Liping Su

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 Activation Induces Mucosal Interleukin-13 Expression to Alter Tight Junction Ion Selectivity

Intestinal barrier function is reduced in inflammatory bowel disease (IBD). Tumor necrosis factor (TNF) and interleukin (IL)-13, which are up-regulated in IBD, induce barrier defects that are associated with myosin light chain kinase (MLCK) activation and increased claudin-2 expression, respectively, in cultured intestinal epithelial monolayers. Here we report that these independent signaling pathways have distinct effects on tight junction barrier properties and interact in vivo. MLCK activation alters size selectivity to enhance paracellular flux of uncharged macromolecules without affecting charge selectivity and can be rapidly reversed by MLCK inhibition. In contrast, IL-13-dependent claudin-2 expression increases paracellular cation flux in vitro and in vivo without altering tight junction size selectivity but is unaffected by MLCK inhibition in vitro. In vivo, MLCK activation increases paracellular flux of uncharged macromolecules and also triggers IL-13 expression, claudin-2 synthesis, and increased paracellular cation flux. We conclude that reversible, MLCK-dependent permeability increases cause mucosal immune activation that, in turn, feeds back on the tight junction to establish long-lasting barrier defects. Interactions between these otherwise distinct tight junction regulatory pathways may contribute to IBD pathogenesis.

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.

Mechanisms and Functional Implications of Intestinal Barrier Defects

Intestinal epithelial barrier defects, or increased paracellular permeability, were first reported in patients with Crohn’s disease (CD) over 25 years ago. Although increased permeability may herald relapse to active disease, suggesting that impaired barrier function may contribute to progression, limited understanding of the mechanisms that create barrier defects in CD has made it impossible to determine whether increased permeability is a cause or effect of disease. It is now clear that inflammatory cytokines trigger intestinal barrier defects acutely, by cytoskeletal contraction, or chronically, via modulation of tight junction protein expression. Both mechanisms cause barrier dysfunction, but their effects on paracellular size and charge selectivity differ. The clinical ramifications of this distinction are not yet clear. Recent data using in vivo models demonstrate that cytoskeletally mediated barrier dysfunction is sufficient to activate innate and adaptive components of mucosal immunity. Consistent with the presence of increased permeability in some healthy first-degree relatives of CD patients, these barrier defects are insufficient to cause disease in the absence of other stimuli. However, cytoskeletally mediated barrier defects are sufficient to accelerate onset and increase severity of experimental inflammatory bowel disease. Thus, inflammatory cytokines can cause barrier defects and, conversely, barrier defects can activate the mucosal immune system. This raises the possibility that restoration of barrier function may be therapeutic in CD. Consistent with this hypothesis, emerging data indicate that inhibition of cytoskeletally mediated barrier dysfunction may be able to prevent disease progression. Barrier restoration may, therefore, represent a non-immunosuppressive approach to achieving or maintaining disease remission.

MAPKAPK-2 is a critical signaling intermediate in NHE3 activation following Na+-glucose cotransport

Villus enterocyte nutrient absorption occurs via precisely orchestrated interactions among multiple transporters. For example, transport by the apical Na+-glucose cotransporter, SGLT1, triggers translocation of NHE3, Na+-H+ antiporter isoform 3, to the plasma membrane. This translocation requires activation of p38 mitogen-activated protein kinase (MAPK), Akt2, and ezrin. Akt2 directly phosphorylates ezrin, but the precise role of p38 MAPK in this process remains to be defined. Sequence analysis suggested that p38 MAPK could not directly phosphorylate Akt2. We hypothesized that MAPKAPK-2 might link p38 MAPK and Akt2 activation. MAPKAPK-2 was phosphorylated after initiation of Na+-glucose cotransport with kinetics that paralleled activation of p38 MAPK, Akt2, and ezrin. MAPKAPK-2, Akt2, and ezrin phosphorylation were all attenuated by p38 MAPK inhibition but were unaffected by dominant negative ezrin expression. Akt2 inhibition blocked ezrin but not p38 MAPK or MAPKAPK-2 phosphorylation, suggesting that MAPKAPK-2 could be an intermediate in p38 MAPK-dependent Akt2 activation. Consistent with this, MAP-KAPK-2 could phosphorylate an Akt2-derived peptide in vitro. siRNA-mediated MAPKAPK-2 knockdown inhibited phosphorylation of Akt2 and ezrin but not p38 MAPK. MAPKAPK-2 knockdown also blocked NHE3 translocation. Thus, MAP-KAPK-2 controls Akt2 phosphorylation. In so doing, MAP-KAPK-2 links p38 MAPK to Akt2, ezrin, and NHE3 activation after SGLT1-mediated transport.