Analysis of intestinal immunity and flora in a collagen-induced mouse arthritis model: differences during arthritis progression.

Intestinal immunity and flora are reported to be associated with the onset of rheumatoid arthritis. However, differences in the intestinal immunity and flora dynamics between the initial peak and relapse of arthritis have not been investigated. Here we analyzed the lymphocyte populations in different lymphoid tissues, the IgA in feces, and the intestinal flora at the initial peak and the relapse phase of arthritis in a collagen-induced arthritis (CIA) mouse model. In this model compared with the control group, the percentage of RORγt+CD4+ T cells in the mesenteric lymph nodes (mLN) was increased at the initial peak but decreased at the relapse stage of arthritis, and the opposite changes were observed in the spleen. The percentage of Foxp3+CD4+ T cells was unchanged at the initial peak in both tissues but increased only in the mLN at the relapse stage. The IgA in feces increased with the progression of arthritis, and bacterial analysis revealed that some specific bacterial families were changed at the peak and relapse stages of arthritis. Finally, the immune dynamics under different arthritic conditions were examined by integrating these factors using principal component analysis (PCA). PCA showed that the immunological and intestinal flora profiles were different between the initial peak and the relapse of the arthritis. Our findings suggest that the intestinal immunity and the environment change drastically with the progress of arthritis.

Interleukin-12p40 variant form reduces Interleukin-12p80 secretion.

IL-12 is a key cytokine for the promotion of CD4+ T cells differentiation to type 1 helper T cells. IL-12 is a heterodimer (IL-12p70) consisting of p40 and p35 subunits, and is mainly secreted from activated antigen-presenting cells, such as macrophages and dendritic cells (DCs). In this study, we found that activated mouse bone marrow-derived DCs (BMDCs) produced a p40 splice variant form mRNA in addition to the conventional p40 mRNA. This p40 variant mRNA was produced by alternative splicing in exon 5, and possessed a premature stop codon. As a result, the p40 variant protein contained 157 amino acids of the N-terminal part of p40 and an additional 10 novel amino acids. When the p40 variant was expressed in HEK-293T cells, it was not secreted from the cells. To investigate the function of the p40 variant, it was co-expressed with p40 and/or p35. The p40 variant did not affect the secretion of IL-12p40 or IL-12p70, or the function of the secreted p70. In contrast, the secretion of IL-12p80, a homodimeric IL-12 with two p40 subunits, was significantly decreased when the p40 variant was expressed. This new splicing variant p40 may act to fine-tune the function of IL-12p80.

IL-21 Enhances the Development of Colitis-Associated Colon Cancer: Possible Involvement of Activation-Induced Cytidine Deaminase Expression.

Inflammatory bowel diseases are known to be the origin of colitis-associated colon cancer (CAC). We previously reported that dextran sulfate sodium (DSS)-induced colitis is exacerbated in mouse-IL-21-isoform transgenic (Tg) mice. In this study, we assessed the CAC development induced by azoxymethane (AOM) and DSS in our Tg mice. AOM-DSS-induced tumor development was dramatically increased in the Tg mice compared with wild-type mice. IL-21 is known to enhance activation-induced cytidine deaminase (AID) expression in B cells and induce Ab class switching. In contrast, the AID expression in cells other than B cells initiates tumor development in many tissues. Therefore, we investigated whether IL-21 induces the AID expression in the large intestinal epithelial cells (IECs) during CAC development. AID gene and protein expression was increased in the IECs of AOM-DSS- or DSS-treated Tg mice compared with those of wild-type mice. Furthermore, we confirmed IL-21 induced AID gene expression in the purified IECs ex vivo. The present study also showed IL-21R gene expression in unstimulated wild-type mouse IECs, and this gene expression was augmented by TNF-α stimulation. The IL-21R expression and IL-21-induced AID gene activation were further confirmed in the Colon-38 cell line. Taken together, IL-21 may be involved in increasing the risk of CAC by enhancing the AID expression in IECs.

Systemic neutrophil migration and rapid consumption of neutrophils in the spleen.

The systemic migration of neutrophils is not fully understood. In this study, we purified neutrophils from rat peripheral blood and labeled them with [51Cr] sodium chromate. The labeled cells were injected into the tail veins of rats, and were traced. Neutrophils were rapidly trapped in the liver and the spleen within 6 h. The migration ratios of neutrophils in the lung and the gut were lower compared with those in the liver and the spleen. Interestingly, migrated cells into the spleen were rapidly phagocytosed by monocytes/macrophages. Therefore, accumulation of intact neutrophils in the spleen may be difficult to measure.

Augmentation of the expression of the eotaxin receptor on duodenal neutrophils by IL-21.

Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193+ neutrophils, which easily migrated into the duodenum. These observations suggested that CD193+ neutrophils increase in number under inflammatory conditions due to chronic IL-21 production.