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1.
J Biol Chem ; 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988243

RESUMO

Glucocorticoids are potent endogenous anti-inflammatory molecules, and their cognate receptor, glucocorticoid receptor (GR), is expressed in nearly all immune cells. Macrophages are heterogeneous immune cells having a central role in both tissue homeostasis and inflammation and also play a role in the pathogenesis of some inflammatory diseases. Paradoxically, glucocorticoids have only a limited efficacy in controlling the resolution of these macrophage-related diseases. Here, we report that the transcriptomes of monocyte-like THP-1 cells and macrophage-like THP-1 cells (THP1-MΦ) have largely conserved gene expression patterns. In contrast, the differentiation to THP1-MΦ significantly altered the sensitivity of gene transcription to glucocorticoids. Among glucocorticoid-regulated genes, we identified the exopeptidase dipeptidyl peptidase-4 (DPP4) as a critical glucocorticoid-responsive gene in THP1-MΦ. We found that GR directly induces DPP4 gene expression by binding to two glucocorticoid-responsive elements (GREs) within the DPP4 promoter. Additionally, we show that glucocorticoid-induced DPP4 expression is blocked by the GR antagonist RU-486 and by GR siRNA transfection and that DPP4 enzyme activity is reduced by DPP4 inhibitors. Of note, glucocorticoids highly stimulated macrophage mobility; unexpectedly, DPP4 mediated the glucocorticoid-induced macrophage migration, and siRNA-mediated knockdowns of GR and DPP4 blocked dexamethasone-induced THP1-MΦ migration. Moreover, glucocorticoid-induced DPP4 activation was also observed in proinflammatory M1-polarized murine macrophages, as well as peritoneal macrophages, and was associated with increased macrophage migration. Our results indicate that glucocorticoids directly up-regulate DPP4 expression and thereby induce migration in macrophages, potentially explaining why glucocorticoid therapy is less effective in controlling macrophage-dominated inflammatory disorders.

2.
Front Immunol ; 10: 2449, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824476

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by mucosa damage associated with an uncontrolled inflammatory response. This immunological impairment leads to altered inflammatory mediators such as IL-33, which is shown to increase in the mucosa of active UC (aUC) patients. MicroRNAs present a distorted feature in inflamed colonic mucosa and are potential IL-33 regulating candidates in UC. Therefore, we studied the microRNA and mRNA profiles in inflamed colonic samples of UC patients, evaluating the effect of a microRNA (selected by in silico analysis and its expression in UC patients), on IL-33 under inflammatory conditions. We found that inflamed mucosa (n = 8) showed increased expression of 40 microRNAs and 2,120 mRNAs, while 49 microRNAs and 1,734 mRNAs were decreased, as determined by microarrays. In particular, IL-33 mRNA showed a 3.8-fold increase and eight members of a microRNA family (miR-378), which targets IL-33 mRNA in the 3'UTR, were decreased (-3.9 to -3.0 times). We selected three members of the miR-378 family (miR-378a-3p, miR-422a, and miR-378c) according to background information and interaction energy analysis, for further correlation analyses with IL-33 expression through qPCR and ELISA, respectively. We determined that aUC (n = 24) showed high IL-33 levels, and decreased expression of miR-378a-3p and miR-422a compared to inactive UC (n = 10) and controls (n = 6). Moreover, both microRNAs were inversely correlated with IL-33 expression, while miR-378c does not show a significant difference. To evaluate the effect of TNFα on the studied microRNAs, aUC patients with anti-TNF therapy were compared to aUC receiving other treatments. The levels of miR-378a-3p and miR-378c were higher in aUC patients with anti-TNF. Based on these findings, we selected miR-378a-3p to exploring the molecular mechanism involved by in vitro assays, showing that over-expression of miR-378a-3p decreased the levels of an IL-33 target sequence ß-gal-reporter gene in HEK293 cells. Stable miR-378a-3p over-expression/inhibition inversely modulated IL-33 content and altered viability of HT-29 cells. Additionally, in an inflammatory context, TNFα decreased miR-378a-3p levels in HT-29 cells enhancing IL-33 expression. Together, our results propose a regulatory mechanism of IL-33 expression exerted by miR-378a-3p in an inflammatory environment, contributing to the understanding of UC pathogenesis.

4.
Front Immunol ; 10: 1394, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281317

RESUMO

In colorectal cancer (CRC), cancer-associated fibroblasts (CAFs) are the most abundant component from the tumor microenvironment (TM). CAFs facilitate tumor progression by inducing angiogenesis, immune suppression and invasion, thus altering the organization/composition of the extracellular matrix (i.e., desmoplasia) and/or activating epithelial-mesenchymal transition (EMT). Soluble factors from the TM can also contribute to cell invasion through secretion of cytokines and recently, IL-33/ST2 pathway has gained huge interest as a protumor alarmin, promoting progression to metastasis by inducing changes in TM. Hence, we analyzed IL-33 and ST2 content in tumor and healthy tissue lysates and plasma from CRC patients. Tissue localization and distribution of these molecules was evaluated by immunohistochemistry (using localization reference markers α-smooth muscle actin or α-SMA and E-cadherin), and clinical/histopathological information was obtained from CRC patients. In vitro experiments were conducted in primary cultures of CAFs and normal fibroblasts (NFs) isolated from tumor and healthy tissue taken from CRC patients. Additionally, migration and proliferation analysis were performed in HT29 and HCT116 cell lines. It was found that IL-33 content increases in left-sided CRC patients with lymphatic metastasis, with localization in tumor epithelia associated with abundant desmoplasia. Although ST2 content showed similarities between tumor and healthy tissue, a decreased immunoreactivity was observed in left-sided tumor stroma, associated to metastasis related factors (advanced stages, abundant desmoplasia, and presence of tumor budding). A principal component analysis (including stromal and epithelial IL-33/ST2 and α-SMA immunoreactivity with extent of desmoplasia) allowed us to distinguish clusters of low, intermediate and abundant desmoplasia, with potential to develop a diagnostic signature with benefits for further therapeutic targets. IL-33 transcript levels from CAFs directly correlated with CRC cell line migration induced by CAFs conditioned media, with rhIL-33 inducing a mesenchymal phenotype in HT29 cells. These results indicate a role of IL-33/ST2 in tumor microenvironment, specifically in the interaction between CAFs and epithelial tumor cells, thus contributing to invasion and metastasis in left-sided CRC, most likely by activating desmoplasia.

5.
Front Immunol ; 9: 1026, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29867993

RESUMO

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.


Assuntos
Doença de Crohn/microbiologia , Dexametasona/farmacologia , Infecções por Escherichia coli/imunologia , Glucocorticoides/farmacologia , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Animais , Aderência Bacteriana , Doença de Crohn/imunologia , Citocinas/imunologia , Escherichia coli/patogenicidade , Humanos , Inflamação , Macrófagos/microbiologia , Camundongos , Camundongos Knockout , Análise em Microsséries , Proteína Adaptadora de Sinalização NOD2/genética , Reação em Cadeia da Polimerase em Tempo Real , Células THP-1 , Fator de Necrose Tumoral alfa/imunologia
6.
World J Gastroenterol ; 23(36): 6628-6638, 2017 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-29085208

RESUMO

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.


Assuntos
Anti-Inflamatórios/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Glucocorticoides/uso terapêutico , Imunossupressores/uso terapêutico , Anti-Inflamatórios/farmacologia , Colite Ulcerativa/epidemiologia , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Doença de Crohn/epidemiologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Sistemas de Liberação de Medicamentos/métodos , Resistência a Medicamentos/genética , Epigênese Genética , Glucocorticoides/farmacologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Imunossupressores/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiopatologia , Prevalência , Resultado do Tratamento
7.
Sci Rep ; 7(1): 10180, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860510

RESUMO

The ST2/IL33 signalling pathway has been associated with ulcerative colitis (UC). ST2, encoded by the IL1RL1 gene, is expressed as both a membrane-anchored receptor (ST2L) activated by IL33 and as a soluble receptor (sST2) with anti-inflammatory properties. In UC patients, sST2 is further increased by corticosteroid treatment; however, the glucocorticoid-mediated molecular regulation remains unknown. We therefore tested whether genetic variants in the IL1RL1 distal promoter are involved in UC and affect glucocorticoid-mediated ST2 expression. Serum ST2 levels and genetic variants in the IL1RL1 distal promoter were examined by ELISA and PCR sequencing in UC patients receiving corticosteroids. Glucocorticoid-mediated ST2 production was evaluated in intestinal mucosa cultures. Molecular regulation of glucocorticoid-mediated ST2 was assessed by RT-qPCR, ChIP assay and luciferase reporter assay. Dexamethasone effect on ST2 transcript expression was analyzed in leukocytes and related to IL1RL1 variants. Sequencing of a distal IL1RL1 promoter region demonstrated that SNPs rs6543115(C) and rs6543116(A) are associated with increased sST2 in UC patients on corticosteroids. Dexamethasone up-regulated sST2 transcription through interaction with the glucocorticoid-response element (GRE) carrying rs6543115(C) variant. Our data indicate that IL1RL1 SNPs rs6543115(C) confer susceptibility to UC and is contained in the GRE, which may modulate glucocorticoid-induced sST2 expression.


Assuntos
Corticosteroides/farmacologia , Colite Ulcerativa/tratamento farmacológico , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Polimorfismo de Nucleotídeo Único , Regulação para Cima , Corticosteroides/uso terapêutico , Adulto , Células Cultivadas , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacos , Análise de Sequência de DNA
8.
Rev. méd. Chile ; 145(9): 1129-1136, set. 2017. tab, graf
Artigo em Espanhol | LILACS-Express | ID: biblio-902597

RESUMO

Background: Different strains of invasive Escherichia coli (E. coli), isolated from intestinal mucosa of patients, are related to the pathogenesis of inflammatory bowel diseases (IBD). Aim: To evaluate an association between intracellular E. coli and IBD; its clinical characteristics and use of steroids. Material and Methods: Sixty one patients with Crohn's disease and 83 with ulcerative colitis were studied. To determine the intracellular E. coli content, colonoscopy biopsies of these patients and 29 control subjects were processed using the gentamicin protection assay. Differences in the bacterial content between patient groups were evaluated using Mann-Whitney test, while the association between presence of E. coli with endoscopic activity, location/extension and use of corticosteroid as anti-inflammatory treatment were evaluated with Fisher's exact test or Chi-square test. Results: E. coli strains were detected in 36.1, 39.3 and 10.3% of patients with ulcerative colitis, Crohn's disease and controls, respectively. The number of bacteria per biopsy in Crohn's disease and ulcerative colitis was significantly higher than in controls (p < 0.01 between patients and controls). In ulcerative colitis, significant associations were found between the presence of bacteria and disease location and use of corticosteroids. In Crohn's disease, no association was found. Conclusions: IBD are associated with the presence of intracellular E. coli strains in the intestinal mucosa, suggesting an alteration in the microbiota or loss of integrity of the epithelial barrier. The association of intracellular E. coli with clinical features and the use of corticosteroids in ulcerative colitis suggests that different factors could promote colonization or proliferation of these bacteria.

9.
Rev Med Chil ; 145(9): 1129-1136, 2017 Sep.
Artigo em Espanhol | MEDLINE | ID: mdl-29424399

RESUMO

BACKGROUND: Different strains of invasive Escherichia coli (E. coli), isolated from intestinal mucosa of patients, are related to the pathogenesis of inflammatory bowel diseases (IBD). AIM: To evaluate an association between intracellular E. coli and IBD; its clinical characteristics and use of steroids. MATERIAL AND METHODS: Sixty one patients with Crohn's disease and 83 with ulcerative colitis were studied. To determine the intracellular E. coli content, colonoscopy biopsies of these patients and 29 control subjects were processed using the gentamicin protection assay. Differences in the bacterial content between patient groups were evaluated using Mann-Whitney test, while the association between presence of E. coli with endoscopic activity, location/extension and use of corticosteroid as anti-inflammatory treatment were evaluated with Fisher's exact test or Chi-square test. RESULTS: E. coli strains were detected in 36.1, 39.3 and 10.3% of patients with ulcerative colitis, Crohn's disease and controls, respectively. The number of bacteria per biopsy in Crohn's disease and ulcerative colitis was significantly higher than in controls (p < 0.01 between patients and controls). In ulcerative colitis, significant associations were found between the presence of bacteria and disease location and use of corticosteroids. In Crohn's disease, no association was found. CONCLUSIONS: IBD are associated with the presence of intracellular E. coli strains in the intestinal mucosa, suggesting an alteration in the microbiota or loss of integrity of the epithelial barrier. The association of intracellular E. coli with clinical features and the use of corticosteroids in ulcerative colitis suggests that different factors could promote colonization or proliferation of these bacteria.


Assuntos
Colite Ulcerativa/microbiologia , Doença de Crohn/microbiologia , Escherichia coli/isolamento & purificação , Mucosa Intestinal/microbiologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios/uso terapêutico , Estudos de Casos e Controles , Colite Ulcerativa/tratamento farmacológico , Contagem de Colônia Microbiana , Doença de Crohn/tratamento farmacológico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Valores de Referência , Estatísticas não Paramétricas , Adulto Jovem
10.
BMC Gastroenterol ; 16: 103, 2016 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-27565556

RESUMO

BACKGROUND: The ST2/IL-33 pathway has been related to ulcerative colitis (UC), and soluble ST2 (sST2), to disease severity. We tested the potential usefulness of sST2 as a predictive marker of treatment response and patients' outcome. METHODS: Twenty-six patients with active UC were prospectively recruited and grouped according to an endoscopic score and therapy response. Colonoscopic biopsies were collected at baseline and 6 months or when patients showed clinical activity. The protocol was reinitiated in patients requiring rescue therapy. Blood and stool were collected at baseline, 1, 3, 6 and 12 months. Serum and mucosal ST2, and fecal calprotectin (FC) content were determined by ELISA and correlated to Mayo clinical and endoscopic subscore. Intestinal ST2 was evaluated by immunofluorescence. Wilcoxon signed rank test and Spearman correlations (Rs) were applied (p <0.05). RESULTS: Follow-up was completed in 24 patients. sST2 levels (median and range) varied from 173.5 [136.6-274.0] to 86.5 [54.6-133.2] in responders (p < 0.05), and 336.3 [211.0-403.2] to 385.3 pg/mL [283.4-517.3] in non-responders at baseline and 6 months, respectively. sST2 levels correlated with Mayo clinical and endoscopic subscore, mucosal ST2 and FC (Rs = 0.57, 0.66, 0.74 and 0.42, respectively; p < 0.0001) and showed a trend similar to that of FC in responders. Non-responders revealed an increased ST2 content, restricted to the lamina propria's cellular infiltrate. CONCLUSIONS: Consecutive sST2 measurement to follow changes in inflammatory activity of UC patients who respond or not to treatment identifies sST2, like FC, as a useful biomarker in predicting clinical outcome of UC patients.


Assuntos
Colite Ulcerativa/sangue , Colite Ulcerativa/patologia , Proteína 1 Semelhante a Receptor de Interleucina-1/análise , Adulto , Biomarcadores/análise , Biópsia/métodos , Colite Ulcerativa/terapia , Colonoscopia , Ensaio de Imunoadsorção Enzimática , Fezes/química , Feminino , Imunofluorescência , Humanos , Mucosa Intestinal/patologia , Complexo Antígeno L1 Leucocitário/análise , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Índice de Gravidade de Doença , Estatísticas não Paramétricas
11.
Rheumatology (Oxford) ; 54(8): 1518-27, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25802401

RESUMO

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-ß, IL-6 and IL-1ß, 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 (SO3-3Galß1-3GlcNAc), 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.


Assuntos
Citocinas/metabolismo , Inflamação/metabolismo , Mucinas/farmacologia , Síndrome de Sjogren/metabolismo , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismo , Receptor 4 Toll-Like/metabolismo , Adulto , Estudos de Casos e Controles , Células Cultivadas , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Feminino , Humanos , Imunidade Inata/fisiologia , Masculino , Pessoa de Meia-Idade , Mucina-5B/metabolismo , Mucinas/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Transdução de Sinais/fisiologia , Síndrome de Sjogren/patologia , Glândula Submandibular/patologia , Fatores de Tempo
12.
PLoS One ; 9(12): e104624, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25531754

RESUMO

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.


Assuntos
Metaloproteases/metabolismo , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/química , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-8/biossíntese , Ligantes , Lipopeptídeos/farmacologia , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Terciária de Proteína , Solubilidade , Receptor 2 Toll-Like/metabolismo
13.
Int J Med Microbiol ; 304(3-4): 384-92, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24581881

RESUMO

Crohn's 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-1ß 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-1ß production may contribute to CD and UC pathogenesis.


Assuntos
Proteínas de Transporte/metabolismo , Escherichia coli/fisiologia , Interações Hospedeiro-Patógeno , Inflamassomos/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Macrófagos/microbiologia , Viabilidade Microbiana , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carga Bacteriana , Biópsia , Linhagem Celular , Citosol/microbiologia , Células Epiteliais/microbiologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Feminino , Genótipo , Humanos , Íleo/microbiologia , Íleo/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fatores de Virulência/genética , Adulto Jovem
14.
Immunobiology ; 217(6): 634-42, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22101184

RESUMO

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.


Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Leucócitos Mononucleares/metabolismo , Membrana Mucosa/metabolismo , Receptor 2 Toll-Like/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Receptor 1 de Quimiocina CX3C , Movimento Celular/imunologia , Quimiocina CX3CL1/genética , Quimiocina CX3CL1/imunologia , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Feminino , Expressão Gênica , Humanos , Imunidade Inata , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Membrana Mucosa/imunologia , Membrana Mucosa/patologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologia , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Solubilidade , Técnicas de Cultura de Tecidos
15.
World J Gastroenterol ; 17(17): 2181-90, 2011 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-21633527

RESUMO

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.


Assuntos
Colite Ulcerativa/sangue , Receptores de Superfície Celular/sangue , Adulto , Biomarcadores , Estudos de Coortes , Colo/química , Citocinas/sangue , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Masculino , Pessoa de Meia-Idade , Curva ROC , Receptores de Superfície Celular/fisiologia , Índice de Gravidade de Doença
16.
Inflamm Bowel Dis ; 16(7): 1097-107, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20014018

RESUMO

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.


Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Interleucinas/metabolismo , Receptores de Superfície Celular/metabolismo , Adolescente , Adulto , Idoso , Western Blotting , Estudos de Casos e Controles , Colite Ulcerativa/genética , Doença de Crohn/genética , Feminino , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Interleucina-33 , Interleucinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
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