D-amino Acids Ameliorate Experimental Colitis and Cholangitis by Inhibiting Growth of Proteobacteria: Potential Therapeutic Role in Inflammatory Bowel Disease.

BACKGROUND & AIMS: D-amino acids, the chiral counterparts of protein L-amino acids, were primarily produced and utilized by microbes, including those in the human gut. However, little was known about how orally administered or microbe-derived D-amino acids affected the gut microbial community or gut disease progression. METHODS: The ratio of D- to L-amino acids was analyzed in feces and blood from patients with ulcerative colitis (UC) and healthy controls. Also, composition of microbe was analyzed from patients with UC. Mice were treated with D-amino acid in dextran sulfate sodium colitis model and liver cholangitis model. RESULTS: The ratio of D- to L-amino acids was lower in the feces of patients with UC than that of healthy controls. Supplementation of D-amino acids ameliorated UC-related experimental colitis and liver cholangitis by inhibiting growth of Proteobacteria. Addition of D-alanine, a major building block for bacterial cell wall formation, to culture medium inhibited expression of the ftsZ gene required for cell fission in the Proteobacteria Escherichia coli and Klebsiella pneumoniae, thereby inhibiting growth. Overexpression of ftsZ restored growth of E. coli even when D-alanine was present. We found that D-alanine not only inhibited invasion of pathological K. pneumoniae into the host via pore formation in intestinal epithelial cells but also inhibited growth of E. coli and generation of antibiotic-resistant strains. CONCLUSIONS: D-amino acids might have potential for use in novel therapeutic approaches targeting Proteobacteria-associated dysbiosis and antibiotic-resistant bacterial diseases by means of their effects on the intestinal microbiota community.

Downregulation of chemokine receptor 9 facilitates CD4+CD8αα+ intraepithelial lymphocyte development.

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4+ T cells can develop into CD4+CD8αα+ IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4+CD8αα+ IELs, but CCR9 deficiency results in CD4+CD8αα+ over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4+CD8αα+ IELs in Ccr9-/- mice. CD4+ T cells isolated from the epithelium of Ccr9-/- mice also display increased expression of Cbfß2, and the genomic occupancy modification of Cbfß2 expression reveals its important function in CD4+CD8αα+ differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4+CD8αα+ IEL differentiation.

Single-cell transcriptomics of human gut T cells identifies cytotoxic CD4+CD8A+ T cells related to mouse CD4 cytotoxic T cells.

Cytotoxic CD4+ T cells (CD4-CTLs) show the presence of cytolytic granules, which include the enzymes granzyme and perforin. The cells have a pathogenic and protective role in various diseases, including cancer, viral infection, and autoimmune disease. In mice, cytotoxic CD4+ T cells express CD8αα+ and reside in the intestine (mouse CD4+CTLs; mCD4-CTLs). The population of cytotoxic CD4+ T cells in the human intestine is currently unknown. Moreover, it is unclear how cytotoxic CD4 T cells change in patients with inflammatory bowel disease (IBD). Here, we aimed to identify cytotoxic CD4+ T cells in the human intestine and analyze the characteristics of the population in patients with IBD using single-cell RNA-seq (scRNA-seq). In CD4+ T cells, granzyme and perforin expression was high in humanMAIT (hMAIT) cells and hCD4+CD8A+ T cell cluster. Both CD4 and CD8A were expressed in hTreg, hMAIT, and hCD4+CD8A+ T cell clusters. Next we performed fast gene set enrichment analysis to identify cell populations that showed homology to mCD4CTLs. The analysis identified the hCD4+CD8A+ T cell cluster (hCTL-like population; hCD4-CTL) similar to mouse CTLs. The percentage of CD4+CD8A+ T cells among the total CD4+ T cells in the inflamed intestine of the patients with Crohn's disease was significantly reduced compared with that in the noninflamed intestine of the patients. In summary, we identified cytotoxic CD4+CD8+ T cells in the small intestine of humans. The integration of the mouse and human sc-RNA-seq data analysis highlight an approach to identify human cell populations related to mouse cell populations, which may help determine the functional properties of several human cell populations in mice.

Aryl hydrocarbon receptor signals in epithelial cells govern the recruitment and location of Helios+ Tregs in the gut.

CD4+Foxp3+ regulatory T cells (Tregs) are essential for homeostasis in the colon, but the mechanism by which local environmental cues determine the localization of colonic Tregs is unclear. Here, we administer indigo naturalis (IN), a nontoxic phytochemical aryl hydrocarbon receptor (AhR) agonist used for treating patients with ulcerative colitis (UC) in Asia, and we show that IN increases Helios+ Tregs and MHC class II+ epithelial cells (ECs) in the colon. Interactions between Tregs and MHC class II+ ECs occur mainly near the crypt bottom in the steady state, whereas Tregs dramatically increase and shift toward the crypt top following IN treatment. Moreover, the number of CD25+ T cells is increased near the surface of ECs in IN-treated UC patients compared with that in patients treated with other therapies. We also highlight additional AhR-signaling mechanisms in intestinal ECs that determine the accumulation and localization of Helios+ Tregs in the colon.

Intracellular metabolic adaptation of intraepithelial CD4+CD8αα+ T lymphocytes.

Intestinal intraepithelial lymphocytes (IELs), the first line of defense against microbial and dietary antigens, are classified as natural or induced based on their origin and receptor expression. Induced CD4+CD8αα+TCRß+ T cells (double positive, DPIELs) originated from CD4+CD8α-TCRß+ T cells (single positive, SPIELs) increase with aging. However, the metabolic requirements and the metabolic-related genes in IEL development remain unclear. We determined that the intraepithelial compartment is hypoxic in the presence of microbes and DPIELs increased more than natural IELs in this location. Moreover, DPIELs consumed less oxygen and glucose and exhibited unique alterations in mitochondria. Using inhibitors and genetically modified mice, we revealed that DPIELs adapt to their surrounding oxygen-deprived environment in peripheral tissues by modulating specific genes, including hypoxia-inducible factor, mammalian target of rapamycin complexes (mTORC), phosphorylated ribosomal protein S6 (pS6), and other glycolytic factors. Our findings provide valuable insight into the metabolic properties of IELs.

Characteristics of the Mucosal Surface on Scanning Electron Microscopy in Patients with Remitting Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) is characterized by chronic intestinal epithelial damage, and previous studies have evaluated the epithelial structure of patients with active UC using electron microscopy. AIMS: This study aimed to assess the intestinal epithelial structure using scanning electron microscopy (SEM) and the features of patients with UC who are in remission. METHODS: In total, eight healthy controls and 20 patients with UC were enrolled, and colonic tissue samples from the cecum and rectum were collected. Then, we compared the epithelial surface structure on SEM between patients with UC who are in clinical remission and healthy controls. RESULTS: In healthy controls, the colonic surface comprises small lobes (termed units), with one crypt located in the middle of each unit. In patients with UC, we found irregular unit and crypt mouth size, double crypt sign (> 1 crypt per unit), and lower number of small vesicles in the intestinal epithelial cells. Compared with healthy controls, patients with UC often presented with irregular unit size, double crypt sign, and irregular crypt mouth size in the rectum. The small vesicles were observed less frequently in patients with UC than in healthy controls. CONCLUSIONS: SEM revealed a unique epithelial structure in patients with UC who are in remission.

Mucosal concentrations of N-acetyl-5-aminosalicylic acid related to endoscopic activity in ulcerative colitis patients with mesalamine.

BACKGROUND AND AIM: 5-Aminosalicylic acid (5-ASA) is a fundamental treatment for mild-to-moderate ulcerative colitis (UC). 5-ASA is taken up into the colonic mucosa and metabolized to N-acetyl-5-ASA (Ac-5-ASA). Few studies have assessed whether mucosal 5-ASA and Ac-5-ASA concentrations are associated with endoscopic remission. This study aimed to investigate differences in 5-ASA and Ac-5-ASA concentrations according to endoscopic activity. METHODS: This single-center, prospective, cross-sectional study was conducted between March 2018 and February 2019. UC patients who were administered with 5-ASA medication for at least 8 weeks before sigmoidoscopy were enrolled. Mucosal 5-ASA and Ac-5-ASA concentrations were measured using liquid chromatography with tandem mass spectrometry. The primary endpoint was defined as the difference in mucosal concentrations of 5-ASA and Ac-5-ASA, according to the Mayo endoscopic subscore (MES). RESULTS: Mucosal concentrations were analyzed in 50 patients. In the sigmoid colon, the median 5-ASA concentration in patients with MES of 0 (17.3 ng/mg) was significantly higher than MES ≥ 1 (6.4 ng/mg) (P = 0.019). The median 5-ASA concentrations in patients with Ulcerative Colitis Endoscopic Index of Severity ≤ 1 (16.4 ng/mg) were also significantly higher than in patients with Ulcerative Colitis Endoscopic Index of Severity ≥ 2 (4.63 ng/mg) (P = 0.047). In the sigmoid colon, the concentration of Ac-5-ASA was higher in patients with MES of 0 (21.2 ng/mg) than in patients with MES ≥ 1 (5.81 ng/mg) (P = 0.022). CONCLUSIONS: The present study showed that mucosal Ac-5-ASA concentrations, as well as 5-ASA concentrations, are higher in UC patients with endoscopic remission. Ac-5-ASA may be useful for a biomarker of 5-ASA efficacy.

Development of an Indigo Naturalis Suppository for Topical Induction Therapy in Patients with Ulcerative Colitis.

BACKGROUND: Indigo naturalis (IN) consists of ligands for the aryl hydrocarbon receptor and exhibits anti-inflammatory effects. Previously, we demonstrated that an 8-week treatment with oral IN is effective in inducing a clinical response in patients with ulcerative colitis (UC). Some UC patients with proctitis are refractory to topical mesalamine or corticosteroids and therefore require an alternative topical treatment. OBJECTIVES: We aimed to prospectively evaluate the safety and efficacy of IN suppositories in UC patients. METHOD: We performed an open-label, single-center, prospective pilot study from February 2018 to October 2018. A total of 10 patients with active UC, who had moderate to severe inflammation from the rectum to the sigmoid colon, were enrolled. The patients received a daily dose of 50 mg IN suppository for 4 weeks. The primary endpoint was safety at week 4. RESULTS: Although 1 patient experienced anal pain, no serious adverse events were observed. At week 4, the rates of clinical remission and mucosal healing were 30 and 40%, respectively. Mayo rectal bleeding subscores significantly improved after treatment (1.80 ± 0.13 vs. 0.90 ± 0.28; p = 0.009). Approximately 80% of the patients with a baseline Mayo endoscopic subscore in the rectum (r-MES) of 2 achieved mucosal healing, but those with a baseline r-MES of 3 did not. CONCLUSIONS: We found that 4 weeks of IN suppository can be tolerated by UC patients, but its efficacy was limited by the severity of the disease. Further investigation will be needed in order to confirm the optimum dose of IN suppository for patients with UC.

[Intestinal immune response is regulated by gut microbe].

  Human Intestine has a diverse population of bacteria which induces pathogens to disrupt not only the intestinal homeostasis but whole body immune systems. Dysbiosis, the abnormal proliferation and reduction of the microbiota, breaks down the homeostasis of the immunity and metabolisms in the host. The evolution of the microbiota analysis technology contributed to reveal the molecular biological complex interaction between the microbiota and its host systemically as well as locally. Because several diseases are caused by the dysbiosis, fecal transplantation would be the new therapeutic target for them. It has been investigated in some intestinal diseases such as CD infection, or inflammatory bowel disease. Here, we review these symbiotic interactions and the current state for the clinical application.