DSS-induced Intestinal Injury Research Articles

Pterostilbene Alleviates Dextran Sodium Sulfate (DSS)-Induced Intestinal Barrier Dysfunction Involving Suppression of a S100A8-TLR-4-NF-κB Signaling Cascade.

Intestinal barrier hemostasis is the key to health. As a resveratrol analogue, pterostilbene (PT) has been reported to prevent dextran sodium sulfate (DSS)-induced intestinal barrier dysfunction mainly associated with the intestinal NF-κB signaling pathway. However, the exact underlying mechanisms are not yet well-defined yet. In this study, we performed RNA-sequencing analysis and unexpectedly found that alarmin S100A8 sensitively responded to DSS-induced intestinal injury. Accordingly, histologic assessments suggested that the high expression of S100A8 was accompanied by increased intestinal infiltration of macrophages, upregulated intestinal epithelial Toll-like receptor 4 (TLR-4), and activated NF-κB signaling pathway. Interestingly, the above phenomena were effectively counteracted upon the addition of PT. Furthermore, by using a coculture system of macrophage THP-1 cells and HT-29 colon cells, we identified macrophage-secreted S100A8 activated intestinal epithelial NF-κB signaling pathway through TLR-4. Taken together, these findings suggested that PT ameliorated DSS-induced intestinal barrier injury through suppression of the macrophage S100A8-intestinal epithelial TLR-4-NF-κB signaling cascade.

Matrine Ameliorates DSS-Induced Colitis by Suppressing Inflammation, Modulating Oxidative Stress and Remodeling the Gut Microbiota.

Matrine (MT) possesses anti-inflammatory, anti-allergic and antioxidative properties. However, the impact and underlying mechanisms of matrine on colitis are unclear. The purpose of this research was to examine the protective impact and regulatory mechanism of matrine on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. MT alleviated DSS-induced UC by inhibiting weight loss, relieving colon shortening and reducing the disease activity index (DAI). Moreover, DSS-induced intestinal injury and the number of goblet cells were reversed by MT, as were alterations in the expression of zonula occludens-1 (ZO-1) and occludin in colon. Simultaneously, matrine not only effectively restored DSS-induced oxidative stress in colonic tissues but also reduced the production of inflammatory cytokines. Furthermore, MT could treat colitis mice by regulating the regulatory T cell (Treg)/T helper 17 (Th17) cell imbalance. We observed further evidence that MT alleviated the decrease in intestinal flora diversity, reduced the proportion of Firmicutes and Bacteroidetes, decreased the proportion of Proteobacteria and increased the relative abundance of Lactobacillus and Akkermansia in colitis mice. In conclusion, these results suggest that MT may mitigate DSS-induced colitis by enhancing the colon barrier integrity, reducing the Treg/Th17 cell imbalance, inhibiting intestinal inflammation, modulating oxidative stress and regulating the gut microbiota. These findings provide strong evidence for the development and application of MT as a dietary treatment for UC.

Protective effect of San Huang Pill and its bioactive compounds against ulcerative colitis in Drosophila via modulation of JAK/STAT, apoptosis, Toll, and Nrf2/Keap1 pathways

Ethnopharmacological relevanceSan Huang Pill (SHP) is a prescription in Dunhuang Ancient Medical Prescription, which has the efficacy of heat-clearing and dampness-drying, and is a traditional formula for the treatment of gastrointestinal diseases. However, its efficacy and mechanism in treating ulcerative colitis (UC) are still unclear. Aim of the studyTo investigate the protective effects of SHP and its bioactive compounds against Dextran Sulfate Sodium (DSS)-induced intestinal damage using the Drosophila melanogaster model, and to detect the molecular mechanism of SHP in the treatment of UC. MethodsSurvival rate, locomotion, feeding, and excretion were used to explore the anti-inflammatory effects of SHP. The pharmacotoxicity of SHP was measured using developmental analysis. Intestinal integrity, intestinal length, intestinal acid-base homeostasis, and Tepan blue assay were used to analyze the protective effect of SHP against DSS-induced intestinal damage. The molecular mechanism of SHP was detected using DHE staining, immunofluorescence, real-time PCR, 16 S rRNA gene sequencing, and network pharmacology analysis. Survival rate, intestinal length, and integrity analysis were used to detect the protective effect of bioactive compounds of SHP against intestinal damage. ResultsSHP supplementation significantly increased the survival rate, restored locomotion, increased metabolic rate, maintained intestinal morphological integrity and intestinal homeostasis, protected intestinal epithelial cells, and alleviated intestinal oxidative damage in adult flies under DSS stimulation. Besides, administration of SHP had no toxic effect on flies. Moreover, SHP supplementation remarkably decreased the expression levels of genes related to JAK/STAT, apoptosis, and Toll signaling pathways, increased the gene expressions of the Nrf2/Keap1 pathway, and also reduced the relative abundance of harmful bacteria in DSS-treated flies. Additionally, the ingredients in SHP (palmatine, berberine, baicalein, wogonin, rhein, and aloeemodin) had protection against DSS-induced intestinal injury, such as prolonging survival rate, increasing intestinal length, and maintaining intestinal barrier integrity. ConclusionSHP had a strong anti-inflammatory function, and remarkably alleviated DSS-induced intestinal morphological damage and intestinal homeostatic imbalance in adult flies by regulating JAK/STAT, apoptosis, Toll and Nrf2/Keap1 signaling pathways, and also gut microbial homeostasis. This suggests that SHP may be a potential complementary and alternative medicine herb therapy for UC, which provides a basis for modern pharmacodynamic evaluation of other prescriptions in Dunhuang ancient medical prescription.

Vitamin D Receptor Mediates Attenuating Effect of Lithocholic Acid on Dextran Sulfate Sodium Induced Colitis in Mice.

Bile acids are major components of bile; they emulsify dietary lipids for efficient digestion and absorption and act as signaling molecules that activate nuclear and membrane receptors. The vitamin D receptor (VDR) is a receptor for the active form of vitamin D and lithocholic acid (LCA), a secondary bile acid produced by the intestinal microflora. Unlike other bile acids that enter the enterohepatic circulation, LCA is poorly absorbed in the intestine. Although vitamin D signaling regulates various physiological functions, including calcium metabolism and inflammation/immunity, LCA signaling remains largely unknown. In this study, we investigated the effect of the oral administration of LCA on colitis in a mouse model using dextran sulfate sodium (DSS). Oral LCA decreased the disease activity of colitis in the early phase, which is a phenotype associated with the suppression of histological injury, such as inflammatory cell infiltration and goblet cell loss. These protective effects of LCA were abolished in VDR-deleted mice. LCA decreased the expression of inflammatory cytokine genes, but this effect was at least partly observed in VDR-deleted mice. The pharmacological effect of LCA on colitis was not associated with hypercalcemia, an adverse effect induced by vitamin D compounds. Therefore, LCA suppresses DSS-induced intestinal injury in its action as a VDR ligand.

Anti-inflammatory and anti-oxidant properties of Melianodiol on DSS-induced ulcerative colitis in mice.

BackgroundUlcerative colitis is a unique inflammatory bowel disease with ulcerative lesions of the colonic mucosa. Melianodiol (MN), a triterpenoid, isolated from the fruits of the Chinese medicinal plant Melia azedarach, possesses significant anti-inflammatory properties.ObjectiveThe present study investigated the protective effects of MN on lipopolysaccharide (LPS)-induced macrophages and DSS-mediated ulcerative colitis in mice.MethodsIn the study, mice were given MN (50, 100, and 200 mg/kg) and 5-ASA (500 mg/kg) daily for 9 days after induction by DSS for 1 week. The progress of the disease was monitored daily by observation of changes in clinical signs and body weight.ResultsThe results showed that MN effectively improved the overproduction of inflammatory factors (IL-6, NO, and TNF-α) and suppressed the activation of the NF-κB signalling cascade in LPS-mediated RAW264.7 cells. For DSS-mediated colitis in mice, MN can reduce weight loss and the disease activity index (DAI) score in UC mice, suppress colon shortening, and alleviate pathological colon injury. Moreover, MN treatment notably up regulated the levels of IL-10 and down regulated those of IL-1β and TNF-α, and inhibited the protein expression of p-JAK2, p-STAT3, iNOS, NF-κB P65, p-P65, p-IKKα/β, and p-IκBα in the colon. After MN treatment, the levels of MDA and NO in colonic tissue were remarkably decreased, whereas the levels of GSH, SOD, Nrf-2, Keap-1, HO-1, IκBα, and eNOS protein expression levels were significantly increased.ConclusionThese results indicate that MN can activate the Nrf-2 signalling pathway and inhibit the JAK/STAT, iNOS/eNOS, and NF-κB signalling cascades, enhance intestinal barrier function, and effectively reduce the LPS-mediated inflammatory response in mouse macrophages and DSS-induced intestinal injury in UC.

Intestinal epithelial cell autophagy deficiency suppresses inflammation-associated colon tumorigenesis

Colitis-associated cancer (CAC) is closely related to chronic inflammation, whose underlying molecular mechanism, however, has not been elaborated comprehensively. In the current study, an investigation was conducted on the role of autophagy in the initiation and progression of azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon tumors, a mouse model for CAC in humans. Mice with the intestinal epithelial cell (IEC)-specific deletion of the autophagy-related gene 7 (Atg7) saw a significant decrease in tumor number, burden, and risk of high-grade dysplasia. The autophagy deficiency of IECs resulted in the accumulation of T cells, especially CD8+ T lymphocytes in colon lamina propria. Furthermore, it was found that autophagy protects against DSS-induced intestinal injury through maintaining epithelial barrier function and promoting the survival and proliferation of IECs. Mechanistically, autophagy in IECs enhanced the activation of epithelial STAT3/ERK to promote the survival and proliferation of colonic epithelial cells during the development of CAC. Therefore, the findings unveil the essential role of autophagy in activating the processes of colonic protection, regeneration, and tumorigenesis.

THE ROLE OF GROUP 3 INNATE LYMPHOID CELLS (ILC3) IN GM-CSF/CSF2RB-DEPENDENT INTESTINAL HOMEOSTASIS IN CROHN’S DISEASE

Abstract Inflammatory Bowel Disease (IBD) is comprised of two subtypes, Crohn’s disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWAS) of IBD identified 240 loci significantly associated with IBD risk. CSF2RB, one of the identified IBD-associated genes and the receptor for GM-CSF signaling, is responsible for maintaining intestinal homeostasis through retinoic acid (RA) production by macrophages and dendritic cells. GM-CSF (Sargramostim) treatment showed a promising result for preventing intestinal injury and inflammation in both a murine and our novel zebrafish model. However, due to GM-CSF-neutralizing antibodies present in patients, Sargramostim failed in a large cohort Phase II clinical trial. This GM-CSF-dependent homeostasis relies on the balance of innate lymphoid cells (ILCs). In human patients, we report that during inflammation in CD, there is a decrease of NCR+ ILC3s while proinflammatory ILC1s rapidly increase. Neutralizing GM-CSF auto-antibodies were detected in the pre-disease stage (ILC1 low & ILC3 low) in CD patients, a different result from CSF2RB frameshift allele carriers (ILC1 low & ILC3 high) (Figure 1). We developed a novel zebrafish model to mimic the acute and chronic nature of IBD disease pathogenicity in vivo. In our model, human GM-CSF treatment significantly alleviated intestinal injury but showed no effect during mucosal healing. With a CRISPR knockout csf2rb zebrafish line, we showed this protective effect is CSF2RB-dependent. Using single-cell technologies, DSS-induced injury increases rorc+ ILC3s. These ILC3 cell proportion changes were diminished in csf2rb-/- zebrafish (Figure 2). Figure 1. Distinct ILC subpopulations between anti-GM-CSF antibodies and CSFRB mutation carriers. Scatter plot of the PC1 values of the ILC3 gene list (ILC3-PC) (y axis) against the PC1 values of the ILC1 gene list (ILC1-PC) (x axis). Both ILC1-PC and ILC3-PC were derived from PCA analysis of signature gene lists of ILC1 and ILC3 cell types against a sub-dataset of the bulk mRNAseq samples from the MSCCR. Red dot indicate patient with anti-GM-CSF antibody. The logarithm antibody titer was shown with the intensity of red color. The green dots indicate the CSF2RB heterozygote carriers. Figure 2. GM-CSF protects against DSS-induced intestinal injury and regulates ILC3. (A-B) Treatment of human GM-CSF significantly reduces intestinal injury in dose-dependent manner in WT (A) but not CSF2RB-/- (B). ***, P< 0.001 (C) Analyzing scRNAseq data of zebrafish intestine cells, RORC+ (ILC3) cell proportion over total immune cells increases with DSS injury and decreases with human GM-CSF co-treatment. There is no RORC+ cell proportion change in CSF2RB-/-. (D) Modeling of GM-CSF in preventing ILC3 expansion and attenuating ILC3-related proinflammatory cytokines during intestinal injury. Our studies illuminate mechanisms of GM-CSF in perpetuating gut homeostasis through maintaining and regulating ILC3s. This novel aspect of regulating IBD pathogenesis through ILCs may lead to novel therapeutic targets.

Enterococcus faecium Alleviates Gut Barrier Injury in C57BL/6 Mice with Dextran Sulfate Sodium-Induced Ulcerative Colitis.

The involvement of gut microbiota composition in ulcerative colitis is strongly supported by previous research. Growing evidence suggests that probiotic therapy protects against inflammatory bowel disease in animal models and patients. However, as a probiotic, the role of Enterococcus faecium (E. faecium) in UC remains unclear. Nevertheless, the potential mechanism of the protective effect of E. faecium remains unknown. In this study, a dextran sulphate sodium-induced (DSS-induced) colitis model was used to detect the underlying mechanism of E. faecium in maintaining gut homeostasis. ELISA was performed to detect the levels of cytokines (TNF-α, IL-1β, IL-6, and IL-10). Furthermore, 454 pyrosequencing was used to investigate the microbiota composition in fecal samples. The results illustrate that E. faecium administration could prevent DSS-induced gut inflammation and intestinal flora imbalance. At the same time, the damage to intestinal mucosal barrier and tight junctions was partially repaired. These results demonstrate the preventive effect of E. faecium in DSS-induced intestinal injury. The present study provides new insights into the medicinal value of E. faecium for UC.

Protective effects of sulfated polysaccharide from Enterobacter cloacae Z0206 against DSS-induced intestinal injury via DNA methylation

We previously obtained and characterized a novel sulfated derivative of the exopolysaccharides from Enterobacter cloacae Z0206 (SEPS). This study aimed at investigating the effects and mechanism of SEPS against dextran sulfate sodium (DSS) induced intestinal injury. The results showed that SEPS increased the proliferation and survival of intestinal epithelial cells during DSS stimulation. Furthermore, SEPS maintained the barrier function and inflammatory response via JAK2 and MAPK signaling to protect against DSS-induced intestinal injury. Mechanistically, SEPS elevated the DNA methylation in the promoter region to negatively regulate the JAK2 and MAPKs expression. Thus, the current study shows the potential effects and mechanism of SEPS on DSS-induced intestinal epithelial cell injury.

Atractylodis macrocephalae polysaccharides protect against DSS-induced intestinal injury through a novel lncRNA ITSN1-OT1

Many dietary polysaccharides have been shown to protect the intestinal barrier integrity against several noxious stimuli. Previously, we have isolated a polysaccharide RAMPtp from Atractylodis macrocephalae Koidz, and analyzed its structure. However, the effects of RAMPtp on intestinal barrier function have not been investigated. Here, we evaluated the protective effects of RAMPtp on Dextran sulfate sodium (DSS)-induced intestinal epithelial cells (IECs) injury. The findings showed that RAMPtp boosted the proliferation and survival of IECs during DSS stimulation. Furthermore, we found that RAMPtp protected the IECs from injury induced by DSS through maintaining the barrier function and inflammation response. Mechanistically, we identified a novel lncRNA ITSN1-OT1, which was induced by RAMPtp during DSS stimulation. It blocked the nuclear import of phosphorylated STAT2 to prevent the DSS induced decreased expression and structural destroy of tight junction proteins. Hence, the study clarified the protective effects and mechanism of polysaccharides RAMPtp on DSS-induced intestinal barrier dysfunction.

Intervention with the crude polysaccharides of Physalis pubescens L. mitigates colitis by preventing oxidative damage, aberrant immune responses, and dysbacteriosis.

In this study, a colitis mouse model induced by dextran sulfate sodium (DSS) was used to investigate the mechanisms of action of an extract of crude polysaccharides (POL) from Physalis pubescens L. as a dietary intervention for colitis. Our results showed that the administration of POL prior to DSS-induced colitis protected the colon mucosal layer; maintained intestinal barrier integrity; alleviated oxidative damage; and lowered neutrophil infiltration by downregulating intercellular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression. More importantly, POL pretreatment reduced the expression of the proinflammatory factors tumor necrosis factor-α, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), thereby modulating the nuclear factor-κB/iNOS-COX-2 signal transduction pathway. In addition, POL reversed DSS-induced gut dysbiosis, accompanied by reducing the relative abundance of Helicobacter, Mucispirillum, and Erysipelatoclostridium. In conclusion, POL ameliorated DSS-induced intestinal injury in mice, indicating that POL could be a useful dietary nutrient to protect against colitis. PRACTICAL APPLICATION: Physalis pubescens L. is an edible fruit. The results of this study show that the intervention with Physalis pubescens L. crude polysaccharides may help prevent ulcerative colitis.

Toll-like receptor 4 (TLR4) deficiency aggravates dextran sulfate sodium (DSS)-induced intestinal injury by down-regulating IL6, CCL2 and CSF3.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) that causes long-lasting inflammation and ulcers in the human digestive tract. The repair role of TLR4 in the intestinal epithelium is still unknown. By comparing to wild-type (WT) mice, Toll-like receptor 4 (TLR4)-knockout mice (TLR4-KO) were used as dextran sulfate sodium (DSS)-induced colitis models to explore the role of TLR4 signaling in intestinal injury. High-throughput RNA-Seq, RT-qPCR and ELISA were performed to screen and verify key differences in gut genes between WT and TLR4-KO mice. Functional study of core dysregulated factors was performed in intestinal cell lines. We found that DSS-induced intestinal injury was aggravated by LPS (TLR4 agonist) and TLR4-KO. When compared to WT mice, IL6, CCL2, CSF3, IL11, Ccnb1, Ccnd1 and TNF-α significantly decreased and Fas and FasL have increased in the gut of TLR4-KO mice. IL6, CCL2, CSF3, Fas and FasL have all increased in CT-26 cells treated with LPS. Combined with the above data and KEGG enrichment, it can be assumed that TLR4-KO might aggravate DSS-induced intestinal damage by attenuating cell cycle, cytokine-cytokine receptor interaction, and Toll-like receptor signaling pathway, and enhancing the apoptosis pathway. In the functional study of core dysregulated factors, it was found that LPS, IL6, IL11, CSF3, CCL2, S100A8, S100A9 and Mmp3 have improved viability of colon cancer cell lines and decreased apoptosis rate of mouse colon cancer cells when these were treated with DSS. However, Jo-2 (Fas agonistic monoclonal antibody) played the opposite role in colon cancer cells treated with DSS. TLR4 had a repairing effect on DSS-induced intestinal damage and it up-regulate IL6, CCL2 and CSF3. Fas and FasL enhanced DSS-induced colon injury in mice, but might have little to do with TLR4 signaling.

Oral delivery of a Lactococcus lactis strain secreting bovine lactoferricin-lactoferrampin alleviates the development of acute colitis in mice.

Ulcerative colitis (UC) is a chronic relapsing disease. Treatment of UC would benefit from specific targeting of therapeutics to the intestine. Previous studies have demonstrated that bovine lactoferricin and lactoferrampin have bactericidal, anti-inflammatory, and immunomodulatory effects. Here, we investigated whether oral administration of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactococcus lactis (LL-LFCA) strain could alleviate experimental colitis. LFCA derived from LL-LFCA inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. In mice, administration of LL-LFCA decreased the disease activity index and attenuated dextran sulfate sodium (DSS)-induced body weight loss and colon shortening. LL-LFCA treatment also ameliorated DSS-induced colon damage, inhibited inflammatory cell infiltration, significantly decreased myeloperoxidase activity, and ameliorated DSS-induced disruption of intestinal permeability and tight junctions. In addition, 16S rDNA sequencing showed that LL-LFCA reversed DSS-induced gut dysbiosis. The production of proinflammatory mediators in serum and the colon was also reduced by administration of LL-LFCA. In vitro, LFCA derived from LL-LFCA decreased the messenger RNA expression of proinflammatory factors. The underlying mechanisms may involve inhibition of the nuclear factor kappa B (NF-κB) pathway. The results demonstrate that LL-LFCA ameliorates DSS-induced intestinal injury in mice, suggesting that LL-LFCA might be an effective drug for the treatment of inflammatory bowel diseases.

Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury.

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell-cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2ΔIEC; DSC2ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2ΔIEC, but not DSC2ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.

Polysaccharide of Hericium erinaceus attenuates colitis in C57BL/6 mice via regulation of oxidative stress, inflammation-related signaling pathways and modulating the composition of the gut microbiota

Inflammatory bowel disease (IBD) is a disease caused by a dysregulated immune with unknown etiology. Hericium erinaceus (H. erinaceus) is a Chinese medicinal fungus, with the effect of prevention and treatment of gastrointestinal disorders. In this study, we have tested the anti-inflammatory effect of polysaccharide of H. erinaceus (HECP, Mw: 86.67 kDa) in the model of dextran sulfate sodium (DSS)-induced colitis in C57BL/6 mice. Our data indicated that HECP could improve clinical symptoms and down-regulate key markers of oxidative stresses, including nitric oxide (NO), malondialdehyde (MDA), total superoxide dismutase (T-SOD), and myeloperoxidase (MPO). HECP also suppressed the secretion of interleukin (IL)-6, interleukin (IL)-1β, tumor necrosis factor (TNF)-α and the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and decreased the expression of related mRNA. Meanwhile, HECP blocked phosphorylation of nuclear factor-κB (NF-κB) p65, NF-κB inhibitor alpha (IκB-α), mitogen-activated protein kinases (MAPK) and Protein kinase B (Akt) in DSS-treated mice. Moreover, HECP reversed DSS-induced gut dysbiosis and maintained intestinal barrier integrity. In conclusion, HECP ameliorates DSS-induced intestinal injury in mice, which suggests that HECP can serve as a protective dietary nutrient against IBD.

NLRP6 function in inflammatory monocytes reduces susceptibility to chemically induced intestinal injury.

NLRP6 is a member of the Nod like receptor family, whose members are involved in the recognition of microbes and/or tissue injury. NLRP6 was previously demonstrated to regulate the production of IL-18 and is important for protecting mice against chemically-induced intestinal injury and colitis-associated colon cancer. However, the cellular mechanisms by which NLRP6 reduces susceptibility to colonic inflammation remain unclear. Here, we determined that NLRP6 expression is specifically upregulated in Ly6Chi inflammatory monocytes that infiltrate into the colon during dextran sulfate sodium (DSS)-induced inflammation. Adoptive transfer of WT Ly6Chi inflammatory monocytes into Nlrp6−/− mice was sufficient to protect them from mortality, significantly reducing intestinal permeability and damage. NLRP6-deficient inflammatory monocytes were defective in TNFα production, which was important for reducing DSS-induced mortality and was dependent on autocrine IL-18 signaling by inflammatory monocytes. Our data reveal a previously unappreciated role for NLRP6 in inflammatory monocytes, which are recruited after DSS-induced intestinal injury to promote barrier function and limit bacteria-driven inflammation. This study highlights the importance of early cytokine responses, particularly NLRP6-dependent and IL-18-dependent TNFα production, in preventing chronic dysregulated inflammation.

Berberine promotes recovery of colitis and inhibits inflammatory responses in colonic macrophages and epithelial cells in DSS-treated mice.

Inflammatory bowel disease (IBD) results from dysregulation of intestinal mucosal immune responses to microflora in genetically susceptible hosts. A major challenge for IBD research is to develop new strategies for treating this disease. Berberine, an alkaloid derived from plants, is an alternative medicine for treating bacterial diarrhea and intestinal parasite infections. Recent studies suggest that berberine exerts several other beneficial effects, including inducing anti-inflammatory responses. This study determined the effect of berberine on treating dextran sulfate sodium (DSS)-induced intestinal injury and colitis in mice. Berberine was administered through gavage to mice with established DSS-induced intestinal injury and colitis. Clinical parameters, intestinal integrity, proinflammatory cytokine production, and signaling pathways in colonic macrophages and epithelial cells were determined. Berberine ameliorated DSS-induced body weight loss, myeloperoxidase activity, shortening of the colon, injury, and inflammation scores. DSS-upregulated proinflammatory cytokine levels in the colon, including TNF, IFN-γ, KC, and IL-17 were reduced by berberine. Berberine decreased DSS-induced disruption of barrier function and apoptosis in the colon epithelium. Furthermore, berberine inhibited proinflammatory cytokine production in colonic macrophages and epithelial cells in DSS-treated mice and promoted apoptosis of colonic macrophages. Activation of signaling pathways involved in stimulation of proinflammatory cytokine production, including MAPK and NF-κB, in colonic macrophages and epithelial cells from DSS-treated mice was decreased by berberine. In summary, berberine promotes recovery of DSS-induced colitis and exerts inhibitory effects on proinflammatory responses in colonic macrophages and epithelial cells. Thus berberine may represent a new therapeutic approach for treating gastrointestinal inflammatory disorders.

Mu Opioid Signaling Protects Against Acute Murine Intestinal Injury in a Manner Involving Stat3 Signaling

Opiates have long been used as analgesics to relieve pain associated with various medical conditions. Here, we evaluated the effect and mechanism of mu opioid signaling on the intestinal wound healing response and assessed downstream pathways known to be protective against intestinal injury. Mice (C57BL/6) were exposed to 3% dextran sodium sulfate (DSS) for 7 days or 4% DSS for 5 days followed by 7 days of water. The mu opioid receptor (MOR)-specific agonist [D-Arg2,Lys4]dermorphin-(1,4)-amide (DALDA) and the antagonist cyprodime were injected s.c. daily for in vivo studies or used for in vitro analysis. We found that MOR activation attenuated DSS-induced histologic and gross intestinal injury and weight loss; diminished Ifng, Tnf, and Il6 mRNA expression; and promoted intestinal healing during recovery. DALDA also enhanced colonocyte proliferation (Ki-67 staining) by 350%. MOR activation increased Stat3 phosphorylation in both DALDA-treated mice and the CMT-93 cell line. Importantly, DALDA-induced colonocyte migration was completely ablated by shStat3 knockdown. Together, this work shows that MOR activation protects against and enhances recovery from DSS-induced intestinal injury. This is associated with an increase in Stat3 activation. Furthermore, Stat3 is required for DALDA-induced colonocyte migration. Consequently, manipulation of MOR signaling may represent a novel means to promote mucosal healing and to maintain intestinal homeostasis after intestinal injury.

Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism

Probiotic bacteria can potentially have beneficial effects on the clinical course of several intestinal disorders, but our understanding of probiotic action is limited. We have identified a probiotic bacteria-derived soluble protein, p40, from Lactobacillus rhamnosus GG (LGG), which prevents cytokine-induced apoptosis in intestinal epithelial cells. In the current study, we analyzed the mechanisms by which p40 regulates cellular responses in intestinal epithelial cells and p40's effects on experimental colitis using mouse models. We show that the recombinant p40 protein activated EGFR, leading to Akt activation. Activation of EGFR by p40 was required for inhibition of cytokine-induced apoptosis in intestinal epithelial cells in vitro and ex vivo. Furthermore, we developed a pectin/zein hydrogel bead system to specifically deliver p40 to the mouse colon, which activated EGFR in colon epithelial cells. Administration of p40-containing beads reduced intestinal epithelial apoptosis and disruption of barrier function in the colon epithelium in an EGFR-dependent manner, thereby preventing and treating DSS-induced intestinal injury and acute colitis. Furthermore, p40 activation of EGFR was required for ameliorating colon epithelial cell apoptosis and chronic inflammation in oxazolone-induced colitis. These data define what we believe to be a previously unrecognized mechanism of probiotic-derived soluble proteins in protecting the intestine from injury and inflammation.

IL-10–Producing Regulatory B10 Cells Inhibit Intestinal Injury in a Mouse Model

B cells mediate multiple functions that influence immune and inflammatory responses. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to immediate intestinal injury. Dextran sulfate sodium-induced intestinal injury serves as an experimental animal model for human ulcerative colitis. The contribution of B cells to DSS-induced intestinal injury is unclear. In this study, we show that DSS-induced intestinal injury was more severe in CD19-deficient (CD19(-/-)) mice than in wild-type mice. These inflammatory responses were negatively regulated by a unique IL-10-producing CD1d(hi)CD5(+) regulatory B cell subset (B10 cells) that was absent in CD19(-/-) mice and represented only 1% to 2% of splenic B220(+) cells in wild-type mice. Remarkably, adoptive transfer of these B10 cells from wild-type mice reduced inflammation in CD19(-/-) mice in an IL-10-dependent manner. These results demonstrate that IL-10 production from regulatory B10 cells regulates DSS-induced intestinal injury. These findings may provide new insights and therapeutic approaches for treating ulcerative colitis.