David Díaz-Jiménez

Characterization of the Novel ST2/IL-33 System in Patients with Inflammatory Bowel Disease

Background: ST2 has been proposed to be a regulator of inflammation and Th1/Th2 balance. ST2L is the IL‐33 membrane receptor and belongs to the IL‐1R family. The soluble variant, ST2s, is identical to the extracellular region of ST2L and competes for IL‐33 binding, inhibiting receptor signaling. Although ST2s has been associated with inflammatory processes in patients with sepsis, trauma, asthma, and autoimmunity, until now there are no reported studies showing the role of ST2/IL‐33 in inflammatory bowel disease (IBD). Methods: Expression of ST2 and IL‐33 was determined in serum and colonic biopsies from IBD patients. ST2 transcript and protein was determined by reverse‐transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA)/immunoblot, respectively, and IL‐33 protein by ELISA. Intestinal mucosa localization of ST2 and IL‐33 was conducted by immunofluorescence. Results: ST2s transcript in the colonic mucosa was mainly expressed in UC patients rather than Crohns disease or control; however, ST2L mRNA remained constant in all samples. Total ST2 protein was significantly higher in mucosa samples from patients with active UC, with a predominant induction of ST2s that strongly correlates with serum ST2 levels. Mucosa IL‐33 levels were higher in UC patients and serum levels were barely detected in all patient groups. ST2 and IL‐33 are both abundantly expressed in the cytoplasm of epithelial cells of control subjects; however, in ulcerative colitis patients ST2 decreases and IL‐33 showed cytoplasm‐nuclear redistribution. Conclusions: The novel association between the ST2/IL‐33 system and IBD seems to identify that variations in this axis might regulate the inflammatory process in these diseases. Inflamm Bowel Dis 2010

Soluble ST2: A new and promising activity marker in ulcerative colitis

AIM To correlate circulating soluble ST2 (sST2) levels with the severity of ulcerative colitis (UC) and serum levels of pro-inflammatory cytokines, and to demonstrate the predictive power of sST2 levels for differentiation between active and inactive UC. METHODS We recruited 153 patients: 82 with UC, 26 with Crohns disease (CD) and 43 disease controls [non-inflammatory bowel disease (IBD)]. Subjects were excluded if they had diagnosis of asthma, autoimmune diseases or hypertension. The serum levels of sST2 and pro-inflammatory cytokines [pg/mL; median (25th-75th)] as well as clinical features, endoscopic and histological features, were subjected to analyses. The sST2 performance for discrimination between active and inactive UC, non-IBD and healthy controls (HC) was determined with regard to sensitivity and specificity, and Spearmans rank correlation coefficient (r). To validate the method, the area under the curve (AUC) of receiver-operator characteristic (ROC) was determined (AUC, 95% CI) and the total ST2 content of the colonic mucosa in UC patients was correlated with circulating levels of sST2. RESULTS The serum sST2 value was significantly higher in patients with active [235.80 (90.65-367.90) pg/mL] rather than inactive UC [33.19 (20.04-65.32) pg/mL], based on clinical, endoscopic and histopathological characteristics, as well as compared with non-IBD and HC (P < 0.001). The median level of sST2 in CD patients was 54.17 (35.02-122.0) pg/mL, significantly higher than that of the HC group only (P < 0.01). The cutoff was set at 74.87 pg/mL to compare active with inactive UC in a multicenter cohort of patients. Values of sensitivity, specificity, and ability to correctly classify UC, according to activity, were 83.33%, 83.33% and 83.33%, respectively. The AUC of the ROC curve to assess the ability of this molecule to discriminate between active vs inactive UC was 0.92 (0.86-0.97, P < 0.0001). The serum levels of sST2 in patients with UC significantly correlated with endoscopic and histopathological scores (r = 0.76 and r = 0.67, P < 0.0001, respectively), and with the pro-inflammatory cytokine, tumor necrosis factor-α (r = 0.69 and r = 0.61, respectively, P < 0.0001). Interestingly, we found a direct correlation between total intestinal ST2 content and serum levels of sST2, adjusted to endoscopic activity score in patients with mild (r = 0.44, P = 0.004), moderate (r = 0.59, P = 0.002) and severe disease (r = 0.82, P = 0.002). Only patients with inactive UC showed no significant correlation (r = 0.45, P = 0.267). CONCLUSION sST2 levels correlated with disease severity and inflammatory cytokines, are able to differentiate active from inactive UC and might have a role as a biomarker.

Escherichia coli isolates from inflammatory bowel diseases patients survive in macrophages and activate NLRP3 inflammasome.

Crohns disease (CD) is a multifactorial pathology associated with the presence of adherent-invasive Escherichia coli (AIEC) and NLRP3 polymorphic variants. The presence of intracellular E. coli in other intestinal pathologies (OIP) and the role of NLRP3-inflammasome in the immune response activated by these bacteria have not been investigated. In this study, we sought to characterize intracellular strains isolated from patients with CD, ulcerative colitis (UC) and OIP, and analyze NLRP3-inflammasome role in the immune response and bactericidal activity induced in macrophages exposed to invasive bacteria. For this, intracellular E. coli isolation from ileal biopsies, using gentamicin-protection assay, revealed a prevalence and CFU/biopsy of E. coli higher in biopsies from CD, UC and OIP patients than in controls. To characterize bacterial isolates, pulsed-field gel electrophoresis (PFGE) patterns, virulence genes, serogroup and phylogenetic group were analyzed. We found out that bacteria isolated from a given patient were closely related and shared virulence factors; however, strains from different patients were genetically heterogeneous. AIEC characteristics in isolated strains, such as invasive and replicative properties, were assessed in epithelial cells and macrophages, respectively. Some strains from CD and UC demonstrated AIEC properties, but not strains from OIP. Furthermore, the role of NLRP3 in pro-inflammatory cytokines production and bacterial elimination was determined in macrophages. E. coli strains induced IL-1β through NLRP3-dependent mechanism; however, their elimination by macrophages was independent of NLRP3. Invasiveness of intracellular E. coli strains into the intestinal mucosa and IL-1β production may contribute to CD and UC pathogenesis.

Increased production of soluble TLR2 by lamina propria mononuclear cells from ulcerative colitis patients.

Toll-like receptor 2 (TLR2) is a type I pattern recognition receptor that has been shown to participate in intestinal homeostasis. Its increased expression in the lamina propria has been associated with the pathogenesis in inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohns disease (CD). Recently, soluble TLR2 (sTLR2) variants have been shown to counteract inflammatory responses driven by the cognate receptor. Despite the evident roles of TLR2 in intestinal immunity, no study has elucidated the production and cellular source of sTLR2 in IBD. Furthermore, an increase in the population of activated macrophages expressing TLR2 that infiltrates the intestine in IBD has been reported. We aimed first to assess the production of the sTLR2 by UC and CD organ culture biopsies and lamina propria mononuclear cells (LPMCs) as well as the levels of sTLR2 in serum, and then characterize the cell population from lamina propria producing the soluble protein. Mucosa explants, LPMCs and serum were obtained from UC, CD patients and control subjects. The level of sTLR2 was higher in conditioned media from organ culture biopsies and LPMCs from UC patients in comparison to CD and controls. Moreover, an inverse correlation between the content of intestinal and serum sTLR2 levels was observed in UC patients. Additionally, when characterizing the cellular source of the increased sTLR2 by LPMCs from UC patients, an increase in TLR2(+)/CD33(+) cell population was found. Also, these cells expressed CX3CR1, which was related to the increased levels of intestinal FKN in UC patients, suggesting that a higher proportion of TLR2(+) mononuclear cells infiltrate the lamina propria. The increased production of sTLR2 suggests that a differential regulating factor of the innate immune system is present in the intestinal mucosa of UC patients.

Metalloproteinase-Dependent TLR2 Ectodomain Shedding is Involved in Soluble Toll-Like Receptor 2 (sTLR2) Production

Toll-like receptor (TLR) 2, a type I membrane receptor that plays a key role in innate immunity, recognizes conserved molecules in pathogens, and triggering an inflammatory response. It has been associated with inflammatory and autoimmune diseases. Soluble TLR2 (sTLR2) variants have been identified in human body fluids, and the TLR2 ectodomain can negatively regulate TLR2 activation by behaving as a decoy receptor. sTLR2 generation does not involve alternative splicing mechanisms, indicating that this process might involve a post-translational modification of the full-length receptor; however, the specific mechanism has not been studied. Using CD14+ peripheral human monocytes and the THP-1 monocytic leukemia-derived cell line, we confirm that sTLR2 generation increases upon treatment with pro-inflammatory agents and requires a post-translational mechanism. We also find that the constitutive and ligand-induced release of sTLR2 is sensitive to pharmacological metalloproteinase activator and inhibitors leading us to conclude that metalloproteinase TLR2 shedding contributes to soluble receptor production. By expressing human TLR2 in ADAM10- or ADAM17-deficient MEF cells, we find both enzymes to be implicated in TLR2 ectodomain shedding. Moreover, using a deletion mutant of the TLR2 juxtamembrane region, we demonstrate that this domain is required for sTLR2 generation. Functional analysis suggests that sTLR2 generated by metalloproteinase activation inhibitsTLR2-induced cytokine production by this monocytic leukemia-derived cell line. The identification of the mechanisms involved in regulating the availability of soluble TLR2 ectodomain and cell surface receptors may contribute further research on TLR2-mediated processes in innate immunity and inflammatory disorders.

Salivary mucins induce a Toll-like receptor 4-mediated pro-inflammatory response in human submandibular salivary cells: are mucins involved in Sjögren’s syndrome?

OBJECTIVES A hallmark characteristic of SS patients is the ectopic presence of the mucins MUC5B and MUC7 in the extracellular matrix of salivary glands that have lost apical-basolateral acinar-cell polarity. This study aims to determine whether exogenous salivary mucins induce gene expression of pro-inflammatory cytokines, as well as to evaluate whether the Toll-like receptor-4 (TLR4) pathway is involved in this response. METHODS Differentiated human submandibular gland (HSG) cells were stimulated with mucins or oligosaccharide residues at different concentrations and for different periods of time. The expression of pro-inflammatory cytokines and their receptors was determined by semi-quantitative real time PCR (sqPCR). TLR4-mediated responses induced by mucin were evaluated with the Toll-IL-1 receptor domain containing adaptor protein (TIRAP) inhibitory peptide or using anti-hTLR4 blocking antibody. TLR4-receptor expression was also determined in SS patients, controls and HSG cells. RESULTS Mucins induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-β, IL-6 and IL-1β, but not B cell activating factor (BAFF). Cytokine induction was mediated by TLR4, as shown using TIRAP or using anti-hTLR4 antibody. Sugar residues present in MUC5B, such as sulpho-Lewis (SO3-3Galβ1-3GlcNAc), also induced cytokines. Unexpectedly, mucins induced MUC5B, but not MUC7 expression. CONCLUSION Salivary mucins were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells to amplify and perpetuate inflammation and thereby contribute to the development of a chronic state characteristic of SS. The ectopic localization of MUC5B and MUC7 in the salivary gland extracellular matrix from SS patients and the current results reveal the importance of salivary epithelial cells in innate immunity, as well as in SS pathogenesis.

Glucocorticosteroid therapy in inflammatory bowel diseases: From clinical practice to molecular biology

Inflammatory bowel diseases (IBDs), such as ulcerative colitis and Crohn’s disease, are chronic pathologies associated with a deregulated immune response in the intestinal mucosa, and they are triggered by environmental factors in genetically susceptible individuals. Exogenous glucocorticoids (GCs) are widely used as anti-inflammatory therapy in IBDs. In the past, patients with moderate or severe states of inflammation received GCs as a first line therapy with an important effectiveness in terms of reduction of the disease activity and the induction of remission. However, this treatment often results in detrimental side effects. This downside drove the development of second generation GCs and more precise (non-systemic) drug-delivery methods. Recent clinical trials show that most of these new treatments have similar effectiveness to first generation GCs with fewer adverse effects. The remaining challenge in successful treatment of IBDs concerns the refractoriness and dependency that some patients encounter during GCs treatment. A deeper understanding of the molecular mechanisms underlying GC response is key to personalizing drug choice for IBDs patients to optimize their response to treatment. In this review, we examine the clinical characteristics of treatment with GCs, followed by an in depth analysis of the proposed molecular mechanisms involved in its resistance and dependence associated with IBDs. This thorough analysis of current clinical and biomedical literature may help guide physicians in determining a course of treatment for IBDs patients and identifies important areas needing further study.

Glucocorticoids Impair Phagocytosis and Inflammatory Response Against Crohn’s Disease-Associated Adherent-Invasive Escherichia coli

Crohn’s disease (CD) is a chronic inflammatory bowel disorder characterized by deregulated inflammation triggered by environmental factors. Notably, adherent-invasive Escherichia coli (AIEC), a bacterium with the ability to survive within macrophages is believed to be one of such factors. Glucocorticoids are the first line treatment for CD and to date, it is unknown how they affect bactericidal and inflammatory properties of macrophages against AIEC. The aim of this study was to evaluate the impact of glucocorticoid treatment on AIEC infected macrophages. First, THP-1 cell-derived macrophages were infected with a CD2-a AIEC strain, in the presence or absence of the glucocorticoid dexamethasone (Dex) and mRNA microarray analysis was performed. Differentially expressed mRNAs were confirmed by TaqMan-qPCR. In addition, an amikacin protection assay was used to evaluate the phagocytic and bactericidal activity of Dex-treated macrophages infected with E. coli strains (CD2-a, HM605, NRG857c, and HB101). Finally, cytokine secretion and the inflammatory phenotype of macrophages were evaluated by ELISA and flow cytometry, respectively. The microarray analysis showed that CD2-a, Dex, and CD2-a + Dex-treated macrophages have differential inflammatory gene profiles. Also, canonical pathway analysis revealed decreased phagocytosis signaling by Dex and anti-inflammatory polarization on CD2-a + Dex macrophages. Moreover, amikacin protection assay showed reduced phagocytosis upon Dex treatment and TaqMan-qPCR confirmed Dex inhibition of three phagocytosis-associated genes. All bacteria strains induced TNF-α, IL-6, IL-23, CD40, and CD80, which was inhibited by Dex. Thus, our data demonstrate that glucocorticoids impair phagocytosis and induce anti-inflammatory polarization after AIEC infection, possibly contributing to the survival of AIEC in infected CD patients.

SAT0372 Ectopically Secreted Mucins Might Perpetuate the Inflammation in Salivary Glands of SjÖgren's Syndrome Patients

Background Loss of apico-basal polarity in acinar cells is a characteristic of labial salivary glands (LSG) of Sjögrens syndrome (SS) patients (1). In these glands, the ectopic presence of salivary mucins in the extracellular matrix has been described (1). The structure of mucins consists of a protein backbone with large number of O-linked oligosaccharides covalently attached. In SS-patients, ectopic mucin oligosaccharides might be recognized as not-self antigens by innate immunity receptors, inducing or increasing a pro-inflammatory response. Objectives To determine whether exogenous salivary mucins or mucin oligosaccharides induce the expression of pro-inflammatory cytokines, to evaluate if the Toll-like receptor-4 (TLR4) is involved in this response and if pro-inflammatory cytokines are able to induce the expression of salivary mucins. Methods Human submandibular gland (HSG) cells were stimulated with mucin from bovine submaxillary glands (BSM), Sulfo-Lewis (SO3-3Galβ1-3GlcNAc) oligosaccharide and human recombinant TNF-α or IFN-γ at different concentrations and for different periods of time. For TLR4 signaling assays, HSG cells were pre-treated with the TIRAP inhibitory peptide TBX2 or with an anti-TLR4 blocking antibody and then stimulated with BSM. The mRNA expression of mucins and cytokines was determined by real time PCR. Results Mucins and Sulfo-Lewis induced a significant increase in CXCL8, TNF-α, IFN-α, IFN-β, IL-6 and IL-1β mRNA levels. The induction of cytokines was mediated by TLR4 as shown by reversion of the effect using TBX2 or the anti-TLR4 antibody. TNF-α and IFN-γ induced in vitro the expression of salivary mucins such as MUC1Y/ and MUC1/SEC, which are overexpressed in LSG of SS-patients. Conclusions Salivary mucins and mucin oligosaccharides were recognized by TLR4 in epithelial cells initiating a pro-inflammatory response that could attract inflammatory cells. The overexpression and aberrant localization of mucins in LSG of SS-patients and their induction upon stimulation with pro-inflammatory cytokines support a self-perpetuating mucin-cytokine signaling loop that may facilitate the maintenance of the inflammatory environment leading to disruption of salivary gland homeostasis in SS-patients. References Barrera MJ, Bahamondes V, Sepúlveda D, Quest AFG, Castro I, Cortés J, Aguilera S, Urzúa U, Molina C, Pérez P, Ewert P, Alliende C, Hermoso MA, González S, Leyton C and González MJ. Sjögrens Syndrome: J Autoimmun. 2013 May;42:7-18. Acknowledgements Fondecyt 1120062 (MJG, SA, CM, SG) and PhD fellowship Conicyt-Chile (MJB and JC). Disclosure of Interest None declared

Identification of Intracellular Escherichia Coli (IEC), Associated With Crohn’s Disease, in Other Intestinal Pathologies: P-260

markers and greater volatility in gut microbiota that eventuated in colitic T5KO mice having a markedly different microbiota than their non-colitic siblings, whose microbiota stabilized to a similar composition as WT mice. One striking feature of gut microbiota of colitic T5KO mice was the transient presence of high levels of Proteobacteria, and especially Enterobacteria species including E. coli, which could be observed in close proximity to the gut epithelium. Administration of a Crohn’s disease-associated E. coli strain induced chronic colitis in T5KO, but not WT, mice that persisted well past the time by which both strains of mice had largely eliminated the bacteria. Of interest, gut microbiota composition analysis revealed modification in T5KO mice compare to WT after LF82 clearance. This reveal that LF82 strain could act as an instigator able to modify microbiota to colitogenic, as suggested by analysis of LPS and flagellin loads. CONCLUSION(S): Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring Proteobacteria can drive and/or instigate chronic colitis.