6 research outputs found
Cholesterol-Ester Transfer Protein Alters M1 and M2 Macrophage Polarization and Worsens Experimental Elastase-Induced Pulmonary Emphysema
Cholesterol-ester transfer protein (CETP) plays a role in atherosclerosis, the inflammatory response to endotoxemia and in experimental and human sepsis. Functional alterations in lipoprotein (LP) metabolism and immune cell populations, including macrophages, occur during sepsis and may be related to comorbidities such as chronic obstructive pulmonary disease (COPD). Macrophages are significantly associated with pulmonary emphysema, and depending on the microenvironment, might exhibit an M1 or M2 phenotype. Macrophages derived from the peritoneum and bone marrow reveal CETP that contributes to its plasma concentration. Here, we evaluated the role of CETP in macrophage polarization and elastase-induced pulmonary emphysema (ELA) in human CETP-expressing transgenic (huCETP) (line 5203, C57BL6/J background) male mice and compared it to their wild type littermates. We showed that bone marrow-derived macrophages from huCETP mice reduce polarization toward the M1 phenotype, but with increased IL-10. Compared to WT, huCETP mice exposed to elastase showed worsened lung function with an increased mean linear intercept (Lm), reflecting airspace enlargement resulting from parenchymal destruction with increased expression of arginase-1 and IL-10, which are M2 markers. The cytokine profile revealed increased IL-6 in plasma and TNF, and IL-10 in bronchoalveolar lavage (BAL), corroborating with the lung immunohistochemistry in the huCETP-ELA group compared to WT-ELA. Elastase treatment in the huCETP group increased VLDL-C and reduced HDL-C. Elastase-induced pulmonary emphysema in huCETP mice promotes lung M2-like phenotype with a deleterious effect in experimental COPD, corroborating the in vitro result in which CETP promoted M2 macrophage polarization. Our results suggest that CETP is associated with inflammatory response and influences the role of macrophages in COPD
Role of mTOR complex 2 (mTORC2) in the neutrophils effector function and metabolism during a sepsis model
Os neutrófilos são importantes células do sistema imune inato e a primeira linha de resposta durante um processo inflamatório. Suas propriedades e funções, assim como seu metabolismo, bastante estudados no passado ganharam uma nova perspectiva com o advento de uma nova área do conhecimento: o imunometabolismo. Com isso, o estudo da via metabólica mTOR se evidenciou nas células imunes. Porém, a participação dessa via nas funções efetoras e no metabolismo dos neutrófilos ainda é pouco esclarecida. De maneira mais restrita, a importância do complexo mTORC2 nessas células é ainda menos explorada, pois não há um inibidor específico desse complexo. Sabendo-se que a via mTOR se conecta com a via PI3K pela quinase AKT, formulamos a hipótese de que a deleção de mTORC2 prejudicaria as funções efetoras dos neutrófilos, uma vez que a PI3K é importante para a quimiotaxia, fagocitose, degranulação e para o burst oxidativo dos neutrófilos. Dessa forma, usando animais deficientes ou não em mTORC2 ativado (LysMRic/ ou LysMRic fl/fl) apenas nas células mieloides, nós investigamos o papel desse complexo sob as funções efetoras e o metabolismo dos neutrófilos. Além disso, verificamos o impacto dessa deleção em modelo experimental de sepse. Ao avaliarmos o estado de ativação da via mTOR, observamos que o estímulo com LPS é capaz de ativar mTORC2 e que a ausência de mTORC2 ativo nos neutrófilos diminui a fosforilação da AKTT308 frente ao LPS. Quanto às funções efetoras dos neutrófilos, verificamos que a ausência de mTORC2 ativo prejudica a quimiotaxia frente ao fMLP, mas não aparenta interferir na capacidade fagocítica dessas células. No entanto, a capacidade microbicida está reduzida nesses neutrófilos. Identificamos que o prejuízo no killing da E. coli é devido a uma menor produção de NO, superóxido e, principalmente, HOCl. Possivelmente, a menor produção de HOCl tenha sido responsável também pela menor produção de NETs nos neutrófilos sem mTORC2 ativo. Ao avaliarmos o metabolismo desses neutrófilos, observamos que na ausência de mTORC2 os neutrófilos são mais glicolíticos frente aos estímulos com fMLP ou LPS, porém a captação de glicose desses neutrófilos é menor quando comparada ao controle com a via intacta. Posteriormente, o impacto da deficiência de mTORC2 em neutrófilos foi avaliado in vivo em modelo de sepse induzida por E. coli . Neste caso, não observamos diferença na sobrevida dos animais, mas os parâmetros bioquímicos e da calorimetria indireta sugerem um quadro de sepse mais grave nos animais LysMRic/. Além disso, a carga bacteriana nos órgãos-alvo da sepse foi maior nos animais LysMRic/, corroborando nossos achados in vitro . Assim, concluímos que a deficiência de mTORC2 nos neutrófilos implica em uma menor produção de oxidantes, principalmente o HOCl, o que leva a um prejuízo na capacidade microbicida e na formação das NETs nesses neutrófilos.Neutrophils are the first line of defense in the innate arm of the immune system. Their functions and metabolism were well explored in the past. However, a new research field, the immunometabolism, brought new perspectives to neutrophils functional properties. Besides, the metabolic pathway mTOR, also became spotlighted with the immunometabolism emergence. Nevertheless, the role of mTOR pathway, mainly the mTORC2, in neutrophils is not clear. It is well known that mTOR pathway is linked to PI3K via AKT. Therefore, we hypothesized that the mTORC2 deletion in neutrophils would impair their effector functions once PI3K is important to neutrophils chemotaxis, phagocytose, degranulation and oxidative burst. In this sense, we used a myeloid-specific Rictor deleted mice (inactive mTORC2) to investigate the role of mTORC2 in neutrophils effector functions and metabolism. Moreover, we evaluated the impact of Rictor deletion in a mouse sepsis model. We observed that LPS can activate mTORC2 and the absence of active mTORC2 decreases the AKTT308 phosphorylation upon LPS stimulation. We demonstrated that inactive mTORC2 impairs neutrophils chemotaxis upon fMLP stimulation, and apparently does not interfere with its phagocytic capacity. However, the neutrophils microbicidal capacity is clearly compromised due to an impairment in NO, superoxide, but mainly in HOCl production. Possibly, the decrease in HOCl production affected the NETs formation. Regarding the neutrophils metabolism, we observed that when mTORC2 is absent the neutrophils are more glycolytic upon fMLP or LPS stimulation, but their glucose uptake is lower than the active mTORC2-neutrophils. After, we investigated the impact of the Rictor deletion in a E. coli-induced sepsis model. Although we did not see statistic difference in the survival curve, the glycemia, urea and indirect calorimetry suggest a poor sepsis outcome in the LysMRic/ animals. Besides, the bacterial burden was increased in the LysMRic/, corroborating our in vitro results. Therefore, we conclude that mTORC2 absence in neutrophils implies in a decrease in oxidants production, mainly HOCl, leanding to an impairment in the microbicidal capacity and NETs formation
Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone
pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10–20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics
Mitochondria as central hub of the immune system
Nearly 130 years after the first insights into the existence of mitochondria, new rolesassociated with these organelles continue to emerge. As essential hubs that dictate cell fate, mitochondria integrate cell physiology, signaling pathways and metabolism. Thus, recent research has focused on understanding how these multifaceted functions can be used to improve inflammatory responses and prevent cellular dysfunction. Here, we describe the role of mitochondria on the development and function of immune cells, highlighting metabolic aspects and pointing out some metabolic- independent features of mitochondria that sustain cell function26CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão temnão tem2014/10910-7; 2015/15626-8; 2015/26682-6; 2016/18031-8; 2017/05264-
Extracellular Vesicles isolated from Mesenchymal Stromal Cells Modulate CD4+ T Lymphocytes Toward a Regulatory Profile
Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by confocal microscopy. Expression of surface markers was detected by flow cytometry or CytoFLEX and cytokines were detected by RT-PCR, FACS and confocal microscopy and a miRNA PCR array was performed. We demonstrated that MSC-EVs were incorporated by lymphocytes in vitro and decreased T cell proliferation and Th1 differentiation. Interestingly, in Th1 polarization, MSC-EVs increased Foxp3 expression and generated a subpopulation of IFN-γ+/Foxp3+T cells with suppressive capacity. A differential expression profile of miRNAs in MSC-EVs-treated Th1 cells was seen, and also a modulation of one of their target genes, TGFbR2. MSC-EVs altered the metabolism of Th1-differentiated T cells, suggesting the involvement of the TGF-β pathway in this metabolic modulation. The addition of MSC-EVs in vivo, in an OVA immunization model, generated cells Foxp3+. Thus, our findings suggest that MSC-EVs are able to specifically modulate activated T cells at an alternative regulatory profile by miRNAs and metabolism shifting