RESUMO
Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder associated with a 5-tenfold decrease in lung levels of alpha-1-antitrypsin (AAT) and an increased risk for obstructive lung disease. α-defensins are cationic broad-spectrum cytotoxic and pro-inflammatory peptides found in the azurophilic granules of neutrophils. The concentration of α-defensins is less than 30 nM in the bronchoalveolar lavage fluid of healthy controls but is up to 6 µM in AATD individuals with significant lung function impairment. Alveolar macrophages are generally classified into pro-inflammatory (M1) or anti-inflammatory (M2) subsets that play distinct roles in the initiation and resolution of inflammation. Therefore, monocyte-macrophage differentiation should be tightly controlled to maintain lung integrity. In this study, we determined the effect of α-defensins on monocyte-macrophage differentiation and identified the molecular mechanism of this effect. The results of this study demonstrate that 2.5 µM of α-defensins inhibit the phosphorylation of ERK1/2 and STAT3 and suppress the expression of M2 macrophage markers, CD163 and CD206. In addition, a scratch assay shows that the high concentration of α-defensins inhibits cell movement by ~ 50%, and the phagocytosis assay using flow cytometry shows that α-defensins significantly reduce the bacterial phagocytosis rate of monocyte-derived macrophages (MDMs). To examine whether exogenous AAT is able to alleviate the inhibitory effect of α-defensins on macrophage function, we incubated MDMs with AAT prior to α-defensin treatment and demonstrate that AAT improves the migratory ability and phagocytic ability of MDMs compared with MDMs incubated only with α-defensins. Taken together, this study suggests that a high concentration of α-defensins inhibits the activation of ERK/STAT3 signaling, negatively regulates the expression of M2 macrophage markers, and impairs innate immune function of macrophages.
Assuntos
Deficiência de alfa 1-Antitripsina , alfa-Defensinas , Humanos , Monócitos/metabolismo , alfa-Defensinas/metabolismo , Macrófagos/metabolismo , Deficiência de alfa 1-Antitripsina/metabolismo , Macrófagos Alveolares/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by a hepatic accumulation of mutant alpha-1 antitrypsin (ZAAT). Individuals with AATD are prone to develop a chronic liver disease that remains undiagnosed until late stage of the disease. Here, we sought to characterize the liver pathophysiology of a human transgenic mouse model for AATD with a manifestation of liver disease compared with normal transgenic mice model. Male and female transgenic mice for normal (Pi*M) and mutant variant (Pi*Z) human alpha-1 antitrypsin at 3 and 6 mo of age were subjected to this study. The progression of hepatic ZAAT accumulation, hepatocyte injury, steatosis, liver inflammation, and fibrotic features were monitored by performing an in vivo study. We have also performed a Next-Gene transcriptomic analysis of the transgenic mice liver tissue 16 h after lipopolysaccharide (LPS) administration to delineate liver inflammatory response in Pi*Z mice as compared with Pi*M. Our results show hepatic ZAAT accumulation, followed by hepatocyte ballooning and liver steatosis developed at 3 mo in Pi*Z mice compared with the mice carrying normal variant of human alpha-1 antitrypsin. We observed higher levels of hepatic immune cell infiltrations in both 3- and 6-mo-old Pi*Z mice compared with Pi*M as an indication of liver inflammation. Liver fibrosis was observed as accumulation of collagen in 6-mo-old Pi*Z liver tissues compared with Pi*M control mice. Furthermore, the transcriptomic analysis revealed a dysregulated liver immune response to LPS in Pi*Z mice compared with Pi*M. Of particular interest for translational work, this study aims to establish a mouse model of AATD with a strong manifestation of liver disease that will be a valuable in vivo tool to study the pathophysiology of AATD-mediated liver disease. Our data suggest that the human transgenic mouse model of AATD could provide a suitable model for the evaluation of therapeutic approaches and preventive reagents against AATD-mediated liver disease.NEW & NOTEWORTHY We have characterized a mouse model of human alpha-1 antitrypsin deficiency with a strong manifestation of liver disease that can be used as an in vivo tool to test preventive and therapeutic reagents. Our data explores the altered immunophenotype of alpha-1 antitrypsin-deficient liver macrophages and suggests a relationship between acute inflammation, immune response, and fibrosis.
Assuntos
Fígado Gorduroso , Deficiência de alfa 1-Antitripsina , Masculino , Feminino , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Lipopolissacarídeos , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Camundongos Transgênicos , Modelos Animais de Doenças , InflamaçãoRESUMO
Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.
Assuntos
Hepatócitos/citologia , Deficiência de alfa 1-Antitripsina/patologia , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Mutação com Ganho de Função , Perfilação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Transporte Proteico , Proteólise , Análise de Sequência de RNA , alfa 1-Antitripsina/química , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismoRESUMO
BACKGROUND: Alpha-1 antitrypsin deficiency (AATD) is a genetic disease caused by misfolding and accumulation of mutant alpha-1 antitrypsin (ZAAT) in the endoplasmic reticulum of hepatocytes. Hepatic ZAAT aggregates acquire a toxic gain-of-function that impacts the endoplasmic reticulum which is theorized to cause liver disease in individuals with AATD who present asymptomatic until late-stage cirrhosis. Currently, there is no treatment for AATD-mediated liver disease except liver transplantation. In our study of mitochondrial RNA, we identified that Sirtuin3 (SIRT3) plays a role in the hepatic phenotype of AATD. METHODS: Utilizing RNA and protein analysis in an in vitro AATD model, we investigated the role of SIRT3 in the pathophysiology of AATD-mediated liver disease while also characterizing our novel, transgenic AATD mouse model. RESULTS: We show lower expression of SIRT3 in ZAAT-expressing hepatocytes. In contrast, the overexpression of SIRT3 increases hepatic ZAAT degradation. ZAAT degradation mediated by SIRT3 appeared independent of proteasomal degradation and regular autophagy pathways. We observed that ZAAT-expressing hepatocytes have aberrant accumulation of lipid droplets, with ZAAT polymers localizing on the lipid droplet surface in a direct interaction with Perilipin2, which coats intracellular lipid droplets. SIRT3 overexpression also induced the degradation of lipid droplets in ZAAT-expressing hepatocytes. We observed that SIRT3 overexpression induces lipophagy by enhancing the interaction of Perilipin2 with HSC70. ZAAT polymers then degrade as a consequence of the mobilization of lipids through this process. CONCLUSIONS: In this context, SIRT3 activation may eliminate the hepatic toxic gain-of-function associated with the polymerization of ZAAT, providing a rationale for a potential novel therapeutic approach to the treatment of AATD-mediated liver disease.
Assuntos
Sirtuína 3 , Deficiência de alfa 1-Antitripsina , alfa 1-Antitripsina , Animais , Camundongos , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/metabolismo , Autofagia/genética , Camundongos Transgênicos , Polímeros , Sirtuína 3/genética , Humanos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMO
Alpha-1 antitrypsin (AAT) deficiency (AATD) is an inherited disease caused by mutations in the serpin family A member 1 (SERPINA1, also known as AAT) gene. The most common variant, PI*Z (Glu342Lys), causes accumulation of aberrantly folded AAT in the endoplasmic reticulum (ER) of hepatocytes that is associated with a toxic gain of function, hepatocellular injury, liver fibrosis, and hepatocellular carcinoma. The unfolded protein response (UPR) is a cellular response to improperly folded proteins meant to alleviate ER stress. It has been unclear whether PI*Z AAT elicits liver cell UPR, due in part to limitations of current cellular and animal models. This study investigates whether UPR is activated in a novel human PI*Z AAT cell line and a new PI*Z human AAT (hAAT) mouse model. A PI*Z AAT hepatocyte cell line (Huh7.5Z) was established using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing of the normal ATT (PI*MM) gene in the Huh7.5 cell line. Additionally, novel full-length genomic DNA PI*Z hAAT and PI*M hAAT transgenic mouse models were established. Using these new models, UPR in Huh7.5Z cells and PI*Z mice were comprehensively determined. Robust activation of UPR was observed in Huh7.5Z cells compared to Huh7.5 cells. Activated caspase cascade and apoptosis markers, increased chaperones, and autophagy markers were also detected in Z hepatocytes. Selective attenuation of UPR signaling branches was observed in PI*Z hAAT mice in which the protein kinase R-like ER kinase and inositol-requiring enzyme1α branches were suppressed while the activating transcription factor 6α branch remained active. This study provides direct evidence that PI*Z AAT triggers canonical UPR and that hepatocytes survive pro-apoptotic UPR by selective suppression of UPR branches. Our data improve understanding of underlying pathological molecular mechanisms of PI*Z AATD liver disease.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Deficiência de alfa 1-Antitripsina , Animais , Carcinoma Hepatocelular/genética , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Resposta a Proteínas não Dobradas/genética , Deficiência de alfa 1-Antitripsina/genéticaRESUMO
For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8⺠cytotoxic T lymphocyte (CTL), CD4⺠CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4⺠than CD8⺠T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8⺠T-cell responses were higher or equivalent to those of CD4⺠T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans.
Assuntos
Epitopos de Linfócito T/imunologia , Síndrome de Imunodeficiência Adquirida Felina/prevenção & controle , Imunogenicidade da Vacina , Peptídeos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Gatos , Reações Cruzadas , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1 , Humanos , Imunidade Celular , Vírus da Imunodeficiência Felina , Ativação Linfocitária , Organismos Livres de Patógenos Específicos , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
The prototype and the commercial dual-subtype feline immunodeficiency virus (FIV) vaccines conferred protection against homologous FIV strains as well as heterologous FIV strains from the vaccine subtypes with closely related envelope (Env) sequences. Such protection was mediated by the FIV neutralizing antibodies (NAbs) induced by the vaccines. Remarkably, both prototype and commercial FIV vaccines also conferred protection against heterologous FIV subtypes with highly divergent Env sequences from the vaccine strains. Such protection was not mediated by the vaccine-induced NAbs but was mediated by a potent FIV-specific T-cell immunity generated by the vaccines (Aranyos et al., Vaccine 34: 1480-1488, 2016). The protective epitopes on the FIV vaccine antigen were identified using feline interleukin-2 (IL-2) and interferon-γ (IFNγ) ELISpot assays with overlapping FIV peptide stimulation of the peripheral blood mononuclear cells (PBMC) from cats immunized with prototype FIV vaccine. Two of the protective FIV peptide epitopes were identified on FIV p24 protein and another two protective peptide epitopes were found on FIV reverse transcriptase. In the current study, the multiple antigenic peptides (MAPs) of the four protective FIV peptides were combined with an adjuvant as the FIV MAP vaccine. The laboratory cats were immunized with the MAP vaccine to evaluate whether significant levels of vaccine-specific cytokine responses can be generated to the FIV MAPs and their peptides at post-second and post-third vaccinations. The PBMC from vaccinated cats and non-vaccinated control cats were tested for IL-2, IFNγ, and IL-10 ELISpot responses to the FIV MAPs and peptides. These results were compared to the results from CD4+ and CD8+ T-cell proliferation to the FIV MAPs and peptides. Current study demonstrates that IL-2 and IFNγ ELISpot responses can be used to detect memory responses of the T cells from vaccinated cats after the second and third vaccinations.
Assuntos
ELISPOT/métodos , Vírus da Imunodeficiência Felina/imunologia , Imunogenicidade da Vacina , Linfócitos T/imunologia , Vacinas Virais/imunologia , Animais , Gatos , Citocinas/metabolismo , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Síndrome de Imunodeficiência Adquirida Felina/prevenção & controle , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Linfócitos T/metabolismo , VacinaçãoRESUMO
The importance of vaccine-induced T-cell immunity in conferring protection with prototype and commercial FIV vaccines is still unclear. Current studies performed adoptive transfer of T cells from prototype FIV-vaccinated cats to partial-to-complete feline leukocyte antigen (FLA)-matched cats a day before either homologous FIVPet or heterologous-subtype pathogenic FIVFC1 challenge. Adoptive-transfer (A-T) conferred a protection rate of 87% (13 of 15, p < 0.001) against FIVPet using the FLA-matched T cells, whereas all 12 control cats were unprotected. Furthermore, A-T conferred protection rate of 50% (6 of 12, p<0.023) against FIVFC1 using FLA-matched T cells, whereas all 8 control cats were unprotected. Transfer of FLA-matched T and B cells demonstrated that T cells are needed to confer A-T protection. In addition, complete FLA-matching and addition of T-cell numbers > 13 × 10(6) cells were required for A-T protection against FIVFC1 strain, reported to be a highly pathogenic virus resistant to vaccine-induced neutralizing-antibodies. The addition of FLA-matched B cells alone was not protective. The poor quality of the anti-FIV T-cell immunity induced by the vaccine likely contributed to the lack of protection in an FLA-matched recipient against FIVFC1. The quality of the immune response was determined by the presence of high mRNA levels of cytolysin (perforin) and cytotoxins (granzymes A, B, and H) and T helper-1 cytokines (interferon-γ [IFNγ] and IL2). Increased cytokine, cytolysin and cytotoxin production was detected in the donors which conferred protection in A-T studies. In addition, the CD4(+) and CD8(+) T-cell proliferation and/or IFNγ responses to FIV p24 and reverse transcriptase increased with each year in cats receiving 1X-3X vaccine boosts over 4 years. These studies demonstrate that anti-FIV T-cell immunity induced by vaccination with a dual-subtype FIV vaccine is essential for prophylactic protection against AIDS lentiviruses such as FIV and potentially HIV-1.