Daniel R. Clayburgh

Epithelial myosin light chain kinase–dependent barrier dysfunction mediates T cell activation–induced diarrhea in vivo

Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

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.

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

The intestinal epithelial barrier is frequently disrupted in inflammatory bowel disease (IBD) and this has been proposed to play a role in disease pathogenesis and reactivation. In vitro studies show that cytokine-induced epithelial barrier dysfunction can be mediated by increased myosin light chain kinase (MLCK) expression and subsequent myosin II regulatory light chain (MLC) phosphorylation. However, this has never been examined in human disease. The aim of these studies, therefore, was to determine whether MLCK is upregulated in the intestinal epithelium of IBD patients. MLCK expression and MLC phosphorylation in human intestinal resection and biopsy specimens were determined by quantitative immunofluorescence microscopy and correlated with clinical and histopathological data. The data show that ileal epithelial MLCK expression was mildly upregulated in inactive IBD. Expression increased further in active disease, with progressive increases in MLCK expression correlating positively with histological disease activity. This correlation between activity and MLCK expression was also seen in individual patients where areas of differing disease activity were analyzed. Colonic epithelial MLCK expression was similarly increased in active IBD and these increases also correlated positively with disease activity, both in individual patients and the overall study group. To evaluate MLCK enzymatic activity, MLC phosphorylation was assessed in snap-frozen colon biopsies. MLC phosphorylation was significantly increased in biopsies with active, but not inactive, IBD. Therefore, these data show that MLCK expression and enzymatic activity are increased in IBD. Moreover, the correlation with disease activity suggests that MLCK upregulation may contribute to barrier dysfunction and IBD pathogenesis.

Coordinated epithelial NHE3 inhibition and barrier dysfunction are required for TNF-mediated diarrhea in vivo

Acute T cell-mediated diarrhea is associated with increased mucosal expression of proinflammatory cytokines, including the TNF superfamily members TNF and LIGHT. While we have previously shown that epithelial barrier dysfunction induced by myosin light chain kinase (MLCK) is required for the development of diarrhea, MLCK inhibition does not completely restore water absorption. In contrast, although TNF-neutralizing antibodies completely restore water absorption after systemic T cell activation, barrier function is only partially corrected. This suggests that, while barrier dysfunction is critical, other processes must be involved in T cell-mediated diarrhea. To define these processes in vivo, we asked whether individual cytokines might regulate different events in T cell-mediated diarrhea. Both TNF and LIGHT caused MLCK-dependent barrier dysfunction. However, while TNF caused diarrhea, LIGHT enhanced intestinal water absorption. Moreover, TNF, but not LIGHT, inhibited Na+ absorption due to TNF-induced internalization of the brush border Na+/H+ exchanger NHE3. LIGHT did not cause NHE3 internalization. PKCalpha activation by TNF was responsible for NHE3 internalization, and pharmacological or genetic PKCalpha inhibition prevented NHE3 internalization, Na+ malabsorption, and diarrhea despite continued barrier dysfunction. These data demonstrate the necessity of coordinated Na+ malabsorption and barrier dysfunction in TNF-induced diarrhea and provide insight into mechanisms of intestinal water transport.

Mechanism underlying inhibition of intestinal apical Cl–/OH– exchange following infection with enteropathogenic E. coli

Enteropathogenic E. coli (EPEC) is a major cause of infantile diarrhea, but the pathophysiology underlying associated diarrhea is poorly understood. We examined the role of the luminal membrane Cl(-)/OH(-) exchange process in EPEC pathogenesis using in vitro and in vivo models. Cl(-)/OH(-) exchange activity was measured as OH(-) gradient-driven (36)Cl(-) uptake. EPEC infection (60 minutes-3 hours) inhibited apical Cl(-)/OH(-) exchange activity in human intestinal Caco-2 and T84 cells. This effect was dependent upon the bacterial type III secretory system (TTSS) and involved secreted effector molecules EspG and EspG2, known to disrupt the host microtubular network. The microtubule-disrupting agent colchicine (100 muM, 3 hours) also inhibited (36)Cl(-) uptake. The plasma membrane expression of major apical anion exchanger DRA (SLC26A3) was considerably reduced in EPEC-infected cells, corresponding with decreased Cl(-)/OH(-) exchange activity. Confocal microscopic studies showed that EPEC infection caused a marked redistribution of DRA from the apical membrane to intracellular compartments. Interestingly, infection of cells with an EPEC mutant deficient in espG significantly attenuated the decrease in surface expression of DRA protein as compared with treatment with wild-type EPEC. EPEC infection in vivo (1 day) also caused marked redistribution of surface DRA protein in the mouse colon. Our data demonstrate that EspG and EspG2 play an important role in contributing to EPEC infection-associated inhibition of luminal membrane chloride transport via modulation of surface DRA expression.

Enteropathogenic E. coli disrupts tight junction barrier function and structure in vivo.

Enteropathogenic Escherichia coli (EPEC) infection disrupts tight junctions (TJs) and perturbs intestinal barrier function in vitro. E. coli secreted protein F (EspF) is, in large part, responsible for these physiological and morphological alterations. We recently reported that the C57BL/6J mouse is a valid in vivo model of EPEC infection as EPEC colonizes the intestinal epithelium and effaces microvilli. Our current aim was to examine the effects of EPEC on TJ structure and barrier function of the mouse intestine and to determine the role of EspF in vivo. C57BL/6J mice were gavaged with ∼2 × 108 EPEC organisms or PBS. At 1 or 5 days postinfection, mice were killed and ileal and colonic tissue was mounted in Üssing chambers to determine barrier function (measured as transepithelial resistance) and short circuit current. TJ structure was analyzed by immunofluorescence microscopy. Wild-type (WT) EPEC significantly diminished the barrier function of ileal and colonic mucosa at 1 and 5 days postinfection. Deficits in barrier function correlated with redistribution of occludin in both tissues. Infection with an EPEC strain deficient of EspF (ΔespF) had no effect on barrier function at 1 day postinfection. Furthermore, ΔespF had no effect on ileal TJ morphology and minor alterations of colonic TJ morphology at 1 day postinfection. In contrast, at 5 days postinfection, WT EPEC and ΔespF had similar effects on barrier function and occludin localization. In both cases this was associated with immune activation, as demonstrated by increased mucosal tumor necrosis factor-α levels 5 days postinfection. In conclusion, these data demonstrate that WT EPEC infection of 6–8-week-old C57BL/6J mice (1) significantly decreases barrier function in the ileum and colon (2) redistributes occludin in the ileum and colon and (3) is dependent upon EspF to induce TJ barrier defects at early, but not late, times postinfection.

A differentiation-dependent splice variant of myosin light chain kinase, MLCK1, regulates epithelial tight junction permeability

Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.

Tumor Necrosis Factor-induced Long Myosin Light Chain Kinase Transcription Is Regulated by Differentiation-dependent Signaling Events CHARACTERIZATION OF THE HUMAN LONG MYOSIN LIGHT CHAIN KINASE PROMOTER

Myosin light chain kinase (MLCK) is expressed as long and short isoforms from unique transcriptional start sites within a single gene. Tumor necrosis factor (TNF) augments intestinal epithelial long MLCK expression, which is critical to cytoskeletal regulation. We found that TNF increases long MLCK mRNA transcription, both in human enterocytes in vitro and murine enterocytes in vivo.5′-RACE identified two novel exons, 1A and 1B, which encode alternative long MLCK transcriptional start sites. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis identified two essential Sp1 sites upstream of the exon 1A long MLCK transcriptional start site. Analysis of deletion and truncation mutants showed that a 102-bp region including these Sp1 sites was necessary for basal transcription. A promoter construct including 4-kb upstream of exon 1A was responsive to TNF, AP-1, or NFκB, but all except NFκB responses were absent in a shorter 2-kb construct, and all responses were absent in a 1-kb construct. Electrophoretic mobility shift assays, ChIP, and site-directed mutagenesis explained these data by identifying three functional AP-1 sites between 2- and 4-kb upstream of exon 1A and two NFκB sites between 1- and 2-kb upstream of exon 1A. Analysis of differentiating epithelia showed that only well differentiated enterocytes activated the 4-kb long MLCK promoter in response to TNF, and consensus promoter reporters demonstrated that TNF-induced NFκB activation decreased during differentiation while TNF-induced AP-1 activation increased. Thus either AP-1 or NFκB can up-regulate long MLCK transcription, but the mechanisms by which TNF up-regulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.

Epithelial NF-κB Enhances Transmucosal Fluid Movement by Altering Tight Junction Protein Composition after T Cell Activation

In inflammatory bowel disease (IBD), aberrant activation of innate and adaptive immune responses enhances mucosal permeability through mechanisms not completely understood. To examine the role of epithelial nuclear factor (NF-kappaB) in IBD-induced enhanced permeability, epithelial-specific IkappaBalpha mutant (NF-kappaB super repressor) transgenic (TG) mice were generated. NF-kB activation was inhibited in TG mice, relative to wild-type mice, following T cell-mediated immune cell activation using an anti-CD3 monoclonal antibody. Furthermore, epithelial NF-kappaB super repressor protein inhibited diarrhea and blocked changes in transepithelial resistance and transmucosal flux of alexa350 (0.35 kDa) and dextran3000 (3 kDa). In vivo perfusion loop studies in TG mice revealed reversed net water secretion and reduced lumenal flux of different molecular probes (bovine serum albumin, alexa350, and dextran3000). Cell-imaging and immunoblotting of low-density, detergent-insoluble membrane fractions confirmed that tight junction proteins (occludin, claudin-1 and zona occludens-1) are internalized through an NF-kappaB-dependent pathway. Taken together, these data suggest that IBD-associated diarrhea results from NF-kappaB-mediated tight junction protein internalization and increased paracellular permeability. Thus, reduction of epithelial NF-kappaB activation in IBD may repair defects in epithelial barrier function, reduce diarrhea, and limit protein (eg, serum albumin) losses. Epithelial NF-kappaB activation induced by mucosal T cells, therefore, actively plays a role in opening paracellular spaces to promote transmucosal fluid effux into the intestinal lumen.

Response to Field

We read with interest the commentary (1) accompanying our article (2) in the October issue of the JCI. We appreciate Michael Field’s opinion that based on our data, the concept of secretory diarrhea must be revised to include the idea that net water secretion, or diarrhea, can occur in the absence of active ion secretion. However, we disagree with his conclusion that TNF must affect capillary permeability or smooth muscle contractility to generate increased interstitial pressure and a hydraulic driving force for water secretion. In contrast to Field’s suggestion, our study of 2 TNF superfamily members, TNF and LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry mediator on T cells), demonstrates that water secretion occurs as a consequence of epithelial Na+ malabsorption and increased epithelial paracellular permeability. This is supported by the observation that although TNF and LIGHT cause quantitatively similar paracellular permeability increases, only TNF causes diarrhea. The key difference that accounts for this is that only TNF activates PKCα, thereby inhibiting Na+/H+ exchanger isoform 3 (NHE3); TNF did not induce diarrhea when PKCα-mediated NHE3 inhibition was prevented by either pharmacological inhibitors or PKCα knockout. Could these approaches to PKCα inhibition have also prevented PKCα-dependent changes in capillary permeability or smooth muscle contractility? To test this we took advantage of the observation that LIGHT does not activate PKCα. LIGHT did cause diarrhea when PKC was activated pharmacologically, confirming that LIGHT was fully able to recapitulate TNF-like diarrhea when coupled with PKC activation. More importantly, direct NHE3 inhibition, either pharmacological or genetic, showed that this was the critical second component necessary for LIGHT to cause diarrhea. This was not due to inhibition of endothelial or smooth muscle NHE3, since neither tissue expresses NHE3. The unmistakable conclusion is that the necessary role of PKCα in TNF-induced diarrhea is to inhibit epithelial NHE3 and not to effect changes in capillary permeability or smooth muscle contractility. Why, then, is NHE3 so critical? Is it simply that Na+ absorption provides an osmotic driving force for water absorption? This alone is an inadequate explanation, as inhibition of absorption is not necessarily equivalent to induction of secretion. We propose that loss of NHE3-mediated Na+ absorption results in dissipation of the normal hyperosmolarity of the upper villus that drives water absorption (3). It then follows that increased paracellular permeability allows Na+ to leak from tissue to lumen; without NHE3, Na+ cannot be reabsorbed. Increased epithelial paracellular permeability may also allow water and other solutes to flow into the lumen, thereby compounding loss of the villous osmotic gradient that drives water absorption. In terms of absorption, the model gains further support from the observation that LIGHT-induced increases in permeability enhance water absorption (when NHE3 is active). Thus, this model fully explains the data without the need to invoke increased interstitial pressure to force water through paracellular or transcellular water channels. Is it therefore impossible for elevated interstitial pressure, secondary to increased capillary permeability or smooth muscle contraction, to contribute to TNF-induced diarrhea? It is not. However, if elevated venous or interstitial pressure were able to cause diarrhea by the mechanism proposed by Yablonski and Lifson (4) that is cited by Field (1), it is surprising that diarrhea is not a common clinical characteristic of patients with portal hypertension and that jejunal water and electrolyte transport are normal in these patients (5). We therefore conclude that TNF causes acute diarrhea via myosin light chain kinase–dependent increases in epithelial paracellular permeability coupled with PKCα-dependent NHE3 inhibition. Although intriguing as a hypothesis, no data support the role of increased interstitial pressure physically squeezing water into the lumen as proposed by Field. However, existing models of epithelial transport do provide an ample explanation for the observed synergy between increased epithelial paracellular permeability and Na+ malabsorption.