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 (TNF)-induced barrier dysfunction, occurs in human IBD. We aimed to determine whether in an intact epithelium, primary dysregulation of the intestinal epithelial barrier by pathophysiologically relevant mechanisms can contribute to development of colitis.

BACKGROUND & AIMS Tight junction dysregulation and epithelial damage contribute to barrier loss in patients with inflammatory bowel disease (IBD). However, the mechanisms that regulate these processes and their relative contributions to disease pathogenesis are incompletely understood. We investigated these processes using colitis models in mice. METHODS We induced colitis by adoptive transfer of CD4+CD45RBhi cells or administration of dextran sulfate sodium (DSS) 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, ELISA, and real-time PCR assays. RESULTS Induction of immune-mediated colitis by CD4+CD45RBhi 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 (MLC). Long MLCK upregulation, MLC phosphorylation, barrier loss, and weight loss were attenuated in TNFR2−/−, but not TNFR1−/−, recipients of wildtype CD4+CD45RBhi cells. Similarly, long MLCK−/− mice had limited increases in MLC phosphorylation, claudin-2 expression, and intestinal permeability and delayed onset of cell transfer-induced colitis. However, coincident with onset of epithelial apoptosis, colitis ultimately developed. 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 DSS colitis. CONCLUSIONS In immune-mediated IBD 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 results 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 may lead to improved IBD therapies.

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. Semi-quantitative 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, dysplasia, IBD-associated dysplasia, but 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 in early stages of transformation in IBD-associated neoplasia.

Compromised intestinal barrier function is a prominent feature of inflammatory bowel disease (IBD). However, links between intestinal barrier loss and disease extend much further, including documented associations with celiac disease, type I diabetes, rheumatoid arthritis, and multiple sclerosis. Intestinal barrier loss has also been proposed to have a critical role in the pathogenesis of graft-versus-host disease (GVHD), a serious, potentially fatal consequence of hematopoietic stem cell transplantation. Experimental evidence has begun to support this view, as barrier loss and its role in initiating and establishing a pathogenic inflammatory cycle in GVHD is emerging. Here we discuss similarities between IBD and GVHD, mechanisms of intestinal barrier loss in these diseases, and the crosstalk between barrier loss and the immune system, with a special focus on natural killer (NK) cells. Unanswered questions and future research directions on the topic are discussed along with implications for treatment.

Previous studies have shown a correlation between pre-transplant conditioning intensity, intestinal barrier loss, and graft-versus-host disease (GVHD) severity. However, since irradiation and other forms of pre-transplant 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 pre-transplant variables. First, intestinal damage was required for the induction of minor mismatch (MHC-matched) GVHD, but was not necessary for major mismatch GVHD, demonstrating fundamental pathogenic distinctions between these forms of disease. Moreover, recipient NK cells prevented minor mismatch GVHD by limiting expansion and target organ infiltration of alloreactive T cells via a perforin-dependent mechanism, revealing a previously unrecognized immunoregulatory function of recipient NK cells in GVHD. Minor mismatch GVHD required MyD88-mediated TLR4 signaling on donor cells, and intestinal damage could be bypassed by parenteral LPS administration, indicating a critical role for the influx of bacterial components triggered by intestinal barrier loss. In all, the data demonstrate that pre-transplant conditioning plays a dual role in promoting minor mismatch GVHD by both depleting recipient NK cells and inducing intestinal barrier loss.

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 + 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.

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Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.