Rodrigo Quera

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

Bacteremia as an adverse event of fecal microbiota transplantation in a patient with Crohn's disease and recurrent Clostridium difficile infection

Dear Sir, A 61-year-old male with Crohns disease (CD) compromising jejunum and terminal ileum managed with mesalamine, azathioprine and infliximab. Ever since 2009 our patient has presented a tortuous evolution based on multiple episodes of active CD, acute diverticulitis, Clostridium difficile infection (CDI) and bacteremia for multidrug-sensitive Escherichia coli (MDSEC) (Fig. 1). Figure 1 Temporal evolution of episodes of bacteremia, infection for Clostridium difficile , diverticulitis and surgery for Crohns disease. FMT: fecal microbiota transplantation. E. coli : Escherichia coli . On September 2009 the patient presented fever, after a thorough work-up was done to exclude primary infection sources, bacteremia was confirmed …

Thromboembolism—an Important Manifestation of Inflammatory Bowel Disease

Thromboembolism is an extraintestinal manifestation and an important cause of mortality in inflammatory bowel disease (IBD). The risk of thromboembolism appears to be multifactorial and related to mucosal inflammatory activity in most patients. Various laboratory markers such as thrombin activatable fibrinolysis inhibitor (TAFI) levels have been linked with thrombophilia in IBD but no single laboratory marker has emerged with sufficient predictive value to identify patients at particular risk. Prospective multifactorial analyses will be required; in the interim, clinicians must be vigilant and address common risk factors for thromboembolism in all patients with IBD.

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.

Small Intestinal Clustered Contractions and Bacterial Overgrowth: A Frequent Finding in Obese Patients

BackgroundSmall intestinal bacterial overgrowth (SIBO) has been observed in several disorders of the gastrointestinal tract. Studies have shown abnormalities of motor function in obese patients, and there is indirect evidence suggesting that SIBO is present in them.AimsTo study small intestinal motility and the prevalence of SIBO in obese patients and to determine whether there was any relationship between both parameters.MethodsThirty-nine patients scheduled for bariatric surgery were subjected to hydrogen breath test with lactulose and to a stationary small intestinal motility study with perfused catheters.ResultsSIBO was observed in 41% of obese patients and was not related to body mass index. Small intestinal manometry showed a marked increase of clustered contractions in obese patients with SIBO compared to obese subjects without SIBO, whereas all the other parameters of fasting cyclic activity were not different.ConclusionsSIBO was a frequent finding in obese patients and was associated with an increased pattern of clustered contractions, which was not observed in absence of SIBO.

Enfermedad inflamatoria intestinal: Una mirada inmunológica

Inflammatory bowel diseases (IBD) are inflammatory diseases with a multifactorial component that involve the intestinal tract. The two relevant IBD syndromes are Crohns disease (CD) and ulcerative colitis (UC). One factor involved in IBD development is a genetic predisposition, associated to NOD2/CARD15 and Toll-like receptor 4 (TLR4) polymorphisms that might favor infectious enterocolitis that is possibly associated to the development of IBD. The identification of specific immunologic alterations in IBD and their relationship to the etiology of the disease is a relevant research topic. The role of intra and extracellular molecules, such as transcription factors and cytokines that are involved in the inflammatory response, needs to be understood. The relevance of immunologic molecules that might drive the immune response to a T helper (Th) 1, Th 2 or the recently described Th 17 phenotype, has been demonstrated in animal models and clinical studies with IBD patients. CD and UC predominantly behave with a Th 1 and Th 2 immune phenotype, respectively. Recently, an association between CD and Th 17 has been reported. The knowledge acquired from immunologic and molecular research will help to develop accurate diagnostic methods and efficient therapies (Rev Med Chile 2008; 136: 367-75). (Key words: Colitis, ulcerative; Crohn disease; T-Lymphocytes, Helper-Inducer; Inflammatory bowel diseases; Nod2 signaling adaptor protein)

IL-23R Arg381Gln polymorphism in Chilean patients with inflammatory bowel disease

UNLABELLED Crohns disease (CD) and ulcerative colitis (UC) are multifactorial diseases with a genetic background. Recent results have shown that a non-synonymous, single nucleotide polymorphism (rs11209026, c.1142G>A, p.Arg381Gln) located in the IL-23R gene is associated with inflammatory bowel disease (IBD). The prevalence of IBD is rapidly rising in Chile and there is no information about the frequency of this polymorphism in the Chilean population. AIM To assess the distribution of DNA variants in the IL-23R gene in Chilean patients with IBD. METHODS We studied 100 IBD patients (38 CD and 62 UC) and 59 healthy controls. IL-23R Arg381Gln (G1142A) was genotyped by the polymerase chain reaction and restriction fragment length polymorphism assay. Clinical and demographic features were characterized. RESULTS The IL-23R genetic variant did not have an association with IBD in Chilean patients. This polymorphism was present in 5.2% of the control group and 5% of IBD patients (7.9% for CD and 3.2% for UC) (p > 0.05). CONCLUSIONS These results suggest that the IL-23R Arg381Gln seems not to be involved in the genetic predisposition to IBD in a Chilean population, and confirms that there are ethnic differences in the genetic background of IBD. Replication studies by independent groups are necessary to elucidate the contribution of susceptibility genes to IBD in different ethnic populations.

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.