The canonical Hippo pathway components modulate the differentiation of lamina propria regulatory T cells and T helper 17-like regulatory T cells in mouse colitis.

Regulatory T cells (Tregs) ameliorate inflammatory bowel diseases. However, their plasticity is not completely understood. In this study using a mouse colitis model, Tregs and T helper 17 (Th17)-like Tregs were detected and sorted using flow cytometry, followed by transcriptome sequencing, real-time RT-PCR, and flow cytometry to analyze the mRNA profiles of these cells. Treg plasticity was evaluated by in vitro differentiation assays. The immunosuppressive activities of Tregs and Th17-like Tregs were assessed in an adoptive transfer assay. We found Tregs-derived Th17-like Tregs in inflamed colonic lamina propria (LP). LP Th17-like Tregs expressed higher Th17-related cytokines and lower immunosuppressive cytokines compared with LP Tregs. Notably, Tregs expressed higher Yes-associated protein 1 (YAP1) but lower transcriptional coactivator with PDZ­binding motif (TAZ) than Th17-like Tregs. Verteporfin-mediated inhibition of YAP1 activity enhanced Th17-like Treg generation, whereas IBS008739-induced TAZ activation did not affect Th17-like Treg generation. Besides, verteporfin enhanced while IBS008739 suppressed the differentiation of Th17-like Tregs into Th17 cells. Furthermore, YAP1 activated STAT5 signaling in Tregs, whereas YAP1 and TAZ activated STAT3 and STAT5 signaling in Th17-like Tregs. Compared with Tregs, Th17-like Tregs were less efficacious in ameliorating colitis. Therefore, YAP1 suppressed Treg differentiation into Th17-like Tregs. Both YAP1 and TAZ inhibited the differentiation of Th17-like Tregs into Th17 cells. Therefore, YAP1 and TAZ probably maintain the immunosuppressive activities of Tregs and Th17-like Tregs in colitis.

Sirtuin2 suppresses the polarization of regulatory T cells toward T helper 17 cells through repressing the expression of signal transducer and activator of transcription 3 in a mouse colitis model.

INTRODUCTION: Regulatory T cells (Tregs) play an important role in inflammatory bowel diseases (IBDs) through modulating intestinal inflammation. However, the factors affecting Treg function and plasticity during IBD progression are not thoroughly disclosed. The current study aims to reveal new molecular mechanisms affecting Treg plasticity. METHODS: A mouse strain, in which tdTomato and enhanced green fluorescent protein were under the control of the Foxp3 promoter and Il17a promoter, was established and subjected to colitis induction with dextran sulfate sodium. The existence of Tregs and IL-17-expressing Tregs (i.e., Treg/T helper 17 [Th17] cells) were observed and sorted from the spleen, mesenteric lymph nodes, and lamina propria by flow cytometry, followed by measuring Sirtuin2 (Sirt2) expression using quantitative reverse transcription polymerase chain reaction and Immunoblotting. Lentivirus-induced Sirt2 silencing was applied to determine the impact of Sirt2 on Treg polarization to Treg/Th17 cells and even Th17 cells. The effect of Sirt2 on Stat3 was analyzed by flow cytometry and immunoblotting. RESULTS: Sirt2 was highly expressed in lamina propria Tregs and it moderately suppressed Foxp3 expression as well as the immunosuppressive function of Tregs. Surprisingly, lentivirus-mediated Sirt2 silencing promoted the generation of Treg/Th17 cells out of Tregs. Sirt2 silencing also enhanced the generation of Th17 cells out of Tregs under the Th17 induction condition. Furthermore, Sirt2 inhibited Th17 induction by suppressing the protein level of the signal transducer and activator of transcription 3. CONCLUSION: Sirt2 suppresses Treg function but also inhibits Treg polarization toward Treg/Th17 cells and Th17 cells. The ultimate effect of Sirt2 on colitis might depend on the balance among Tregs, Treg/Th17 cells, and Th17 cells.

Contribution of CD4+ T cell-mediated inflammation to diarrhea in patients with COVID-19.

OBJECTIVES: This study aimed to explore the role of CD4+ T cells in the mechanisms of COVID-19 related diarrhea. METHODS: We analyzed lymphocyte subsets in patients with COVID-19 and the expression of angiotensin-converting enzyme 2 (ACE2), the transmembrane protease serine 2, and CD4+ T cell-related indicators in the colon were compared between patients with and without diarrhea. Correlation analyses were performed for ACE2 and other indicators to identify the relationship between SARS-CoV-2 infection and CD4+ mediated inflammation. The expression and distribution of CD4+ T cell-associated chemokines and their receptors were detected to determine the possibility of migration of CD4+ T cells to inflammation sites. RESULTS: The CD4+ T cell counts and percentages and CD4/CD8 ratio showed the most significant differences between the 2 groups. The diarrhea group expressed higher levels of ACE2, T-box expressed in T cells (Tbet), and tumor necrosis factor-alpha (TNFα) at both the mRNA and protein levels, with no difference from the nondiarrhea group for the percentage of ACE2+TNFα+ cells, indicating an indirect association between ACE2 and TNFα. The mRNA expression of CXCL10, CXCL11, and CXCR3 and the number of CD4+CXCR3+T cells were increased in the diarrhea group. CONCLUSIONS: CD4+ T cell-mediated inflammation may contribute to COVID-19 related diarrhea. CXCR3+ mediated migration of CD4+ T cells into the gut may perpetuate inflammation.

Hypoxic colorectal cancer-secreted exosomes deliver miR-210-3p to normoxic tumor cells to elicit a protumoral effect.

Hypoxia, the most common feature in the tumor microenvironment, is closely related to tumor malignant progression and poor patient's prognosis. Exosomes, initially recognized as cellular "garbage dumpsters", are now known to be important mediums for mediating cellular communication in tumor microenvironment. However, the mechanisms of hypoxic tumor cell-derived exosomes facilitate colorectal cancer progression still need further exploration. In the present study, we found that exosomes from hypoxic colorectal cancer cells (H-Exos) promoted G1-S cycle transition and proliferation while preventing the apoptosis of colorectal cancer cells by transmitting miR-210-3p to normoxic tumor cells. Mechanistic investigation indicated that miR-210-3p from H-Exos elicited its protumoral effect via suppressing CELF2 expression. A preclinical study further confirmed that H-Exos could promote tumorigenesis in vivo. Clinically, the expression of miR-210-3p in circulating plasma exosomes was markedly upregulated in colorectal cancer patients, which were closely associated with multiple unfavorable clinicopathological features. Taken together, these results suggest that hypoxia may stimulate colorectal cancer cells to secrete miR-210-3p-enriched exosomes in tumor microenvironment, which elicit protumoral effects by inhibiting CELF2 expression. These findings provide new insights on the mechanism of colorectal cancer progression and potential therapeutic targets for colorectal cancer.

Sestrin3 enhances macrophage-mediated generation of T helper 1 and T helper 17 cells in a mouse colitis model.

Intestinal macrophages participate in the pathogenesis of inflammatory bowel diseases (IBDs) through secreting pro-inflammatory and tissue-damaging factors as well as inducing the differentiation of T helper 1 (Th1) and T helper 17 (Th17) cells. Elucidating the regulatory mechanisms of intestinal macrophage activity in IBDs is important for developing new therapeutic approaches. In the current study, the expression of Sestrins in myeloid cells and lymphocytes in colonic lamina propria (LP) was evaluated in a murine acute colitis model. We found that Sestrin3 was significantly up-regulated in LP macrophages by the colonic LP microenvironment. In the in vitro experiments, lentivirus-mediated Sestrin3 knockdown significantly reduced the production of IL-12 and IL-23 in activated macrophages, in addition to decreasing the expression of classical pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. Additionally, Sestrin3 knockdown impaired macrophage-mediated generation of Th1 and Th17 cells from CD4+ T cells, probably through up-regulating the phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) in macrophages. In the in vivo experiments, adoptive transfer of Sestrin3-deficient macrophages alleviated the generation of Th1 and Th17 cells in the colonic LP and mesenteric lymph nodes. Furthermore, the adoptive transfer mitigated the severity of colitis, as demonstrated by lower production of pro-inflammatory cytokines and fewer tissue lesions in the colon. Our study suggests that Sestrin3 might be crucial for macrophage-mediated generation of pathogenic Th1 and Th17 cells in IBDs.

Meta-analysis: total parenteral nutrition versus total enteral nutrition in predicted severe acute pancreatitis.

BACKGROUND: Total parenteral nutrition (TPN) as a traditional mode of treatment in severe acute pancreatitis was still used widely in clinical work. In addition, enteral nutrition treatment methods have developed; early enteral nutrition has already been highlighted for severe acute pancreatitis, but the therapeutic risks versus benefits need to be studied. AIMS AND OBJECTIVE: To compare total parenteral nutrition with total enteral nutrition (TEN) in patients with severe acute pancreatitis by performing a meta-analysis. MATERIALS AND METHODS: Electronic databases including PubMed, EMBASE, Science Citation Index, were searched to find relevant randomized controlled trials. Two reviewers independently identified relevant trials evaluating the effect of total parenteral nutrition and early enteral nutrion. Outcome measures were the mortality, hospital length of stay, infectious complications, duration of nutrition, organ failure and surgical intervention. RESULTS: Eight randomized controlled trials (RCTs) including 381 patients were identified. Meta-analysis demonstrated that TEN was significantly superior to TPN when considering mortality [p=0.001, 95%CI 0.37(0.21-0.68)], infectious complications [p=0.004, 95%CI 0.46(0.27-0.78)], organ failure [p=0.02, 95%CI 0.44(0.22-0.88)] and surgical intervention [p=0.003, 95%CI 0.41(0.23-0.74)].While no difference between TEN and TPN when considering the hospital length of stay [p=0.22, 95%CI -14.10(-36.48-8.26)] and as for duration of nutrition [p=0.72, 95%CI -1.50(-9.56-6.56)] there was not enough data to compare the differences. CONCLUSION: Total enteral nutritional support is associated with lower mortality, fewer infectious complications, decreased organ failure and surgical intervention rate compared to parenteral nutritional support.

Association of cytotoxic T lymphocyte associated antigen-4 gene (rs60872763) polymorphism with Crohn's disease and high levels of serum sCTLA-4 in Crohn's disease.

BACKGROUND AND AIM: Our aim was to evaluate cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene polymorphisms in Crohn's disease (CD) and explore soluble CTLA-4 (sCTLA-4) levels in serum of CD patients in central China. METHODS: A total of 126 Chinese CD patients and 300 healthy controls were enrolled in this study. CTLA-4 (AT)n repeat polymorphism was genotyped by a semiautomatic fluorescently labeled polymerase chain reaction (PCR) method, and CTLA-4 -1661A/G and -1722T/C polymorphisms were genotyped by DNA sequencing. Serum sCTLA-4 and C-reactive protein (CRP) levels were determined by enzyme linked immunosorbent assay (ELISA) and immunonephelometry, respectively. RESULTS: The frequency of 84 bp allele of CTLA-4 (AT)n repeats was lower in CD patients than in the healthy controls (22.2% vs 33.2%, P = 0.001, odds ratio = 0.58, 95% confidence interval: 0.41-0.81). The 84 bp allele carriers of (AT)n repeats were associated with non-stricturing and non-penetrating disease behavior in CD patients (P = 0.007). Serum sCTLA-4 levels were more elevated in CD patients than in the healthy controls (P < 0.001). Among CD patients, serum sCTLA-4 levels were increased in active disease compared with inactive disease (P = 0.015), and were correlated with CRP levels (r = 0.524, P < 0.001). Serum sCTLA-4 levels were higher in CD patients with stricturing disease behavior than in patients with other disease behaviors (P = 0.009). CONCLUSIONS: 84 bp allele of CTLA-4 (AT)n repeat polymorphism was associated with CD in central China. sCTLA-4 levels were highly expressed in CD, especially in active disease, and were correlated with CRP levels and disease behavior in CD patients.

[CTLA-4 gene polymorphism regulates CTLA-4 mRNA stability and protein level in patients with ulcerative colitis].

OBJECTIVE: To investigate the effect of (AT)n repeat polymorphism of the 3'untranslated region in cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) gene on CTLA-4 mRNA stability and full length (flCTLA-4) and soluble CTLA4 (sCTLA-4) expression in ulcerative colitis (UC). METHODS: flCTLA-4 mRNA in colonic biopsies and sCTLA-4 mRNA stability in peripheral blood mononuclear cells of UC patients were measured by quantitative PCR and half-life, respectively. The protein expression of flCTLA-4 in colonic biopsies and sCTLA-4 in sera of UC patients were determined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The polymorphism of CTLA-4 (AT)n repeats in 300 UC and 700 age and sex matched healthy controls was genotyped by fluorescent PCR. RESULTS: Among the UC patients, sCTLA-4 mRNA expression levels were decreased in active disease compared to non-active disease (P= 0.004). Carriers of the longer alleles of the (AT)n repeats expressed lower levels of flCTLA-4 and sCTLA-4 mRNA and sCTLA-4 protein than those of the shorter alleles in UC (all P< 0.01), and mRNA with long (AT)n repeat alleles has shorter half-life than mRNA with short alleles and, hence, are unstable. The frequency of long allele carriers of CTLA-4 (AT)n repeats was significantly higher in UC patients than in the healthy controls (22.0% vs. 6.3%, P< 0.01, OR= 4.21, 95% CI: 2.79-6.33), and associated with extensive colitis (P= 0.008). CONCLUSION: CTLA-4 gene expression levels were associated with (AT)n repeat polymorphisms in UC patients. The expression of CTLA-4 mRNA and protein were decreased in carriers of the longer alleles of the (AT)n repeats of CTLA-4 gene. This study suggests that CTLA-4 plays an important role in genetic risk and pathophysiology for UC in central China.

Genetic polymorphism of methylenetetrahydrofolate reductase G1793A, hyperhomocysteinemia, and folate deficiency correlate with ulcerative colitis in central China.

BACKGROUND AND AIMS: Methylenetetrahydrofolate reductase (MTHFR) encoding genes were associated with ulcerative colitis in Chinese in our previous study. We further studied association of a new polymorphism of MTHFR G1793A with ulcerative colitis and assessed relationship of this polymorphism with hyperhomocysteinemia (HHcy, > or = 15 mmol/L) and deficiency of folate (< or = 7 nmol/L) and vitamin B(12) (< or = 150 pmol/L) in a cohort of patients with ulcerative colitis in central China. METHODS: A total of 252 patients and 654 healthy controls were recruited. Polymorphism of MTHFR G1793A was examined using a polymerase chain reaction-restriction fragment length polymorphism method. Plasma levels of homocysteine (Hcy), folate and vitamin B(12) were determined by enzymatic cycling assay and corpuscle immune chemiluminescence assay, respectively. RESULTS: Frequencies of alleles and genotypes in MTHFR G1793A gene differed significantly between ulcerative colitis patients and the healthy controls (20.83% vs 10.47%, 95% confidence interval [CI]: 1.703-2.972, P = 0.0006; 40.48% vs 19.88%, 95% CI: 1.997-3.761, P = 0.0002, respectively). Plasma Hcy levels were higher and folate and vitamin B(12) concentrations were lower in the patients than in the healthy controls (21.72 +/- 6.59 vs 12.47 +/- 5.02, 95% CI: -10.93--7.58, P < 0.0001; 11.25 +/- 6.19 vs 15.28 +/- 7.72, 95% CI: 2.03-6.04; P < 0.001; 322.81 +/- 128.47 vs 442.59 +/- 129.36, 95% CI: 62.61-136.95, P < 0.0001, respectively). HHcy and folate deficiency were more prevalent in patients with ulcerative colitis (45.32% vs 26.17%, 95% CI: 1.285-4.378, P = 0.005; 30.68% vs 13.0%, 95% CI: 1.416-6.197, P = 0.003, respectively). CONCLUSIONS: MTHFR G1793A gene polymorphism, HHcy, folate deficiency and low vitamin B(12) concentration were associated with ulcerative colitis in central China. Our findings demonstrate that the Hcy-related gene and metabolites are involved in pathogenesis of ulcerative colitis.

MICB0106 gene polymorphism is associated with ulcerative colitis in central China.

BACKGROUND: The highly polymorphic nonclassical MHC class I chain-related genes A and B (MICA and MICB) encode stress-inducible glycoproteins expressed on various epithelial cells including intestinal epithelial cells. MICA and MICB gene polymorphisms and expressions are associated with autoimmune diseases but not known in ulcerative colitis (UC). AIMS: To investigate the association of MICB exon 2-4 polymorphisms and soluble MICA (sMICA) expression with the susceptibility of UC in central China. MATERIALS AND METHODS: Genomic DNA was isolated from peripheral blood. The allele frequencies of MICB exon 2-4 were genotyped in 105 UC patients and 213 healthy controls by PCR single-stranded conformation polymorphism method. Thirty-two patients and 32 controls were selected for determining serum sMICA expression by ELISA. RESULTS: Allele frequency of MICB0106 was significantly higher in UC patients than in healthy controls (19.0% vs. 8.9%, corrected P (Pc) = 0.0006), especially in patients with extensive colitis (24.4% vs. 8.9%, Pc = 0.0006), moderate and severe disease (24.1% vs. 8.9%, Pc = 0.0006), extraintestinal manifestations (20.5% vs. 8.9%, Pc = 0.012), male patients (22.1% vs. 8.0%, Pc = 0.006), and patients over the age of 40 years (28.8% vs. 8.3%, Pc = 0.0006). The sMICA level was significantly higher in UC than in healthy controls (604.41 +/- 480.43 pg/ml vs. 175.37 +/- 28.31 pg/ml, P = 0.0001) but not associated with the MICB0106 genotypes. CONCLUSIONS: Overall, MICB0106 allele was positively associated with UC in the Han Chinese in central China. sMICA was highly expressed in UC but not associated with the MICB0106 genotype.

[Association of cytotoxic T lymphocyte antigen-4 promoter C-658T polymorphism with ulcerative colitis in Chinese].

OBJECTIVE: To investigate the association of gene polymorphism of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) with ulcerative colitis (UC) in Chinese. METHODS: One hundred and seventeen patients with UC and 246 healthy controls were genotyped for the polymorphisms of C-658T in the promoter and C61T at the 3' untranslated region of the CTLA-4 gene using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), respectively. The genotype and allele frequencies of the two groups were calculated and compared by chi square test. RESULTS: The frequency of TT+CT genotype at the CTLA-4 gene C-658T in the promoter was significantly higher in UC patients than that in healthy controls (P=0.015). The frequency of the T allele at this locus was also significantly higher in UC patients than that in the controls (P=0.033). The frequencies of TT genotype and T allele at the C-658T locus were highly associated with extensive colitis in UC patients (P=0.037, and P=0.0067, respectively). CONCLUSION: The T allele of CTLA-4 promoter C-658T locus was highly associated with UC in Chinese Han of central China.

Role of major histocompatibility complex class I-related molecules A*A5.1 allele in ulcerative colitis in Chinese patients.

The major histocompatibility complex (MHC) class I-related molecules A (MICA) is a stress-inducible cell surface antigen that is recognized by intestinal epithelial Vdelta1 gammadelta T cells, natural killer (NK) cells and CD8(+) T cells with NKG2D receptor participating in the immunological reaction in the intestinal mucosa. The present study aimed to investigate the functions of the MICA*A5.1 allele in the development of ulcerative colitis (UC) in the Chinese population. The microsatellite polymorphisms of MICA were genotyped in 124 unrelated Chinese patients with UC and 172 ethnically matched healthy controls using a semiautomatic fluorescently labelled polymerase chain reaction. MICA*A5.1-expressing Raji cells were generated by gene transfection. Cytotoxicity of NK cells to Raji cells expressing different MICA molecules was detected using the lactate dehydrogenase method. Soluble MICA in the culture supernatant was detected by enzyme-linked immunosorbent assay. The frequency of MICA*A5.1 was significantly higher in UC patients compared with the healthy controls (29.0% versus 17.4%, P = 0.001, corrected P = 0.005, OR = 1.936, 95% CI 1.310-2.863) and the frequency of a MICA*A5.1/A5.1 homozygous genotype was increased in UC patients (18.5% versus 7% in healthy controls, P = 0.0032, corrected P = 0.048, OR = 3.036, 95% CI 1.447-6.372). Raji cells with MICA*A5.1 expression produced more soluble MICA (t = 5.75, P < 0.01) than Raji cells with full-length MICA expression in culture supernatant. Raji cells with MICA*A5.1 expression were more resistant to killing by NK cells than Raji cells with full-length MICA expression. The MICA*A5.1 allele and MICA*A5.1/A5.1 genotype are significantly associated with Chinese UC patients in central China. MICA*A5.1 may play a role in the development of UC by producing more soluble MICA and resistance to NK cells.

Cytotoxic T lymphocyte antigen-4 promoter -658CT gene polymorphism is associated with ulcerative colitis in Chinese patients.

BACKGROUND AND AIM: Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a role in the downregulation of T cell activation. The present study aimed to examine an association between the CTLA-4 gene polymorphisms and ulcerative colitis (UC) in the Han Chinese in central China. MATERIALS AND METHODS: One hundred seventeen patients with UC and 246 healthy controls were genotyped for CTLA-4 gene -658CT in the promoter and CT61 at the 3' untranslated region using a method of polymerase chain reaction-based restriction fragment length polymorphism and single-strand conformation polymorphism, respectively. The distributions of the genotypes and the allele frequencies of the CTLA-4 gene in UC patients and healthy controls were compared by chi-square test. RESULTS: The frequency of the T/T+C/T genotype at the CTLA-4 gene -658CT in the promoter was significantly higher in UC patients than in healthy controls (26.5% vs 15.4%, chi (2) = 6.287, P = 0.015, OR = 1.973, 95%CI = 1.153-3.375). The frequency of the T allele at the CTLA-4 -658CT was also significantly higher in UC patients than in the controls (13.2% vs 8.1%, chi (2) = 4.707, P = 0.033, OR = 1.726, 95%CI = 1.049-2.838). The frequency of the T/T genotype at the -658 locus was highly associated with extensive colitis in UC patients (P = 0.037, OR = 3.955, 95%CI = 1.068-14.647). The frequency of the T allele at the -658 locus was highly associated with extensive colitis in UC patients (P = 0.0067, OR = 3.05, 95%CI = 1.320-7.048). CONCLUSION: The T allele of CTLA-4 -658 polymorphism in the promoter of CTLA-4 gene was highly associated with UC in the Han Chinese in central China.

[An association between MICB 0106 allele and ulcerative colitis in Chinese Han in Hubei province].

OBJECTIVE: To investigate the association between the exon 2, 3, 4 of MHC class I chain-related gene-B (MICB) and ulcerative colitis (UC) in Chinese Han. METHODS: Using polymerase chain reaction single-stranded conformation polymorphism, allele frequency of MICB exon 2, 3 and 4 in 105 patients with UC and 213 age and sex matched healthy controls were genotyped. All of the studied individuals were Chinese Han. RESULTS: Allele frequency of MICB 0106 was increased in patients with UC as compared with normal controls (19.0% vs 8.9%, P = 0.000, Pc < 0.001, OR = 2.402, 95% CI: 1.488-3.879). The frequency of MICB 0106 was increased significantly in patients with extensive colitis (24.4% vs 8.9%, P = 0.000, Pc < 0.001, OR = 3.294, 95% CI: 1.800-6.027), moderate and severe disease (24.1% vs 8.9%, P = 0.000, Pc < 0.001, OR = 3.294, 95% CI: 1.893-5.576) and in those with extra intestinal manifestations (20.5% vs 8.9%, P = 0.002, Pc = 0.012, OR = 2.626, 95% CI: 1. 18 4. 61). Furthermore, MICB 0106 allele was higher in frequency in the male patients with UC (22. % vs 8. % P = 0. 01, Pc =0 . 06, OR =3 . 76, 95% CI:1 . 37 6. 78) and the patients more than4 0 years old (28.8% vs 8.3% P = 0.000, Pc <0 .001, OR= 4 .500, 95% I:2 . 81 8.504) as compared with healthy controls. CONCLUSION: MICB 0106 allele is positively associated with UC, especially with extensive colitis, moderate and severe disease, presence of extra intestinal manifestations, male gender and age of more than 40 years in Chinese Han in Hubei province.