| Ulcerative colitis (UC) is a long-term multifactorial disease, characterized by chronic ulcers in the innermost lining of the colon and rectum, which often results in abdominal pain, cramping, diarrhea, and/or hemofecia, with the possibility of developing life-threatening complications. Also, the long-term UC will have the possibility to develop into colorectal cancer (CRC). Symptom treatment plays critical roles in UC, because it is easy to operate clinically, and is widely accepted by patients and clinicians, However, the treatments are not very satisfactory. For UC susceptible individuals, the dysregulated immune system may cause inappropriate responses to antigen secreted by gut bacteria. The improper immune responses result in expression of proinflammatory factors, interleukins (IL), and tumor necrosis factor alpha(TNF-α). The mechanism of UC is extremely complicated and is thought to involve in immune, metabolic, hereditary and environmental factors. Also, the exact cause of UC progressing to CRC is still unclear. Studies have shown that Glucagon-like peptide-2(GLP-2), interleukin 35 (IL-35), and CD8~+ T cells play important roles in human immune system and metabolic system. GLP-2 is a gut hormone, and increase gut digestion, absorption, and barrier function. IL-35 is a new number of IL-12 family,and play important roles in inducing inflammation. The major functions of CD8~+ T cells are control infect and suppress tumor progress. CD8~+ T cells can differentiate into cytotoxic T cells and express various proinflammatory factors.Based on these studies, we hypothesize that dysregulation of GLP-2. IL-35.and CD8~+ T cells may be correlated with UC and CRC. Here, we investigated changes and mechanisms of GLP-2, IL-35, and CD8~+ T cells in UC. We also preliminary examined these factors in CRC.The research contains two parts.1. To investigate changes and roles of GLP-2, IL-35, and CD8~+ T cells in UC.Purpose: to understand the changes and roles of GLP-2, IL-35, and CD8~+ T cells in UC, and further learn the effect of IL-35 on T cells and B cells in UC.Methods: UC rat model was established, and levels of GLP-2 and IL-35 in serum as well as CD8~+ T cells in peripheral blood were examined. In addition, we recruited 18 active UC patients and 18 healthy controls, and examined levels of GLP-2 and IL-35 in serum as well as CD8~+ T cells in peripheral blood. The patient group was divided into 3 subsets according to the severity of the disease. CD8~+ T cells and B cells were isolated from PBMCs using the Human CD8~+ T cell enrichment kit and the Human B cell enrichment kit, which employed negative magnetic sorting strategy. CD4+CD25+Treg cells were positively sorted by using the Human CD4+CD25+ T cell isolation kit.We further investigated the role of IL-35 in T cells and B cells, and related pathways through in vitro experiments. Comparisons between two datasets were performed using Welch’s t test (parametric) or Mann-Whitney test (nonparametric). Comparisons between multiple datasets were performed using one-way or two-way ANOVAs,followed by Bonferroni’s (parametric) or Dunn’s (nonparametric) post-test. All tests were performed by PRISM 5 software.Results: Level of GLP-2 in UC rat model remained unchanged compared with controls, whereas IL-35 level significantly decreased and the proportion of CD8~+ T cells slightly increased. Level of GLP-2 in human UC patients was dispersed, and was similar among patients with different severity. IL-35 level in patients was significantly decreased, and was correlated with disease severity. The proportion of CD8~+ T cells in the peripheral blood of UC patients was slightly elevated, whereas CD8~+ T cells in severe patients presented higher percentage than those in healthy controls. Further study of the role of IL-35 showed that pre-treating peripheral blood mononuclear cells (PBMCs) with IL-35 resulted in significantly elevated IL-10 production from whole PBMCs as well as B cells, whereas pre-treating PBMCs with IL-12 or IL-27 did not demonstrate a similar effect. IL-35 suppressed the proliferation of CD4+CD25- conventional T cells, CD4+CD25+ regulatory T cells (Tregs), and CD8~+ T cells, but did not inhibit the proliferation of B cells. IL-35-mediated IL-10 secretion in B cells did not require the presence of Tregs. To examine whether the enhancement in CD4+CD25- and CD8~+ T cell suppression by IL-35-treated B cells was restricted to UC patients, we repeated these experiments using T cells and B cells harvested from healthy controls. We found that IL-35-treated B cells from healthy controls significantly suppressed IFN-y, IL-17, and TNF-a secretion from CD4+CD25- T cells, as well as IFN-y and TNF-a production from CD8~+ T cells.Furthermore, the proliferation CD4+CD25- T cells and CD8~+ T cells was suppressed by IL-35-treated B cells, a function blocked by IL-10 and IL-10R monoclonal antibodies. After treatment with IL-35, B cells from UC patients presented significantly enhanced regulatory function, characterized by inhibiting cell proliferation and interferon (IFN)-γ, IL-17 and tumor necrosis factor (TNF)-a secretion from autologous CD4+CD25- T cells and CD8~+ T cells, which was dependent on IL-10 signaling. However, IL-35-treatment did not demonstrate an effect on regulating IL-5 and IL-13 responses. These discoveries identified a Thl,Th17 and CD8~+ T cell-targeting role of IL-35 in UC patients. Next, we examined the IL-35 expression in the intestinal mucosal in UC patients. Data showed that both non-inflamed and inflamed tissues from UC patients presented significantly lower IL-35 secretion compared to healthy control tissues, which was associated with suppressed p35 transcription. UC patients with higher IL-35 also presented higher IL-10 secretion in gut mucosa.Conclusions: GLP-2 did not present clear changes, whereas IL-35 significantly decreased in the peripheral blood of UC, and was correlated with disease progression.Percentage of CD8~+ T cells was only significantly elevated in severe patients. Our study identified that IL-35 could mediate antiinflammatory function through promoting regulatory B cell functions, but this effect was suppressed in UC patients.2. To preliminary study changes and roles of GLP-2, IL-35, and subtype of CD8~+T cells in CRC.Purpose: Colorectal cancer is the third most prevalent cancer type worldwide and contributes to a significant percentage of cancer-related mortality.GLP-2, IL-35, and CD8~+ T cells play import roles in the metabolism, inflammation, and immunity of intestinal system. It is possible that these factors are correlated with the development of CRC. In addition, CD8~+ T cell is one of the most important cells in antitumor immunity. Recent studies have shown that the CXCR5+CD8~+ T cells present more potent proinflammatory function, and it is possible that the cells may affect the progression of CRC. This study aimed at understanding the changes of GLP-2, IL-35,and CD8~+ T cells in CRC patients,as well as the role of CXCR5+CD8~+ T cells in the cancer.Methods: we examined levels of GLP-2 and IL-35 in serum as well as CD8~+ T cells in peripheral blood of 7 CRC patients and 7 controls. For the detection of CXCR5+CD8~+ T cell frequency, freshly isolated PBMCs, tumor-infiltrating lymphocytes or tumor-associated lymph node-infiltrating lymphocytes were incubated with Aqua Dead Cell stain and anti-human CD3, CD4, CD8 and CXCR5. For intracellular cytokine or transcription factor detection, after surface staining, cells were treated with Cytofix/Cytoperm buffer set or Transcription Factor buffer set according to the manufacturer’s instructions. We further investigated the number of CXCR5+CD8~+ T cells in CRC tumor and PBMCs. Function of CXCR5+CD8~+ T cells was examined through in vitro experiments. Data were analyzed in GraphPad Prism.Some data were analyzed using the one-way ANOVA with Tukey’s multiple comparison test, some using the unpaired t test with Welch’s correction,and some using the two-way ANOVA with Sidak’s multiple comparison test. Data were considered significant when P< 0.05.Results: GLP-2 and IL-35 did not change in CRC, whereas level of CD8~+ T cell was relatively increased and was dispersed in CRC. We demonstrated that CXCR5+CD8~+T cells were rarely present in the PBMCs from healthy and colorectal cancer individuals, but the proportion of the cells was significantly increased in resected tumors and tumor-associated lymph nodes. Under ex vivo condition, the CXCR5+CD8~+ T cells demonstrated lower degranulation, TNFα expression and IFNγexpression than CXCR5-CD8~+ T cells. However after PMA + ionomycin stimulation,the degranulation and TNFa expression by CXCR5+CD8~+ T cells were significantly elevated to a level comparable with CXCR5-CD8~+ T cells, whereas the IFNy expression by PMA + ionomycin-stimulated CXCR5+CD8~+ T cells were significantly higher than that by CXCR5-CD8~+ T cells. Following long-term TCR-stimulation,CXCR5+CD8~+ T cells demonstrated significantly more potent proliferation capacity and higher IFNy expression than CXCR5-CD8~+ T cells. TCR-stimulated CXCR5+CD8~+ T cells also showed a gradual downregulation in CXCR5 expression.CXCR5+CD8~+ T cells also enhanced the IgG expression by autologous B cells.Conclusions: GLP-2 and IL-35 did not obviously change, whereas CD8~+ T cells moderately elevated in CRC. CXCR5+CD8~+ T cells may play roles in the tumor microenvironment and the tumor-associated lymph nodes by secreting cytokines and increasing B cell immunities, and have the potential to contribute to antitumor immunity in colorectal cancer.Significance and novelty of the study: This study clarified the changes of GLP-2,IL-35 and CD8~+ T cells in UC as wells as CRC. and particularly learned the role of IL-35 in UC as well as the role of CXCR5+CD8~+ T cells in CRC. This knowledge will shed lights on developing IL-35 or CXCR5+CD8~+ T cells targeted novel immunotherapies on these diseases. |
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