RESUMO
For over 50 years, patients with drug-sensitive and drug-resistant tuberculosis have undergone long, arduous, and complex treatment processes with several antimicrobials. With the prevalence of drug-resistant strains on the rise and new therapies for tuberculosis urgently required, we assessed whether manipulating iron levels in macrophages infected with mycobacteria offered some insight into improving current antimicrobials that are used to treat drug-resistant tuberculosis. We investigated if the iron chelator, desferrioxamine, can support the function of human macrophages treated with an array of second-line antimicrobials, including moxifloxacin, bedaquiline, amikacin, clofazimine, linezolid and cycloserine. Primary human monocyte-derived macrophages were infected with Bacillus Calmette-Guérin (BCG), which is pyrazinamide-resistant, and concomitantly treated for 5 days with desferrioxamine in combination with each one of the second-line tuberculosis antimicrobials. Our data indicate that desferrioxamine used as an adjunctive treatment to bedaquiline significantly reduced the bacterial load in human macrophages infected with BCG. Our findings also reveal a link between enhanced bactericidal activity and increases in specific cytokines, as the addition of desferrioxamine increased levels of IFN-γ, IL-6, and IL-1ß in BCG-infected human monocyte-derived macrophages (hMDMs) treated with bedaquiline. These results provide insight, and an in vitro proof-of-concept, that iron chelators may prove an effective adjunctive therapy in combination with current tuberculosis antimicrobials.
Assuntos
Antituberculosos/farmacologia , Desferroxamina/farmacologia , Diarilquinolinas/farmacologia , Quelantes de Ferro/farmacologia , Ferro/metabolismo , Macrófagos/efeitos dos fármacos , Mycobacterium bovis/efeitos dos fármacos , Amicacina/farmacologia , Carga Bacteriana/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Clofazimina/farmacologia , Ciclosserina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Sinergismo Farmacológico , Expressão Gênica , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Linezolida/farmacologia , Macrófagos/imunologia , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana , Moxifloxacina/farmacologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/metabolismo , Cultura Primária de Células , Pirazinamida/farmacologiaRESUMO
Tuberculosis (TB) remains a global health challenge. Patients with drug-sensitive and drug-resistant TB undergo long, arduous, and complex treatment regimens, often involving multiple antimicrobials. While these drugs were initially implemented based on their bactericidal effects, some studies show that TB antimicrobials can also directly affect cells of the immune system, altering their immune function. As use of these antimicrobials has been the mainstay of TB therapy for over fifty years now, it is more important than ever to understand how these antimicrobials affect key pathways of the immune system. One such central pathway, which underpins the immune response to a variety of infections, is immunometabolism, namely glycolysis and oxidative phosphorylation (OXPHOS). We hypothesise that in addition to their direct bactericidal effect on Mycobacterium tuberculosis (Mtb), current TB antimicrobials can modulate immunometabolic profiles and alter mitochondrial function in primary human macrophages. Human monocyte-derived macrophages (hMDMs) were differentiated from PBMCs isolated from healthy blood donors, and treated with four first-line and six second-line TB antimicrobials three hours post stimulation with either iH37Rv-Mtb or lipopolysaccharide (LPS). 24 h post stimulation, baseline metabolism and mitochondrial function were determined using the Seahorse Extracellular Flux Analyser. The effect of these antimicrobials on cytokine and chemokine production was also assayed using Meso Scale Discovery Multi-Array technology. We show that some of the TB antimicrobials tested can significantly alter OXPHOS and glycolysis in uninfected, iH37Rv-Mtb, and LPS-stimulated hMDMs. We also demonstrate how these antimicrobial-induced immunometabolic effects are linked with alterations in mitochondrial function. Our results show that TB antimicrobials, specifically clofazimine, can modify host immunometabolism and mitochondrial function. Moreover, clofazimine significantly increased the production of IL-6 in human macrophages that were stimulated with iH37Rv-Mtb. This provides further insight into the use of some of these TB antimicrobials as potential host-directed therapies in patients with early and active disease, which could help to inform TB treatment strategies in the future.
Assuntos
Antituberculosos/imunologia , Antituberculosos/farmacologia , Macrófagos/imunologia , Macrófagos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Clofazimina/farmacologia , Citocinas/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Mitocôndrias/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de CélulasRESUMO
Introduction: Tuberculosis (TB) still kills over 1 million people annually. The only approved vaccine, BCG, prevents disseminated disease in children but shows low efficacy at preventing pulmonary TB. Myeloid dendritic cells (mDCs) are promising targets for vaccines and immunotherapies to combat infectious diseases due to their essential role in linking innate and adaptive immune responses. DCs undergo metabolic reprogramming following exposure to TLR agonists, which is thought to be a prerequisite for a successful host response to infection. We hypothesized that metabolic rewiring also plays a vital role in the maturation and migration of DCs stimulated with BCG. Consequently, we investigated the role of glycolysis in the activation of primary human myeloid CD1c+ DCs in response to BCG. Methods/results: We show that CD1c+ mDC mature and acquire a more energetic phenotype upon challenge with BCG. Pharmacological inhibition of glycolysis with 2-deoxy-D-glucose (2-DG) decreased cytokine secretion and altered cell surface expression of both CD40 and CCR7 on BCG-challenged, compared to untreated, mDCs. Furthermore, inhibition of glycolysis had differential effects on infected and uninfected bystander mDCs in BCG-challenged cultures. For example, CCR7 expression was increased by 2-DG treatment following challenge with BCG and this increase in expression was seen only in BCG-infected mDCs. Moreover, although 2-DG treatment inhibited CCR7-mediated migration of bystander CD1C+ DCs in a transwell assay, migration of BCG-infected cells proceeded independently of glycolysis. Discussion: Our results provide the first evidence that glycolysis plays divergent roles in the maturation and migration of human CD1c+ mDC exposed to BCG, segregating with infection status. Further investigation of cellular metabolism in DC subsets will be required to determine whether glycolysis can be targeted to elicit better protective immunity against Mtb.
Assuntos
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Criança , Humanos , Vacina BCG , Receptores CCR7/metabolismo , Citocinas/metabolismo , Células DendríticasRESUMO
In order to mount an appropriate immune response to infection, the macrophage must alter its metabolism by increasing aerobic glycolysis and concomitantly decreasing oxidative phosphorylation; a process known as the Warburg effect. Consequently, lactate, the end-product of glycolysis, accumulates in the extracellular environment. The subsequent effect of lactate on surrounding macrophages is poorly understood. Mycobacterium tuberculosis (Mtb), the causative organism of Tuberculosis (TB), is phagocytosed by macrophages in the airways. Mtb infected macrophages upregulate aerobic glycolysis and effector functions to try to kill the bacteria. Our lab has previously shown that human macrophages produce lactate in response to infection with Mtb. Although lactate has largely been considered a waste product of aerobic glycolysis, we hypothesised that the presence of extracellular lactate would impact subsequent immunometabolic responses and modulate macrophage function. We demonstrate that the presence of exogenous lactate has an immediate effect on the cellular metabolism of resting human macrophages; causing a decrease in extracellular acidification rate (ECAR; analogous to the rate of glycolysis) and an increase in the oxygen consumption rate (OCR; analogous to oxidative phosphorylation). When lactate-treated macrophages were stimulated with Mtb or LPS, glycolysis proceeds to increase immediately upon stimulation but oxidative phosphorylation remains stable compared with untreated cells that display a decrease in OCR. This resulted in a significantly reduced ECAR/OCR ratio early in response to stimulation. Since altered metabolism is intrinsically linked to macrophage function, we examined the effect of lactate on macrophage cytokine production and ability to kill Mtb. Lactate significantly reduced the concentrations of TNF and IL-1ß produced by human macrophages in response to Mtb but did not alter IL-10 and IL-6 production. In addition, lactate significantly improved bacillary clearance in human macrophages infected with Mtb, through a mechanism that is, at least in part, mediated by promoting autophagy. These data indicate that lactate, the product of glycolysis, has a negative feedback effect on macrophages resulting in an attenuated glycolytic shift upon subsequent stimulation and reduced pro-inflammatory cytokine production. Interestingly, this pro-resolution effect of lactate is associated with increased capacity to kill Mtb.
Assuntos
Glicólise/efeitos dos fármacos , Ácido Láctico/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Células Cultivadas , Citocinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Ácido Láctico/metabolismo , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Viabilidade Microbiana , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Fosforilação Oxidativa/efeitos dos fármacosRESUMO
Tuberculosis (TB) is the leading infectious killer in the world. Mycobacterium tuberculosis (Mtb), the bacteria that causes the disease, is phagocytosed by alveolar macrophages (AM) and infiltrating monocyte-derived macrophages (MDM) in the lung. Infected macrophages then upregulate effector functions through epigenetic modifications to make DNA accessible for transcription. The metabolic switch to glycolysis and the production of proinflammatory cytokines are key effector functions, governed by epigenetic changes, that are integral to the ability of the macrophage to mount an effective immune response against Mtb. We hypothesised that suberanilohydroxamic acid (SAHA), an FDA-approved histone deacetylase inhibitor (HDACi), can modulate epigenetic changes upstream of the metabolic switch and support immune responses during Mtb infection. The rate of glycolysis in human MDM, infected with Mtb and treated with SAHA, was tracked in real time on the Seahorse XFe24 Analyzer. SAHA promoted glycolysis early in the response to Mtb. This was associated with significantly increased production of IL-1ß and significantly reduced IL-10 in human MDM and AM. Since innate immune function directs downstream adaptive immune responses, we used SAHA-treated Mtb-infected AM or MDM in a co-culture system to stimulate T cells. Mtb-infected macrophages that had previously been treated with SAHA promoted IFN-γ, GM-CSF, and TNF co-production in responding T helper cells but did not affect cytotoxic T cells. These results indicate that SAHA promoted the early switch to glycolysis, increased IL-1ß, and reduced IL-10 production in human macrophages infected with Mtb. Moreover, the elevated proinflammatory function of SAHA-treated macrophages resulted in enhanced T helper cell cytokine polyfunctionality. These data provide an in vitro proof-of-concept for the use of HDACi to modulate human immunometabolic processes in macrophages to promote innate and subsequent adaptive proinflammatory responses.