RESUMO
Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ-induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pathways. Interestingly, mTORC-1 regulated SM-induced cell death independent of M1 differentiation. In contrast, SM-induced cell death in polarized M1 macrophages is regulated by the mTORC-2 pathway. Moreover, SM-induced cell death is regulated by cellular IAP (cIAP)-2, receptor-interacting protein kinase (RIPK)-1, and RIPK-3 degradation through mTORC activation during differentiation into M1 macrophages and in polarized M1 macrophages. In contrast to cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-α, as well as the NF-κB pathway. Collectively, selective induction of cell death in human M1 macrophages by SMs may be mediated by cIAP-2, RIPK-1, and RIPK-3 degradation through mTORC activation. Moreover, blocking cIAP-1/2, mTORC, or IFN regulatory factor-1 may represent a promising therapeutic strategy to control M1-associated diseases.
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Artrite Reumatoide/imunologia , Biomimética/métodos , Doenças Inflamatórias Intestinais/imunologia , Macrófagos/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas Reguladoras de Apoptose/genética , Morte Celular , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Fator Regulador 1 de Interferon/metabolismo , Proteínas Mitocondriais/genética , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Células Th1/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Hypoxia-inducible factor-1α (HIF-1α) is an important regulator of glucose metabolism and inflammatory cytokine production in innate immune responses. Viruses modulate HIF-1α to support viral replication and the survival of infected cells, but it is unclear if this transcription factor also plays an important role in regulating antiviral immune responses. In this study, we found that short and long dsRNA differentially engage TLR3, inducing distinct levels of proinflammatory cytokine production (TNF-α and IL-6) in bone marrow-derived macrophages from C57BL/6 mice. These responses are associated with differential accumulation of HIF-1α, which augments NF-κB activation. Unlike TLR4 responses, increased HIF-1α following TLR3 engagement is not associated with significant alterations in glycolytic activity and was more pronounced in low glucose conditions. We also show that the mechanisms supporting HIF-1α stabilization may differ following stimulation with short versus long dsRNA and that pyruvate kinase M2 and mitochondrial reactive oxygen species play a central role in these processes. Collectively, this work suggests that HIF-1α may fine-tune proinflammatory cytokine production during early antiviral immune responses, particularly when there is limited glucose availability or under other conditions of stress. Our findings also suggest we may be able to regulate the magnitude of proinflammatory cytokine production during antiviral responses by targeting proteins or molecules that contribute to HIF-1α stabilization.
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Citocinas/biossíntese , Glucose/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Macrófagos/imunologia , Ácidos Nucleicos/imunologia , Receptor 3 Toll-Like/imunologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/imunologiaRESUMO
IFN-γ, a proinflammatory cytokine produced primarily by T cells and NK cells, activates macrophages and engages mechanisms to control pathogens. Although there is evidence of IFN-γ production by murine macrophages, IFN-γ production by normal human macrophages and their subsets remains unknown. Herein, we show that human M1 macrophages generated by IFN-γ and IL-12- and IL-18-stimulated monocyte-derived macrophages (M0) produce significant levels of IFN-γ. Further stimulation of IL-12/IL-18-primed macrophages or M1 macrophages with agonists for TLR-2, TLR-3, or TLR-4 significantly enhanced IFN-γ production in contrast to the similarly stimulated M0, M2a, M2b, and M2c macrophages. Similarly, M1 macrophages generated from COVID-19-infected patients' macrophages produced IFN-γ that was enhanced following LPS stimulation. The inhibition of M1 differentiation by Jak inhibitors reversed LPS-induced IFN-γ production, suggesting that differentiation with IFN-γ plays a key role in IFN-γ induction. We subsequently investigated the signaling pathway(s) responsible for TLR-4-induced IFN-γ production in M1 macrophages. Our results show that TLR-4-induced IFN-γ production is regulated by the ribosomal protein S6 kinase (p70S6K) through the activation of PI3K, the mammalian target of rapamycin complex 1/2 (mTORC1/2), and the JNK MAPK pathways. These results suggest that M1-derived IFN-γ may play a key role in inflammation that may be augmented following bacterial/viral infections. Moreover, blocking the mTORC1/2, PI3K, and JNK MAPKs in macrophages may be of potential translational significance in preventing macrophage-mediated inflammatory diseases.
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Interferon gama/biossíntese , Macrófagos/efeitos dos fármacos , Poli I-C/farmacologia , COVID-19/imunologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/imunologia , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/imunologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/agonistasRESUMO
BACKGROUND: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. METHODS: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments. RESULTS: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. CONCLUSIONS: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.
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Apoptose/genética , Proteínas de Fluorescência Verde , Infecções por HIV , Macrófagos , RNA Interferente Pequeno , Linfócitos T CD4-Positivos/virologia , Estudo de Associação Genômica Ampla , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Linfócitos TRESUMO
Small interfering RNA (siRNA) is a critical loss-of-function tool for elucidating the role of genes in biomedical studies. The effective use of siRNA needs transfection technology that delivers siRNA into the correct location of target cells, especially those which are extremely difficult to transfect. Macrophages, which play an important role in the pathogenesis of many diseases, are known to be extremely hard to transfect. Thus, to elucidate the functions of genes in human macrophage biology, it is essential to devise technology for efficient siRNA transfection. However, a fast and efficient method for siRNA transfection in primary human macrophages has not been reported. The siRNA transfection is a tug-of-war between transfection rate and cytotoxicity. A higher transfection rate is generally accompanied with increased cytotoxicity, therefore, choosing a transfection reagent that limits cell death while maintain a desirable transfection rate is important. In this study, we employed auto-analysis function of the IncuCyte® to devise a fast and cost-saving technology for efficient transfection of adherent cells and particularly human macrophages. We show that DharmaFECT3 transfection reagent from Dharmacon was the most efficient in transfecting primary human monocyte-derived macrophages and PMA-differentiated U937 cells, whereas other transfection reagents tested were cytotoxic. This method exhibited approximately 85% transfection efficiency in human macrophages. Moreover, siRNA silencing of Bax with this technique effectively protected primary human macrophages and PMA-differentiated U937 cells against Resveratrol-induced cell death. In addition, this method inherently takes the balance between transfection rate and cytotoxicity of siRNA transfection reagents into consideration.
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Apoptose/efeitos dos fármacos , Apoptose/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , Resveratrol/farmacologia , Proteína X Associada a bcl-2/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Dosagem de Genes , Expressão Gênica , Humanos , Macrófagos/citologia , TransfecçãoRESUMO
With the advent of antiretroviral therapy (ART), HIV-infected individuals are now living longer and healthier lives. However, ART does not completely restore health and treated individuals are experiencing increased rates of noncommunicable diseases such as dyslipidemia, insulin resistance, type 2 diabetes, cardiovascular disease, and nonalcoholic fatty liver disease. While it is well known that persistent immune activation and inflammation contribute to the development of these comorbid diseases, the mechanisms underlying this chronic activation remain incompletely understood. In this review, we will discuss emerging evidence that suggests that alterations in cellular metabolism may play a central role in driving this immune dysfunction in HIV patients on ART.
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Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Infecções por HIV/metabolismo , Humanos , Inflamação/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/imunologia , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
Emerging evidence suggests that cellular metabolism plays a critical role in regulating immune activation. Alterations in energy and lipid and amino acid metabolism have been shown to contribute to type I interferon (IFN) responses in macrophages, but the relationship between metabolic reprogramming and the establishment of early antiviral function remains poorly defined. Here, we used transcriptional profiling datasets to develop global metabolic signatures associated with early IFN-α responses in two primary macrophage model systems: mouse bone marrow-derived macrophages (BMM) and human monocyte-derived macrophages (MDM). Short-term stimulation with IFN-α (<4 hours) was associated with significant metabolic rewiring, with >500 metabolic genes altered in mouse and human macrophage models. Pathway and network analysis identified alterations in genes associated with cellular bioenergetics, cellular oxidant status, cAMP/AMP and cGMP/GMP ratios, branched chain amino acid catabolism, cell membrane composition, fatty acid synthesis, and ß-oxidation as key features of early IFN-α responses. These changes may have important implications for initial establishment of antiviral function in these cells.
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Interferon-alfa/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Humanos , Interferon Tipo I/farmacologia , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Intestinal macrophages are key regulators of inflammatory responses to the gut microbiome and play a central role in maintaining tissue homeostasis and epithelial integrity. However, little is known about the role of these cells in HIV infection, a disease fuelled by intestinal inflammation, a loss of epithelial barrier function and increased microbial translocation (MT). METHODS: Phenotypic and functional characterization of intestinal macrophages was performed for 23 African AIDS patients with chronic diarrhea and/or weight loss and 11 HIV-negative Africans with and without inflammatory bowel disease (IBD). AIDS patients were treated with cotrimoxazole for the prevention of opportunistic infections (OIs). Macrophage phenotype was assessed by flow cytometry and immuno-histochemistry (IHC); production of proinflammatory mediators by IHC and Qiagen PCR Arrays; in vitro secretion of cytokines by the Bio-Plex Suspension Array System. Statistical analyses were performed using Spearman's correlation and Wilcoxon matched-pair tests. Results between groups were analyzed using the Kruskal-Wallis with Dunn's post-test and the Mann-Whitney U tests. RESULTS: None of the study participants had evidence of enteric co-infections as assessed by stool analysis and histology. Compared to healthy HIV-negative controls, the colon of AIDS patients was highly inflamed with increased infiltration of inflammatory cells and increased mRNA expression of proinflammatory cytokine (tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IFN-γ, and IL-18), chemokines (chemokine (C-C motif) ligand (CCL)2 and chemokine (C-X-C) motif ligand (CXCL)10) and transcription factors (TNF receptor-associated factor (TRAF)6 and T-box (TXB)21). IHC revealed significant co-localization of TNF-α and IL-1ß with CD68(+) cells. As in IBD, HIV was associated with a marked increase in macrophages expressing innate response receptors including CD14, the co-receptor for lipopolysaccharide (LPS). The frequency of CD14(+) macrophages correlated positively with plasma LPS, a marker of MT. Total unfractionated mucosal mononuclear cells (MMC) isolated from the colon of AIDS patients, but not MMC depleted of CD14(+) cells, secreted increased levels of proinflammatory cytokines ex vivo in response to LPS. CONCLUSIONS: Intestinal macrophages, in the absence of overt OIs, play an important role in driving persistent inflammation in HIV patients with late-stage disease and diarrhea. These results suggest intensified treatment strategies that target inflammatory processes in intestinal macrophages may be highly beneficial in restoring the epithelial barrier and limiting MT in HIV-infected patients.
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Síndrome da Imunodeficiência Adquirida/patologia , Colo/imunologia , Citocinas/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/imunologia , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Quimiocinas/genética , Quimiocinas/metabolismo , Coinfecção/patologia , Colo/microbiologia , Colo/patologia , Citocinas/genética , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Inflamação , Doenças Inflamatórias Intestinais/patologia , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Mucosa/metabolismo , RNA Mensageiro/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Fator de Necrose Tumoral alfaRESUMO
Few studies have examined immune activation profiles in patients with advanced HIV-1 subtype C infection or assessed their potential to predict responsiveness to HAART. BioPlex, ELISA, and nephelometric procedures were used to measure plasma levels of inflammatory biomarkers in HIV-1 subtype C-infected patients sampled before and after 6 months of successful HAART (n = 20); in patients failing HAART (n = 30); and in uninfected controls (n = 8). Prior to HAART, CXCL9, CXCL10, ß 2M, sTNF-R1, TGF- ß 1, IFN- γ , IL-6, TNF, and sCD14 were significantly elevated in HIV-1-infected patients compared to controls (P < 0.01). All of these markers, with the exception of sTNF-R1, were also elevated in patients failing HAART (P < 0.05). The persistently elevated levels of CXCL9, CXCL10, and ß 2M in patients failing therapy in the setting of a marked reduction in these markers in patients on successful HAART suggest that they may be useful not only to monitor immune activation during HAART, but also to distinguish between good and poor responders. In the case of sCD14 and TGF- ß 1, the levels of these biomarkers remained persistently elevated despite HAART-induced virological suppression, a finding that is consistent with ongoing monocyte-macrophage activation, underscoring a potential role for adjuvant anti-inflammatory therapy.
Assuntos
Biomarcadores/sangue , Infecções por HIV/sangue , HIV-1/efeitos dos fármacos , Adulto , Terapia Antirretroviral de Alta Atividade , Quimiocina CXCL10/sangue , Quimiocina CXCL9/sangue , Feminino , HIV-1/patogenicidade , Humanos , Interferon gama/sangue , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Fator de Crescimento Transformador beta1/sangue , Adulto JovemRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection is associated with a massive depletion of intestinal CD4(+) T cells that is only partially reversed by combination antiretroviral therapy (cART). Here, we assessed the ability of nucleoside reverse-transcriptase inhibitor/nonnucleoside reverse-transcriptase inhibitor treatment to restore the CD4(+) T-cell populations in the intestine of South African patients with AIDS. METHODS: Thirty-eight patients with advanced HIV-1 infection who had chronic diarrhea (duration, >4 weeks) and/or unintentional weight loss (>10% decrease from baseline) of uncertain etiology were enrolled. Blood specimens were collected monthly, and gastrointestinal tract biopsy specimens were collected before cART initiation (from the duodenum, jejunum, ileum, and colon), 3 months after cART initiation (from the duodenum), and 6 months after cART initiation (from the duodenum and colon). CD4(+), CD8(+), and CD38(+)CD8(+) T cells were quantified by flow cytometry and immunohistochemistry analyses, and the HIV-1 RNA load was determined by the Nuclisens assay. RESULTS: CD4(+) T-cell and HIV-1 RNA levels were significantly lower, whereas CD8(+) T-cell levels, including activated CD38(+)CD8(+) T cell levels, were higher in the duodenum and jejunum, compared with the colon. After 6 months of cART, a significant but incomplete recovery of CD4(+) T cells was detected in the colon and peripheral blood but not in the duodenum. Failed restoration of the CD4(+) T-cell count in the duodenum was associated with nonspecific enteritis and CD8(+) T-cell activation. CONCLUSIONS: Strategies that target inflammation and immune activation in the small intestine may be required to expedite CD4(+) T-cell recovery and improve therapeutic outcomes.
Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Mucosa Intestinal/imunologia , Intestino Grosso/imunologia , Intestino Delgado/imunologia , Adulto , Terapia Antirretroviral de Alta Atividade/métodos , Biópsia , Linfócitos T CD8-Positivos/imunologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , África do Sul , Resultado do Tratamento , Adulto JovemRESUMO
We report the draft genome sequence and antimicrobial resistance gene profile of a methicillin-resistant Staphylococcus aureus (MRSA) clinical isolate recovered from a chronic pressure injury wound infection of an adult female patient. The draft genome sequence included a 2.86-Mb chromosome, which was accompanied by a 27-kb plasmid containing blaZ.
RESUMO
Oxidizing agents are low-molecular-weight molecules that oxidize other substances by accepting electrons from them. They include reactive oxygen species (ROS), such as superoxide anions (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (HO-), and reactive chlorine species (RCS) including sodium hypochlorite (NaOCl) and its active ingredient hypochlorous acid (HOCl), and chloramines. Bacteria encounter oxidizing agents in many different environments and from diverse sources. Among them, they can be produced endogenously by aerobic respiration or exogenously by the use of disinfectants and cleaning agents, as well as by the mammalian immune system. Furthermore, human activities like industrial effluent pollution, agricultural runoff, and environmental activities like volcanic eruptions and photosynthesis are also sources of oxidants. Despite their antimicrobial effects, bacteria have developed many mechanisms to resist the damage caused by these toxic molecules. Previous research has demonstrated that growing as a biofilm particularly enhances bacterial survival against oxidizing agents. This review aims to summarize the current knowledge on the resistance mechanisms employed by bacterial biofilms against ROS and RCS, focussing on the most important mechanisms, including the formation of biofilms in response to oxidative stressors, the biofilm matrix as a protective barrier, the importance of detoxifying enzymes, and increased protection within multi-species biofilm communities. Understanding the complexity of bacterial responses against oxidative stress will provide valuable insights for potential therapeutic interventions and biofilm control strategies in diverse bacterial species.
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Pseudomonas aeruginosa is well-known for its antimicrobial resistance and the ability to survive in harsh environmental conditions due to an abundance of resistance mechanisms, including the formation of biofilms and the production of exopolysaccharides. Exopolysaccharides are among the major components of the extracellular matrix in biofilms and aggregates of P. aeruginosa. Although their contribution to antibiotic resistance has been previously shown, their roles in resistance to oxidative stressors remain largely elusive. Here, we studied the function of the exopolysaccharides Psl and Pel in the resistance of P. aeruginosa to the commonly used disinfectants and strong oxidizing agents NaOCl and H2O2. We observed that the simultaneous inactivation of Psl and Pel in P. aeruginosa PAO1 mutant strain ∆pslA pelF resulted in a significant increase in susceptibility to both NaOCl and H2O2. Further analyses revealed that Pel is more important for oxidative stress resistance in P. aeruginosa and that the form of Pel (i.e., cell-associated or cell-free) did not affect NaOCl susceptibility. Additionally, we show that Psl/Pel-negative strains are protected against oxidative stress in co-culture biofilms with P. aeruginosa PAO1 WT. Taken together, our results demonstrate that the EPS matrix and, more specifically, Pel exhibit protective functions against oxidative stressors such as NaOCl and H2O2 in P. aeruginosa. IMPORTANCE: Biofilms are microbial communities of cells embedded in a self-produced polymeric matrix composed of polysaccharides, proteins, lipids, and extracellular DNA. Biofilm bacteria have been shown to possess unique characteristics, including increased stress resistance and higher antimicrobial tolerance, leading to failures in bacterial eradication during chronic infections or in technical settings, including drinking and wastewater industries. Previous studies have shown that in addition to conferring structure and stability to biofilms, the polysaccharides Psl and Pel are also involved in antibiotic resistance. This work provides evidence that these biofilm matrix components also contribute to the resistance of Pseudomonas aeruginosa to oxidative stressors including the widely used disinfectant NaOCl. Understanding the mechanisms by which bacteria escape antimicrobial agents, including strong oxidants, is urgently needed in the fight against antimicrobial resistance and will help in developing new strategies to eliminate resistant strains in any environmental, industrial, and clinical setting.
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BACKGROUND: Metabolic abnormalities are common in HIV-infected individuals on antiretroviral therapy (ART), but the biochemical details and underlying mechanisms of these disorders have not been defined. METHODS: Untargeted metabolomic profiling of plasma was performed for 32 HIV patients with low nadir CD4 counts (<300 cells/ul) on protease inhibitor (PI)-based ART and 20 healthy controls using liquid or gas chromatography and mass spectrometry. Effects of Hepatitis C (HCV) co-infection and relationships between altered lipid metabolites and markers of inflammation, microbial translocation, and hepatic function were examined. Unsupervised hierarchical clustering, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), Random forest, pathway mapping, and metabolite set enrichment analysis (MSEA) were performed using dChip, Metaboanalyst, and MSEA software. RESULTS: A 35-metabolite signature mapping to lipid, amino acid, and nucleotide metabolism distinguished HIV patients with advanced disease on PI-based ART from controls regardless of HCV serostatus (p<0.05, false discovery rate (FDR)<0.1). Many altered lipids, including bile acids, sulfated steroids, polyunsaturated fatty acids, and eicosanoids, were ligands of nuclear receptors that regulate metabolism and inflammation. Distinct clusters of altered lipids correlated with markers of inflammation (interferon-α and interleukin-6), microbial translocation (lipopolysaccharide (LPS) and LPS-binding protein), and hepatic function (bilirubin) (p<0.05). Lipid alterations showed substantial overlap with those reported in non-alcoholic fatty liver disease (NALFD). Increased bile acids were associated with noninvasive markers of hepatic fibrosis (FIB-4, APRI, and YKL-40) and correlated with acylcarnitines, a marker of mitochondrial dysfunction. CONCLUSIONS: Lipid alterations in HIV patients receiving PI-based ART are linked to markers of inflammation, microbial translocation, and hepatic function, suggesting that therapeutic strategies attenuating dysregulated innate immune activation and hepatic dysfunction may be beneficial for prevention and treatment of metabolic disorders in HIV patients.
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Fígado Gorduroso/metabolismo , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Inflamação/metabolismo , Transtornos do Metabolismo dos Lipídeos/metabolismo , Adulto , Ácidos e Sais Biliares/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Estudos de Coortes , Fígado Gorduroso/sangue , Fígado Gorduroso/virologia , Feminino , Infecções por HIV/enzimologia , Hepatite C/enzimologia , Hepatite C/metabolismo , Hepatite C/virologia , Humanos , Inflamação/sangue , Inflamação/complicações , Análise dos Mínimos Quadrados , Transtornos do Metabolismo dos Lipídeos/sangue , Transtornos do Metabolismo dos Lipídeos/virologia , Lipídeos/sangue , Masculino , Redes e Vias Metabólicas , Metaboloma , Metabolômica , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica , Análise de Componente Principal , Inibidores de Proteases/uso terapêuticoRESUMO
Several studies over the last decade have identified intimate links between cellular metabolism and macrophage function. Metabolism has been shown to both drive and regulate macrophage function by producing bioenergetic and biosynthetic precursors as well as metabolites (and other bioactive molecules) that regulate gene expression and signal transduction. Many studies have focused on lipopolysaccharide-induced reprogramming, assuming that it is representative of most inflammatory responses. However, emerging evidence suggests that diverse pathogen-associated molecular patterns (PAMPs) are associated with unique metabolic profiles, which may drive pathogen specific immune responses. Further, these metabolic pathways and processes may act as a rheostat to regulate the magnitude of an inflammatory response based on the biochemical features of the local microenvironment. In this review, we will discuss recent work examining the relationship between cellular metabolism and macrophage responses to viral PAMPs and describe how these processes differ from lipopolysaccharide-associated responses. We will also discuss how an improved understanding of the specificity of these processes may offer new insights to fine-tune macrophage function during viral infections or when using viral PAMPs as therapeutics.
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Antivirais , Viroses , Humanos , Imunidade Inata , Moléculas com Motivos Associados a Patógenos/metabolismo , LipopolissacarídeosRESUMO
Significance: Altered lipid metabolism of cancer cells has been implicated in increased radiation resistance. A better understanding of this phenomenon may lead to improved radiation treatment planning. Stimulated Raman scattering (SRS) microscopy enables label-free and quantitative imaging of cellular lipids but has never been applied in this domain. Aim: We sought to investigate the radiobiological response in human breast cancer MCF7 cells using SRS microscopy, focusing on how radiation affects lipid droplet (LD) distribution and cellular morphology. Approach: MCF7 breast cancer cells were exposed to either 0 or 30 Gy (X-ray) ionizing radiation and imaged using a spectrally focused SRS microscope every 24 hrs over a 72-hr time period. Images were analyzed to quantify changes in LD area per cell, lipid and protein content per cell, and cellular morphology. Cell viability and confluency were measured using a live cell imaging system while radiation-induced lipid peroxidation was assessed using BODIPY C11 staining and flow cytometry. Results: The LD area per cell and total lipid and protein intensities per cell were found to increase significantly for irradiated cells compared to control cells from 48 to 72 hrs post irradiation. Increased cell size, vacuole formation, and multinucleation were observed as well. No significant cell death was observed due to irradiation, but lipid peroxidation was found to be greater in the irradiated cells than control cells at 72 hrs. Conclusions: This pilot study demonstrates the potential of SRS imaging for investigating ionizing radiation-induced changes in cancer cells without the use of fluorescent labels.
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Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/diagnóstico por imagem , Projetos Piloto , Microscopia Óptica não Linear , Radiação Ionizante , Lipídeos , Análise Espectral Raman/métodosRESUMO
The capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines and other extracellular stimuli. In this study, we demonstrate that cytokine-induced polarization of human monocyte-derived macrophage (MDM) into either classical (M1) or alternatively activated (M2a) MDM is associated with a reduced capacity to support productive CCR5-dependent (R5) HIV-1 infection. M1 polarization was associated with a significant down-regulation of CD4 receptors, increased secretion of CCR5-binding chemokines (CCL3, CCL4, and CCL5), and a >90% decrease in HIV-1 DNA levels 48-h postinfection, suggesting that the inhibition occurred at an early preintegration step in the viral life cycle. In contrast, M2a polarization had no effect on either HIV-1 DNA or protein expression levels, indicating that inhibition occurred at a late/postintegration level in the viral life cycle. M2a inhibition was sustained for up to 72-h postinfection, whereas M1-effects were more short-lived. Most phenotypic and functional changes were fully reversible 7 days after removal of the polarizing stimulus, and a reciprocal down-regulation of M1-related chemokines and cytokines was observed in M2a MDM and vice versa. Since reversion to a nonpolarized MDM state was associated with a renewed capacity to support HIV replication to control levels, M1/M2a polarization may represent a mechanism that allows macrophages to cycle between latent and productive HIV-1 infection.
Assuntos
Diferenciação Celular/imunologia , HIV-1/fisiologia , Ativação de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/virologia , Replicação Viral/fisiologia , Antígenos CD4/biossíntese , Antígenos CD4/imunologia , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Interferon gama/imunologia , Macrófagos/imunologia , Receptores CCR5/biossíntese , Receptores CCR5/imunologia , Receptores CXCR4/biossíntese , Receptores CXCR4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Macrophages are terminally differentiated cells of the mononuclear phagocyte system that also encompasses dendritic cells, circulating blood monocytes, and committed myeloid progenitor cells in the bone marrow. Both macrophages and their monocytic precursors can change their functional state in response to microenvironmental cues exhibiting a marked heterogeneity. However, there are still uncertainties regarding distinct expression patterns of surface markers that clearly define macrophage subsets, particularly in the case of human macrophages. In addition to their tissue distribution, macrophages can be functionally polarized into M1 (proinflammatory) and M2 (alternatively activated) as well as regulatory cells in response to both exogenous infections and solid tumors as well as by systems biology approaches.
Assuntos
Polaridade Celular , Ativação de Macrófagos , Macrófagos/imunologia , Animais , Antígenos de Superfície/imunologia , Diferenciação Celular , Microambiente Celular , Citocinas/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Imunidade Inata , Macrófagos/patologia , Macrófagos/virologia , Neoplasias/imunologia , Neoplasias/patologia , Biologia de SistemasRESUMO
BACKGROUND: Microbial translocation contributes to immune activation and disease progression during chronic human immunodeficiency virus type 1 (HIV-1) infection. However, its role in the African AIDS epidemic remains controversial. Here, we investigated the relationship between markers of monocyte activation, plasma lipopolysaccharide (LPS), and HIV-1 RNA in South Africans prioritized to receive combination antiretroviral therapy (cART). METHODS: Ten HIV-1-negative African controls and 80 HIV-1-infected patients with CD4 T cell counts <200 cells/microL were sampled prior to (n=60) or during (n=20) receipt of effective cART. Viral load was measured by Nuclisens; LPS by the Limulus amoebocyte lysate assay; monocyte and T cell subsets by flow cytometry; and soluble CD14, cytokines, and chemokines by enzyme-linked immunosorbent assay and customized Bio-Plex plates. RESULTS: Three distinct sets of markers were identified. CCL2, CXCL10, and CD14(+)CD16(+) monocyte levels were positively correlated with HIV-1 viremia. This finding, together with cART-induced normalization of these markers, suggests that their upregulation was driven by HIV-1. Plasma interleukin-6 was associated with the presence of opportunistic coinfections. Soluble CD14 and tumor necrosis factor were linked to plasma LPS levels and, as observed for LPS, remained elevated in patients receiving effective cART. CONCLUSIONS: Microbial translocation is a major force driving chronic inflammation in HIV-infected Africans receiving cART. Prevention of monocyte activation may be especially effective at enhancing therapeutic outcomes.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Lipopolissacarídeos/sangue , Monócitos/imunologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Adulto , Contagem de Linfócito CD4 , Quimioterapia Combinada , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , Carga ViralRESUMO
Pseudomonas aeruginosa is a Gram-negative environmental and human opportunistic pathogen highly adapted to many different environmental conditions. It can cause a wide range of serious infections, including wounds, lungs, the urinary tract, and systemic infections. The high versatility and pathogenicity of this bacterium is attributed to its genomic complexity, the expression of several virulence factors, and its intrinsic resistance to various antimicrobials. However, to thrive and establish infection, P. aeruginosa must overcome several barriers. One of these barriers is the presence of oxidizing agents (e.g., hydrogen peroxide, superoxide, and hypochlorous acid) produced by the host immune system or that are commonly used as disinfectants in a variety of different environments including hospitals. These agents damage several cellular molecules and can cause cell death. Therefore, bacteria adapt to these harsh conditions by altering gene expression and eliciting several stress responses to survive under oxidative stress. Here, we used PubMed to evaluate the current knowledge on the oxidative stress responses adopted by P. aeruginosa. We will describe the genes that are often differently expressed under oxidative stress conditions, the pathways and proteins employed to sense and respond to oxidative stress, and how these changes in gene expression influence pathogenicity and the virulence of P. aeruginosa. Understanding these responses and changes in gene expression is critical to controlling bacterial pathogenicity and developing new therapeutic agents.