Lactobacillus rhamnosus GG increases cyclooxygenase-2 expression and prostaglandin E2 secretion in colonic myofibroblasts via a MyD88-dependent mechanism during homeostasis.
ABSTRACT: Prostaglandin E2 (PGE2 ) plays a critical role in intestinal mucosal tolerance and barrier integrity. Cyclooxygenase-2 (COX-2)-dependent PGE2 production involves mobilisation of arachidonic acid. Lactobacillus rhamnosus GG (LbGG) is one of the most widely used probiotics reported to colonise the colonic mucosa. LbGG contributes to the protection of the small intestine against radiation injury through the repositioning of mucosal COX-2 expressing cells. However, it is unknown if LbGG modulates PGE2 production in the colonic mucosa under homeostasis and the major cellular elements involved in these processes. Colonic epithelial and CD90+ mesenchymal stromal cells, also known as (myo) fibroblasts (CMFs), are abundant innate immune cells in normal colonic mucosa able to produce PGE2 . Herein, we tested the hypothesis that under colonic mucosal homeostasis, LbGG modulates the eicosanoid pathway resulting in increased PGE2 production in both epithelial and stromal cells. Among the five tested human colonic epithelial cell lines, only exposure of Caco-2 to LbGG for 24 hr led to the mobilisation of arachidonic acid with concomitant increase in the components within the leukotriene and COX-2-dependent PGE2 pathways. By contrast, CMFs isolated from the normal human colonic mucosa responded to LbGG with increased expression of COX-2 and PGE2 in the prostaglandin pathway, but not 5-LO in the leukotriene pathway. Oral gavage of C57BL/6 mice for 5 days with LbGG (5 × 108 Colony-Forming Unit (CFU)/dose) increased COX-2 expression in the colonic mucosa. The majority of cells upregulating COX-2 protein expression were located in the colonic lamina propria and colocalised with ?-SMA+ cells corresponding to the CMF phenotype. This process was myeloid differentiation factor-88-dependent, because silencing of myeloid differentiation factor-88 expression in CMFs abrogated LbGG-induced upregulation of COX-2 in culture and in vivo. Taken together, our data suggest that LbGG increases release of COX-2-mediated PGE2 , contributing to the maintenance of mucosal homeostasis in the colon and CMFs are among the major contributors to this process.
Project description:Signaling via programmed death ligand-1 (PD-L1) and PD-L2 is crucial for maintaining peripheral tolerance. CD90(+) myofibroblasts/fibroblasts (CMFs) are major programmed cell death-1 (PD-1) ligand-expressing cells in normal human colonic mucosa. CMFs suppress activated CD4(+) T cell proliferation via PD-1 ligands. It is not known whether signaling through TLRs contribute to the regulation PD-1 ligands on CMFs upon colonic mucosal tolerance. In this study, we demonstrated that stimulation of TLR4 on human CMFs upregulates PD-L1, but not PD-L2, and reinforces CMF-mediated suppression of CD4(+) T cell proliferation and IFN-? production. TLR4-mediated upregulation of PD-L1 on CMFs involved NF-?B pathways and was JAK2 and MyD88 dependent. MyD88-dependent stimulation of TLR1/2 and TLR5 also upregulated PD-L1 expression on CMFs in culture. PD-L1 expression was drastically decreased in vivo in the colonic mucosa of mice devoid of MyD88. Induction of MyD88 deficiency in CMFs in fibroblast-specific MyD88 conditional knockout mice resulted in a strong increase in a mucosal IFN-? expression concomitantly with the abrogation of PD-L1 expression in CMFs under homeostasis and epithelial injury induced by dextran sodium sulfate. Together, these data suggest that MyD88-dependent TLR stimulation of CMFs in the normal colonic mucosa may reinforce these cells' anti-inflammatory capacity and thus contribute to the maintenance of mucosal tolerance.
Project description:Regulatory T (Treg) cells (CD4+ CD25high FoxP3+) regulate mucosal tolerance; their adoptive transfer prevents or reduces symptoms of colitis in mouse models of inflammatory bowel disease. Colonic CD90+ mesenchymal myofibroblasts and fibroblasts (CMFs) are abundant, nonprofessional antigen-presenting cells in the normal human colonic mucosa that suppress proliferation of activated CD4+ effector T cells. We studied CMF suppressive capacity and evaluated the ability of CMF to induce Treg cells.Allogeneic cocultures of CD4+ T cells and CMFs, derived from normal mucosa of patients undergoing colectomy for colon cancer or inflamed colonic tissues from patients with ulcerative colitis or Crohn's disease, were used to assess activation of the Treg cells.Coculture of normal CMF with resting or naïve CD4+ T cells led to development of cells with a Treg phenotype; it also induced proliferation of a CD25+ CD127- FoxP3+ T cells, which expressed CTLA-4, interleukin-10, and transforming growth factor-? and had suppressive activities. In contrast to dendritic cells, normal CMFs required exogenous interleukin-2 to induce proliferation of naturally occurring Treg cells. Induction of Treg cells by normal CMFs required major histocompatibility complex class II and prostaglandin E2. CMFs from patients with inflammatory bowel diseases had reduced capacity to induce active Treg cells and increased capacity to transiently generate CD4+CD25+/- CD127+ T cells that express low levels of FoxP3.CMFs suppress the immune response in normal colon tissue and might therefore help maintain colonic mucosal tolerance. Alterations in CMF-mediated induction of Treg cells might promote pathogenesis of inflammatory bowel diseases.
Project description:Background and Aims:The role of programmed cell death protein 1 (PD-1) and its ligands in the dysregulation of T helper immune responses observed in the inflammatory bowel disease (IBD) is unclear. Recently, a novel concept emerged that CD90+ colonic (myo)fibroblasts (CMFs), also known as stromal cells, act as immunosuppressors, and are among the key regulators of acute and chronic inflammation. The objective of this study was to determine if the level of the PD-1 ligands is changed in the IBD inflamed colonic mucosa and to test the hypothesis that changes in IBD-CMF-mediated PD-1 ligand-linked immunosuppression is a mechanism promoting the dysregulation of Th1?cell responses. Methods:Tissues and cells derived from Crohn's disease (CD), ulcerative colitis (UC), and healthy individuals (N) were studied in situ, ex vivo, and in culture. Results:A significant increase in programmed death-ligand 1 (PD-L1) was observed in the inflamed UC colonic mucosa when compared to the non-inflamed matched tissue samples, CD, and healthy controls. UC-CMFs were among the major populations in the colonic mucosa contributing to the enhanced PD-L1 expression. In contrast, PD-L1 expression was decreased in CD-CMFs. When compared to CD-CMFs and N-CMFs, UC-CMFs demonstrated stronger suppression of IL-2, Th1 transcriptional factor Tbet, and IFN-? expression by CD3/CD28-activated CD4+ T cells, and this process was PD-L1 dependent. Similar observations were made when differentiated Th1?cells were cocultured with UC-CMFs. In contrast, CD-CMFs showed reduced capacity to suppress Th1?cell activity and addition of recombinant PD-L1 Fc to CD-CMF:T cell cocultures partially restored the suppression of the Th1 type responses. Conclusion:We present evidence showing that increased PD-L1 expression suppresses Th1?cell activity in UC. In contrast, loss of PD-L1 expression observed in CD contributes to the persistence of the Th1 inflammatory milieu in CD. Our data suggest that dysregulation of the Th1 responses in the inflamed colonic mucosa of IBD patients is promoted by the alterations in PD-L1 expression in the mucosal mesenchymal stromal cell compartment.
Project description:Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc (?14/+) in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc (?14/+) mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc (?14/+) mice as PGE2 contributes to the growth and expansion of the early cryptal structures.
Project description:Colorectal cancer (CRC) remains a significant cause of mortality. Inhibitors of cyclooxygenase (COX) and thus prostaglandin E2, are promising CRC preventives, but have significant toxicities. Ginger has been shown to inhibit COX, to decrease the incidence and multiplicity of adenomas, and decrease PGE2 concentrations in subjects at normal risk for CRC. This study was conducted to determine the effects of 2.0?g/d of ginger given orally on the levels of PGE2, leukotriene B4 (LTB4), 13-hydroxy-octadecadienoic acids, and 5-, 12-, & 15-hydroxyeicosatetraenoic acid, in the colonic mucosa of subjects at increased risk for CRC. We randomized 20 subjects to 2.0?g/d ginger or placebo for 28 d. At baseline and Day 28, a flexible sigmoidoscopy was used to obtain colon biopsies. A liquid chromatography mass spectrometry method was used to determine eicosanoid levels in the biopsies, and levels were expressed per amount of protein or free arachidonic acid (AA). There was a significant decrease in AA between baseline and Day 28 (P?=?0.05) and significant increase in LTB4 (P?=?0.04) when normalized to protein, in subjects treated with ginger versus placebo. No other changes in eicosanoids were observed. There was no difference between the groups in total adverse events (AE; P?=?0.06). Ginger lacks the ability to decrease eicosanoid levels in people at increased risk for CRC. Ginger did appear to be both tolerable and safe; and could have chemopreventive effects through other mechanisms. Further investigation should focus on other markers of CRC risk in those at increased CRC risk.
Project description:Candida albicans is a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE2), but the physiological significance of fungus-derived PGE2 remains elusive. Here we report that a C. albicans mutant deficient in PGE2 production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE2 supplementation mitigated this fitness defect. Impaired fungal PGE2 production affected neither the in vitro fitness of C. albicans nor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Instead, fungal production of PGE2 was associated with enhanced fungal survival within phagocytes. Consequently, ablation of colonic phagocytes abrogated the intra-GI fitness boost conferred by fungal PGE2. These observations suggest that C. albicans has evolved the capacity to produce PGE2 from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.
Project description:Portal hypertensive gastropathy (PHG) is a serious complication of liver cirrhosis and a potential cause of bleeding in patients with cirrhosis. Suppressed mucosal epithelial proliferation is a crucial pathological characteristic of PHG. Our studies demonstrated an important role for PGE2 and its EP4 receptor in the promotion of mucosal proliferation. However, whether ?-arrestin1 (?-arr1), a well-established mediator of GPCRs, is involved in the PGE2 /EP4 receptor-mediated mucosal proliferation complex in PHG remains unclear. The aim of the study was to investigate whether ?-arr1 participated in PGE2 /EP4 receptor-mediated mucosal proliferation by recruiting the Src/EGF receptor (EGFR) complex to activate Akt/proliferating cell nuclear antigen (PCNA) signalling in PHG.Gastric mucosal proliferation was examined in patients with PHG and the PHG model of ?-arr1-knockout (?-arr1-KO) and ?-arr1-wild type (?-arr1-WT) mice. The induction of ?-arr1 and EP4 receptor expression and the Src/EGFR signalling elements was investigated, and the mechanisms underlying PGE2 -regulated gastric mucosal proliferation were analysed.Portal hypertension suppressed COX-1 but not COX-2, which was accompanied by a down-regulation of PGE2 generation and EP4 receptor levels in the mucosa of patients with PHG. PGE2 administration markedly promoted mucosal proliferation in a mouse model of PHG. Targeted deletion of ?-arr1 abolished PGE2 /EP4 receptor-mediated gastric proliferation in PHG by repressing the Src/EGFR/Akt/PCNA signalling network.These results indicate that ?-arr1 regulates PGE2 /EP4 receptor-mediated mucosal proliferation by promoting activation of the Src/EGFR/Akt/PCNA signalling pathway, and thus, this network is a potential therapeutic target for PHG.
Project description:Release of the free fatty acid arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2) and its subsequent metabolism by the cyclooxygenase and lipoxygenase enzymes produces a broad panel of eicosanoids including prostaglandins (PGs). This study sought to investigate the roles of these mediators in experimental models of inflammation and inflammation-associated intestinal tumorigenesis. Using the dextran sodium sulfate (DSS) model of experimental colitis, we first investigated how a global reduction in eicosanoid production would impact intestinal injury by utilizing cPLA2 knockout mice. cPLA2 deletion enhanced colonic injury, reflected by increased mucosal ulceration and pro-inflammatory cytokine expression. Increased disease severity was associated with a significant reduction in the levels of several eicosanoid metabolites, including PGE2. We further assessed the precise role of PGE2 synthesis on mucosal injury and repair by utilizing mice with a genetic deletion of microsomal PGE synthase-1 (mPGES-1), the terminal synthase in the formation of inducible PGE2. DSS exposure caused more extensive acute injury as well as impaired recovery in knockout mice compared to wild-type littermates. Increased intestinal damage was associated with both reduced PGE2 levels as well as altered levels of other eicosanoids including PGD2. To determine whether this metabolic redirection impacted inflammation-associated intestinal tumorigenesis, Apc(Min/+) and Apc(Min/+):mPGES-1(-/-) mice were exposed to DSS. DSS administration caused a reduction in the number of intestinal polyps only in Apc(Min/+):mPGES-1(-/-) mice. These results demonstrate the importance of the balance of prostaglandins produced in the intestinal tract for maintaining intestinal homeostasis and impacting tumor development.
Project description:BACKGROUND: Leukotriene (LT) B4 concentrations are increased and prostaglandin (PG) E2 concentrations are decreased in exhaled breath condensate (EBC) in patients with chronic obstructive pulmonary disease (COPD). A study was undertaken to investigate the short term effects of cyclo-oxygenase (COX) inhibition on exhaled LTB4 and PGE2 concentrations in patients with COPD and to identify the COX isoform responsible for exhaled PGE2 production. METHODS: Two studies were performed. A double blind, crossover, randomised, placebo controlled study with ibuprofen (400 mg qid for 2 days), a non-selective COX inhibitor, was undertaken in 14 patients with stable COPD, and an open label study with oral rofecoxib (25 mg once a day for 5 days), a selective COX-2 inhibitor, was undertaken in a different group of 16 COPD patients. EBC was collected before and after drug treatment. Exhaled LTB4 and PGE2 concentrations were measured with specific immunoassays. RESULTS: All patients complied with treatment as indicated by a reduction in ex vivo serum thromboxane B2 concentrations (ibuprofen) and a reduction in lipopolysaccharide induced increase in ex vivo plasma PGE2 values (rofecoxib) of more than 80%. Exhaled LTB4 was increased after ibuprofen (median 175.5 (interquartile range 128.8-231.5) pg/ml v 84.0 (70.0-98.5) pg/ml, p < 0.001) and exhaled PGE2 was reduced (93.5 (84.0-105-5) pg/ml v 22.0 (15.0-25.5) pg/ml, p < 0.0001). Rofecoxib had no effect on exhaled LTB4 (p = 0.53) or PGE2 (p = 0.23). CONCLUSIONS: Non-selective COX inhibition decreases PGE2 and increases LTB4 in EBC, whereas selective COX-2 inhibition has no effect on these eicosanoids. PGE2 in EBC is primarily derived from COX-1 activity, and COX inhibition may redirect arachidonic acid metabolism towards the 5-lipoxygenase pathway.
Project description:The intestinal protozoan parasite Entamoeba histolytica (Eh) causes amebiasis associated with severe diarrhea and/or liver abscess. Eh pathogenesis is multifactorial requiring both parasite virulent molecules and host-induced innate immune responses. Eh-induced host pro-inflammatory responses plays a critical role in disease pathogenesis by causing damage to tissues allowing parasites access to systemic sites. Eh cyclooxygenase (EhCox) derived prostaglandin E2 stimulates the chemokine IL-8 from mucosal epithelial cells that recruits neutrophils to the site of infection to exacerbate disease. At present, it is not known how EhCox is regulated or whether it affects the expression of other proteins in Eh. In this study, we found that gene silencing of EhCox (EhCoxgs) markedly increased endogenous cysteine protease (CP) protein expression and virulence without altering CP gene transcripts. Live virulent Eh pretreated with arachidonic acid substrate to enhance PGE2 production or aspirin to inhibit EhCox enzyme activity or addition of exogenous PGE2 to Eh had no effect on EhCP activity. Increased CP enzyme activity in EhCoxgs was stable and significantly enhanced erythrophagocytosis, cytopathic effects on colonic epithelial cells and elicited pro-inflammatory cytokines in mice colonic loops. Acute infection with EhCoxgs in colonic loops increased inflammation associated with high levels of myeloperoxidase activity. This study has identified EhCox protein as one of the important endogenous regulators of cysteine protease activity. Alterations of CP activity in response to Cox gene silencing may be a negative feedback mechanism in Eh to limit proteolytic activity during colonization that can inadvertently trigger inflammation in the gut.