RESUMO
Early increase in the level of endothelial progenitor cells (EPCs) in the systemic circulation occurs in patients with septic infection/sepsis. The significance and underlying mechanisms of this response remain unclear. This study investigated the bone marrow EPC response in adult mice with septic infection induced by intravenous injection (i.v.) of Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor cells (SPCs) including lineage(lin)-stem cell factor receptor (c-kit)+stem cell antigen-1 (Sca-1)-, lin-c-kit+, and lin- cells were cultured with or without lipopolysaccharides (LPSs) and recombinant murine vascular endothelial growth factor (VEGF) in the absence and presence of anti-Sca-1 crosslinking antibodies. In a separate set of experiments, marrow lin-c-kit+ cells from green fluorescence protein (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for 24 h, were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded rapidly following septic infection, which was supported by both proliferative activation and phenotypic conversion of marrow stem/progenitor cells. Increase in marrow EPCs and their reprogramming for enhancing angiogenic activity correlated with cell-marked upregulation of Sca-1 expression. Sca-1 was coupled with Ras-related C3 botulinum toxin substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and SDF-1. The early increase in circulating EPCs was accompanied by their active homing and incorporation into pulmonary microvasculature. These results demonstrate that the marrow EPC response is a critical component of the host defense system. Sca-1 signaling plays a pivotal role in the regulation of EPC response in mice with septic infection.
Assuntos
Células Progenitoras Endoteliais , Proteínas de Membrana , Sepse , Animais , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/imunologia , Sepse/imunologia , Sepse/metabolismo , Camundongos , Camundongos Knockout , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Camundongos Endogâmicos C57BL , Fator A de Crescimento do Endotélio Vascular/metabolismo , Antígenos Ly/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/imunologia , Células Cultivadas , MasculinoRESUMO
People living with HIV (PLWH) are at increased risk for noncommunicable diseases such as lung disease in part due to opportunistic infections including pneumonia. HIV infection is associated with increased prevalence of impaired lung function and abnormal gas exchange. Alcohol use disorder (AUD) is exceedingly common in PLWH and is associated with higher risk of pneumonia in PLWH. Alcohol use may lead to lung damage through several mechanisms. Data on the long-term effect of AUD on pulmonary function in PLWH are sparse and conflicting. To evaluate this relationship, we conducted a cross-sectional analysis of adult PLWH in care in Louisiana. We hypothesized that chronic alcohol use would be associated with subsequent pulmonary dysfunction in a dose-dependent fashion. All participants performed standardized spirometry on study entry. In total, 350 participants with acceptable spirometry were included in this analysis. Thirty-one percent of participants were female. Women reported less lifetime alcohol use and less smoking; however, they reported more chronic respiratory symptoms. In adjusted models, total lifetime alcohol use was not associated with spirometry measures of pulmonary function. HIV-related variables (CD4 count and viral load) were also not associated with measures of pulmonary function. We then conducted sex-stratified analyses to eliminate residual confounding of sex and similarly found no association of total lifetime alcohol use and pulmonary function. We found no association of AUDIT score or early life alcohol use and pulmonary function. In latent class factor analysis, current heavy alcohol use was associated with lower measures of pulmonary function as compared to former heavy alcohol use. In summary, in this cohort of New Orleanian men and women living with HIV with robust measures of alcohol use, though total lifetime alcohol use and early life alcohol use were not associated with pulmonary function, current heavy alcohol use was associated with impaired pulmonary function.
Assuntos
Alcoolismo , Infecções por HIV , Pneumopatias , Pneumonia , Adulto , Alcoolismo/epidemiologia , Contagem de Linfócito CD4 , Estudos Transversais , Feminino , Infecções por HIV/complicações , Infecções por HIV/epidemiologia , Humanos , Pulmão , MasculinoRESUMO
Intracellular reduction-oxidation (RedOx) status mediates a myriad of critical biological processes. Importantly, RedOx status regulates the differentiation of hematopoietic stem and progenitor cells (HSPCs), mesenchymal stromal cells (MSCs) and maturation of CD8+ T Lymphocytes. In most cells, mitochondria are the greatest contributors of intracellular reactive oxygen species (ROS). Excess ROS leads to mitochondrial DNA (mtDNA) damage and protein depletion. We have developed a fluorescence-activated cell sorting (FACS)-based protocol to simultaneously analyze RedOx status and mtDNA integrity. This simultaneous analysis includes measurements of ROS (reduced glutathione (GSH)), ATP5H (nuclear encoded protein), MTCO1 (mitochondrial DNA encoded protein), and cell surface markers to allow discrimination of different cell populations. Using the ratio of MTCO1 to ATP5H median fluorescence intensity (MFI), we can gain an understanding of mtDNA genomic stability, since MTCO1 levels are decreased when mtDNA becomes significantly damaged. Furthermore, this workflow can be optimized for sorting cells, using any of the above parameters, allowing for downstream quantification of mtDNA genome copies/nucleus by quantitative PCR (qPCR). This unique methodology can be used to enhance analyses of the impacts of pharmacological interventions, as well as physiological and pathophysiological processes on RedOx status along with mitochondrial dynamics in most cell types.
RESUMO
Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of â¼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an â¼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.
Assuntos
Suscetibilidade a Doenças/induzido quimicamente , Disbiose/microbiologia , Etanol/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Infecções por HIV/complicações , Pneumonia Pneumocócica/etiologia , Animais , Transplante de Medula Óssea , Contagem de Linfócito CD4 , Modelos Animais de Doenças , Suscetibilidade a Doenças/microbiologia , Suscetibilidade a Doenças/virologia , Disbiose/virologia , Feminino , Microbioma Gastrointestinal/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Transplante de Fígado , Camundongos , RNA Ribossômico 16S/genética , Timo/transplante , Transplante Heterólogo , Carga Viral/efeitos dos fármacosRESUMO
BACKGROUND: Chronic alcohol intoxication suppresses immune function and increases osteoporosis risk suggesting bone-tissue cytotoxicity. Human immunodeficiency virus infection leads to similar impairments. This study investigated the effects of chronic alcohol administration during the early stage of simian immunodeficiency virus (SIV) infection on hematopoietic stem and progenitor cells (HSPCs) and their differentiated progeny in the bone marrow and peripheral blood of rhesus macaques. METHODS: Rhesus macaques were administered alcohol or sucrose daily for a period of 3 months prior to intrarectal inoculation with 250 TCID50 of SIVmac251 . Bone marrow aspirates and blood samples were taken prior to and 2 weeks after SIV infection. Bone marrow cells (BMCs) were assessed using flow cytometric phenotyping for upstream HSPCs and for differentiated cells of the monocyte-granulocyte lineages. Likewise, cells were quantitated in peripheral blood. RESULTS: Of the bone marrow HSPCs, only the common lymphoid progenitor (CLP) was altered by alcohol administration pre-SIV (38 ± 9.4/10(6) BMCs vs. 226 ± 64.1/10(6) BMCs, sucrose vs. alcohol). Post-SIV, the frequency of CLPs in the bone marrow of alcohol-administered macaques decreased compared with the sucrose-administered macaques (107 ± 47.6/10(6) BMCs vs. 43 ± 16.3/10(6) BMCs). However, marrow mature cells of the monocyte lineage, specifically macrophages and osteoclast progenitors, were increased by both chronic alcohol administration and SIV infection (287% and 662%, respectively). As expected, mature cells such as granulocytes (polymorphonuclear cells), B cells, and CD4+ T cells in the peripheral blood were decreased by SIV infection (37 to 62% decline from preinfection), but not affected after 3 months of chronic alcohol administration. CONCLUSIONS: Chronic alcohol administration disrupts myelomonocytic development in the bone marrow during the early period of SIV infection promoting macrophage and osteoclast lineages. We predict this shift in CLP:macrophage/osteoclast balance creates an environment that favors bone resorption and immunosuppression.
Assuntos
Alcoolismo/patologia , Alcoolismo/fisiopatologia , Etanol/administração & dosagem , Etanol/efeitos adversos , Mielopoese/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Alcoolismo/sangue , Alcoolismo/complicações , Animais , Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Macaca mulatta , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/sangueRESUMO
Effects of tobacco smoke on hematologic derangements have received little attention. This study employed a mouse model of cigarette smoke exposure to explore the effects on bone marrow niche function. While lung cancer is the most widely studied consequence of tobacco smoke exposure, other malignancies, including leukemia, are associated with tobacco smoke exposure. Animals received cigarette smoke exposure for 6 h/day, 5 days/week for 9 months. Results reveal that the hematopoietic stem and progenitor cell (HSPC) pool size is reduced by cigarette smoke exposure. We next examined the effect of cigarette smoke exposure on one supporting cell type of the niche, the mesenchymal stromal cells (MSCs). Smoke exposure decreased the number of MSCs. Transplantation of naïve HSPCs into irradiated mice with cigarette smoke exposure yielded fewer numbers of engrafted HSPCs. This result suggests that smoke-exposed mice possess dysfunctional niches, resulting in abnormal hematopoiesis. Co-culture experiments using MSCs isolated from control or cigarette smoke-exposed mice with naïve HSPCs in vitro showed that MSCs from cigarette smoke-exposed mice generated marked expansion of naïve HSPCs. These data show that cigarette smoke exposure decreases in vivo MSC and HSC number and also increases pro-proliferative gene expression by cigarette smoke-exposed MSCs, which may stimulate HSPC expansion. These results of this investigation are clinically relevant to both bone marrow donors with a history of smoking and bone marrow transplant (BMT) recipients with a history of smoking.
RESUMO
In response to severe bacterial infection, bone marrow hematopoietic activity shifts toward promoting granulopoiesis. The underlying cell signaling mechanisms remain obscure. To study the role of Toll-like receptor 4 (TLR4)/stem cell antigen-1 (Sca-1) signaling in this process, bacteremia was induced in mice by intravenous injection of Escherichia coli. A subgroup of animals also received intravenous 5-bromo-2-deoxyuridine (BrdU). In a separate set of experiments, bone marrow lineage-negative (lin(-)) stem cell growth factor receptor-positive (c-kit(+)) Sca-1(-) cells containing primarily common myeloid progenitors were cultured in vitro without or with E. coli lipopolysaccharide (LPS). In genotypic background control mice, bacteremia significantly upregulated Sca-1 expression by lin(-) c-kit(+) cells, as reflected by a marked increase in BrdU-negative lin(-) c-kit(+) Sca-1(+) cells in the bone marrow. In mice with the TLR4 gene deletion, this bacteremia-evoked Sca-1 response was blocked. In vitro, LPS induced a dose-dependent increase in Sca-1 expression by cultured marrow lin(-) c-kit(+) Sca-1(-) cells. LPS-induced upregulation of Sca-1 expression was regulated at the transcriptional level. Inhibition of c-Jun N-terminal kinase/stress-activated protein kinase (JNK) activity with the specific inhibitor SP600125 suppressed LPS-induced upregulation of Sca-1 expression by marrow lin(-) c-kit(+) Sca-1(-) cells. Engagement of Sca-1 with anti-Sca-1 antibodies enhanced the expression of Sfpi1 spleen focus-forming virus (SFFV) proviral integration 1 (PU.1) in marrow lin(-) c-kit(+) Sca-1(-) cells cultured with LPS. Sca-1 null mice failed to maintain the marrow pool of granulopoietic cells following bacteremia. These results demonstrate that TLR4/Sca-1 signaling plays an important role in the regulation of hematopoietic precursor cell programming and their enhancement of granulocyte lineage commitment in response to E. coli bacteremia.
Assuntos
Antígenos Ly/metabolismo , Bacteriemia/patologia , Infecções por Escherichia coli/patologia , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Membrana/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos Ly/genética , Escherichia coli , Regulação da Expressão Gênica , Granulócitos/fisiologia , Células-Tronco Hematopoéticas/fisiologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/genéticaRESUMO
Enhancement of stem cell Ag-1 (Sca-1) expression by myeloid precursors promotes the granulopoietic response to bacterial infection. However, the underlying mechanisms remain unclear. ERK pathway activation strongly enhances proliferation of hematopoietic progenitor cells. In this study, we investigated the role of Sca-1 in promoting ERK-dependent myeloid lineage proliferation and the effects of alcohol on this process. Thirty minutes after i.p. injection of alcohol, mice received i.v. challenge with 5 × 10(7) Escherichia coli for 8 or 24 h. A subset of mice received i.v. BrdU injection 20 h after challenge. Bacteremia increased Sca-1 expression, ERK activation, and proliferation of myeloid and granulopoietic precursors. Alcohol administration suppressed this response and impaired granulocyte production. Sca-1 expression positively correlated with ERK activation and cell cycling, but negatively correlated with myeloperoxidase content in granulopoietic precursors. Alcohol intoxication suppressed ERK activation in granulopoietic precursors and proliferation of these cells during bacteremia. Granulopoietic precursors in Sca-1(-/-) mice failed to activate ERK signaling and could not increase granulomacrophagic CFU activity following bacteremia. These data indicate that Sca-1 expression promotes ERK-dependent myeloid cell proliferation during bacteremia. Suppression of this response could represent an underlying mechanism for developing myelosuppression in alcohol-abusing hosts with severe bacterial infection.
Assuntos
Intoxicação Alcoólica/imunologia , Antígenos Ly/imunologia , Bacteriemia/imunologia , Etanol/farmacologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/imunologia , Células Mieloides/metabolismo , Animais , Antígenos Ly/biossíntese , Antígenos Ly/metabolismo , Proliferação de Células/efeitos dos fármacos , Escherichia coli/imunologia , Escherichia coli/patogenicidade , Infecções por Escherichia coli/imunologia , Granulócitos/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Peroxidase/biossíntese , Transdução de SinaisRESUMO
OBJECTIVE: Granulocytopenia frequently occurs in alcohol abusers with severe bacterial infection, which strongly correlates with poor clinical outcome. Knowledge of the molecular mechanisms underlying the granulopoietic response to bacterial infection remains limited. This study investigated the involvement of stem cell antigen-1 expression by granulocyte lineage-committed progenitors in the granulopoietic response to septicemia and how alcohol affected this response. DESIGN: : Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Balb/c mice. INTERVENTIONS: Thirty mins after intraperitoneal injection of alcohol (20% ethanol in saline at 5 g of ethanol/kg) or saline, mice received an intravenous Escherichia coli challenge. MEASUREMENTS AND MAIN RESULTS: E. coli septicemia activated stem cell antigen-1 expression by marrow immature granulocyte differentiation antigen-1 precursors which correlated with an increase in proliferation, granulocyte macrophage colony-forming unit production, and expansion of this granulopoietic precursor cell pool. Acute alcohol treatment suppressed stem cell antigen-1 activation and inhibited the infection-induced increases in proliferation, granulocyte macrophage colony-forming unit production, and expansion the of immature granulocyte differentiation antigen-1 precursor cell population. Consequently, recovery of the marrow mature granulocyte differentiation antigen-1 cell population after E. coli challenge was impaired. Stem cell antigen-1 was induced in sorted granulocyte differentiation antigen-1, stem cell antigen-1' cells by lipopolysaccharide-stimulated C-Jun kinase activation that was also inhibited by alcohol. Furthermore, stem cell antigen-1 knockout mice failed to expand the marrow immature granulocyte differentiation antigen-1 cell pool and demonstrated fewer newly produced granulocytes in the circulation after the E. coli challenge. CONCLUSIONS: Alcohol suppresses the stem cell antigen-1 response in granulocyte lineage-committed precursors and restricts granulocyte production during septicemia, which may serve as a novel mechanism underlying impaired host defense in alcohol abusers.
Assuntos
Agranulocitose/induzido quimicamente , Antígenos Ly/fisiologia , Etanol/farmacologia , Proteínas de Membrana/fisiologia , Sepse/imunologia , Agranulocitose/metabolismo , Agranulocitose/fisiopatologia , Animais , Western Blotting , Células da Medula Óssea/fisiologia , Infecções por Escherichia coli/imunologia , Citometria de Fluxo , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. G-CSF produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27(Kip1) pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the cytochrome P450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27(Kip1) signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.
Assuntos
Regulação para Baixo/imunologia , Etanol/administração & dosagem , Fator Estimulador de Colônias de Granulócitos/fisiologia , Granulócitos/imunologia , Pneumonia Pneumocócica/imunologia , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/imunologia , Regulação para Cima/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Regulação para Baixo/efeitos dos fármacos , Granulócitos/microbiologia , Granulócitos/patologia , Hematopoese/efeitos dos fármacos , Hematopoese/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/patologia , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Alcohol abuse is associated with an increased incidence and severity of pneumonia. In both the general population and individuals consuming excess alcohol, Streptococcus pneumoniae is the most frequent lung infection pathogen. Alcoholic patients with pneumonia frequently present with granulocytopenia, which is predictive of increased mortality. The mechanisms underlying this impaired granulopoietic response to pneumococcal pneumonia have yet to be elucidated. METHODS: Acute alcohol intoxication was induced in mice 30 minutes before intrapulmonary infection with S. pneumoniae. Bone marrow, lung, and blood samples were collected. Bone marrow cells were also isolated from naïve mice and treated in vitro with plasma from mice infected with S. pneumoniae. RESULTS: Alcohol intoxication impaired the pneumococcal-induced increase in granulocyte recruitment into the alveolar space, decreased bacterial clearance from the lung, and increased mortality. Pneumococcal pneumonia significantly increased bone marrow lineage(-) c-Kit(+) Sca-1(+) (LKS) cell number and colony-forming unit-granulocytes and monocyte (CFU-GM) activity of these cells. Both enhanced proliferation of LKS cells and re-expression of Sca-1 surface protein on downstream progenitor cells bearing lineage(-) c-Kit(+) Sca-1(-) surface markers accounted for the expansion of marrow LSK cells during pneumonia. Alcohol intoxication impaired these 2 mechanisms of LKS cell population expansion and was associated with a relative granulocytopenia during pneumococcal lung infection. CONCLUSIONS: Alcohol inhibits the hematopoietic precursor cell response to pneumonia, which may serve as a mechanism underlying the granulocytopenia and impaired host defense in alcohol abusers with bacterial pneumonia.
Assuntos
Intoxicação Alcoólica/fisiopatologia , Células-Tronco Hematopoéticas/fisiologia , Pneumonia Pneumocócica/fisiopatologia , Agranulocitose/induzido quimicamente , Agranulocitose/complicações , Intoxicação Alcoólica/complicações , Animais , Antígenos Ly/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células-Tronco Hematopoéticas/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Pneumonia Pneumocócica/complicações , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/mortalidadeRESUMO
BACKGROUND: Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. METHODS: Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL-4 (10 ng/ml) and GM-CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL-1beta (18 ng), IL-6 (1800 U), TNF-alpha (18 ng), and PGE(2) (1.8 microg) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. RESULTS: EtOH-treated animals had significantly lower numbers of myeloid DCs (lineage-HLA-DR+CD11c+CD123-) in both the PBMCs and BMCs compared to controls (5,654 +/- 1,273/10(6) vs. 2,353 +/- 660/10(6) PBMCs and 503 +/- 34 vs. 195 +/- 44/10(6) BMCs). Under culture conditions, the number of lineage-HLA-DR+CD83+ cells was low in control wells (0.38 +/- 0.08%). Alcohol inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in iDC wells (2.30 +/- 0.79% vs. 5.73 +/- 1.40%). Alcohol also inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in mature DC wells (1.23 +/- 0.15% vs. 4.13 +/- 0.62%). CONCLUSIONS: Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T-cell expansion.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Depressores do Sistema Nervoso Central/toxicidade , Células Dendríticas/efeitos dos fármacos , Etanol/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Células Cultivadas , Citometria de Fluxo , Macaca mulatta , Proteínas de Membrana/biossíntese , Monócitos/efeitos dos fármacosRESUMO
Alcohol abuse predisposes the host to bacterial infections. In response to bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for enhancing host defense. This study investigated the hematopoietic precursor cell response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in BALB/c mice 30 min before initiation of Escherichia coli bacteremia. Bacteremia caused a significant increase in the number of bone marrow lineage (lin(-))-c-kit(+)Sca-1(+) cells. Marrow lin(-)c-kit(+)Sca-1(+) cells isolated from bacteremic mice showed an increase in CFU-granulocyte/macrophage activity compared with controls. In addition to enhanced proliferation of lin(-)c-kit(+)Sca-1(+) cells as reflected by BrdU incorporation, phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells primarily accounted for the rapid increase in marrow lin(-)c-kit(+)Sca-1(+) cells following bacteremia. Bacteremia increased plasma concentration of TNF-alpha. Culture of marrow lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells with murine rTNF-alpha for 24 h caused a dose-dependent increase in conversion of these cells to lin(-)c-kit(+)Sca-1(+) cells. Sca-1 mRNA expression by the cultured cells was also up-regulated following TNF-alpha stimulation. Acute alcohol intoxication inhibited the increase in the number of lin(-)c-kit(+)Sca-1(+) cells in the bone marrow after E. coli infection. Alcohol impeded the increase in BrdU incorporation into marrow lin(-)c-kit(+)Sca-1(+) cells in response to bacteremia. Alcohol also suppressed the plasma TNF-alpha response to bacteremia and inhibited TNF-alpha-induced phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells in vitro. These data show that alcohol inhibits the hematopoietic precursor cell response to bacteremia, which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections.
Assuntos
Intoxicação Alcoólica/imunologia , Bacteriemia/imunologia , Linhagem da Célula/imunologia , Infecções por Escherichia coli/imunologia , Células-Tronco Hematopoéticas/imunologia , Terapia de Imunossupressão , Proteínas de Membrana/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Intoxicação Alcoólica/microbiologia , Intoxicação Alcoólica/patologia , Animais , Antígenos Ly/biossíntese , Antígenos Ly/fisiologia , Bacteriemia/microbiologia , Bacteriemia/patologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Técnicas de Cultura de Células , Diferenciação Celular/imunologia , Citocinas/sangue , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Imunofenotipagem , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-kit/biossínteseRESUMO
Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kit-positive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1alpha, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.
Assuntos
Vasos Coronários/fisiologia , Mioblastos Cardíacos/fisiologia , Neovascularização Fisiológica , Regeneração , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Quimiocina CXCL12/metabolismo , Vasos Coronários/citologia , Células Endoteliais/citologia , Feminino , Fator de Crescimento de Hepatócito/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/transplante , Isquemia Miocárdica/metabolismo , Miócitos de Músculo Liso/citologia , Proteínas Proto-Oncogênicas c-kit/análise , Ratos , Ratos Endogâmicos F344 , Transplante de Células-Tronco , Células-Tronco/química , Células-Tronco/efeitos dos fármacosRESUMO
The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.