RESUMO
BACKGROUND: The developmental immaturity of the innate immune system helps explains the increased risk of infection in the neonatal period. Importantly, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for the prevention of hepatocyte apoptosis in adult animals, yet whether developmental immaturity of these pathways increases the risk of hepatic injury in the neonatal period is unknown. METHODS: Using a murine model of endotoxemia (LPS 5 mg/kg IP x 1) in neonatal (P3) and adult mice, we evaluated histologic evidence of hepatic injury and apoptosis, presence of p65/NFκB and c-Jun/AP1 activation and associated transcriptional regulation of apoptotic genes. RESULTS: We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis. This is associated with absent hepatic p65/NFκB signaling and impaired expression of anti-apoptotic target genes. Hepatic c-Jun/AP1 activity was attenuated in endotoxemic P3 mice, with resulting upregulation of pro-apoptotic factors. CONCLUSIONS: These results demonstrate that developmental absence of innate immune p65/NFκB and c-Jun/AP1 signaling, and target gene expression is associated with apoptotic injury in neonatal mice. More work is needed to determine if this contributes to long-term hepatic dysfunction, and whether immunomodulatory approaches can prevent this injury. IMPACT: Various aspects of developmental immaturity of the innate immune system may help explain the increased risk of infection in the neonatal period. In adult models of inflammation and infection, innate immune signaling pathways such as p65/NFκB and c-Jun/AP1 are responsible for a protective, pro-inflammatory transcriptome and regulation of apoptosis. We demonstrate that in contrast to adults, endotoxemic neonatal (P3) mice exhibit a significant increase in hepatic apoptosis associated with absent hepatic p65/NFκB signaling and c-Jun/AP1 activity. We believe that these results may explain in part hepatic dysfunction with neonatal sepsis, and that there may be unrecognized developmental and long-term hepatic implications of early life exposure to systemic inflammatory stress.
Assuntos
Animais Recém-Nascidos , Apoptose , Endotoxemia , Imunidade Inata , Fígado , Transdução de Sinais , Fator de Transcrição AP-1 , Animais , Fator de Transcrição AP-1/metabolismo , Fígado/metabolismo , Fígado/imunologia , Fígado/patologia , Camundongos , Endotoxemia/imunologia , Endotoxemia/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-jun/metabolismo , Modelos Animais de DoençasRESUMO
Maintenance of hepatocyte homeostasis plays an important role in mediating the pathogenesis of many diseases. A growing body of literature has established a critical role played by tumor necrosis factor-α (TNFα) in maintaining hepatocyte homeostasis; however, the transcriptional mechanisms underlying constitutive Tnf expression are unknown. Whole liver fractions and primary hepatocytes from adult control C57BL/6 mice and the murine hepatocyte cell line AML12 were assessed for constitutive Tnf expression. Impacts of glycogen synthase kinase-3 ß (GSK3ß) and nuclear factor κB (NF-κB) inhibition on constitutive Tnf expression were assessed in AML12 cells. Finally, AML12 cell proliferation following GSK3ß and NF-κB inhibition was evaluated. Constitutive Tnf gene expression is present in whole liver, primary hepatocytes, and cultured AML12 hepatocytes. Cytokine-induced Tnf gene expression is regulated by NF-κB activation. Pharmacological inhibition of GSK3ß resulted in a time- and dose-dependent inhibition of Tnf gene expression. GSK3ß inhibition decreased nuclear levels of the NF-κB subunits p65 and p50. We determined that NF-κB transcription factor subunit p65 binds to consensus sequence elements present in the murine TNFα promoter and inhibition of GSK3ß decreases binding and subsequent Tnf expression. Finally, AML12 cell growth was significantly reduced following GSK3ß and NF-κB inhibition. These results demonstrate that GSK3ß and NF-κB are essential for mediating Tnf expression and constitutive hepatocyte cell growth. These findings add to a growing body of literature on TNFα mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in mediating response to various disease states in the liver.NEW & NOTEWORTHY Maintenance of hepatocyte homeostasis plays an important role in controlling the pathogenesis of many diseases. Our findings add to a growing body of literature on tumor necrosis factor-α (TNFα)-mediated hepatocyte homeostasis and identify novel molecular mechanisms involved in regulating this response.
Assuntos
NF-kappa B , Fator de Transcrição RelA , Fator de Necrose Tumoral alfa , Animais , Camundongos , Expressão Gênica , Glicogênio Sintase Quinase 3 beta , Hepatócitos/metabolismo , Homeostase , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.
Assuntos
Endotoxemia , NF-kappa B , Masculino , Feminino , Animais , Camundongos , NF-kappa B/metabolismo , Proteína C-Reativa/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Imunidade InataRESUMO
Decreased selenium (Se) levels during childhood and infancy are associated with worse respiratory health. Se is biologically active after incorporation into Se-containing antioxidant enzymes (AOE) and proteins. It is unknown how decreased maternal Se during pregnancy and lactation impacts neonatal pulmonary selenoproteins, growth, and lung development. Using a model of neonatal Se deficiency that limits Se intake to the dam during pregnancy and lactation, we evaluated which neonatal pulmonary selenoproteins are decreased in both the saccular (postnatal day 0, P0) and early alveolar (postnatal day 7, P7) stages of lung development. We found that Se deficient (SeD) pups weigh less and exhibit impaired alveolar development compared to Se sufficient (SeS) pups at P7. The activity levels of glutathione peroxidase (GPx) and thioredoxin reductase (Txnrd) were decreased at P0 and P7 in SeD lungs compared to SeS lungs. Protein content of GPx1, GPx3 and Txnrd1 were decreased in SeD lungs at P0 and P7, whereas Txnrd2 content was unaltered compared to SeS controls. The expression of NRF-2 dependent genes and several non-Se containing AOE were similar between SeS and SeD lungs. SeD lungs exhibited a decrease in selenoprotein N, an endoplasmic reticulum protein implicated in alveolar development, at both time points. We conclude that exposure to Se deficiency during pregnancy and lactation impairs weight gain and lung growth in offspring. Our data identify multiple selenoproteins in the neonatal lung that are vulnerable to decreased Se intake, which may impact oxidative stress and cell signaling under physiologic conditions as well as after oxidative stressors.
RESUMO
Acetaminophen (N-acetyl-p-aminophenol [APAP]) toxicity is a common cause of acute liver failure. Innate immune signaling and specifically NFκB activation play a complex role in mediating the hepatic response to toxic APAP exposures. While inflammatory innate immune responses contribute to APAP-induced injury, these same pathways play a role in regeneration and repair. Previous studies have shown that attenuating IκBß/NFκB signaling downstream of TLR4 activation can limit injury, but whether this pathway contributes to APAP-induced hepatic injury is unknown. We hypothesized that the absence of IκBß/NFκB signaling in the setting of toxic APAP exposure would attenuate APAP-induced hepatic injury. To test this, we exposed adult male WT and IκBß-/- mice to APAP (280 mg/kg, IP) and evaluated liver histology at early (2-24 hr) and late (48-72 hr) time points. Furthermore, we interrogated the hepatic expression of NFκB inflammatory (Cxcl1, Tnf, Il1b, Il6, Ptgs2, and Ccl2), anti-inflammatory (Il10, Tnfaip3, and Nfkbia), and Nrf2/antioxidant (Gclc, Hmox, and Nqo1) target genes previously demonstrated to play a role in APAP-induced injury. Conflicting with our hypothesis, we found that hepatic injury was similar in WT and IκBß-/- mice. Acutely, the induced expression of some target genes was similar in WT and IκBß-/- mice (Tnfaip3, Nfkbia, and Gclc), while others were either not induced (Cxcl1, Tnf, Ptgs2, and Il10) or significantly attenuated (Ccl2) in IκBß-/- mice. At later time points, APAP-induced hepatic expression of Il1b, Il6, and Gclc was significantly attenuated in IκBß-/- mice. Based on these findings, the therapeutic potential of targeting IκBß/NFκB signaling to treat toxic APAP-induced hepatic injury is likely limited.
RESUMO
Acetaminophen (APAP) overdose results in high morbidity and mortality, with limited treatment options. Increased understanding of the cellular signaling pathways activated in response to toxic APAP exposure is needed to provide insight into novel therapeutic strategies. Toxic APAP exposure induces hepatic nuclear factor kappa B (NFκB) activation. NFκB signaling has been identified to mediate the proinflammatory response but also induces a prosurvival and regenerative response. It is currently unknown whether potentiating NFkB activation would be injurious or advantageous after APAP overdose. The NFκB inhibitory protein beta (IκBß) dictates the duration and degree of the NFκB response following exposure to oxidative injuries. Thus, we sought to determine whether IκBß/NFκB signaling contributes to APAP-induced hepatic injury. At late time points (24 h) following toxic APAP exposures, mice expressing only IκBß knock-in mice (AKBI mice) exhibited increased serologic evidence of hepatic injury. This corresponded with increased histologic injury, specifically related to sinusoidal dilatation. When compared with wild type mice, AKBI mice demonstrated sustained hepatic nuclear translocation of the NFκB subunits p65 and p50, and enhanced NFκB target gene expression. This included increased expression of interleukin-6 (Il-6), a known contributor to hepatic sinusoidal dilation. This transcriptional response corresponded with increased plasma protein content of Il-6, as well as increased activation of signal transducer and activator of transcription 3.