Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells.

Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-ß1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFß1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the ß7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.

[Filmarray GI TM panel for detection of enteric pathogens in stool samples: preliminary experience]. / Panel Filmarray GI® en la detección de patógenos entéricos en deposiciones: experiencia preliminar.

INTRODUCTION: Gastrointestinal infections remain a major public health problem worldwide and its etiologic diagnosis is one of the main challenges. In molecular diagnostic techniques recently developed, the Filmarray GI® panel allows detection of 23 pathogens (14 bacteria, virus 5 and 4 parasites) within an hour. OBJECTIVE: To describe the experience of Filmarray GI® panel in the Molecular Biology Laboratory of Clinica Las Condes. METHOD: A cross-sectional observational study that includes the results of 305 stool samples tested by Filmarray GI® panel was performed. RESULTS: Of the 305 tests requested 99 (32.5%) were negative and 206 were positive (67.5%). Of the positive samples, in 107 samples (51.9%) one pathogen was detected and in 99 samples (48.1%) more than one pathogen was found. CONCLUSIONS: Describe the experience of using FilmArray GI® panel in stool specimens, which highlights the large number of positive samples for a microorganism and co-detection of enteric pathogens.

Neisseria gonorrhoeae induces a tolerogenic phenotype in macrophages to modulate host immunity.

Neisseria gonorrhoeae is the etiological agent of gonorrhoea, which is a sexually transmitted disease widespread throughout the world. N. gonorrhoeae does not improve immune response in patients with reinfection, suggesting that gonococcus displays several mechanisms to evade immune response and survive in the host. N. gonorrhoeae is able to suppress the protective immune response at different levels, such as B and T lymphocytes and dendritic cells. In this study, we determined whether N. gonorrhoeae directly conditions the phenotype of RAW 264.7 murine macrophage cell line and its response. We established that gonococcus was effectively phagocytosed by the RAW 264.7 cells and upregulates production of immunoregulatory cytokines (IL-10 and TGF- ß 1) but not the production of proinflammatory cytokine TNF- α , indicating that gonococcus induces a shift towards anti-inflammatory cytokine production. Moreover, N. gonorrhoeae did not induce significant upregulation of costimulatory CD86 and MHC class II molecules. We also showed that N. gonorrhoeae infected macrophage cell line fails to elicit proliferative CD4+ response. This implies that macrophage that can phagocytose gonococcus do not display proper antigen-presenting functions. These results indicate that N. gonorrhoeae induces a tolerogenic phenotype in antigen-presenting cells, which seems to be one of the mechanisms to induce evasion of immune response.

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 Crohn's 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.

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 Crohn's 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 Spearman's 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.

Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation.

Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam(3) -Cys-Ser-Lys(4) . These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478-511) and exposed to Pam(3) -Cys-Ser-Lys(4) in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

Constant TCR triggering suggests that the TCR expressed on intestinal intraepithelial γδ T cells is functional in vivo.

Intestinal intraepithelial lymphocytes carrying the γδ TCR (γδ iIEL) are involved in the maintenance of epithelial integrity. γδ iIEL have an activated phenotype, characterized by CD69 expression and increased cell size compared with systemic T lymphocytes. As an additional activation marker, the majority of γδ iIEL express the CD8αα homodimer. However, our knowledge about cognate ligands for most γδ TCR remains fragmentary and recent advances show that γδ T cells including iIEL may be directly activated by cytokines or through NK-receptors, TLR and other pattern recognition receptors. We therefore asked whether the TCR of γδ iIEL was functional beyond its role during thymic selection. Using TcrdH2BeGFP (Tcrd, T-cell receptor δ locus; H2B, histone 2B) reporter mice to identify γδ T cells, we measured their intracellular free calcium concentration in response to TCR-crosslinking. In contrast to systemic γδ T cells, CD8αα(+) γδ iIEL showed high basal calcium levels and were refractory to TCR-dependent calcium-flux induction; however, they readily produced CC chemokine ligand 4 (CCL4) and IFN-γ upon TCR triggering in vitro. Notably, in vivo blocking of the γδ TCR with specific mAb led to a decrease of basal calcium levels in CD8αα(+) γδ iIEL. This suggests that the γδ TCR of CD8αα(+) γδ iIEL is constantly being triggered and therefore functional in vivo.

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 Crohn's 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.

Peripheral cytokine profile in Chilean patients with Crohn's disease and ulcerative colitis.

Crohn's disease (CD) and ulcerative colitis (UC) belong to the group of inflammatory bowel diseases (IBD), with complex ethiopathogenic factors that include an unbalanced immune and inflammatory response to commensal and food antigens. The differential diagnosis between CD and UC is performed using clinical, endoscopic, histopathological, serological and radiological methods; however between 10-15% of IBD patients are diagnosed as "unclassified colitis". Further research into IBD is necessary in order to develop additional diagnostic tools. The aim of this work was to see if the Th1, Th17 or Th2 immune pattern, represented by CD4+ lymphocytes producing IFN-gamma, IL-17 and IL-5 or IL-13, respectively (CD4/IFN-gamma+, CD4/IL-17+,CD4/IL-5+ or, CD4/IL13+), are useful peripheral markers which can be used to differentiate between UC and CD. Peripheral blood samples were taken from IBD patients from the Clinic Hospital of the University of Chile. The percentage of IFN-gamma-, IL-17-, IL-5- or IL-13-expressing CD4+ cells was determined by flow cytometry in phorbol ester- (PMA) and calcymycin-activated blood samples. The percentages of the CD4+ cell populations producing each cytokine were compared between UC and CD. IFN-gamma production by CD4+ lymphocytes was significantly higher in CD compared to UC and the control. The percentage of IL-17-expressing cells was significantly higher in CD patients compared to to the control; however, there were no differences between UC and CD; or between UC and healthy individuals. No significant differences were observed between the different groups as regards the representative Th2 cytokines. This study suggests that, under pathogenic conditions, several immune profiles may be operating, in the development of IBD. Although peripheral IFN-gamma and IL-17 expression, as indicators of the immune pattern, may help in the diagnosis of IBD, other cytokines and adaptive immune markers should be analyzed to allow better differentiation between the two entities.

Regulatory T cells are locally induced during intravaginal infection of mice with Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a gram-negative diplococcus that in human beings produces gonorrhea. Much clinical evidence has led to the conclusion that gonococcus has important mechanisms to evade host immune functions; however, these mechanisms are only now beginning to be elucidated. In this study, we determined that the BALB/c mouse is a good animal model to study gonococcus infection and examined the immune response against the bacteria. We determined that after intravaginal inoculation of mice with Neisseria gonorrhoeae, the bacteria reached and invaded the upper female reproductive tissues and elicited a T-cell-specific immune response associated with a very weak humoral response, altogether resembling gonococcus infection and disease in women. Remarkably, in the draining lymph nodes of the genital tracts of infected mice, we found an increase of regulatory T lymphocytes, namely, transforming growth factor beta1-positive CD4(+) T cells and CD4(+) CD25(+) Foxp3(+) T cells. Altogether, results indicate that N. gonorrhoeae induces regulatory T cells, which might be related to the local survival of the pathogen and the establishment of a chronic asymptomatic infection.