Sam C. Nalle

Targeted Epithelial Tight Junction Dysfunction Causes Immune Activation and Contributes to Development of Experimental Colitis

BACKGROUND & AIMS Inflammatory bowel disease (IBD) is a multifactorial disease thought to be caused by alterations in epithelial function, innate and adaptive immunity, and luminal microbiota. The specific role of epithelial barrier function remains undefined, although increased activity of intestinal epithelial myosin light chain kinase (MLCK), which is the primary mechanism of tumor necrosis factor-induced barrier dysfunction, occurs in human IBD. Our aim was to determine whether, in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis. METHODS We developed transgenic (Tg) mice that express constitutively active MLCK (CA-MLCK) specifically within intestinal epithelia. Their physiology, immune status, and susceptibility to disease were assessed and compared with non-Tg littermate controls. RESULTS CA-MLCK Tg mice demonstrated significant barrier loss but grew and gained weight normally and did not develop spontaneous disease. CA-MLCK Tg mice did, however, develop mucosal immune activation demonstrated by increased numbers of lamina propria CD4(+)lymphocytes, redistribution of CD11c+cells, increased production of interferon-gamma and tumor necrosis factor, as well as increased expression of epithelial major histocompatibility complex class I. When challenged with CD4+CD45+Rb(hi) lymphocytes, Tg mice developed an accelerated and more severe form of colitis and had shorter survival times than non-Tg littermates. CONCLUSIONS Primary pathophysiologically relevant intestinal epithelial barrier dysfunction is insufficient to cause experimental intestinal disease but can broadly activate mucosal immune responses and accelerate the onset and severity of immune-mediated colitis.

Claudin-1 and claudin-2 expression is elevated in inflammatory bowel disease and may contribute to early neoplastic transformation

Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal adenocarcinoma. The factors that result in IBD-associated carcinogenesis are not understood. We hypothesized that altered expression of intestinal epithelial tight junction proteins might contribute to neoplastic progression. Semiquantitative immunohistochemical staining of human biopsies was used to assess expression of the tight junction proteins claudin-1, claudin-2, claudin-4, and occludin in IBD, IBD-associated dysplasia, acute, self-limited colitis (ASLC), and sporadic adenomas. Claudin-1 and claudin-2 expression was elevated in active IBD, adenomas, and IBD-associated dysplasia, but not ASLC. In contrast, claudin-4 expression was elevated in both active IBD and ASLC. Occludin expression was similar to control in all cases. Importantly, in IBD, claudin-1 and claudin-2 expression correlated positively with inflammatory activity. To investigate mechanisms underlying altered claudin expression, β-catenin activation was assessed as nuclear localization. Like claudin-1 and claudin-2, β-catenin was markedly activated in IBD, sporadic dysplasia, and IBD-associated dysplasia, but was only slightly activated in ASLC. Taken together, these data suggest that β-catenin transcriptional activity is elevated in chronic injury and that this may contribute to increased claudin-1 and claudin-2 expression. We speculate that increased claudin-1 and claudin-2 expression may be involved at early stages of transformation in IBD-associated neoplasia.

TNFR2 activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated barrier loss and experimental colitis.

BACKGROUND & AIMS Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease. However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are not completely understood. We investigated these processes using colitis models in mice. METHODS We induced colitis by adoptive transfer of CD4(+)CD45RB(hi) cells or administration of dextran sulfate sodium to mice, including those deficient in tumor necrosis factor receptor (TNFR) 1, TNFR2, or the long isoform of myosin light chain kinase (MLCK). Intestinal tissues and isolated epithelial cells were analyzed by immunoblot, immunofluorescence, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction assays. RESULTS Induction of immune-mediated colitis by CD4(+)CD45RB(hi) adoptive transfer increased intestinal permeability, epithelial expression of claudin-2, the long isoform of MLCK, and TNFR2 (but not TNFR1) and phosphorylation of the myosin II light chain. Long MLCK upregulation, myosin II light chain phosphorylation, barrier loss, and weight loss were attenuated in TNFR2(-/-) , but not TNFR1(-/-) , recipients of wild-type CD4(+)CD45RB(hi) cells. Similarly, long MLCK(-/-) mice had limited increases in myosin II light chain phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of adoptive transfer-induced colitis. However, coincident with onset of epithelial apoptosis, long MLCK(-/-) mice ultimately developed colitis. This indicates that disease progresses via apoptosis in the absence of MLCK-dependent tight junction regulation. In support of this conclusion, long MLCK(-/-) mice were not protected from epithelial apoptosis-mediated, damage-dependent dextran sulfate sodium colitis. CONCLUSIONS In immune-mediated inflammatory bowel disease models, TNFR2 signaling increases long MLCK expression, resulting in tight junction dysregulation, barrier loss, and induction of colitis. At advanced stages, colitis progresses by apoptosis and mucosal damage that result in tight junction- and MLCK-independent barrier loss. Therefore, barrier loss in immune-mediated colitis occurs via two temporally and morphologically distinct mechanisms. Differential targeting of these mechanisms can lead to improved inflammatory bowel disease therapies.

Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease

Pretransplant conditioning promotes minor mismatch GVHD by causing intestinal barrier loss and depleting NK cells. Double Trouble Before Transplant Graft-versus-host disease, a condition where transplanted immune cells attack the body of the transplant recipient, is a common complication of bone marrow or stem cell transplant. Although there are treatments available for treating graft-versus-host disease, they don’t always work well. Nalle et al. have now developed several different mouse models of this condition to provide insight into its causes. The authors used these mouse models to pinpoint two different ways that pretransplant conditioning contributes to graft-versus-host disease. This knowledge should help scientists search for new treatments against this disease, as well as identify safer ways to prepare patients for transplant. Previous studies have shown a correlation between pretransplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, because irradiation and other forms of pretransplant conditioning have pleiotropic effects, the precise role of intestinal barrier loss in GVHD pathogenesis remains unclear. We developed GVHD models that allowed us to isolate the specific contributions of distinct pretransplant variables. Intestinal damage was required for the induction of minor mismatch [major histocompatibility complex (MHC)–matched] GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient natural killer (NK) cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing an immunoregulatory function of MHC-matched recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated Toll-like receptor 4 (TLR4) signaling on donor cells, and intestinal damage could be bypassed by parenteral lipopolysaccharide (LPS) administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pretransplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

Endothelial and Epithelial Barriers in Graft-Versus-Host Disease

Endothelial and epithelial cells form selectively permeable barriers that separate tissue compartments. These cells coordinate movement between the lumen and tissue via the transcellular and paracellular pathways. The primary determinant of paracellular permeability is the tight junction, which forms an apical belt-like structure around endothelial and epithelial cells. This chapter discusses endothelial and epithelial barriers in graft-versus-host disease after allogeneic bone marrow transplantation, with a focus on the tight junction and its role in regulating paracellular permeability. Recent studies suggest that in graft-versus-host disease, pathological increases in paracellular permeability, or barrier dysfunction, are initiated by pretransplant conditioning and sustained by alloreactive cells and the proinflammatory milieu. The intestinal epithelium is a significant focus, as it is a target organ of graft-versus-host disease, and the mechanisms of barrier regulation in intestinal epithelium have been well characterized. Finally, we propose a model that incorporates endothelial and epithelial barrier dysfunction in graft-versus-host disease and discuss modulating barrier properties as a therapeutic approach.

Epithelium, Tear Down This Wall!

The presence of bacteria within epithelial-lined compartments, such as the lung, elicits rapid recruitment of innate immune cells. In this issue of Cell Host & Microbe, Chun and Prince (2009) report that epithelial cells facilitate this process by reorganizing their intercellular junctions to enhance immune cell transmigration.

Ménétrier’s Disease Therapy: Rebooting Mucosal Signaling

EGFR neutralization provides hope of sustained remission in Ménétrier’s disease patients. Ménétrier’s disease, a rare hyperproliferative disorder of the stomach, is associated with chronic abdominal pain, vomiting, weight loss, and edema, as well as an increased risk of gastric cancer. Therapy, other than surgical resection of the stomach, is limited to supportive measures and reflects the limited understanding of Ménétrier’s disease pathogenesis. Data reported in this issue describe a promising targeted therapeutic approach and provide new insight into the causes of Ménétrier’s disease.