Article

The ERM protein, Ezrin, regulates neutrophil transmigration by modulating the apical localization of MRP2 in response to the SipA effector protein during Salmonella Typhimurium infection

Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Cellular Microbiology (Impact Factor: 4.82). 09/2011; 13(12):2007-21. DOI: 10.1111/j.1462-5822.2011.01693.x
Source: PubMed

ABSTRACT In human disease induced by Salmonella enterica serovar Typhimurium (S. Typhimurium), transepithelial migration of neutrophils rapidly follows attachment of the bacteria to the epithelial apical membrane. We have previously shown that during S. Typhimurium infection the multidrug resistance-associated protein 2 (MRP2) is highly expressed at the apical surface of the intestinal epithelia, and that it functions as an efflux pump for the potent neutrophil chemoattractant hepoxilin A(3) . However, the molecular mechanisms regulating its apical localization during active states of inflammation remain unknown. Thus, our objective was to determine the mechanistic basis for the translocation of MRP2 to the apical surface of intestinal epithelial cells during S. Typhimurium infection. We show that suppression of ezrin, through either RNAi or truncation of the C-terminus, results not only in a decrease in S. Typhimurium-induced neutrophil transmigration but also significantly attenuates the apical membrane expression of MRP2 during Salmonella infection. In addition, we determined that S. Typhimurium induces the activation of ezrin via a PKC-α-dependent pathway and that ezrin activation is coupled to apical localization of MRP2. Based on these results we propose that activation of ezrin is required for the apical localization of MRP2 during S. Typhimurium infection.

0 Followers
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The human intestinal epithelium consists of a single layer of epithelial cells that forms a barrier against food antigens and the resident microbiota within the lumen. This delicately balanced organ functions in a highly sophisticated manner to uphold the fidelity of the intestinal epithelium and to eliminate pathogenic microorganisms. On the luminal side, this barrier is fortified by a thick mucus layer, and on the serosal side exists the lamina propria containing a resident population of immune cells. Pathogens that are able to breach this barrier disrupt the healthy epithelial lining by interfering with the regulatory mechanisms that govern the normal balance of intestinal architecture and function. This disruption results in a coordinated innate immune response deployed to eliminate the intruder that includes the release of antimicrobial peptides, activation of pattern-recognition receptors, and recruitment of a variety of immune cells. In the case of Salmonella enterica serovar typhimurium (S. typhimurium) infection, induction of an inflammatory response has been linked to its virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host's cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. As the role of the intestinal epithelium in initiating an immune response has been increasingly realized, this review will highlight recent research that details progress made in understanding mechanisms underlying the mucosal inflammatory response to Salmonella infection, and how such inflammatory responses impact pathogenic fitness of this organism.
    Frontiers in Immunology 07/2014; 5:311. DOI:10.3389/fimmu.2014.00311
  • [Show abstract] [Hide abstract]
    ABSTRACT: Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12-myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction-mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. After preincubation of T84 cells with short-chain β2→1-fructans, the decrease in TEER as induced by PMA (62.3 ± 5.2%, P < 0.001) was strongly attenuated (15.2 ± 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short-chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), confirming that β2→1-fructans are specific ligands for TLR2. To conclude, β2→1-fructans exert time-dependent and chain length-dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan-mediated health effects.
    Journal of Nutrition 04/2014; 144(7). DOI:10.3945/jn.114.191643 · 4.23 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNAs (miRNAs) are a class of naturally occurring noncoding RNAs of 17–30 nucleotides, which are known to regulate gene expression by inducing translational inhibition and cleavage of their target mRNAs [10]. miRNAs have been shown to elicit several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control, and other cellular functions [11–13]. Recent investigations have linked aberrant miRNA expression with acute myeloid leukemia [14–16]. A recent study evaluated miRNA expression in 122 newly diagnosed adult acute myeloid leukemia (AML) cases using a microarray platform [17]. This study identified a subset of miRNAs that were significantly downregulated in AML patients with respect to CD34+ cells obtained from 10 healthy donors. Interestingly, via a miRNA prediction algorithm (TargetScan), the authors identified PODXL as a highly conserved target of miR-199b, a significantly downregulated miRNA in AML (Fig. 1C). Experimental validation via transfecting hsa-miR-199b-5p mimics revealed that PODXL is indeed a bona fide target of miR-199b-5p (Fig. 2A). Further analyses of miR-199b-5p's predicted targets revealed that Discoidin domain receptor 1 (DDR1) is also a highly conserved target of miR-199b-5p (Fig. 2B). DDR1, is a class of collagen receptor, expressed on human leukocytes, including neutrophils, monocytes, lymphocytes, and podocytes [18]. This subfamily is distinct from other members of the large receptor tyrosine kinase group due to a homology domain to discoidin, a lectin first described during the cell aggregation process of the slime mold Dictyostelium discoideum [18]. DDR1′s autophosphorylation is achieved by all collagens tested (type I to type VI). Previous in situ hybridization studies and Northern blot analysis have shown that the expression of DDR1 is restricted to epithelial cells, particularly in the kidney, lung, gastrointestinal tract, and brain [18]. Importantly, similar to PODXL, DDR1 is also significantly overexpressed in several human tumors of breast, ovarian, esophageal, and brain origin [18]. Though considerable upregulation of DDR1 gene expression in cytokine-activated neutrophils has been reported [18], its elevated expression is not reported in any subset (or form) of leukemia including AML. Significantly, our IHC analyses in AML samples via a similar TMA approach revealed that DDR1 levels were upregulated in the same AML cases where PODXL levels were increased (Fig. 2C and D). Previously, overexpression of DDR1 in lung cancer cells resulted in a significant increase of cell motility and invasiveness, where DDR1–collagen interactions facilitated the invasiveness and importantly, was associated with poor prognosis of non-small cell lung cancer [19]. Further, in hepatocellular carcinoma cells and glioma cells (glioblastoma multiforme), overexpression of DDR1 promoted invasion and migration of tumor cells [20, 21]. Taken together, the presently observed increase of DDR1 expression on leukemic blasts suggests that upregulation of DDR1 may contribute toward migration and invasion of leukemic blasts to extramedullary sites. Interestingly, further comparative analyses of the IHC results revealed that PODXL and DDR1 were co-expressed at high levels in n = 9 AML patients (Fig. 3A). Interestingly, 55% of these cases were patients with persistent AML. Significantly, both PODXL and DDR1 have highly overlapping critical functional roles in maintaining slit diaphragms in the kidney. PODXL knockout mice fail to form foot processes and slit diaphragms and exhibit post-natal lethality with anuria, omphalocele, and perinatal lethality [3, 22]. Interestingly, DDR1-deficient podocytes also exhibits loss of cell–matrix communication that results in disturbed anchorage of foot processes and disruption of the slit diaphragm [23]. Further experimental investigations via transfection of miR-199b-5p mimics revealed that DDR1 was also a specific target of miR-199b-5p (Fig. 3B). To conclusively prove that miR-199b-5p targets PODXL and DDR1, the authors generated respective 3′UTR–luciferase constructs (both wild type and mutants that block miR-199b-5p binding) and co-transfected with miR-199b-5p mimics. The results clearly revealed that miR-199b-5p specifically targets PODXL and DDR1 via its predicted binding sites in respective 3′UTRs (Fig. 3C). Our results demonstrates for the first time that miR-199b-5p regulates two proteins with analogous biological functions and strongly implies that decreased miR-199b-5p expression in AML may lead to concurrent upregulation of PODXL and DDR1.
    American Journal of Hematology 04/2012; 87(4):442-6. DOI:10.1002/ajh.23129 · 3.48 Impact Factor

Preview