Search

Intestinal barrier leakage constitutes a potential therapeutic target for many inflammatory diseases and represents a disease progression marker during chronic viral infections. However, the causes of altered gut barrier remain mostly unknown. Using murine infection with lymphocytic choriomeningitis virus, we demonstrate that, in contrast to an acute viral strain, a persistent viral isolate leads to long-term viral replication in hematopoietic and mesenchymal cells, but not epithelial cells (IECs), in the intestine. Viral persistence drove sustained intestinal epithelial barrier leakage, which was characterized by increased paracellular flux of small molecules and was associated with enhanced colitis susceptibility. Type I IFN signaling caused tight junction dysregulation in IECs, promoted gut microbiome shifts and enhanced intestinal CD8 T cell responses. Notably, both type I IFN receptor blockade and CD8 T cell depletion prevented infection-induced barrier leakage. Our study demonstrates that infection with a virus that persistently replicates in the intestinal mucosa increases epithelial barrier permeability and reveals type I IFNs and CD8 T cells as causative factors of intestinal leakage during chronic infections.

Background & aimsSLC26A3 (DRA) is an electroneutral Cl-/HCO3- exchanger that is present in the apical domain of multiple intestinal segments. An area that has continued to be poorly understood is related to DRA regulation in acute adenosine 3',5'-cyclic monophosphate (cAMP)-related diarrheas, in which DRA appears to be both inhibited as part of NaCl absorption and stimulated to contribute to increased HCO3- secretion. Different cell models expressing DRA have shown that cAMP inhibits, stimulates, or does not affect its activity.MethodsThis study re-evaluated cAMP regulation of DRA using new tools, including a successful knockout cell model, a specific DRA inhibitor (DRAinh-A250), specific antibodies, and a transport assay that did not rely on nonspecific inhibitors. The studies compared DRA regulation in colonoids made from normal human colon with regulation in the colon cancer cell line, Caco-2.ResultsDRA is an apical protein in human proximal colon, differentiated colonoid monolayers, and Caco-2 cells. It is glycosylated and appears as 2 bands. cAMP (forskolin) acutely stimulated DRA activity in human colonoids and Caco-2 cells. In these cells, DRA is the predominant apical Cl-/HCO3- exchanger and is inhibited by DRAinh-A250 with a median inhibitory concentration of 0.5 and 0.2 μmol/L, respectively. However, there was no effect of cAMP in HEK293/DRA cells that lacked a cystic fibrosis transmembrane conductance regulator (CFTR). When CFTR was expressed in HEK293/DRA cells, cAMP also stimulated DRA activity. In all cases, cAMP stimulation of DRA was not inhibited by CFTRinh-172.ConclusionsDRA is acutely stimulated by cAMP by a process that is CFTR-dependent, but appears to be one of multiple regulatory effects of CFTR that does not require CFTR activity.

Mesenteric adipose tissue hyperplasia is a hallmark of Crohn's disease (CD). Recently, we showed that mesenteric adipose-derived stromal cells (ADSCs) from CD, ulcerative colitis, and control patients synthesize and release adipokines in a disease-dependent manner. Here we examined the expression profiles of CD and control patient-derived mesenteric ADSCs and studied the effects of their extracellular mediators on colonocyte signaling in vitro and experimental colitis in vivo. ADSCs were isolated from mesenteric fat of control and CD patients. Microarray profiling and network analysis were performed in ADSCs and human colonocytes treated with conditioned media from cultured ADSCs. Mice with acute colitis received daily injections of conditioned media from patient-derived ADSCs, vehicle, or apolactoferrin. Proliferative responses were evaluated in conditioned media-treated colonocytes and mouse colonic epithelium. Total protein was isolated from cultured colonocytes after treatment with apolactoferrin for Western blot analysis of phosphorylated intracellular signaling kinases. Microarray profiling revealed differential mRNA expression in CD patient-derived ADSCs compared with controls, including lactoferrin. Administration of CD patient-derived medium or apolactoferrin increased colonocyte proliferation compared with controls. Conditioned media from CD patient-derived ADSCs or apolactoferrin attenuated colitis severity in mice and enhanced colonocyte proliferation in vivo. ADSCs from control and CD patients show disease-dependent inflammatory responses and alter colonic epithelial cell signaling in vitro and in vivo. Furthermore, we demonstrate lactoferrin production by adipose tissue, specifically mesenteric ADSCs. We suggest that mesenteric ADSC-derived lactoferrin may mediate protective effects and participate in the pathophysiology of CD by promoting colonocyte proliferation and the resolution of inflammation.

The tight junction protein claudin-2 is upregulated in disease. Although many studies have linked intestinal barrier loss to local and systemic disease, these have relied on macromolecular probes. In vitro analyses show, however, that these probes cannot be accommodated by size- and charge-selective claudin-2 channels. We sought to define the impact of claudin-2 channels on disease. Transgenic claudin-2 overexpression or IL-13-induced claudin-2 upregulation increased intestinal small cation permeability in vivo. IL-13 did not, however, affect permeability in claudin-2-knockout mice. Claudin-2 is therefore necessary and sufficient to effect size- and charge-selective permeability increases in vivo. In chronic disease, T cell transfer colitis severity was augmented or diminished in claudin-2-transgenic or -knockout mice, respectively. We translated the in vitro observation that casein kinase-2 (CK2) inhibition blocks claudin-2 channel function to prevent acute, IL-13-induced, claudin-2-mediated permeability increases in vivo. In chronic immune-mediated colitis, CK2 inhibition attenuated progression in claudin-2-sufficient, but not claudin-2-knockout, mice, i.e., the effect was claudin-2 dependent. Paracellular flux mediated by claudin-2 channels can therefore promote immune-mediated colitis progression. Although the mechanisms by which claudin-2 channels intensify disease remain to be defined, these data suggest that claudin-2 may be an accessible target in immune-mediated disorders, including inflammatory bowel disease., Introduction Barriers that define distinct tissue compartments and separate self from non-self are necessary for survival of multicellular organisms. In some organs, for example the integument and bladder, the barrier [...]

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1[beta], a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor, ERN1/IRE1[alpha]. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free [Ern2.sup.-/-] mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free WT mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense., Introduction Epithelial cells lining mucosal surfaces of the gastrointestinal (GI) and respiratory tracts express 2 paralogues of the most evolutionarily conserved endoplasmic reticulum (ER) stress sensor IRE1. While all cell [...]

Background & aimsSubstance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions.MethodsMesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients.ResultsTacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients.ConclusionsPreadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

Background & aimsSubstance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions.MethodsMesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients.ResultsTacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients.ConclusionsPreadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

In inflammatory bowel disease (IBD), obesity is associated with worsening of the course of disease. Here, we examined the role of obesity in the development of colitis and studied mesenteric fat-epithelial cell interactions in patients with IBD. We combined the diet-induce obesity with the trinitrobenzene sulfonic acid (TNBS) colitis mouse model to create groups with obesity, colitis, and their combination. Changes in the mesenteric fat and intestine were assessed by histology, myeloperoxidase assay, and cytokine mRNA expression by real-time PCR. Medium from human mesenteric fat and cultured preadipocytes was obtained from obese patients and those with IBD. Histological analysis showed inflammatory cell infiltrate and increased histological damage in the intestine and mesenteric fat of obese mice with colitis compared with all other groups. Obesity also increased the expression of proinflammatory cytokines including IL-1β, TNF-α, monocyte chemoattractant protein 1, and keratinocyte-derived chemokine, while it decreased the TNBS-induced increases in IL-2 and IFN-γ in mesenteric adipose and intestinal tissues. Human mesenteric fat isolated from obese patients and those with and IBD demonstrated differential release of adipokines and growth factors compared with controls. Fat-conditioned media reduced adiponectin receptor 1 (AdipoR1) expression in human NCM460 colonic epithelial cells. AdipoR1 intracolonic silencing in mice exacerbated TNBS-induced colitis. In conclusion, obesity worsens the outcome of experimental colitis, and obesity- and IBD-associated changes in adipose tissue promote differential mediator release in mesenteric fat that modulates colonocyte responses and may affect the course of colitis. Our results also suggest an important role for AdipoR1 for the fat-intestinal axis in the regulation of inflammation during colitis.

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients. [ABSTRACT FROM AUTHOR]

In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ~62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)-deficient enhanced green fluorescent protein (EGFP)-occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludin(ΔOCEL). Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1-binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK-binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.

Graft-versus-host disease (GVHD) is a complication of hematopoietic stem cell transplantation (HSCT) that affects multiple organs. GVHD-associated intestinal damage can be separated into two distinct phases, initiation and propagation, which correspond to conditioning-induced damage and effector T cell activation and infiltration, respectively. Substantial evidence indicates that intestinal damage induced by pretransplant conditioning is a key driver of GVHD initiation. Here, we aimed to determine the impact of dysregulated intestinal permeability on the subsequent GVHD propagation phase. The initiation phase of GVHD was unchanged in mice lacking long MLCK (MLCK210), an established regulator of epithelial tight junction permeability. However, MLCK210-deficient mice were protected from sustained barrier loss and exhibited limited GVHD propagation, as indicated by reduced histopathology, fewer [CD8.sup.+] effector T cells in the gut, and improved overall survival. Consistent with these findings, intestinal epithelial MLCK210 expression and enzymatic activity were similarly increased in human and mouse GVHD biopsies. Intestinal epithelial barrier loss mediated by MLCK210 is therefore a key driver of the GVHD propagation. These data suggest that inhibition of MLCK210-dependent barrier regulation may be an effective approach to limiting GVHD progression., Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is used in the treatment of neoplastic and immune disorders. Graft-versus-host disease (GVHD), in which donor-derived T cells target recipient alloantigens to cause [...]

The use of model systems is of obvious importance for understanding human physiology and pathophysiology. However, the value of using human models for drug development has only relatively recently been [...]