyclines, and used this system to visualize cell division within defined timeframes post-inoculation. revTetR mCherry signal did not appear enriched in a particular spatial location within replicating centers of bacteria. However, the addition of a NO-sensing reporter (Phmp::gfp) showed that heightened NO exposure correlated with heightened mCherry signal, suggesting decreased cell division within this subpopulation. This revTetR reporter will provide a critical tool for future studies to identify and isolate slowly replicating bacterial subpopulations from host tissues.