Circadian clock component PER2 negatively regulates CD4+ T cell IFN-γ production in ulcerative colitis.

Period circadian clock 2 (PER2) is involved in the pathogenesis of various inflammatory and autoimmune diseases. However, there are gaps in our understanding of the role of PER2 in regulating CD4+ T cells beyond its time-keeping function in ulcerative colitis (UC) pathogenesis. Our findings revealed PER2 was predominantly expressed in CD4+ T cells, while it was significantly decreased in the inflamed mucosa and peripheral blood CD4+ T cells of UC patients compared with that in Crohn's disease (CD) patients and healthy controls (HC). Notably, PER2 expression was significantly recovered in UC patients in remission (R-UC) compared to that in active UC patients (A-UC) but not in CD patients. It was negatively correlated with the Ulcerative Colitis Endoscopic Index of Severity (UCEIS), Crohn's Disease Activity Index (CDAI), Simple Endoscopic Score for Crohn's disease (SES-CD), and C-reactive protein (CRP), respectively. Overexpression of PER2 markedly inhibited IFN-γ production in UC CD4+ T cells. RNA-seq analysis showed that overexpression of PER2 could repress the expression of a disintegrin and metalloproteinase 12 (ADAM12), a costimulatory molecule that determines Th1 cell fate. Mechanistically, cleavage under targets and tagmentation (CUT&Tag) analysis revealed that PER2 down-regulated ADAM12 expression by reducing its binding activity, thereby suppressing IFN-γ production in UC CD4+ T cells. Additionally, our data further demonstrated that ADAM12 was upregulated in CD4+ T cells and inflamed mucosa of A-UC patients compared to HC. Our study reveals a critical role of PER2 in regulating CD4+ T cell differentiation and highlights its potential as a therapeutic target for UC treatment.

Intestinal epithelial pH-sensing receptor GPR65 maintains mucosal homeostasis via regulating antimicrobial defense and restrains gut inflammation in inflammatory bowel disease.

Intestinal epithelial cell (IEC) regulation of barrier function and mucosal homeostasis enables the establishment of a harmonious gut microenvironment. However, host-derived regulatory networks that modulate intestinal antimicrobial defenses have not been fully defined. Herein we generated mice with IEC-specific deletion of Gpr65 (Gpr65ΔIEC) and investigated the role of epithelial GPR65 using DSS- and C. rodentium-induced murine colitis models. RNA sequencing analysis was conducted on colonic IECs from Gpr65fl/fl and Gpr65ΔIEC mice, and colonoids and colonic epithelial cell lines were used to evaluate the pH-sensing effect of GPR65. The expression of GPR65 was determined in IECs from patients with inflammatory bowel disease (IBD) and DSS colitis mice by qRT-PCR, Western blot, and immunohistochemistry, respectively. We observed that the absence of GPR65 in IECs abrogated homeostatic antimicrobial programs, including the production of antimicrobial peptides (AMPs) and defense response-associated proteins. Gpr65ΔIEC mice displayed dysbiosis of the gut microbiota and were prone to DSS- and C. rodentium-induced colitis, as characterized by significantly disrupted epithelial antimicrobial responses, pathogen invasion, and increased inflammatory infiltrates in the inflamed colon. RNA sequencing analysis revealed that deletion of GPR65 in IECs provoked dramatic transcriptome changes with respect to the downregulation of immune and defense responses to bacteria. Forced AMP induction assays conducted in vivo or in ex vivo colonoids revealed that IEC-intrinsic GPR65 signaling drove antimicrobial defense. Mechanistically, GPR65 signaling promoted STAT3 phosphorylation to optimize mucosal defense responses. Epithelial cell line and colonoid assays further confirmed that epithelial GPR65 sensing pH synergized with IL-22 to facilitate antimicrobial responses. Finally, the expression of GPR65 was markedly decreased in the inflamed epithelia of IBD patients and DSS colitis mice. Our findings define an important role of epithelial GPR65 in regulating intestinal homeostasis and mucosal inflammation and point toward a potential therapeutic approach by targeting GPR65 in the treatment of IBD.

A retrospective analysis of vitamin B6 deficiency and associated changes of gut microbes in Crohn's disease.

BACKGROUND: Patients with inflammatory bowel diseases (IBD) are at risk of micronutrient deficiencies, particularly during flares. Vitamin B6 is required for the proper development of brain, nerves, and many other parts of the body. However, limited studies are available to describe the prevalence, relevance and consequences of vitamin B6 deficiencies in IBD. We aim to estimate the prevalence of vitamin B6 deficiencies in Crohn's disease (CD) patients, to identify associated risk factors and to explore the alteration of intestinal microbiota related to vitamin B6 status. METHODS: A total of 360 CD patients and 55 ulcerative colitis (UC) patients from Shanghai Tenth People's Hospital of Tongji University were included. Serum vitamin B6 concentrations were collected from the computerized laboratory data. The logistic regression was used for statistical analysis. Fecal-associated microbiota was also analyzed using 16S rRNA sequencing in another 20 CD patients (10 of vitamin B6 normal, 10 of vitamin B6 deficiency). RESULTS: The prevalence of vitamin B6 abnormality was significantly higher in CD than in UC patients. Logistic regression analysis showed that small bowel lesion, ileocolonic lesion (L3), extraintestinal manifestations, ileal resection, and usage of immunosuppressor were independently associated with abnormal vitamin B6 in CD. Interestingly, the microbial structure presented significant differences between two CD groups. PICRUSt2 prediction revealed that some enzymes and metabolic pathways between the two groups were significantly different. CONCLUSIONS: Collectively, our analysis showed that vitamin B6 reduction occurred frequently in patients with CD and affected the intestinal flora of patients.

Multimodal metagenomic analysis reveals microbial single nucleotide variants as superior biomarkers for early detection of colorectal cancer.

Microbial signatures show remarkable potentials in predicting colorectal cancer (CRC). This study aimed to evaluate the diagnostic powers of multimodal microbial signatures, multi-kingdom species, genes, and single-nucleotide variants (SNVs) for detecting precancerous adenomas. We performed cross-cohort analyses on whole metagenome sequencing data of 750 samples via xMarkerFinder to identify adenoma-associated microbial multimodal signatures. Our data revealed that fungal species outperformed species from other kingdoms with an area under the ROC curve (AUC) of 0.71 in distinguishing adenomas from controls. The microbial SNVs, including dark SNVs with synonymous mutations, displayed the strongest diagnostic capability with an AUC value of 0.89, sensitivity of 0.79, specificity of 0.85, and Matthews correlation coefficient (MCC) of 0.74. SNV biomarkers also exhibited outstanding performances in three independent validation cohorts (AUCs = 0.83, 0.82, 0.76; sensitivity = 1.0, 0.72, 0.93; specificity = 0.67, 0.81, 0.67, MCCs = 0.69, 0.83, 0.72) with high disease specificity for adenoma. In further support of the above results, functional analyses revealed more frequent inter-kingdom associations between bacteria and fungi, and abnormalities in quorum sensing, purine and butanoate metabolism in adenoma, which were validated in a newly recruited cohort via qRT-PCR. Therefore, these data extend our understanding of adenoma-associated multimodal alterations in the gut microbiome and provide a rationale of microbial SNVs for the early detection of CRC.

Microbial genes outperform species and SNVs as diagnostic markers for Crohn's disease on multicohort fecal metagenomes empowered by artificial intelligence.

Dysbiosis of gut microbial community is associated with the pathogenesis of CD and may serve as a promising noninvasive diagnostic tool. We aimed to compare the performances of the microbial markers of different biological levels by conducting a multidimensional analysis on the microbial metagenomes of CD. We collected fecal metagenomic datasets generated from eight cohorts that altogether include 870 CD patients and 548 healthy controls. Microbial alterations in CD patients were assessed at multidimensional levels including species, gene, and SNV level, and then diagnostic models were constructed using artificial intelligence algorithm. A total of 227 species, 1047 microbial genes, and 21,877 microbial SNVs were identified that differed between CD and controls. The species, gene, and SNV models achieved an average AUC of 0.97, 0.95, and 0.77, respectively. Notably, the gene model exhibited superior diagnostic capability, achieving an average AUC of 0.89 and 0.91 for internal and external validations, respectively. Moreover, the gene model was specific for CD against other microbiome-related diseases. Furthermore, we found that phosphotransferase system (PTS) contributed substantially to the diagnostic capability of the gene model. The outstanding performance of PTS was mainly explained by genes celB and manY, which demonstrated high predictabilities for CD with metagenomic datasets and was validated in an independent cohort by qRT-PCR analysis. Our global metagenomic analysis unravels the multidimensional alterations of the microbial communities in CD and identifies microbial genes as robust diagnostic biomarkers across geographically and culturally distinct cohorts.

Dysregulation of CD177+ neutrophils on intraepithelial lymphocytes exacerbates gut inflammation via decreasing microbiota-derived DMF.

Neutrophils synergize with intestinal resident intraepithelial lymphocytes (IELs) to serve as the first-line defense and maintain intestinal homeostasis. However, the underlying mechanisms whereby neutrophils regulate IELs to inhibit intestinal inflammation are still not completely understood. Here, we found that depletion of neutrophils (especially CD177+ subset) caused expansion of colitogenic TCRγδ+CD8αα+ IELs, increased intestinal inflammation, and dysbiosis after dextran sulfate sodium exposure or Citrobacter rodentium infection in mice. scRNA-seq analysis revealed a pyroptosis-related gene signature and hyperresponsiveness to microbiota in TCRγδ+CD8αα+ IELs from colitic Cd177-/- mice. Microbiota-derived fumarate and its derivative dimethyl fumarate (DMF), as well as fumarate-producing microbiotas, decreased in the feces of colitic Cd177-/- mice. Elimination of dysbiosis by antibiotics treatment or co-housing procedure and DMF supplementation restrained TCRγδ+CD8αα+ IEL activation. Consistently, DMF significantly alleviated intestinal mucosal inflammation in mice through restricting gasdermin D (GSDMD)-induced pyroptosis of TCRγδ+CD8αα+ IELs. Therefore, our data reveal that neutrophils inhibit intestinal inflammation by promoting microbiota-derived DMF to regulate TCRγδ+CD8αα+ IEL activation in a GSDMD-mediated pyroptosis-dependent manner, and that DMF may serve as a therapeutic target for the management of intestinal inflammation.

Distinct alterations of fecal microbiota refer to the efficacy of adalimumab in Crohn's disease.

Background and Aims: Anti-tumor necrosis factor mAb (i.e., adalimumab, ADA) is currently used in the treatment of patients with Crohn's disease (CD). However, its regulation on fecal microbiota is still not fully understood. Methods: A retrospective analysis was conducted on 115 patients with CD who received treatment with ADA for 12 weeks at the Inflammatory Bowel Disease Center in Shanghai Tenth People's Hospital and Department of Gastroenterology in Shanghai General Hospital. The Crohn's disease activity index (CDAI) evaluation was applied to patients before ADA therapy at week 0, 4, 8, and 12. Clinical remission (CR) was defined as the CDAI < 150. All patients underwent ileocolonoscopy or enteroscopy at baseline (week 0) and week 12. Crohn's Disease Endoscopic Index of Severity (CDEIS) scores were calculated by two experienced physicians to assess endoscopic activity. Mucosal healing (MH) was assigned a CDEIS score between 0 and 3. Fecal samples were collected from eight CD patients at baseline and week 12, and the microbiota was analyzed by using 16S RNA sequencing. Results: At week 12, CR was achieved in 70.6% (72/102) of the patients with active CD. A total of 47.1% (48/102) of the patients with active CD attained MH, among which, 56.6% (30/53) of the patients with mildly active CD (3 ≤ CDEIS <9) and 48.0% (12/25) of the moderately active CD patients (9 ≤ CDEIS <12) attained MH, but only 25.0% (6/24) achieved MH in severely active CD patients (CDEIS ≥12). The efficacy of ADA was not associated with lesion locations (χ 2 = 0.409, p = 0.815). Unexpectedly, we found an increase in protective microbiota at the genus level (e.g., Barnesiella, Anaerostipes, Tyzzerella, Lachnoclostridium, and Lachnospiraceae_unclassified) but a decrease in pathogenic bacteria (Escherichia-Shigella) in fecal samples of the ADA-responsive group (ADA-R) when compared with those in the ADA-nonresponsive group (ADA-NR). Notably, the gene bglX coding ß-glucosidase and gph encoding phosphoglycolate phosphatase were enriched in fecal samples of ADA-R. Conversely, the abundance of genes coding ATP-binding cassette (ABC) transporter system proteins was significantly enriched in fecal samples of ADA-NR when compared with that of the ADA-R. Conclusion: This study reveals that ADA markedly improves clinical remission and induces MH in mildly to moderately active CD patients and that distinct changes in the gut microbiota can be used to predict the efficacy of ADA.

Critical roles of G protein-coupled receptors in regulating intestinal homeostasis and inflammatory bowel disease.

G protein-coupled receptors (GPCRs) are a group of membrane proteins that mediate most of the physiological responses to various signaling molecules such as hormones, neurotransmitters, and environmental stimulants. Inflammatory bowel disease (IBD) is a chronic relapsing disorder of the gastrointestinal tract and presents a spectrum of heterogeneous disorders falling under two main clinical subtypes including Crohn's disease (CD) and ulcerative colitis (UC). The pathogenesis of IBD is multifactorial and is related to a genetically dysregulated mucosal immune response to environmental drivers, mainly microbiotas. Although many drugs, such as 5-aminosalicylic acid, glucocorticoids, immunosuppressants, and biological agents, have been approved for IBD treatment, none can cure IBD permanently. Emerging evidence indicates significant associations between GPCRs and the pathogenesis of IBD. Here, we provide an overview of the essential physiological functions and signaling pathways of GPCRs and their roles in mucosal immunity and IBD regulation.

A Prognostic Model Based on Nine DNA Methylation-Driven Genes Predicts Overall Survival for Colorectal Cancer.

Background: Colorectal cancer (CRC) is the third most frequently diagnosed malignancy and the fourth leading cause of cancer-related death among common tumors in the world. We aimed to establish and validate a risk assessment model to predict overall survival (OS) for the CRC patients. Methods: DNA methylation-driven genes were identified by integrating DNA methylation profile and transcriptome data from The Cancer Genome Atlas (TCGA) CRC cohort. Then, a risk score model was built based on LASSO, univariable Cox and multivariable Cox regression analysis. After analyzing the clinicopathological factors, a nomogram was constructed and assessed. Another cohort from GEO was used for external validation. Afterward, the molecular and immune characteristics in the two risk score groups were analyzed. Results: In total, 705 methylation-driven genes were identified. Based on the LASSO and Cox regression analyses, nine genes, i.e., LINC01555, GSTM1, HSPA1A, VWDE, MAGEA12, ARHGAP, PTPRD, ABHD12B and TMEM88, were selected for the development of a risk score model. The Kaplan-Meier curve indicated that patients in the low-risk group had considerably better OS (P = 2e-08). The verification performed in subgroups demonstrated the validity of the model. Then, we established an OS-associated nomogram that included the risk score and significant clinicopathological factors. The concordance index of the nomogram was 0.81. A comprehensive molecular and immune characteristics analysis showed that the high-risk group was associated with tumor invasion, infiltration of immune cells executing pro-tumor suppression (such as myeloid-derived suppressor cells, regulatory T cells, immature dendritic cells) and higher expression of common inhibitory checkpoint molecules (ICPs). Conclusion: Our nine-gene associated risk assessment model is a promising signature to distinguish the prognosis for CRC patients. It is expected to serve as a predictive tool with high sensitivity and specificity for individualized prediction of OS in the patients with CRC.