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1.
PLoS Pathog ; 16(3): e1008340, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226027

RESUMO

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied.


Assuntos
Anticorpos/administração & dosagem , Melanoma/imunologia , Melanoma/terapia , Infecções por Retroviridae/imunologia , Linfócitos T Reguladores/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Vírus da Leucemia Murina de Friend/fisiologia , Humanos , Imunoterapia/efeitos adversos , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Retroviridae/patologia , Infecções por Retroviridae/virologia , Infecções Tumorais por Vírus/patologia , Infecções Tumorais por Vírus/virologia
2.
J Immunol ; 203(6): 1636-1649, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31383741

RESUMO

Balanced control of innate immune signaling in the intestine represents an important host defense mechanism to avoid inappropriate responses that may exacerbate mucosal injury in acute inflammation. In this study, we report that TRIM58, a RING E3-ubiquitin ligase, associates with TLR2. The interaction was found in a yeast two-hybrid screen (human leukocyte and mononuclear library) and confirmed by coimmunoprecipitation of tagged and endogenous proteins. TRIM58 was predominantly expressed by murine and human myeloid-derived cells. Stimulation with a TLR2 ligand modulated TRIM58 synthesis in myeloid cells. Overexpression of TRIM58, but only in presence of the RING domain, promoted proteasome-dependent degradation of TLR2, inhibiting its signaling activity. Genetic deletion of Trim58 in mice (Trim58 -/-) led to impaired resolution of acute dextran sodium sulfate-induced colitis, which was characterized by delayed recovery from colonic injury and associated with enhanced expression of TLR2 protein and proinflammatory cyto/chemokine production in inflamed colons. Using myeloid cell-specific deletion of Trim58 in mice, we demonstrated that the myeloid cell compartment was responsible for early colitis acceleration in Trim58 deficiency. In vitro studies revealed that Trim58 -/- myeloid cells, which showed constitutive upregulation of TLR2 protein, overreacted to a proinflammatory milieu (TNF-α and IFN-γ) with increased IL-1ß protein production, which mechanistically depended on Tlr2 Finally, we found that TRIM58 mRNA and protein expression levels were reduced in colonic specimens from patients with ulcerative colitis. In conclusion, we identify TRIM58 as a novel negative mediator of innate immune control and mucosal homeostasis via TLR2 signaling. Dysfunction of TRIM58 in myeloid cells may contribute to ulcerative colitis pathogenesis.


Assuntos
Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Células Mieloides/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Animais , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Colite Ulcerativa/metabolismo , Colo/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Células HL-60 , Humanos , Interferon gama/metabolismo , Intestinos/patologia , Masculino , Camundongos , Mucosite/metabolismo , Transdução de Sinais/fisiologia , Células THP-1
3.
Sci Rep ; 7(1): 7629, 2017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28794502

RESUMO

Remote ischemic preconditioning (RIPC) by repeated brief cycles of limb ischemia/reperfusion reduces myocardial ischemia/reperfusion injury. In left ventricular (LV) biopsies from patients undergoing coronary artery bypass grafting (CABG), only the activation of signal transducer and activator of transcription 5 was associated with RIPC's cardioprotection. We have now used an unbiased, non-hypothesis-driven proteomics and phosphoproteomics approach to analyze LV biopsies from patients undergoing CABG and from pigs undergoing coronary occlusion/reperfusion without (sham) and with RIPC. False discovery rate-based statistics identified a higher prostaglandin reductase 2 expression at early reperfusion with RIPC than with sham in patients. In pigs, the phosphorylation of 116 proteins was different between baseline and early reperfusion with RIPC and/or with sham. The identified proteins were not identical for patients and pigs, but in-silico pathway analysis of proteins with ≥2-fold higher expression/phosphorylation at early reperfusion with RIPC in comparison to sham revealed a relation to mitochondria and cytoskeleton in both species. Apart from limitations of the proteomics analysis per se, the small cohorts, the sampling/sample processing and the number of uncharacterized/unverifiable porcine proteins may have contributed to this largely unsatisfactory result.


Assuntos
Ponte de Artéria Coronária , Oclusão Coronária/patologia , Ventrículos do Coração/patologia , Precondicionamento Isquêmico Miocárdico , Reperfusão Miocárdica , Fosfoproteínas/análise , Proteoma/análise , Idoso , Animais , Biópsia , Biologia Computacional , Feminino , Humanos , Masculino , Modelos Animais , Proteômica , Suínos
4.
PLoS One ; 12(7): e0180834, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28686677

RESUMO

Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Linfócitos B/imunologia , Colite Ulcerativa/imunologia , Neoplasias Colorretais/imunologia , Regulação Neoplásica da Expressão Gênica , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Linfócitos B/patologia , Carcinogênese/genética , Carcinogênese/imunologia , Carcinogênese/patologia , Quimiotaxia , Colite Ulcerativa/complicações , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Neoplasias Colorretais/complicações , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Epirregulina/genética , Epirregulina/imunologia , Genes de Cadeia Leve de Imunoglobulina/genética , Humanos , Interleucina-11/genética , Interleucina-11/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Quimioatraentes de Monócitos/genética , Proteínas Quimioatraentes de Monócitos/imunologia , Transdução de Sinais , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/imunologia
5.
Oncotarget ; 7(46): 74415-74426, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27769070

RESUMO

Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies.


Assuntos
Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Glucagon/biossíntese , Proteína Proto-Oncogênica N-Myc/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Proteína Glial Fibrilar Ácida/metabolismo , Glucagonoma/genética , Glucagonoma/metabolismo , Glucagonoma/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica N-Myc/metabolismo , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/patologia , Neoplasias Hipofisárias/patologia , Transcriptoma
6.
PLoS One ; 11(6): e0156871, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27271572

RESUMO

Mucinous adenocarcinoma (MAC) represents a distinct histopathological entity of colorectal cancer (CRC), which is associated with disease progression and poor prognosis. Here, we found that expression levels of miR-205 and miR-373 were specifically upregulated only in patients with mucinous colon cancers, but not in CRC that lack mucinous components. To investigate the effects of miR-205 and miR-373 on intestinal epithelial cell (IEC) biology by gain- and loss-of-function experiments in a proof-of-concept approach, we chose previously established in-vitro human Caco-2-based models of differentiated, non-invasive (expressing TLR4 wild-type; termed Caco-2[WT]) versus undifferentiated, invasive (expressing TLR4 mutant D299G; termed Caco-2[D299G]) IEC. Enterocyte-like Caco-2[WT] showed low levels of miR-205 and miR-373 expression, while both miRNAs were significantly upregulated in colorectal carcinoma-like Caco-2[D299G], thus resembling the miRNA expression pattern of paired normal versus tumor samples from MAC patients. Using stable transfection, we generated miR-205- or miR-373-expressing and miR-205- or miR-373-inhibiting subclones of these IEC lines. We found that introduction of miR-205 into Caco-2[WT] led to expansion of mucus-secreting goblet cell-like cells, which was associated with induction of KLF4, MUC2 and TGFß1 expression. Activation of miR-205 in Caco-2[WT] induced chemoresistance, while inhibition of miR-205 in Caco-2[D299G] promoted chemosensitivity. Caco-2[WT] overexpressing miR-373 showed mitotic abnormalities and underwent morphologic changes (loss of epithelial polarity, cytoskeletal reorganization, and junctional disruption) associated with epithelial-mesenchymal transition and progression to inflammation-associated colonic carcinoma, which correlated with induction of phosphorylated STAT3 and N-CADHERIN expression. Functionally, introduction of miR-373 into Caco-2[WT] mediated loss of cell-cell adhesion and increased proliferation and invasion. Reversely, inhibition of miR-373 allowed mesenchymal IEC to regain epithelial properties, which correlated with absence of neoplastic progression. Using xenografts in mice demonstrated miR-373-mediated acceleration of malignant intestinal tumor growth. In conclusion, our results provide first evidence that miR-205 and miR-373 may differentially contribute to the aggressive phenotype of MAC in CRC.


Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma Mucinoso/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , MicroRNAs/fisiologia , Animais , Células CACO-2 , Células Cultivadas , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Nus , Invasividade Neoplásica
7.
Curr Opin Support Palliat Care ; 10(2): 157-64, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26986508

RESUMO

PURPOSE OF REVIEW: Intestinal mucositis represents a common complication and dose-limiting toxicity of cancer chemotherapy. So far chemotherapy-induced intestinal mucositis remains poorly treatable resulting in significant morbidity and reduced quality of life in cancer patients. This review discusses recent insights into the pathophysiology of chemotherapy-induced intestinal mucositis. Novel mechanisms linking gut microbiota, host innate immunity and anticancer drug metabolism are highlighted. RECENT FINDINGS: Gut microbiota may affect xenobiotic metabolism by direct and indirect mechanisms, critically modulating gut toxicity of chemotherapy drugs. Composition and metabolic function of the gut microbiome as well as innate immune responses of the intestinal mucosa are severely altered during chemotherapy. Commensal-mediated innate immune signaling via Toll-like receptors (TLRs) ambiguously shapes chemotherapy-induced genotoxic damage in the gastrointestinal tract. TLR2 may accelerate host detoxification by activating the multidrug transporter ATP-binding cassette 1 (ABCB1)/MDR1 P-glycoprotein to efflux harmful drugs, thus controlling the severity of cancer therapy-induced mucosal damage in the gastrointestinal tract. In contrast, selective chemotherapy drugs may drive LPS hyperresponsiveness via TLR4, which exacerbates mucosal injury through aberrant cytokine storms. Broad-spectrum antibiotic treatment does not seem to represent a valid therapeutic option, as drastic reduction in global gut microbiota may enhance risk of gastrointestinal toxicity and reduce efficacy of some chemotherapy drugs, at least in murine models. SUMMARY: Several variables (environment, metabolism, dysbiosis, infections and/or genetics) influence the outcome of mucosal TLR signaling during cancer treatment. Differences in innate immune responses also reflect chemotherapy drug-specific effects. Future studies must investigate in more detail whether manipulating the delicate balance between gut microbiota and host immune responses by either monotherapy or combinations of different TLR agonists and antagonists may be indeed useful to limit the toxic side-effects of complex chemotherapy regimens, accelerate mucosal tissue regeneration and improve the anticancer treatment response.


Assuntos
Antineoplásicos/efeitos adversos , Mucosa Intestinal/fisiopatologia , Mucosite/induzido quimicamente , Mucosite/fisiopatologia , Receptores Toll-Like/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Disbiose/metabolismo , Meio Ambiente , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Humanos , Imunidade Inata/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Xenobióticos/metabolismo
8.
J Immunol ; 194(4): 1983-95, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25589072

RESUMO

Intestinal mucositis represents the most common complication of intensive chemotherapy, which has a severe adverse impact on quality of life of cancer patients. However, the precise pathophysiology remains to be clarified, and there is so far no successful therapeutic intervention. In this study, we investigated the role of innate immunity through TLR signaling in modulating genotoxic chemotherapy-induced small intestinal injury in vitro and in vivo. Genetic deletion of TLR2, but not MD-2, in mice resulted in severe chemotherapy-induced intestinal mucositis in the proximal jejunum with villous atrophy, accumulation of damaged DNA, CD11b(+)-myeloid cell infiltration, and significant gene alterations in xenobiotic metabolism, including a decrease in ABCB1/multidrug resistance (MDR)1 p-glycoprotein (p-gp) expression. Functionally, stimulation of TLR2 induced synthesis and drug efflux activity of ABCB1/MDR1 p-gp in murine and human CD11b(+)-myeloid cells, thus inhibiting chemotherapy-mediated cytotoxicity. Conversely, TLR2 activation failed to protect small intestinal tissues genetically deficient in MDR1A against DNA-damaging drug-induced apoptosis. Gut microbiota depletion by antibiotics led to increased susceptibility to chemotherapy-induced mucosal injury in wild-type mice, which was suppressed by administration of a TLR2 ligand, preserving ABCB1/MDR1 p-gp expression. Findings were confirmed in a preclinical model of human chemotherapy-induced intestinal mucositis using duodenal biopsies by demonstrating that TLR2 activation limited the toxic-inflammatory reaction and maintained assembly of the drug transporter p-gp. In conclusion, this study identifies a novel molecular link between innate immunity and xenobiotic metabolism. TLR2 acts as a central regulator of xenobiotic defense via the multidrug transporter ABCB1/MDR1 p-gp. Targeting TLR2 may represent a novel therapeutic approach in chemotherapy-induced intestinal mucositis.


Assuntos
Antineoplásicos/efeitos adversos , Mucosite/imunologia , Mucosite/microbiologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Immunoblotting , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microbiota , Mucosite/induzido quimicamente , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Receptor 2 Toll-Like/imunologia
9.
Oncoimmunology ; 2(7): e24890, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24073372

RESUMO

Homeostatic TLR4 signaling protects the intestinal epithelium in health. Evidence suggests that perturbed TLR4 signaling is linked to carcinogenesis. We have recently demonstrated that the common human TLR4 variant D299G exerts pro-inflammatory effects and drives malignant tumor progression in human colon cancer.

10.
J Immunol ; 190(11): 5676-88, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23636052

RESUMO

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1ß activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1ß production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1ß, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Receptor 2 Toll-Like/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11b , Caspase 1/metabolismo , Morte Celular/genética , Morte Celular/imunologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Progressão da Doença , Deleção de Genes , Humanos , Interleucina-1beta/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Antígeno 96 de Linfócito/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/deficiência
11.
Curr Opin Gastroenterol ; 29(1): 85-91, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23207600

RESUMO

PURPOSE OF REVIEW: This review focuses on recent advances and novel insights into the mechanistic events that may link commensal microbiota and host innate immunity in the pathophysiology of intestinal inflammation and neoplasia. Unanswered questions are discussed and future perspectives in the field are highlighted. RECENT FINDINGS: Commensal microbiota, host innate immunity, and genetics form a multidimensional network that controls homeostasis of the mucosal barrier in the intestine. Large-scale sequencing projects have begun to catalog the healthy human microbiome. Converging evidence suggests that alterations in the regulation of the complex host environment [e.g., dysbiosis and overgrowth of select commensal bacterial species, dietary factors, copresence of facultative pathogens (including viruses), and changes in mucus characteristics] may trigger aberrant innate immune signaling, thereby contributing to the development of intestinal inflammation and associated colon cancer in the susceptible individual. Genetically determined innate immune malfunction may create an inflammatory environment that promotes tumor progression (such as the TLR4-D299G mutation). SUMMARY: The next challenging steps to be taken are to decipher changes in the human microbiome (and virome) during well defined diseased states, and relate them to intestinal mucosal immune functions and host genotypes.


Assuntos
Enterocolite/imunologia , Imunidade Inata , Neoplasias Intestinais/imunologia , Metagenoma/imunologia , Interações Microbianas , Enterocolite/microbiologia , Humanos , Neoplasias Intestinais/microbiologia , Transdução de Sinais
12.
Dig Dis ; 30(4): 334-40, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22796793

RESUMO

Commensal microbiota plays a key role in the health and disease of the host. The innate immune system comprises an essential functional component of the intestinal mucosal barrier, maintaining hyporesponsiveness to omnipresent harmless commensals in the lumen, but rapidly recognizing and combating invading bacteria through diverse antimicrobial mechanisms. Interactions between commensals and innate immune cells are constant, multidimensional and entirely context-dependent. Environment, genetics and host defense differentially modulate commensal-innate immune effects and functions in the intestinal mucosa. In IBD, dysbiosis, mucus layer disruption, impairment in bacterial clearance, intestinal epithelial cell barrier dysfunction and/or immune cell deregulation may lead to commensal-innate immune miscommunication, which critically drives mucosal inflammation and associated cancer.


Assuntos
Bactérias/imunologia , Imunidade/imunologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Epitélio/imunologia , Epitélio/microbiologia , Epitélio/patologia , Humanos , Muco/imunologia , Muco/microbiologia
13.
Gastroenterology ; 141(6): 2154-65, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21920464

RESUMO

BACKGROUND & AIMS: The Toll-like receptor (TLR) 4 mediates homeostasis of the intestinal epithelial cell (IEC) barrier. We investigated the effects of TLR4-D299G on IEC functions. METHODS: We engineered IECs (Caco-2) to stably overexpress hemagglutinin-tagged wild-type TLR4, TLR4-D299G, or TLR4-T399I. We performed gene expression profiling using DNA microarray analysis. Findings were confirmed by real-time, quantitative, reverse-transcriptase polymerase chain reaction, immunoblot, enzyme-linked immunosorbent assay, confocal immunofluorescence, and functional analyses. Tumorigenicity was tested using the CD1 nu/nu mice xenograft model. Human colon cancer specimens (N = 214) were genotyped and assessed for disease stage. RESULTS: Caco-2 cells that expressed TLR4-D299G underwent the epithelial-mesenchymal transition and morphologic changes associated with tumor progression, whereas cells that expressed wild-type TLR4 or TLR4-T399I did not. Caco-2 cells that expressed TLR4-D299G had significant increases in expression levels of genes and proteins associated with inflammation and/or tumorigenesis compared with cells that expressed other forms of TLR4. The invasive activity of TLR4-D299G Caco-2 cells required Wnt-dependent activation of STAT3. In mice, intestinal xenograft tumors grew from Caco-2 cells that expressed TLR4-D299G, but not cells that expressed other forms of TLR4; tumor growth was blocked by a specific inhibitor of STAT3. Human colon adenocarcinomas from patients with TLR4-D299G were more frequently of an advanced stage (International Union Against Cancer [UICC] ≥III, 70% vs 46%; P = .0142) with metastasis (UICC IV, 42% vs 19%; P = .0065) than those with wild-type TLR4. Expression of STAT3 messenger RNA was higher among colonic adenocarcinomas with TLR4-D299G than those with wild-type TLR4. CONCLUSIONS: TLR4-D299G induces features of neoplastic progression in intestinal epithelial Caco-2 cells and associates with aggressive colon cancer in humans, implying a novel link between aberrant innate immunity and colonic cancerogenesis.


Assuntos
Adenocarcinoma/patologia , Neoplasias do Colo/patologia , Progressão da Doença , Mucosa Intestinal/efeitos dos fármacos , Receptor 4 Toll-Like/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células CACO-2 , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Ensaio de Imunoadsorção Enzimática , Transição Epitelial-Mesenquimal , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting , Inflamação/imunologia , Masculino , Camundongos , Microscopia de Fluorescência , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/metabolismo , Via de Sinalização Wnt
14.
Inflamm Bowel Dis ; 16(9): 1583-97, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20803699

RESUMO

Differential alteration of Toll-like receptor (TLR) expression in inflammatory bowel disease (IBD) was first described 10 years ago. Since then, studies from many groups have led to the current concept that TLRs represent key mediators of innate host defense in the intestine, involved in maintaining mucosal as well as commensal homeostasis. Recent findings in diverse murine models of colitis have helped to reveal the mechanistic importance of TLR dysfunction in IBD pathogenesis. It has become evident that environment, genetics, and host immunity form a multidimensional and highly interactive regulatory triad that controls TLR function in the intestinal mucosa. Imbalanced relationships within this triad may promote aberrant TLR signaling, critically contributing to acute and chronic intestinal inflammatory processes in IBD colitis and associated cancer.


Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Receptores Toll-Like/metabolismo , Animais , Humanos , Doenças Inflamatórias Intestinais/patologia
15.
Curr Opin Gastroenterol ; 26(6): 583-90, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20664345

RESUMO

PURPOSE OF REVIEW: The intestinal epithelium serves as a highly dynamic immunologic frontier - exhibiting both innate and adaptive immune features. This review focuses on recent advances and novel insights into key intrinsic processes of the intestinal epithelium to closely monitor its intracellular and extracellular environment, communicate messages to neighbouring cells and rapidly initiate active defensive and repair measures, if necessary. RECENT FINDINGS: The intestinal epithelium is uniquely equipped with a vast array of features to control immune barrier homeostasis at the gates of the healthy intestinal mucosa. Deficient Toll-like receptor or NOD-like receptor signalling in the intestinal epithelium may imbalance commensal-dependent homeostasis, facilitating mucosal injury and leading to inflammatory disease. Dysfunction of the NLRP3 inflammasome may trigger aggravation of mucosal inflammation and cancer and has been associated with human inflammatory bowel diseases. Deregulated autophagy may alter inflammasome activity. SUMMARY: Exciting progress has been made in better understanding the complex diversity of physiological functions of innate immune responses in the intestinal epithelial barrier. Regulatory platforms of signalling mechanisms exist which are closely related and interact. However, many questions remain to be answered and more puzzles have arisen which are highlighted here.


Assuntos
Imunidade Inata/fisiologia , Inflamassomos/imunologia , Mucosa Intestinal/imunologia , Animais , Comunicação Celular , Homeostase/imunologia , Humanos , Imunidade nas Mucosas , Inflamassomos/fisiologia , Inflamação/imunologia , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/fisiologia , Transdução de Sinais , Receptores Toll-Like/imunologia
16.
J Biol Chem ; 284(33): 22332-22343, 2009 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-19528242

RESUMO

Gap junctional intercellular communication (GJIC) coordinates cellular functions essential for sustaining tissue homeostasis; yet its regulation in the intestine is not well understood. Here, we identify a novel physiological link between Toll-like receptor (TLR) 2 and GJIC through modulation of Connexin-43 (Cx43) during acute and chronic inflammatory injury of the intestinal epithelial cell (IEC) barrier. Data from in vitro studies reveal that TLR2 activation modulates Cx43 synthesis and increases GJIC via Cx43 during IEC injury. The ulcerative colitis-associated TLR2-R753Q mutant targets Cx43 for increased proteasomal degradation, impairing TLR2-mediated GJIC during intestinal epithelial wounding. In vivo studies using mucosal RNA interference show that TLR2-mediated mucosal healing depends functionally on intestinal epithelial Cx43 during acute inflammatory stress-induced damage. Mice deficient in TLR2 exhibit IEC-specific alterations in Cx43, whereas administration of a TLR2 agonist protects GJIC by blocking accumulation of Cx43 and its hyperphosphorylation at Ser368 to prevent spontaneous chronic colitis in MDR1alpha-deficient mice. Finally, adding the TLR2 agonist to three-dimensional intestinal mucosa-like cultures of human biopsies preserves intestinal epithelial Cx43 integrity and polarization ex vivo. In conclusion, Cx43 plays an important role in innate immune control of commensal-mediated intestinal epithelial wound repair.


Assuntos
Conexina 43/fisiologia , Regulação da Expressão Gênica , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/fisiologia , Animais , Células CACO-2 , Conexina 43/metabolismo , Células Epiteliais/metabolismo , Junções Comunicantes , Humanos , Sistema Imunitário , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Cicatrização
17.
Curr Opin Gastroenterol ; 24(6): 725-32, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19122523

RESUMO

PURPOSE OF REVIEW: Emerging evidence underscores that inappropriate innate immune responses driven by commensals contribute to the pathogenesis of chronic inflammatory bowel diseases in genetically susceptible hosts. The present review focuses on defining the recently described mechanistic functions through which the innate immune signalling apparatus shapes mucosal homeostasis of the intestine in health and disease. RECENT FINDINGS: Commensal-induced innate immune signalling actively drives at least six major interdependent functions to control homeostasis in the healthy intestinal mucosa: 1) barrier preservation, 2) inhibition of apoptosis and inflammation, 3) acceleration of wound repair and tissue regeneration, 4) exclusion of harmful pathogens through autophagy and other antimicrobial defenses, while 5) maintaining immune tolerance towards harmless commensals, and 6) linkage to adaptive immunity. Any disturbance of this peaceful and mutually beneficial host-commensal relationship may imbalance innate immune signalling, which predisposes to chronic intestinal inflammation and associated tumourigenesis in inflammatory bowel diseases. SUMMARY: Recent advances have highlighted the complex mechanistics and functional diversity of innate immunity that paradoxically mediate both protective and destructive responses in the intestinal mucosa. Related signalling targets may offer novel therapeutic approaches in the treatment of inflammatory bowel diseases and inflammation-related cancer.


Assuntos
Imunidade Inata/fisiologia , Mucosa Intestinal/imunologia , Animais , Homeostase , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Transdução de Sinais/imunologia
18.
J Immunol ; 176(7): 4258-66, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16547263

RESUMO

Intestinal epithelial cells (IEC) are constantly exposed to both high concentrations of the bacterial ligand LPS and the serine protease trypsin. MD-2, which contains multiple trypsin cleavage sites, is an essential accessory glycoprotein required for LPS recognition and signaling through TLR4. The aim of this study was to characterize the expression and subcellular distribution of intestinal epithelial MD-2 and to delineate potential functional interactions with trypsin and then alteration in inflammatory bowel disease (IBD). Although MD-2 protein expression was minimal in primary IEC of normal colonic or ileal mucosa, expression was significantly increased in IEC from patients with active IBD colitis, but not in ileal areas from patients with severe Crohn's disease. Endogenous MD-2 was predominantly retained in the calnexin-calreticulin cycle of the endoplasmic reticulum; only a small fraction was exported to the Golgi. MD-2 expression correlated inversely with trypsin activity. Biochemical evidence and in vitro experiments demonstrated that trypsin exposure resulted in extensive proteolysis of endogenous and soluble MD-2 protein, but not of TLR4 in IEC, and was associated with desensitization of IEC to LPS. In conclusion, the present study suggests that endoplasmic reticulum-associated MD-2 expression in IBD may be altered by ileal protease in inflammation, leading to impaired LPS recognition and hyporesponsiveness through MD-2 proteolysis in IEC, thus implying a physiologic mechanism that helps maintain LPS tolerance in the intestine.


Assuntos
Células Epiteliais/metabolismo , Tolerância Imunológica/imunologia , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/metabolismo , Tripsina/metabolismo , Linhagem Celular , Colite/metabolismo , Colite/patologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Tolerância Imunológica/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Chaperonas Moleculares/metabolismo , Ligação Proteica , Regulação para Cima
19.
Mol Immunol ; 42(8): 887-93, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15829278

RESUMO

This brief review summarizes the current understanding of Toll-like receptor (TLRs) mediated intestinal epithelial mechanisms of commensal tolerance versus intolerance and provides an update on the downstream negative control of signaling responses through decreased surface expression, interregulation with NOD2, overexpression of Tollip, various inhibitors of NF-kappaB as well as soluble tolerizing mediators present in lumen and serum which all may maintain or--when dysregulated--impair mucosal homeostasis in health or disease, respectively.


Assuntos
Tolerância Imunológica/imunologia , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Mucosa Intestinal/imunologia , Animais , Proteínas de Ligação a DNA , Humanos , Tolerância Imunológica/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2 , Proteínas Nucleares , PPAR gama/metabolismo , Proteínas/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores Toll-Like , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
20.
Gastroenterology ; 126(4): 1054-70, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15057745

RESUMO

BACKGROUND & AIMS: Despite the ability to participate in immune responses and the continuous presence of bacteria and bacterial products, functional responses of intestinal epithelial cells (IEC) seem to be muted. Previously, tolerance to Toll-like receptors (TLRs) ligands has been described in monocytic cells. However, mechanisms in the intestine are unknown. METHODS: The effect of purified lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on expression and function of TLRs in intestinal epithelial cells (Colo205, SW480, T84) was assessed by Northern and Western blot and FACS analysis, kinase activity assays, immunohistochemistry, and ELISA. RESULTS: Expression of TLRs except 10 was detected in primary IEC and TLR1-10 in the cultured cells. Short-term stimulation with LPS or LTA activated proinflammatory signaling cascades in IEC, including phosphorylation of IRAK and MAP kinases and increased IL-8 secretion, whereas prolonged incubation resulted in a state of hyporesponsiveness with no reactivation of the cells by a second challenge with either substance detected. The cells remained responsive to tumor necrosis factor (TNF). Hyporesponsive cells showed no alteration in expression of TLR or signaling molecules but revealed a decrease in TLR surface expression and IRAK activity. Toll-interacting protein (Tollip) mRNA and protein expression were increased in hyporesponsive cells, and overexpression of Tollip in IEC resulted in a significantly decreased proinflammatory response. CONCLUSIONS: Continuous presence of specific bacterial components results in a status of hyporesponsiveness in otherwise reactive IEC. Down-regulation of TLR surface expression and up-regulation of inhibitory Tollip with decreased phosphorylation of IRAK might all contribute to this hyporesponsiveness.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Bactérias/metabolismo , Aderência Bacteriana , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo , Regulação para Baixo , Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1 , Interleucina-8/genética , Interleucina-8/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ácidos Teicoicos/farmacologia , Receptor 1 Toll-Like , Receptores Toll-Like
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