Probiotic effect of Lactococcus lactis subsp. cremoris RPG-HL-0136 on intestinal mucosal immunity in mice

Lactococcus lactis subsp. cremoris is a lactic acid bacterium commonly used in the cheese manufacturing industry. It is known to produce antibacterial peptides and has recently received attention for its role as a probiotic strain. Here, we report the isolation of a new strain, Lactococcus lactis subsp. cremoris RPG-HL-0136 (RPG0136) from dried compost, which exhibits strong antibacterial activity. When RPG0136 was fed to mice, it increased the intestinal population of two beneficial bacteria, Lactobacillus and Bifidobacterium, whereas it decreased the intestinal population of two harmful bacteria, Bacteroides and Enterobacter. In addition, it increased the concentration of short-chain fatty acids, including acetic acid, propionic acid, and butyric acid, with a simultaneous decrease in pH, and accelerated the catabolic degradation of proteins, lipids, and starch. Lastly, RPG0136 increased the plasma IgG and intestinal mucosal SIgA concentrations and upregulated Reg3r, MUC1, and MUC2 expression to improve the intestinal mucosal immune function. The results of this study suggest that RPG0136 is a potential probiotic strain that supports the growth of a beneficial microbiome by promoting the synthesis of organic acids and enhancing intestinal immune function.

Autophagy is required during high MUC2 mucin biosynthesis in colonic goblet cells to contend metabolic stress

Goblet cells are specialized for the production and secretion of MUC2 glycoproteins that forms a thick layer covering the mucosal epithelium as a protective barrier against noxious substances and invading microbes. High MUC2 mucin biosynthesis induces endoplasmic reticulum (ER) stress and apoptosis in goblet cells during inflammatory and infectious diseases. Autophagy is an intracellular degradation process required for maintenance of intestinal homeostasis. In this study, we hypothesized that autophagy was triggered during high MUC2 mucin biosynthesis from colonic goblet cells to cope with metabolic stress. To interrogate this, we analyzed the autophagy process in high MUC2-producing human HT29-H and a clone HT29-L silenced for MUC2 expression by lentivirus-mediated shRNA, and WT and CRISPR/Cas9 MUC2 KO LS174T cells. Autophagy was constitutively increased in high MUC2 producing cells characterized by elevated pULK1S555 expression and increased numbers of autophagosomes as compared to MUC2 silenced or gene edited cells. Similarly, colonoids from Muc2+/+ but not Muc2-/- littermates differentiated into goblet cells showed increased autophagy. IL-22 treatment corrected misfolded MUC2 protein and alleviated the autophagy process in LS174T cells. This study highlights that autophagy plays an essential role in goblet cells to survive during high mucin biosynthesis by regulating cellular homeostasis.

Upregulated MUC2 Is an Unfavorable Prognostic Indicator for Rectal Cancer Patients Undergoing Preoperative CCRT

For locally advanced rectal cancer patients, introducing neoadjuvant concurrent chemoradiotherapy (CCRT) before radical resection allows tumor downstaging and increases the rate of anus retention. Since accurate staging before surgery and sensitivity prediction to CCRT remain challenging, a more precise genetic biomarker is urgently needed to enhance the management of such situations. The epithelial mucous barrier can protect the gut lumen, but aberrant mucin synthesis may defend against drug penetration. In this study, we focused on genes related to maintenance of gastrointestinal epithelium (GO: 0030277) and identified mucin 2 (MUC2) as the most significantly upregulated gene correlated with CCRT resistance through a public rectal cancer transcriptome dataset (GSE35452). We retrieved 172 records of rectal cancer patients undergoing CCRT accompanied by radical resection from our biobank. We also assessed the expression level of MUC2 using immunohistochemistry. The results showed that upregulated MUC2 immunoexpression was considerably correlated with the pre-CCRT and post-CCRT positive nodal status (p = 0.001 and p < 0.001), advanced pre-CCRT and post-CCRT tumor status (p = 0.022 and p < 0.001), vascular invasion (p = 0.015), and no or little response to CCRT (p = 0.006). Upregulated MUC2 immunoexpression was adversely prognostic for all three endpoints, disease-specific survival (DSS), local recurrence-free survival (LRFS), and metastasis-free survival (MeFS) (all p < 0.0001), at the univariate level. Moreover, upregulated MUC2 immunoexpression was an independent prognostic factor for worse DSS (p < 0.001), LRFS (p = 0.008), and MeFS (p = 0.003) at the multivariate level. Collectively, these results imply that upregulated MUC2 expression is characterized by a more advanced clinical course and treatment resistance in rectal cancer patients undergoing CCRT, revealing the potential prognostic utility of MUC2 expression.

The Bile Acids Specifically Modulate Colonic MUC2 and Tight Junction Protein Expression in the Human Colon Cancer Cell Line

Objectives Alterations to mucin secretion and epithelial tight junctions can compromise the ability of the epithelium to act as a barrier for the host to prevent pathogenic attack. Bile acids are synthesized in hepatocytes and released into the intestine, further modified by gut bacteria. Although many studies have investigated the changes of intestinal bile acids in the pathogenesis of various immune disorders, there are few reports about its function in preventing or interventing the dysfunction of the intestinal barrier. In this study, we sought to investigate the effects of the colonic bile acids on MUC2 and tight junction protein expression, which are crucial to colonic barrier. Methods Regulation of MUC2 and tight junction protein expression was assayed in the human colon cancer LS174T and T84 cells. The cells were treated with deoxycholic acid (DCA), lithocholic acid (LCA), 3-oxo-DCA, 3-oxo-LCA, isoDCA and isoLCA (100 μM or 200 μM), respectively. Proliferation of the cells was investigated with the MTT assay. mRNA expression of MUC2, ZO-1, occludin, claudin1 were measured by RT-PCR. Nuclear bile acid receptor FXR and TGR5, toll-like receptors and TLR adaptor MyD88, and genes (CDX2, AGR2, MyD88) related to mucin synthesis and secretion were also measured. Results In comparison with the untreated control, DCA, 3-oxo-DCA, isoDCA and isoLCA (100 μM) significantly upregulated the ZO-1, occludin and bile acid receptor FXR gene expression in the T84 cell. LCA, 3-oxo-LCA and isoLCA upregulated MUC2 expression at 200 μM, but showed no significant effect at 100 μM. DCA only significantly upregulated MUC2 expression at 200 μM, but isoDCA upregulated MUC2 expression independent of concentration in the LS174T cell. The expression of CDX2, AGR2, MyD88 was consistent with MUC2. Conclusions Bile acids at various concentrations specifically modulate MUC2 and tight junction protein expression, and thereby alter the colonic barrier function. This regulatory effect of bile acids could be mediated by activating bile acid receptors FXR. Funding Sources This work was financially supported by Ministry of Science and Technology of China (10000 Talent Project).

Therapeutic effect of yinchenhao decoction on cholelithiasis via mucin from gallbladder-intestine

Background: Cholelithiasis, known as gallstone, was a common and frequently occurring disease worldwide. Mucus was a viscoelastic protective layer lining the surface of mucosa and synthesized by specialized epithelial cells. Yinchenhao decoction was a classic prescription, first documented in Treatise on Febrile Disease. It had a definite and significant therapeutic effect on cholelithiasis, used as a complement to surgery. Our study aimed to investigate the pathological mechanism of cholelithiasis and the therapeutic mechanism of yinchenhao decoction via mucin from gallbladder-intestine.Methods: The solubility of cholesterol in fasted state simulated intestinal fluid (FaSSIF) without and with mucin was tested. The experiment of supersaturation stability was designed by solvent-shift method. The animal experiment was performed by cholelithiasis model of high cholesterol diet. The stones were observed and the related lipid was tested by automatic biochemical analyzer. The mucin was detected by PCR and western blot. Statistics was analyzed using χ2-tests and t-tests.Results: There was no significant difference in the solubility of cholesterol between FaSSIF without and with mucin. Tss or AUC significantly increased with addition of mucin to FaSSIF (p<0.05). A significant difference was observed in stone rate between the normal group and the model group (p<0.05). Stone rate in the model group showed a significant difference from the yinchenhao decoction group (aspirin group) (p<0.05). The level of related lipid showed a significant increase between the normal group and the model group (p<0.05), while there was a significant decrease between the model group and the yinchenhao decoction group (aspirin group) (p<0.05). A significant increase in the MUC5AC or MUC2 expression was observed between the normal group and the model group (p<0.01). The yinchenhao decoction group (aspirin group) caused a significant decrease in the MUC5AC or MUC2 expression, compared with the model group (p<0.01).Conclusions: In cholelithiasis, the mucin in gallbladder (MUC5AC) highly expressed, shortened cholesterol supersaturation, and promoted cholesterol crystallization; the mucin in small intestine (MUC2) highly expressed, prolonged cholesterol supersaturation, and promoted cholesterol absorption. The yinchenhao decoction inhibited the expression of mucin from gallbladder-intestine for the treatment of cholelithiasis.

Alteration of Colonic Mucin Composition and Cytokine Expression in Acute Swine Dysentery

Swine dysentery (SD) is an enteric disease associated with strongly β-hemolytic Brachyspira spp. that cause mucohemorrhagic diarrhea primarily in grower-finisher pigs. We characterized alteration of colonic mucin composition and local cytokine expression in the colon of pigs with acute SD after B. hyodysenteriae (Bhyo) infection and fed either a diet containing 30% distillers dried grains with solubles (DDGS) or a control diet. Colonic tissue samples from 9 noninoculated pigs (Control, N = 4; DDGS, N = 5) and 10 inoculated pigs experiencing acute SD (Bhyo, N = 4; Bhyo-DDGS, N = 6) were evaluated. At the apex of the spiral colon, histochemical staining with high-iron diamine–Alcian blue revealed increased sialomucin ( P = .008) and decreased sulfomucin ( P = .027) in Bhyo pigs relative to controls, with a dietary effect for sulfomucin. Noninoculated pigs fed DDGS had greater expression of sulfomucin ( P = .002) compared to pigs fed the control diet. Immunohistochemically, there was de novo expression of mucin 5AC (MUC5AC) in the Bhyo group while mucin 2 (MUC2) expression was not significantly different between groups. RNA in situ hybridization to detect the pro-inflammatory cytokine IL-1β often showed increased expression in the Bhyo group although without statistical significance, and this was not correlated with MUC5AC or MUC2 expression, suggesting IL-1β is not a major regulator of their secretion in acute SD. Expression of the anti-inflammatory cytokine TGF-β1 was significantly suppressed in the Bhyo group compared to controls ( P = .005). This study reveals mucin and cytokine alterations in the colon of pigs with experimentally induced SD and related dietary effects of DDGS.

ELEVATED ADENOMATOUS POLYPOSIS COLI IN GOBLET CELLS IS ASSOCIATED WITH INFLAMMATION IN MOUSE AND HUMAN COLON

Adenomatous Polyposis Coli (APC) functions as an essential colon tumor suppressor. Roles for APC in other disease states such as Inflammatory Bowel Disease (IBD) remain less defined. In the process of characterizing Apc protein in gastrointestinal tissues from human patients with active IBD, we found a subset of goblet cells with elevated Apc staining intensity. While normal colon tissue was only sparsely populated with these “APChigh” goblet cells, tissue from patients with Ulcerative Colitis and Crohn’s Disease contained entire regions where almost every goblet cell was APChigh. In the descending colon, the APChigh goblet cells were prominent in patients with active Ulcerative Colitis but were mostly lacking in normal samples. In mice, APChigh cells were observed only in the small intestines and proximal/medial colons. Upon induction of colitis with Dextran Sodium Sulfate (DSS), the APChigh goblet cells remained in the proximal and medial colon, but also appeared in the distal colon. To better understand the link between APC expression, goblet cells, and inflammation, cultured human colon cells derived from normal tissue were depleted of APC and RNAseq analysis was performed. APC depletion resulted in reduced expression of mRNAs encoding IL1 signaling pathway components IL1b and IL1R, known regulators of Muc2 expression. Muc2 is the main constituent of goblet cell mucus, which is secreted to form a protective barrier for the epithelial cells lining the large intestine. We found that treating cancer cells lacking wild-type APC with IL1b resulted in increased IL1R and MUC2 expression. Induction of full-length APC in these cells led to a similar phenotype. Combining IL1β treatment with APC induction led to an increase of MUC2 expression greater than expected for additive affects, suggesting that APC sensitized these cells to IL1 signaling. These results illuminate a novel role for APC in goblet cells under conditions of inflammation. We postulate that the higher amount of APC in a subset of goblet cells might sensitize them to IL-1 signaling. During times of inflammation, such as active Ulcerative Colitis, goblet cells with higher APC levels would be more sensitive to IL-1-induced MUC2 expression than other goblet cells, perhaps protecting the intestinal lining from further barrier breaches.

Helicobacter pylori Infection and the Patterns of Gastric Mucin Expression in Children

Background: The updated model for the mechanism of gastric carcinogenesis demonstrates that Helicobacter pylori (H. pylori) is a risk factor in every step of the process. The expression of certain gastric mucins is altered by H. pylori infection in adult patients. The aim of our research was to assess the impact of H. pylori infection on the expression of secretory mucins in the pediatric antral mucosa. Methods: Slides were stained with monoclonal antibodies for MUC5AC, MUC6 and MUC2, digitalized and scored using both a semiquantitative and a quantitative approach. Results: The expression of MUC5AC was significantly lower in infected children. Also, MUC2 expression was more pronounced in infected children. MUC6 expression did not differentiate between infected and noninfected children. Additionally, the presence of chronic inflammation significantly altered the expression of MUC6 and MUC2. The expression of MUC6 was significantly higher in patients with gastric atrophy. Conclusion: The minor differences in mucin expression at distinct ages might stem from different H. pylori exposure periods. Further research is needed to determine the particular patterns of expression according to age and to evaluate the effects of the interaction between H. pylori and mucins in the progression of the gastric carcinogenesis cascade.

All-Trans Retinoic Acid, A Derivative of Vitamin A, Improved Intestinal Epithelial Barrier Function Through PFKP

Objective: Mucosal Healing, relied on the coordinated activity of IECs for improvement of intestinal barrier function, is the critical goal in treatment of IBD. All-trans retinoic acid (ATRA) is known to regulates cell proliferation and differentiation. The aims of the present study were to investigate the effects of ATRA on the intestinal differentiation. Methods: We collected the clinical sample from the patients to analyze the vitamin A, TEER, western blotting and real-time PCR were performed to detect the effect of vitamin A on PFKP expression and intestinal epithelial cell differentiation. Results: In this study, we showed that increased TEER and decrease paracellular permeability of IECs were induced by ATRA in dose-dependent manner, which is attributed to enhanced MUC2 expression, a marker of goblet cell by western blotting, real-time PCR and TEER assay, while no significant difference of villin was found to alter. The further results show that ATRA suppressed PFKP expression in IECs, while overexpression of PFKP could reverse the promotion of ATRA on MUC2 expression, implying ATRA-induced MUC2 expression in PFKP-dependent manner and in dose-dependent way. The clinical sample analysis suggested vitamin A is not significant associated with gender, and replenishment of vitamin A is critical for intestinal differentiation. Conclusion: ATRA improved intestinal epithelial differentiation via PFKP-mediated Muc2 expression. The findings suggest that ATRA could serve as a novel therapeutic agent to ameliorate development of inflammatory bowel disease. Keywords : All-trans Retinoic Acid; Muc2; Differentiation; PFKP; Intestinal Barrier Function