RESUMO
Acetaminophen (APAP) overdosing is a major cause of acute liver failure worldwide and an established model for drug-induced acute liver injury (ALI). While studying gene expression during murine APAP-induced ALI by 3'mRNA sequencing (massive analysis of cDNA ends, MACE), we observed splenic mRNA accumulation encoding for the neutrophil serine proteases cathepsin G, neutrophil elastase, and proteinase-3 - all are hierarchically activated by cathepsin C (CtsC). This, along with increased serum levels of these proteases in diseased mice, concurs with the established phenomenon of myeloid cell mobilization during APAP intoxication. Objective: In order to functionally characterize CtsC in murine APAP-induced ALI, effects of its genetic or pharmacological inhibition were investigated. Methods and Results: We report on substantially reduced APAP toxicity in CtsC deficient mice. Alleviation of disease was likewise observed by treating mice with the CtsC inhibitor AZD7986, both in short-term prophylactic and therapeutic protocols. This latter observation indicates a mode of action beyond inhibition of granule-associated serine proteases. Protection in CtsC knockout or AZD7986-treated wildtype mice was unrelated to APAP metabolization but, as revealed by MACE, realtime PCR, or ELISA, associated with impaired expression of inflammatory genes with proven pathogenic roles in ALI. Genes consistently downregulated in protocols tested herein included cxcl2, mmp9, and angpt2. Moreover, ptpn22, a positive regulator of the toll-like receptor/interferon-axis, was reduced by targeting CtsC. Conclusions: This work suggests CtsC as promising therapeutic target for the treatment of ALI, among others paradigmatic APAP-induced ALI. Being also currently evaluated in phase III clinical trials for bronchiectasis, successful application of AZD7986 in experimental APAP intoxication emphasizes the translational potential of this latter therapeutic approach.
Assuntos
Acetaminofen , Catepsina C , Doença Hepática Induzida por Substâncias e Drogas , Animais , Masculino , Camundongos , Acetaminofen/efeitos adversos , Catepsina C/metabolismo , Catepsina C/genética , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Murine acetaminophen-induced acute liver injury (ALI) serves as paradigmatic model for drug-induced hepatic injury and regeneration. As major cause of ALI, acetaminophen overdosing is a persistent therapeutic challenge with N-acetylcysteine clinically used to ameliorate parenchymal necrosis. To identify further treatment strategies that serve patients with poor N-acetylcysteine responses, hepatic 3'mRNA sequencing was performed in the initial resolution phase at 24 h/48 h after sublethal overdosing. This approach disclosed 45 genes upregulated (≥5-fold) within this time frame. Focusing on C5aR1, we observed in C5aR1-deficient mice disease aggravation during resolution of intoxication as evidenced by increased liver necrosis and serum alanine aminotransferase. Moreover, decreased hepatocyte compensatory proliferation and increased caspase-3 activation at the surroundings of necrotic cores were detectable in C5aR1-deficient mice. Using a non-hypothesis-driven approach, herein pro-regenerative/-resolving effects of C5aR1 were identified during late acetaminophen-induced ALI. Data concur with protection by the C5a/C5aR1-axis during hepatectomy and emphasize the complex role of inflammation during hepatic regeneration and repair.
RESUMO
Gaining detailed knowledge about sex-related immunoregulation remains a crucial prerequisite for the development of adequate disease models and therapeutic strategies enabling personalized medicine. Here, the key parameter of the production of cytokines mediating disease resolution was investigated. Among these cytokines, STAT3-activating interleukin (IL)-22 is principally associated with recovery from tissue injury. By investigating paradigmatic acetaminophen-induced liver injury, we demonstrated that IL-22 expression is enhanced in female mice. Increased female IL-22 was confirmed at a cellular level using murine splenocytes stimulated by lipopolysaccharide or αCD3/CD28 to model innate or adaptive immunoactivation. Interestingly, testosterone or dihydrotestosterone reduced IL-22 production by female but not by male splenocytes. Mechanistic studies on PMA/PHA-stimulated T-cell-lymphoma EL-4 cells verified the capability of testosterone/dihydrotestosterone to reduce IL-22 production. Moreover, we demonstrated by chromatin immunoprecipitation that testosterone impairs binding of the aryl hydrocarbon receptor to xenobiotic responsive elements within the murine IL-22 promoter. Overall, female mice undergoing acute liver injury and cultured female splenocytes upon inflammatory activation display increased IL-22. This observation is likely related to the immunosuppressive effects of androgens in males. The data presented concur with more pronounced immunological alertness demonstrable in females, which may relate to the sex-specific course of some immunological disorders.
Assuntos
Acetaminofen/efeitos adversos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Expressão Gênica/efeitos dos fármacos , Interleucinas/genética , Interleucinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acetaminofen/sangue , Imunidade Adaptativa/efeitos dos fármacos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Doença Hepática Induzida por Substâncias e Drogas/sangue , Doença Hepática Induzida por Substâncias e Drogas/genética , Di-Hidrotestosterona/farmacologia , Modelos Animais de Doenças , Feminino , Imunidade Inata/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/imunologia , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores Sexuais , Baço/citologia , Baço/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Testosterona/farmacologia , Interleucina 22RESUMO
Unresolved inflammation maintained by release of danger-associated molecular patterns, particularly high-mobility group box-1 (HMGB1), is crucial for hepatocellular carcinoma (HCC) pathogenesis. To further characterize interactions between leucocytes and necrotic cancerous tissue, a cellular model of necroinflammation was studied in which murine Raw 264.7 macrophages or primary splenocytes were exposed to necrotic lysates (N-lys) of murine hepatoma cells or primary hepatocytes. In comparison to those derived from primary hepatocytes, N-lys from hepatoma cells were highly active-inducing in macrophages efficient expression of inflammatory cytokines like C-X-C motif ligand-2 , tumor necrosis factor-α, interleukin (IL)-6 and IL-23-p19. This activity associated with higher levels of HMGB1 in hepatoma cells and was curbed by pharmacological blockage of the receptor for advanced glycation end product (RAGE)/HMGB1 axis or the mitogen-activated protein kinases ERK1/2 pathway. Analysis of murine splenocytes furthermore demonstrated that N-lys did not comprise of functionally relevant amounts of TLR4 agonists. Finally, N-lys derived from hepatoma cells supported inflammatory splenic Th17 and Th1 polarization as detected by IL-17, IL-22 or interferon-γ production. Altogether, a straightforward applicable model was established which allows for biochemical characterization of immunoregulation by HCC necrosis in cell culture. Data presented indicate a remarkably inflammatory capacity of necrotic hepatoma cells that, at least partly, depends on the RAGE/HMGB1 axis and may shape immunological properties of the HCC microenvironment.