RESUMO
Mechanistic target of rapamycin (mTOR) complex (mTORC)1 and mTORC2 regulate the differentiation and function of immune cells. While inhibition of mTORC1 antagonizes dendritic cell (DC) differentiation and suppresses graft rejection, the role of mTORC2 in DCs in determining host responses to transplanted tissue remains undefined. Using a mouse model in which mTORC2 was deleted specifically in CD11c+ DCs (TORC2DC-/- ), we show that the transplant of minor histocompatibility Ag (HY)-mismatched skin grafts from TORC2DC-/- donors into wild-type recipients results in accelerated rejection characterized by enhanced CD8+ T cell responses in the graft and regional lymphoid tissue [Correction added on January 9, 2019, after first online publication: in the previous sentence, major was changed to minor]. Similar enhancement of CD8+ effector T cell responses was observed in MHC-mismatched recipients of TORC2DC-/- grafts. Augmented CD8+ T cell responses were also observed in a delayed-type hypersensitivity model in which mTORC2 was absent in cutaneous DCs. These elevated responses could be ascribed to an increased T cell stimulatory phenotype of TORC2DC-/- and not to enhanced lymph node homing of the cells. In contrast, rejection of ovalbumin transgenic skin grafts in TORC2DC-/- recipients was unaffected. These findings suggest that mTORC2 in skin DCs restrains effector CD8+ T cell responses and have implications for understanding of the influence of mTOR inhibitors that target mTORC2 in transplant.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Rejeição de Enxerto/etiologia , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/fisiologia , Transplante de Pele/efeitos adversos , Pele/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pele/metabolismo , Pele/patologiaRESUMO
The IL-17 family cytokines IL-17A and IL-17C drive the pathogenesis of psoriatic skin inflammation, and anti-IL-17A Abs were recently approved to treat human psoriasis. Little is known about mechanisms that restrain IL-17 cytokine-mediated signaling, particularly IL-17C. In this article, we show that the endoribonuclease MCP-1-induced protein 1 (MCPIP1; also known as regnase-1) is markedly upregulated in human psoriatic skin lesions. Similarly, MCPIP1 was overexpressed in the imiquimod (IMQ)-driven mouse model of cutaneous inflammation. Mice with an MCPIP1 deficiency (Zc3h12a+/-) displayed no baseline skin inflammation, but they showed exacerbated pathology following IMQ treatment. Pathology in Zc3h12a+/- mice was associated with elevated expression of IL-17A- and IL-17C-dependent genes, as well as with increased accumulation of neutrophils in skin. However, IL-17A and IL-17C expression was unaltered, suggesting that the increased inflammation in Zc3h12a+/- mice was due to enhanced downstream IL-17R signaling. Radiation chimeras demonstrated that MCPIP1 in nonhematopoietic cells is responsible for controlling skin pathology. Moreover, Zc3h12a+/-Il17ra-/- mice given IMQ showed almost no disease. To identify which IL-17RA ligand was essential, Zc3h12a+/-Il17a-/- and Zc3h12a+/-Il17c-/- mice were given IMQ; these mice had reduced but not fully abrogated pathology, indicating that MCPIP1 inhibits IL-17A and IL-17C signaling. Confirming this hypothesis, Zc3h12a-/- keratinocytes showed increased responsiveness to IL-17A and IL-17C stimulation. Thus, MCPIP1 is a potent negative regulator of psoriatic skin inflammation through IL-17A and IL-17C. Moreover, to our knowledge, MCPIP1 is the first described negative regulator of IL-17C signaling.
Assuntos
Dermatite/imunologia , Psoríase/imunologia , Ribonucleases/imunologia , Fatores de Transcrição/imunologia , Animais , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interleucina-17/imunologia , Queratinócitos/imunologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Human cutaneous dendritic cells (DCs) have the ability to prime and bias Th17 lymphocytes. However, the factors that stimulate cutaneous DCs to induce Th17 responses are not well known. Alarmins, such as ATP, likely play a pivotal role in the induction and maintenance of cutaneous immune responses by stimulating DC maturation, chemotaxis, and secretion of IL-1ß and IL-6, Th17-biasing cytokines. In this study, using a well-established human skin model, we have demonstrated that signaling purinergic receptors, predominantly the P2X7 receptor (P2X7R), via an ATP analog initiate innate proinflammatory inflammation, DC17 differentiation, and the subsequent induction of Th17-biased immunity. Moreover, our results suggest a potential role for P2X7R signaling in the initiation of psoriasis pathogenesis, a Th17-dependent autoimmune disease. In support of this, we observed the increased presence of P2X7R in nonlesional and lesional psoriatic skin compared with normal healthy tissues. Interestingly, there was also a P2X7R variant that was highly expressed in lesional psoriatic skin compared with nonlesional psoriatic and normal healthy skin. Furthermore, we demonstrated that psoriatic responses could be initiated via P2X7R signaling in nonlesional skin following treatment with a P2X7R agonist. Mechanistic studies revealed a P2X7R-dependent mir-21 angiogenesis pathway that leads to the expression of vascular endothelial growth factor and IL-6 and that may be involved in the development of psoriatic lesions. In conclusion, we have established that purinergic signaling in the skin induces innate inflammation, leading to the differentiation of human Th17 responses, which have implications in the pathogenesis and potential treatment of psoriasis.
Assuntos
Trifosfato de Adenosina/fisiologia , Psoríase/imunologia , Receptores Purinérgicos P2X7/fisiologia , Transdução de Sinais/imunologia , Pele/imunologia , Células Th17/imunologia , Imunidade Adaptativa , Células Cultivadas , Humanos , Imunidade Inata , MicroRNAs/fisiologia , Neovascularização Patológica/imunologia , Psoríase/etiologia , Psoríase/metabolismo , Psoríase/patologia , Pele/metabolismo , Pele/patologia , Células Th17/metabolismo , Células Th17/patologia , Técnicas de Cultura de TecidosRESUMO
Psoriasis is a chronic inflammatory skin disease with no cure. Although the origin of psoriasis and its underlying pathophysiology remain incompletely understood, inflammation is a central mediator of disease progression. In this regard, electrophilic nitro-fatty acids (NO2-FAs) exert potent anti-inflammatory effects in several in vivo murine models of inflammatory diseases, such as chronic kidney disease and cardiovascular disease. To examine the therapeutic potential of NO2-FAs on psoriasiform dermatitis, we employed multiple murine models of psoriasis. Our studies demonstrate that oral treatment with nitro oleic acid (OA-NO2) has both preventative and therapeutic effects on psoriasiform inflammation. In line with this finding, oral OA-NO2 downregulated the production of inflammatory cytokines in the skin. In vitro experiments demonstrate that OA-NO2 decreased both basal IL-6 levels and IL-17A-induced expression of IL-6 in human dermal fibroblasts through the inhibition of NF-κB phosphorylation. Importantly, OA-NO2 diminished STAT3 phosphorylation and nuclear translocation via nitroalkylation of STAT3, which inhibited keratinocyte proliferation. Overall, our results affirm the critical role of both NF-κB and STAT3 in the incitement of psoriasiform dermatitis and highlight the pharmacologic potential of small molecule nitroalkenes for the treatment of cutaneous inflammatory diseases, such as psoriasis.
Assuntos
Dermatite , Ácidos Graxos , Animais , Modelos Animais de Doenças , Humanos , Inflamação , Camundongos , NF-kappa B/metabolismo , Fator de Transcrição STAT3 , Pele/metabolismoRESUMO
Liver dysfunction secondary to severe inflammation is associated with the release of enzymes normally sequestered within hepatocytes. The purpose of these studies was to test the hypothesis that these enzymes are released, at least in part, to modulate potentially deleterious inflammatory processes in distant tissues like the gut. Human Caco-2(BBe) enterocyte-like cells were exposed to cytomix (IFN-gamma, TNF-alpha, and IL-1beta) in the absence or presence of human liver cytosol (LC). Nitric oxide (NO(*)) and inducible nitric oxide synthase (iNOS) protein production were measured by the Griess assay and Western analysis, respectively. Cytomix induced the expression of iNOS and release of NO(*). LC protein (400 microg/ml) added to the basal compartment but not apical compartment completely blocked the release of NO(*) but only slightly decreased the magnitude of iNOS protein induction. Ultrafiltration and ultracentrifugation studies demonstrated that microsome-associated arginase-1 activity was the iNOS-suppressing activity in LC. Liver arginase required activation by a <10-kDa factor that was present in supernatants of cytomix-stimulated cells. The selective iNOS inhibitor l-N(6)-(1-iminoethyl)-lysine.2HCl prevented production of this factor. The biotin switch assay detected increased S-nitrosylation of arginase-1 after incubation with supernatants from immunostimulated Caco-2 cells. Serum from endotoxemic mice contained significantly greater arginase activity compared with serum from control mice. Furthermore, the ratio of mucosal monomeric to dimeric iNOS increased in endotoxemic mice compared with controls. Thus reciprocal activation of arginase-1 and modulation of mucosal iNOS activity may be protective because it would be expected to decrease NO(*)-dependent intestinal barrier dysfunction on that basis.
Assuntos
Arginase/metabolismo , Enterócitos/enzimologia , Inflamação/enzimologia , Fígado/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Western Blotting , Células CACO-2 , Modelos Animais de Doenças , Regulação para Baixo , Enterócitos/efeitos dos fármacos , Enterócitos/imunologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Inflamação/induzido quimicamente , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Lipopolysaccharide (LPS) is an important factor in sepsis. LPS given by intraperitoneal injection induces intestinal hyperpermeability and bacterial translocation in animals and stimulates hepatic Kupffer cells to release TNF-alpha into the bile. This study aims to test the hypothesis that in response to LPS stimulation, hepatic Kupffer cells and extrahepatic macrophages release a large amount of the inflammatory cytokine high-mobility group box 1 (HMGB1) into the bile and that bile containing HMGB1 contributes to gut barrier dysfunction in experimental endotoxemia. To test this, rat common bile ducts were catheterized and bile flow rate was monitored before and during the LPS administration. Eight hours after LPS challenge, anti-HMGB1 neutralizing antibody or nonimmune (sham) IgG was injected into the duodenal lumen of endotoxemic rats; normal mice were also gavaged with normal or endotoxemic rat bile (bile collected from LPS-treated rats). We found that after LPS challenge, the bile flow rate in rats was significantly decreased at the 4- to 12-h time points, TNF-alpha concentration in the bile was markedly elevated at the 3- to 4-h time points, and bile HMGB1 levels were significantly increased at the 8- to 12-h time points. Duodenal injection with anti-HMGB1 antibody reversed LPS-induced gut barrier dysfunction in rats. In addition, feeding endotoxemic rat bile to normal mice significantly increased both mucosal permeability and bacterial translocation. The increase in permeability and bacterial translocation was reversible following removal of HMGB1 from the endotoxemic rat bile. These findings document that bile HMGB1 mediates gut barrier dysfunction in experimental endotoxemia.
Assuntos
Bile/metabolismo , Endotoxemia/fisiopatologia , Proteína HMGB1/metabolismo , Mucosa Intestinal/fisiopatologia , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Claudina-1 , Dextranos/metabolismo , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Proteína HMGB1/sangue , Proteína HMGB1/imunologia , Íleo/efeitos dos fármacos , Íleo/metabolismo , Íleo/microbiologia , Íleo/fisiopatologia , Interleucina-6/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ocludina , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Ethyl pyruvate (EP) is capable of significantly decreasing serum alanine aminotransferase and reducing hepatic necrosis in a murine model of severe acute pancreatitis (SAP); however, the working mechanism is still unclear. This study aims to elucidate the underlying mechanism of EP solution ameliorating SAP-induced liver injury and provide a new therapeutic agent to treat liver injury. MATERIALS AND METHODS: Acute necrotizing pancreatitis was induced in C57Bl/6 male mice by feeding the animals a choline-deficient diet supplemented with 0.5% ethionine for 24 h; then the animals were challenged with 7 hourly 50 mug/kg cerulein i.p. injections and a single i.p. injection of Escherichia coli lipopolysaccharide (4 mg/kg). Two hours after the injection of lipopolysaccharide, 40 mg/kg EP, the same volume of Ringers lactate solution (RLS), or saline solution were i.p. injected to animals of EP, RLS, and control groups every 6 h for a total 48-h period. RESULTS: When mice were treated with EP, hepatic mRNA expression of tumor necrosis factor-alpha, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 was significantly lower than that in pancreatitis mice treated with RLS. Compared to RLS treatment, treatment with EP significantly decreased the number of inflammatory cell infiltration and markedly inhibited hepatic nuclear factor-kappa B DNA binding; EP therapy dramatically inhibited high motility group B1 release from inflamed hepatic tissue and significantly decreased the concentration of hepatic tissue malondialdehyde, an oxidative stress parameter. EP treatment also significantly improved body circulating blood volume. CONCLUSION: EP is a potent anti-inflammatory and anti-oxidative agent to ameliorate hepatic local inflammatory response and resultantly decreases liver injury secondary to SAP.
Assuntos
Hepatopatias/etiologia , Pancreatite Necrosante Aguda/complicações , Piruvatos/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Volume Sanguíneo/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Proteína HMGB1/metabolismo , Heme Oxigenase-1/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Peroxidação de Lipídeos , Fígado/metabolismo , Hepatopatias/metabolismo , Hepatopatias/prevenção & controle , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Piruvatos/farmacologia , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP. METHODS: ALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours. RESULTS: Seventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-kappaB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration. CONCLUSIONS: Prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.
Assuntos
Acetaminofen/intoxicação , Analgésicos não Narcóticos/intoxicação , Antídotos/uso terapêutico , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Cistina/análogos & derivados , Overdose de Drogas/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Antídotos/administração & dosagem , Antídotos/farmacologia , Aspartato Aminotransferases/sangue , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Cistina/administração & dosagem , Cistina/farmacologia , Cistina/uso terapêutico , Modelos Animais de Doenças , Glutationa/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Tempo , Resultado do TratamentoRESUMO
Psoriasis is a chronic inflammatory skin disease dependent on the IL-23/IL-17 axis, a potent inflammatory pathway involved in pathogen clearance and autoimmunity. Several triggers have been proposed as initiators for psoriasis, including alarmins such as adenosine triphosphate. However, the role of alarmins in psoriasis pathogenesis and cutaneous inflammation has not been well addressed. Studies show that signaling through the P2X7 receptor (P2X7R) pathway underlies the development of psoriasiform inflammation. In this regard, psoriasiform dermatitis induced by IL-23 is dependent on P2X7R signaling. Furthermore, direct activation of the P2X7R is sufficient to induce a well-characterized psoriasiform dermatitis. Mechanistic studies determined that P2X7R-induced inflammation is largely dependent on the IL-1ß/NLRP3 inflammasome pathway and neutrophils. In conclusion, this work provides basic mechanistic insight into local inflammatory circuits induced after purinergic P2X7R signaling that are likely involved in the pathogenesis of many inflammatory diseases, such as psoriasis.
Assuntos
Dermatite/imunologia , Interleucina-23/metabolismo , Neutrófilos/imunologia , Psoríase/imunologia , Receptores Purinérgicos P2X7/metabolismo , Pele/patologia , Trifosfato de Adenosina/metabolismo , Animais , Modelos Animais de Doenças , Espaço Extracelular/metabolismo , Feminino , Humanos , Imiquimode , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Interleucina-1beta/metabolismo , Interleucina-23/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Psoríase/induzido quimicamente , Receptores Purinérgicos P2X7/genética , Transdução de SinaisRESUMO
Endogenous electrophilic fatty acids mediate anti-inflammatory responses by modulating metabolic and inflammatory signal transduction and gene expression. Nitro-fatty acids and other electrophilic fatty acids may thus be useful for the prevention and treatment of immune-mediated diseases, including inflammatory skin disorders. In this regard, subcutaneous (SC) injections of nitro oleic acid (OA-NO2), an exemplary nitro-fatty acid, inhibit skin inflammation in a model of allergic contact dermatitis (ACD). Given the nitration of unsaturated fatty acids during metabolic and inflammatory processes and the growing use of fatty acids in topical formulations, we sought to further study the effect of nitro-fatty acids on cutaneous inflammation. To accomplish this, the effect of topically applied OA-NO2 on skin inflammation was evaluated using established murine models of contact hypersensitivity (CHS). In contrast to the effects of subcutaneously injected OA-NO2, topical OA-NO2 potentiated hapten-dependent inflammation inducing a sustained neutrophil-dependent inflammatory response characterized by psoriasiform histological features, increased angiogenesis, and an inflammatory infiltrate that included neutrophils, inflammatory monocytes, and γδ T cells. Consistent with these results, HPLC-MS/MS analysis of skin from psoriasis patients displayed a 56% increase in nitro-conjugated linoleic acid (CLA-NO2) levels in lesional skin compared to non-lesional skin. These results suggest that nitro-fatty acids in the skin microenvironment are products of cutaneous inflammatory responses and, in high local concentrations, may exacerbate inflammatory skin diseases.
Assuntos
Dermatite Alérgica de Contato/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Ácidos Oleicos/imunologia , Psoríase/imunologia , Dermatopatias/imunologia , Pele/patologia , Administração Tópica , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Ethyl pyruvate (EP) is a simple aliphatic ester derived from the endogenous metabolite, pyruvic acid. EP has been shown to decrease the expression of various pro-inflammatory mediators, including nitric oxide (NO*), tumor necrosis factor (TNF), cyclooxygenase-2, and interleukin (IL)-6, in a variety of in vitro and in vivo model systems. In an effort to better understand the chemical features that might explain the anti-inflammatory properties of EP, we screened 15 commercially available compounds for cytoprotective or anti-inflammatory effects using two in vitro assay systems: TNF and NO* production by lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage-like cells and changes in the permeability of Caco-2 human enterocyte-like monolayers stimulated with a cocktail of pro-inflammatory cytokines called cytomix (1000U/ml IFN-gamma plus 10ng/ml TNF-alpha plus 1ng/ml IL-1beta). Two compounds, namely diethyl oxaloproprionate (DEOP) and 2-acetamidoacrylate (2AA), demonstrated consistent anti-inflammatory or cytoprotective pharmacological properties in this screening process. Treatment of mice with either of these compounds ameliorated LPS-induced ileal mucosal hyperpermeability to the fluorescent probe, fluorescein isothiocyanate-labeled dextran (average molecular mass 4kDa), and bacterial translocation to mesenteric lymph nodes. Treatment with either of these compounds also improved survival in mice challenged with a lethal dose of LPS. Finally, in a study that compared 2AA to its methyl ester, we showed that methyl-2-acetamidoacrylate is at least 100-fold more potent than the parent carboxylate as an inhibitor of LPS-induced NO* production by RAW 264.7 cells. Collectively, these data are consistent with the view that anti-inflammatory activity is demonstrable for a number of compounds that either incorporate an olefinic linkage conjugated to a carbonyl moiety or are capable of undergoing tautomeric rearrangement to form such a structure. Moreover, our findings suggest that esters with these general characteristics, perhaps because of their greater lipophilicity or electrophilicity, are more potent anti-inflammatory agents than are the parent carboxylates.
Assuntos
Acrilatos/farmacologia , Alanina/análogos & derivados , Anti-Inflamatórios/farmacologia , Ésteres/farmacologia , Acrilatos/química , Alanina/química , Alanina/farmacologia , Animais , Anti-Inflamatórios/química , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Ésteres/química , Glutationa/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Propionatos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with "cytomix" (a mixture of TNF, IL-1beta, and IFN-gamma) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.
Assuntos
Células CACO-2 , Permeabilidade da Membrana Celular , Citocinas/imunologia , Enterócitos/imunologia , Proteína HMGB1/metabolismo , Animais , Comunicação Autócrina , Células Cultivadas , Enterócitos/citologia , Enterócitos/ultraestrutura , Proteína HMGB1/genética , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The diuretic ethacrynic acid (EA) has been shown to inhibit signaling by the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB). Accordingly, we sought to determine whether this compound is capable of inhibiting the release of cytokines [interleukin (IL)-6 and IL-10] and NO from RAW 264.7 murine macrophage-like cells stimulated with lipopolysaccharide (LPS). Additionally, we sought to determine whether EA can inhibit secretion of high-mobility group box 1 (HMGB1), a nuclear protein that is secreted by immunostimulated macrophages and functions in the extracellular milieu as a proinflammatory mediator. In a concentration-dependent manner, EA inhibited secretion of IL-6, IL-10, nitric oxide, and HMGB1. As expected, EA inhibited NF-kappaB DNA binding in LPS-stimulated RAW 264.7 cells. Treating these cells with pyrrolidine dithiocarbamate, SN50 (amino acid sequence AAVALLPAVLLALLAPVQRKRQKLMP) or 5-(thien-3-yl)-3-aminothiophene-2-carboxamide (SC-514) also inhibited LPS-induced NF-kappaB DNA binding, but these compounds failed to inhibit LPS-induced HMGB1 secretion. These findings suggested that inhibition of HMGB1 secretion by EA might occur via a mechanism unrelated to the NF-kappaB signaling pathway. Because EA is an electrophilic compound that is known to be capable of inducing expression of so-called phase 2 proteins, we sought to determine whether two other phase 2 enzyme inducers, oltipraz and DL-sulforaphane, also are capable of inhibiting HMGB1 release from immunostimulated macrophages. Incubating RAW 264.7 cells with either oltipraz or DL-sulforaphane inhibited LPS-induced HMGB1 secretion. Moreover, both EA and DL-sulforaphane inhibited relocalization of nuclear HMGB1 into the cytoplasm of LPS-stimulated RAW 264.7 cells. These data suggest that phase 2 inducers may exert anti-inflammatory effects by inhibiting secretion of the cytokine-like nuclear protein HMGB1.