Hans-Christian Reinecker Laboratory

Lab Members:

Seiji Arihiro
Atsuki Fukazawa
Bayasi Guleng
Sonal Gupta

Our Address:

Massachusetts General Hospital
Jackson 711
55 Fruit Street
Boston, MA 02114

Phone: (617) 724-2172
E-Mail: hreinecker@partners.org
hans-christian_reinecker@hms.harvard.edu

Our Mission:

Our laboratory is interested in the discovery of genes and the molecular mechanisms of their function in the regulation of the intestinal epithelial cell barrier function, intestinal microbial recognition and mucosal host defense mechanisms.

The intestinal mucosa functions as an immunological organ, which plays a major role in the development of oral tolerance and host-defense mechanisms. Antigens must cross the intestinal epithelium in a controlled manner to interact with dendritic antigen-presenting cells, since bacteria or their products are a primary risk factor for the development of intestinal inflammation. Therefore, the regulation of the intestinal epithelial cell barrier is central to the development of intestinal immunity and inflammation, but the involved mechanisms are largely unknown. Disturbance in the recognition of pathogens and the alteration of the intestinal immune responses may contribute to intestinal cancer and Inflammatory Bowel Disease.

We study the intestinal specification of the epithelial barrier by characterizing the nuclear factors involved in regulation of claudin gene expression in intestinal epithelial cells and by studying the roles of distinct claudins in the intestinal segment specific regulation of barrier function and ion transport in homeostasis and in inflammation. A related interest is in the characterization of the role of commensal bacteria and pathogens in the regulation of intestinal epithelial cell development and barrier function.

A second major area of interest of the laboratory is in elucidating the mechanisms by which intestinal epithelial cell are integrated into the key mechanisms of the innate an adaptive immunity. We are studying chemokine and cytokine receptor signaling to understand how the intestinal epithelium is integrated into host defense mechanisms. In these studies, we currently aim to elucidate the mechanisms of membrane and vesicle compartmentalization upon cytokine receptor activation. We are using genetic approaches in combination with intestinal gene expression profiling with macro- and micro-arrays to identify novel epithelial cell derived factors and signaling pathways required for the recruitment and functional differentiation of immune competent cells in the intestinal mucosa.

Current Projects

Cloning of the human claudin-2 5'-flanking region revealed a TATA-less promoter with conserved binding sites in mouse and human for caudal-related homeodomain proteins and hepatocyte nuclear factor-1alpha.

Claudin-2 is a structural component of tight junctions in the kidneys, liver, and intestine, but the mechanisms regulating its expression have not been defined. The 5'-flanking region of the claudin-2 gene contains binding sites for intestine-specific Cdx homeodomain proteins and hepatocyte nuclear factor (HNF)-1, which are conserved in human and mouse. Both Cdx1 and Cdx2 activated the claudin-2 promoter in the human intestinal epithelial cell line Caco-2. HNF-1alpha augmented the Cdx2-induced but not Cdx1-induced transcriptional activation of the human claudin-2 promoter. In mice, HNF-1alpha was required for claudin-2 expression in the villus epithelium of the ileum and within the liver but not in the kidneys, indicating an organ-specific function of HNF-1alpha in the regulation of claudin-2 gene expression. Tight junction structural components, which determine epithelial polarization and intestinal barrier function, can be regulated by homeodomain proteins that control the differentiation of the intestinal epithelium. J Biol Chem 2002 Jun 14;277(24):21361-70

Interleukin-2 receptor beta subunit-dependent and -independent regulation of intestinal epithelial tight junctions.

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rbeta subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rbeta subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rbeta subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rbeta subunit. Recruitment of claudins and hyperphoshorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rbeta-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex. J Biol Chem 2001 Sep 21;276(38):35571-80

Claudins regulate the intestinal barrier in response to immune mediators.

BACKGROUND & AIMS: To determine the functional role of immune mediators in the formation of the intestinal barrier, we have examined the regulation of claudin expression by interleukin (IL)-17 in human intestinal epithelial cells. METHODS: Expression of claudins, extracellular signal-related (ERK) mitogen-activated protein kinases (MAPKs), and activated ERK MAPKs was determined by immunoblotting. Claudin membrane association was assessed by immunohistochemistry and claudin messenger RNA expression by Northern blot analysis. Intestinal epithelial barrier function was characterized through transepithelial electrical resistance and mannitol tracer flux. RESULTS: IL-17 induced the development of a paracellular barrier of T84 cell monolayers. Inhibition of ERK activation with the MEK inhibitor PD98059 blocked IL-17 as well as basal development of tight junctions in T84 cells. IL-17 induced formation of tight junctions correlated with up-regulation of claudin-1 and claudin-2 gene transcription. Inhibition of MEK reduced the activated and basal expression of claudin-2 messenger RNA and protein expression. Functional MEK was required for the expression and membrane association of claudin-2 but not claudin-1 in T84 cells. CONCLUSIONS: MEK activity is required for claudin-mediated formation of tight junctions. IL-17 is able to regulate the intestinal barrier through the ERK MAPK pathway. Gastroenterology 2000 Jun;118(6):1001-11

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells.

Shigella spp. are a group of Gram-negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype-specific mechanisms, which targets not only expression, but also cellular distri-bution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction-associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin-1 from Triton X-insoluble protein fractions upon apical exposure to T-84 cell monolayers. S. flexneri serotype 2a and 5, but not the non-invasive Escherichia coli strain F-18, share the ability to regulate expression of ZO-1, ZO-2, E-cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat-stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction-associated proteins. Cell Microbiol 2002 Jun;4(6):367-81

Fractalkine-mediated signals regulate cell-survival and immune-modulatory responses in intestinal epithelial cells.

BACKGROUND & AIMS: In this study, we determined the signal transduction and functional consequences after ligand-specific activation of the fractalkine receptor CX3CR1 in human intestinal epithelial cells. METHODS: CX3CR1 expression in human colonic tissues and intestinal epithelial cell lines was determined by immunohistochemistry, immunoblotting, and reverse-transcription polymerase chain reaction. The regulation of mitogen-activated protein kinase (MAPK) activation was assessed by immunoblotting. Regulation of chemokine messenger RNA (mRNA) expression was determined by Northern blotting. NF-kappa B and p53 activation was assessed by electromobility shift assays. RESULTS: Fractalkine mediated the MEK-1 and G alpha i-dependent but phosphatidylinositol-3-kinase-independent activation of extracellular signal-regulated kinase-MAPK. Fractalkine activated NF-kappa B and p53 resulting in interleukin 8 and fractalkine mRNA expression. CX3CR1-mediated activation of intestinal epithelial cells was able to induce migration of human neutrophils into but not through the intestinal epithelial cell monolayer. CONCLUSIONS: CX3CR1 mediates distinct functional responses in intestinal epithelial cells, which include the autocrine regulation of cell-survival signals and activation of immune modulators, indicating a role of CX3CR1 in host defense mechanisms originating from the intestinal epithelium. Gastroenterology 2002 Jan;122(1):166-77

NF-kappa B-inducing kinase is a common mediator of IL-17-, TNF-alpha-, and IL-1 beta-induced chemokine promoter activation in intestinal epithelial cells.

IL-17 expression is restricted to activated T cells, whereas the IL-17R is expressed in a variety of cell types including intestinal epithelial cells. However, the functional responses of intestinal epithelial cells to stimulation with IL-17 are unknown. Moreover, the signal transduction pathways activated by the IL-17R have not been characterized. IL-17 induced NF-kappa B protein-DNA complexes consisting of p65/p50 heterodimers in the rat intestinal epithelial cell line IEC-6. The induction of NF-kappa B correlated with the induction of CXC and CC chemokine mRNA expression in IEC-6 cells. IL-17 acted in a synergistic fashion with IL-1 beta to induce the NF-kappa B site-dependent CINC promoter. Induction of the CINC promoter by IL-17 in IEC-6 cells was TNF receptor-associated factor-6 (TRAF6), but not TRAF2, dependent. Furthermore, IL-17 induction of the CINC promoter could be inhibited by kinase-negative mutants of NF-kappa B-inducing kinase and I kappa B kinase-alpha. In addition to activation of the NF-kappa B, IL-17 regulated the activities of extracellular regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases in IEC-6 cells. Whereas the IL-17-mediated activation of extracellular regulated kinase mitogen-activated protein kinases was mediated through ras, c-Jun N-terminal kinase activation was dependent on functional TRAF6. These data suggest that NF-kappa B-inducing kinase serves as the common mediator in the NF-kappa B signaling cascades triggered by IL-17, TNF-alpha, and IL-1 beta in intestinal epithelial cells. J Immunol 1999 May 1;162(9):5337-44

Regulation of epithelial membrane compartmentalization and polarization

The membrane dynamics responsible for vesicular transport and protein sorting are fundamental to the structure and function of polarized intestinal epithelial cells and to their ability to maintain a selective barrier required for efficient solute transport and host defense. Many microbial agents co-opt the mechanics of membrane internalization to enter the host, or to breech the epithelial barrier. In other well described instances, Immunoglobulins IgG and sIgA bind to specific trafficking receptors to move across the epithelial barrier as intact and fully folded proteins. In doing so, IgG and sIgA act in the intestinal lumen to participate in host defense and immune surveillance at this mucosal surface. Almost all aspects of epithelial cell physiology and biology depend on the proper sorting of membrane proteins and the establishment and maintenance of intracellular membrane compartments exhibiting specific structure and function. These fundamental aspects of cell biology dictate the phenotype of the mucosal barrier in health and disease. Such mechanisms of membrane trafficking also account for antigen processing essential to the host immune response. Both membrane lipids and membrane proteins follow specific and regulated trafficking pathways.

Intestinal gene expression profiling

A new project aims to establish an intestinal gene expression profiling database from surveillance data from various model systems, intestinal diseases, and treatments trials in human and mice. Cross-referencing and compiling these into databases to obtain experiment numbers high enough for statistical analysis, will greatly aid the identification of intestinal candidate genes in inflammatory bowel disease and clarify unspecific inflammatory responses. Identification of additional factors is likely to permit development of better murine models for Inflammtory Bowel disease, which can be used to study mechanisms of pathogenesis and microbial regulation as well as to develop new therapeutic strategies.

Fractalkine is an epithelial and endothelial cell-derived chemoattractant for intraepithelial lymphocytes in the small intestinal mucosa.

Fractalkine is a unique chemokine that combines properties of both chemoattractants and adhesion molecules. Fractalkine mRNA expression has been observed in the intestine. However, the role of fractalkine in the healthy intestine and during inflammatory mucosal responses is not known. Studies were undertaken to determine the expression and function of fractalkine and the fractalkine receptor CX3CR1 in the human small intestinal mucosa. We identified intestinal epithelial cells as a novel source of fractalkine. The basal expression of fractalkine mRNA and protein in the intestinal epithelial cell line T-84 was under the control of the inflammatory mediator IL-1beta. Fractalkine was shed from intestinal epithelial cell surface upon stimulation with IL-1beta. Fractalkine localized with caveolin-1 in detergent-insoluble glycolipid-enriched membrane microdomains in T-84 cells. Cellular distribution of fractalkine was regulated during polarization of T-84 cells. A subpopulation of isolated human intestinal intraepithelial lymphocytes expressed the fractalkine receptor CX3CR1 and migrated specifically along fractalkine gradients after activation with IL-2. Immunohistochemistry demonstrated fractalkine expression in intestinal epithelial cells and endothelial cells in normal small intestine and in active Crohn's disease mucosa. Furthermore, fractalkine mRNA expression was significantly up-regulated in the intestine during active Crohn's disease. This study demonstrates that fractalkine-CX3CR1-mediated mechanism may direct lymphocyte chemoattraction and adhesion within the healthy and diseased human small intestinal mucosa. J Immunol 2000 Mar 15;164(6):3368-76

Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine.

The chemokine receptors CCR2 and CCR5 and their respective ligands regulate leukocyte chemotaxis and activation. To determine the role of these chemokine receptors in the regulation of the intestinal immune response, we induced colitis in CCR2- and CCR5-deficient mice by continuous oral administration of dextran sodium sulfate (DSS). Both CCR2- and CCR5-deficient mice were susceptible to DSS-induced intestinal inflammation. The lack of CCR2 or CCR5 did not reduce the DSS-induced migration of macrophages into the colonic lamina propria. However, both CCR5-deficient mice and, to a lesser degree, CCR2-deficient mice were protected from DSS-induced intestinal adhesions and mucosal ulcerations. CCR5-deficient mice were characterized by a greater relative infiltration of CD4+ and NK1.1+ lymphocyte in the colonic lamina propria when compared to wild-type and CCR2-deficient mice. In CCR5-deficient mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN-gamma was decreased, corresponding to a Th2 pattern of T cell activation. In CCR2-deficient mice, the infiltration of Th2-type T cells in the lamina propria was absent, but increased levels of IL-10 and decreased levels of IFN-gamma may have down regulated mucosal inflammation. Our data indicate that CCR5 may be critical for the promotion of intestinal Th1-type immune responses in mice. J Immunol 2000 Jun 15;164(12):6303-12

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