Cristiano Rumio

Unique role of junctional adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease.

BACKGROUND & AIMS Junctional adhesion molecule-A (JAM-A) is localized at the tight junctions and controls leukocyte migration into the tissues. However, its functional role in inflammatory bowel disease (IBD) is unexplored. METHODS Control, Crohns disease (CD), and ulcerative colitis (UC) tissue specimens were studied for JAM-A expression, as well as the colon of mice given dextran sodium sulfate (DSS). Wild-type and JAM-A(-/-), Tie-2-Cre-JAM-A(-/-) (endothelial/hematopoietic-specific JAM inactivation) mice were studied for susceptibility to DSS. Disease activity and colonic inflammation were assessed using a disease activity index histology and endoscopy, and mucosal cytokines were measured by enzyme-linked immunosorbent assay. JAM-A function was investigated by RNA silencing in epithelial cells, and apoptosis was measured. RESULTS In both CD and UC, as well as in experimental colitis, there is a loss of epithelial but not endothelial JAM-A expression. Deletion of JAM-A results in a dramatic increase in susceptibility to DSS colitis, as assessed by weight loss, disease activity index, histologic and endoscopic severity, and strikingly high mortality rates. This is not caused by the absence of JAM-A in the endothelial or hematopoietic compartments because Tie-2-Cre-JAM-A(-/-) mice are no more susceptible to DSS colitis than wild-type animals. JAM-A(-/-) mice displayed increased intestinal permeability and inflammatory cytokine production, and marked epithelial apoptosis. Silencing of JAM-A in intestinal epithelial cells resulted in increased permeability in vitro. CONCLUSIONS Our results show a nonredundant and novel role of JAM-A in controlling mucosal homeostasis by regulating the integrity and permeability of epithelial barrier function.

Degranulation of Paneth Cells via Toll-Like Receptor 9

The release of antimicrobial peptides and growth factors by Paneth cells is thought to play an important role in protecting the small intestine, but the mechanisms involved have remained obscure. Immunohistochemistry and immunofluorescence showed that Paneth cells express Toll-like receptor 9 (TLR9) in the granules. Injection of mice with oligonucleotides containing CpG sequence (CpG-ODNs) led to a down-modulation of TLR9 and a striking decrease in the number of large secretory granules, consistent with degranulation. Moreover CpG-ODN treatment increased resistance to oral challenge with virulent Salmonella typhimurium. Moreover, our findings demonstrate a sentinel role for Paneth cells through TLR9.

Toll-like Receptors 3, 4, and 7 Are Expressed in the Enteric Nervous System and Dorsal Root Ganglia

The aim of the present study was to evaluate the expression of innate immunity receptors belonging to the Toll-like family in the neural plexuses of the different tracts of murine intestine, of the human ileum, and in lower dorsal root ganglia (DRGs) from where extrinsic afferents to these plexuses originate. Results obtained by immunohistochemistry and immunofluorescence on paraffin-embedded tissue and whole-mount preparations show that Toll-like receptors (TLRs) −3 and −7, recognizing viral RNA, and TLR4, recognizing lipopolysaccharide (membrane component of Gram-negative bacteria), are expressed in the myenteric and submucous plexuses of murine intestine and human ileum, and in DRGs primary sensory neurons. They also show that TLR4 immunostaining is stronger in murine distal large bowel. In murine tissue, expression of TLRs was present in both neurons and glial cells. These observations indicate that the enteric neural network might be directly activated by bacterial and viral components and is therefore more in the forefront than previously envisaged in defense responses of the intestinal wall and in the cross-talk with intestinal microbiota. They also highlight the presence of a peripheral neural network that by way of hardwired neurotransmission could potentially convey to the central nervous system specific information on our microbial counterpart and invading or potentially invading pathogens.

Low Molecular Weight Hyaluronic Acid Increases the Self-Defense of Skin Epithelium by Induction of β-Defensin 2 via TLR2 and TLR4

In sites of inflammation or tissue injury, hyaluronic acid (HA), ubiquitous in the extracellular matrix, is broken down into low m.w. HA (LMW-HA) fragments that have been reported to activate immunocompetent cells. We found that LMW-HA induces activation of keratinocytes, which respond by producing β-defensin 2. This production is mediated by TLR2 and TLR4 activation and involves a c-Fos-mediated, protein kinase C-dependent signaling pathway. LMW-HA-induced activation of keratinocytes seems not to be accompanied by an inflammatory response, because no production of IL-8, TNF-α, IL-1β, or IL-6 was observed. Ex vivo and in vivo treatments of murine skin with LMW-HA showed a release of mouse β-defensin 2 in all layers of the epidermal compartment. Therefore, the breakdown of extracellular matrix components, for example after injury, stimulates keratinocytes to release β-defensin 2, which protects cutaneous tissue at a time when it is particularly vulnerable to infection. In addition, our observation might be important to open new perspectives in the development of possible topical products containing LMW-HA to improve the release of β-defensins by keratinocytes, thus ameliorating the self-defense of the skin for the protection of cutaneous tissue from infection by microorganisms.

Macrophage-Derived SPARC Bridges Tumor Cell-Extracellular Matrix Interactions toward Metastasis

Other than genetic imprinting and epithelial to mesenchymal transition, cancer cells need interaction with the nearby stroma toward metastasis. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein known to regulate extracellular matrix (ECM) deposition and cell-ECM interaction. Gene expression profiles associate SPARC to malignant progression. Using reciprocal bone marrow chimeras between SPARC knockout and wild-type mice, we show that SPARC produced by inflammatory cells is necessary for spontaneous, but not experimental, i.v. metastasis. Macrophage-derived SPARC induces cancer cell migration and enhances their migration to other ECM proteins at least through alpha(v)beta(5) integrin. Indeed, RNA interference knockdown of beta(5) integrin expression reduces cell migration in vitro and metastasis in vivo. Together these results show that macrophage-derived SPARC takes part in metastasis, acting at the step of integrin-mediated migration of invasive cells.

Activation of Enteroendocrine Cells via TLRs Induces Hormone, Chemokine, and Defensin Secretion

Enteroendocrine cells are known primarily for their production of hormones that affect digestion, but they might also be implicated in sensing and neutralizing or expelling pathogens. We evaluate the expression of TLRs and the response to specific agonists in terms of cytokines, defensins, and hormones in enteroendocrine cells. The mouse enteroendocrine cell line STC-1 and C57BL/6 mice are used for in vitro and in vivo studies, respectively. The presence of TLR4, 5, and 9 is investigated by RT-PCR, Western blot, and immunofluorescence analyses. Activation of these receptors is studied evaluating keratinocyte-derived chemokine, defensins, and cholecystokinin production in response to their specific agonists. In this study, we show that the intestinal enteroendocrine cell line STC-1 expresses TLR4, 5, and 9 and releases cholecystokinin upon stimulation with the respective receptor agonists LPS, flagellin, and CpG-containing oligodeoxynucleotides. Release of keratinocyte-derived chemokine and β-defensin 2 was also observed after stimulation of STC-1 cells with the three TLR agonists, but not with fatty acids. Consistent with these in vitro data, mice showed increased serum cholecystokinin levels after oral challenge with LPS, flagellin, or CpG oligodeoxynucleotides. In addition to their response to food stimuli, enteroendocrine cells sense the presence of bacterial Ags through TLRs and are involved in neutralizing intestinal bacteria by releasing chemokines and defensins, and maybe in removing them by releasing hormones such as cholecystokinin, which induces contraction of the muscular tunica, favoring the emptying of the distal small intestine.

Critical Role of TLR9 in Acute Graft-versus-Host Disease

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9−/−) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4−/−) mice. The reduced mortality and morbidity in TLR9−/− mice related with reduced stimulatory activity of TLR9−/− spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9+/+ into TLR9−/− and TLR9−/− into TLR9+/+ chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9+/+ cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4.

The cell types of the gut expressing Toll‐like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll‐like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor‐kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin‐8 (IL‐8). We can conclude that Toll‐like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll‐like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll‐like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen. J. Cell. Physiol.

Proximity of TPR and NTRK1 rearranging loci in human thyrocytes

Chromosomal rearrangements are frequently associated with cancer; the mechanisms underlying their cell-type specificity are poorly understood. Papillary thyroid carcinomas are marked by a high frequency of chromosome rearrangements involving the RET and NTRK1 tyrosine kinase receptor genes and producing RET and TRK oncogenes. An explanation for the propensity of thyrocytes to undergo gene rearrangements has been recently proposed by Nikiforova and colleagues, who showed that the recombination between RET and H4 is favored by the loci proximity in interphase nuclei. We investigated whether the spatial proximity is a contributing factor also in the generation of the thyroid-specific TRK oncogenes. The distance between NTRK1 and its oncogenic partner TPR was determined by two-color fluorescence in situ hybridization and two-dimensional microscopy. A three-dimensional reconstruction of the data was also done. We show that the two loci in thyrocytes nuclei display a distance reduced with respect to peripheral blood lymphocytes, thus supporting the notion that spatial proximity of translocation-prone gene loci may favor gene rearrangements.

Therapeutic Synergism of Gemcitabine and CpG-Oligodeoxynucleotides in an Orthotopic Human Pancreatic Carcinoma Xenograft

CpG-oligodeoxynucleotides (CpG-ODN) exhibit potent immunostimulatory activity by binding with Toll-like receptor 9 (TLR9). Based on the finding that TLR9 is highly expressed and functional in pancreatic tissue, we evaluated the antitumor effects of chemotherapy combined with CpG-ODNs in the orthotopic mouse model of a human pancreatic tumor xenograft. Chemotherapy consisted of the maximum tolerated dose of gemcitabine (i.v., 100 mg/kg, q3dx4). CpG-ODNs were delivered (i.p., 20 microg/mouse), weekly, after the end of chemotherapy. CpG-ODNs alone had little effect on tumor growth, whereas gemcitabine alone significantly delayed the median time of disease onset (palpable i.p. tumor) and of bulky disease development (extensive peritoneal tumor burden), but did not enhance survival time. When the gemcitabine regimen was followed by administration of the immunostimulator, development of bulky disease was delayed, survival time was significantly improved (median survival time, 106 days; P < 0.02 versus gemcitabine-treated mice). Autoptic examination showed that tumor spread in the peritoneal cavity was reduced to a greater extent than with gemcitabine alone. All treatment regimens were well-tolerated. The use of nude mice excluded a T cell-mediated immune response, whereas the high pancreatic expression of TLR9 might have contributed to the tumor response. The clear improvement of survival observed in an orthotopic murine model of human pancreatic cancer by the combined use of CpG-ODNs with chemotherapy suggests the promise of this therapeutic regimen in the clinical setting.