Activation Of Macrophage Autophagy Research Articles

Gamma-aminobutyric acid type A receptor alpha 4 coordinates autophagy, inflammation, and immunometabolism to promote innate immune activation

Gamma-aminobutyric acid type A receptor (GABAAR), the ionotropic receptor of GABA, is expressed in macrophages and in the nervous system; however, its role in innate immunity is unknown. Herein, we identified myeloid GABAAR subunit α4 (Gabra4) as a critical regulator of autophagy and a promoter of host innate defense during infection and inflammation. Myeloid Gabra4 deficiency led to defective mycobacterial clearance during infection and increased susceptibility to septic shock. Gabra4 deletion exaggerated inflammatory responses and suppressed the activation of autophagy in macrophages upon infectious and inflammatory stimuli. Mechanistically, Gabra4-mediated signaling led to upregulation of autophagy in macrophages via intracellular calcium release and AMP-activated protein kinase (AMPK) signaling activation, which was required for linking autophagy and antimicrobial responses. Additionally, Gabra4 was required to generate mitochondrial reactive oxygen species, thereby triggering autophagy and antimicrobial responses to mycobacteria. Metabolomics analysis showed that Gabra4 was critical for glucose metabolism and aerobic glycolysis in macrophages. Our findings demonstrate that myeloid Gabra4 coordinates autophagy, inflammation, and immunometabolism to promote innate host defense against pathogenic and dangerous stimuli.Abbreviation AM: Alveolar macrophage; AMPK: AMP-activated protein kinase; ASC: Apoptosis-associated speck-like protein containing a CARD; ATP: Adenosine 5’-triphosphate; BAL: Bronchoalveolar lavage; BCG: Mycobacterium bovis Bacillus Calmette–Guérin; BMDM: Bone marrow-derived macrophage; CCL: CC motif chemokine ligand; CFU: Colony forming unit; CKO: Conditional knock out; CXCL: C-X-C motif ligand; Dpi: Days post-infection; ECAR: Extracellular acidification rate; EGFP: Enhanced green fluorescent protein; FOXO3: Forkhead box O3; GABA: Gamma-aminobutyric acid; GABAAR: GABA type A receptor; Gabarap: GABA type A receptor-associated protein; Gabarapl1: GABA type A receptor-associated protein like 1; GABRA4: Gamma-aminobutyric acid type A receptor subunit alpha4; HIF-1α: Hypoxia-inducible factor-1 alpha; IL: Interleukin; i.n.: Intranasal; i.p.: Intraperitoneal; LDHA: Lactate dehydrogenase A; LPS: Lipopolysaccharide; Mabc: Mycobacteroides abscessus subsp. abscessus; MOI: Multiplicities of infection; Mtb: Mycobacterium tuberculosis; mtROS: Mitochondrial reactive oxygen species; OCR: Oxygen consumption rate; OXPHOS: Oxidative phosphorylation; PM: Peritoneal macrophage; TNF: Tumor necrosis factor; WT: Wild type

Atheroprotective Effects and Molecular Mechanism of Berberine.

Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide. Atherosclerosis is the main pathological basis of cardiovascular diseases and it is closely associated with hyperlipidemia, endothelial injury, macrophage-derived foam cells formation, proliferation and migration of vascular smooth muscle cells (VSMCs), platelet aggregation, and altered gut microbiota. Various symptomatic treatments, that are currently used to inhibit atherosclerosis, need to be administered in long term and their adverse effects cannot be ignored. Berberine (BBR) has beneficial effects on atherosclerosis through regulating multiple aspects of its progression. This review highlights the recent advances in understanding the anti-atherosclerosis mechanism of BBR. BBR alleviated atherosclerosis by attenuation of dyslipidemia, correction of endothelial dysfunction, inhibition of macrophage inflammation and foam cell formation, activation of macrophage autophagy, regulation of the proliferation and migration of VSMCs, attenuation of platelet aggregation, and modulation of gut microbiota. This review would provide a modern scientific perspective to further understanding the molecular mechanism of BBR attenuating atherosclerosis and supply new ideas for atherosclerosis management.

Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis

Macrophages, with diverse functions and variable phenotypes, are considered as an important executor of inflammatory diseases. And it has been proved that autophagy is deeply connected with the development of inflammation, while the exact regulatory mechanism still remains unclear, and the application of autophagy regulators in anti-inflammation needs to be further confirmed. Here, we firstly verified that neochromine S5 (hereinafter referred to as S5) significantly inhibited M1-like macrophage polarization with decrease of the proinflammatory cytokines and downregulation of NF-κB and STAT1 signals. Then, in vivo experiments demonstrated S5 improved cecal ligation and puncture (CLP)-induced sepsis specially based on the regulation of M1-like macrophages. Mechanistic studies indicated that S5 treatment dramatically upregulated cellular autophagy in M1-like macrophage. Furthermore, by multiple methods, S5 was revealed to directly bind with ubiquitin-specific proteases 14 (USP14) at Ser404, Phe405, and Cys414 by hydrogen bond to inhibit its deubiquitinating activity, and block USP14–TRAF6 (TNF receptor associated factor 6) interaction, subsequently promoting ubiquitination of Beclin1, interrupting Beclin1–Bcl2 interaction, and accumulating the autophagosome in macrophages, which finally resulted in the blockade of M1-like macrophage polarization. Animal experiments also confirmed the protection of S5 in CLP mice was dependent on activation of macrophage autophagy. What’s more, as a novel USP14 inhibitor, S5 exhibited higher efficiency and safety than IU1, the known USP14 inhibitor. Therefore, this study has demonstrated that typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis. Moreover, we provide a new candidate compound, S5, for sensitizing autophagy to interfere with the macrophage inflammation.

Myeloid atg5 deletion impairs n-3 PUFA-mediated atheroprotection

Background and aimsDietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. MethodsWe generated myeloid-specific autophagy-deficient and control mice in the Ldlr−/− background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr−/− recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. ResultsCompared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr−/− mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr−/− mice. ConclusionsDeficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.

Dietary PUFAs attenuate NLRP3 inflammasome activation via enhancing macrophage autophagy

Dietary PUFAs reduce atherosclerosis and macrophage inflammation, but how nucleotide-binding oligomerization domain leucine-rich repeat-containing receptor protein (NLRP3) inflammasome activation and autophagy influence PUFA-mediated atheroprotection is poorly understood. We fed Ldlr−/− mice diets containing 10% (calories) palm oil (PO) and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, fish oil (FO), echium oil (EO, containing 18:4 n-3), or borage oil (BO, containing 18:3 n-6). Inflammasome activation, autophagic flux, and mitochondrial function were measured in peritoneal macrophages, blood monocytes, or liver from diet-fed mice. Compared with PO, dietary PUFAs (FO, EO, or BO) markedly inhibited inflammasome activation, shown by 1) less macrophage IL-1β secretion and caspase-1 cleavage in response to NLRP3 inflammasome activators, 2) less IL-1β secretion and caspase-1 cleavage from liver or hepatocytes in response to lipopolysaccharide (LPS), and 3) attenuated caspase-1 activity in blood monocytes. Furthermore, PUFA-enriched diets increased LC3-II expression in macrophage, aorta, and liver samples and reduced numbers of dysfunctional mitochondria in macrophages in response to LPS and palmitate, suggesting enhanced autophagic activation. Dietary PUFAs did not attenuate NLRP3 inflammasome activation in atg5-deficient macrophages, indicating that autophagic activation is critical for the PUFA-mediated inflammasome inactivation. In conclusion, dietary PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.

MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2

Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis.

Activation of autophagy in macrophages by pro-resolving lipid mediators

The resolution of inflammation is an active process driven by specialized pro-resolving lipid mediators, such as 15-epi-LXA4 and resolvin D1 (RvD1), that promote tissue regeneration. Macrophages regulate the innate immune response being key players during the resolution phase to avoid chronic inflammatory pathologies. Their half-life is tightly regulated to accomplish its phagocytic function, allowing the complete cleaning of the affected area. The balance between apoptosis and autophagy appears to be essential to control the survival of these immune cells within the inflammatory context. In the present work, we demonstrate that 15-epi-LXA4 and RvD1 at nanomolar concentrations promote autophagy in murine and human macrophages. Both compounds induced the MAP1LC3-I to MAP1LC3-II processing and the degradation of SQSTM1 as well as the formation of MAP1LC3+ autophagosomes, a typical signature of autophagy. Furthermore, 15-epi-LXA4 and RvD1 treatment favored the fusion of the autophagosomes with lysosomes, allowing the final processing of the autophagic vesicles. This autophagic response involves the activation of MAPK1 and NFE2L2 pathways, but by an MTOR-independent mechanism. Moreover, these pro-resolving lipids improved the phagocytic activity of macrophages via NFE2L2. Therefore, 15-epi-LXA4 and RvD1 improved both survival and functionality of macrophages, which likely supports the recovery of tissue homeostasis and avoiding chronic inflammatory diseases.

Abstract 176: Dietary PUFAs Enhance Macrophage Autophagy, Attenuate NLRP3 Inflammasome Activation, and Reduce Atherosclerosis

Progression of atherosclerosis leads to progressive macrophage autophagy deficiency, which enhances inflammasome activation and atherosclerosis. We previously showed that dietary enrichment with n-3 and n-6 polyunsaturated fatty acids (PUFAs) derived from botanical oils (Echium oil (EO) containing 18:4 n-3 and borage oil (BO) containing 18:3 n-6), reduced atherosclerosis similar to that observed with fish oil (FO) consumption, compared to the saturated/monounsaturated-enriched botanical palm oil (PO). We hypothesized that dietary PUFAs enhance autophagy, which in turn, dampens macrophage NLRP3 (Nucleotide-binding oligomerization domain leucine-rich repeat containing receptor protein 3) inflammasome activation, reducing macrophage inflammation and atherosclerosis. To test the hypothesis, we fed female low density lipoprotein receptor (LDLr) knockout mice diets containing 10% (calories) PO and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, EO, BO or FO for 10-16 weeks before measurement of atherosclerosis, autophagy and inflammasome activation. Compared to their PO-fed counterparts, mice fed PUFAs enriched diets (EO, BO or FO) had increased LC3-II expression in peritoneal macrophage, aorta and liver, suggesting autophagic activation. Consistent with enhanced autophagy, plasma reactive oxygen species (ROS) were significantly lower in PUFA diet-fed mice, relative to PO-fed animals. Since ROS activates, but autophagy attenuates, NLRP3 inflammasome activation, we examined whether dietary PUFAs attenuate NLRP3 inflammasome activation in mice. We found that dietary PUFAs markedly inhibited inflammasome activation, shown by: 1) less IL-1β secretion from peritoneal macrophages after ATP, oxidized LDL, or palmitate-induced NLRP3 inflammasome activation, 2) less IL-1β secretion from liver explants in response to lipopolysaccharide (LPS), and 3) deceased caspase-1 cleavage in peritoneal macrophages and liver and attenuated caspase-1 activity in blood monocytes. In conclusion, our data suggest that dietary n-3 and n-6 PUFAs are equally effective in reducing atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.

Autophagy in pulmonary macrophages mediates lung inflammatory injury via NLRP3 inflammasome activation during mechanical ventilation.

The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced lung injury. Autophagy is an essential, homeostatic process by which cells break down their own components. We explored the role of autophagy in the mechanisms of mechanical ventilation-induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage-depleted mice via a jugular venous cannula 30 min before the start of the ventilation protocol. In some experiments, mice were ventilated in the absence and presence of autophagy inhibitors 3-methyladenine (15 mg/kg ip) or trichostatin A (1 mg/kg ip). Mechanical ventilation with a high tidal volume caused rapid (within minutes) activation of autophagy in the lung. Conventional transmission electron microscopic examination of lung sections showed that mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the inflammatory responses caused by lung hyperinflation. The activation of autophagy in macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.

Adiponectin Suppresses Angiotensin II-Induced Inflammation and Cardiac Fibrosis through Activation of Macrophage Autophagy

Previous studies have indicated that adiponectin (APN) protects against cardiac remodeling, but the underlying mechanism remains unclear. The present study aimed to elucidate how APN regulates inflammatory responses and cardiac fibrosis in response to angiotensin II (Ang II). Male APN knockout (APN KO) mice and wild-type (WT) C57BL/6 littermates were sc infused with Ang II at 750 ng/kg per minute. Seven days after Ang II infusion, both APN KO and WT mice developed equally high blood pressure levels. However, APN KO mice developed more severe cardiac fibrosis and inflammation compared with WT mice. This finding was demonstrated by the up-regulation of collagen I, α-smooth muscle actin, IL-1β, and TNF-α and increased macrophage infiltration in APN KO mice. Moreover, there were substantially fewer microtubule-associated protein 1 light chain 3-positive autophagosomes in macrophages in the hearts of Ang II-infused APN KO mice. Additional in vitro studies also revealed that globular APN treatment induced autophagy, inhibited Ang II-induced nuclear factor-κB activity, and enhanced the expression of antiinflammatory cytokines, including IL-10, macrophage galactose N-acetyl-galactosamine specific lectin 2, found in inflammatory zone 1, and type-1 arginase in macrophages. In contrast, APN-induced autophagy and antiinflammatory cytokine expression was diminished in Atg5-knockdown macrophages or by Compound C, an inhibitor of adenosine 5'-monophosphate-activated protein kinase. Our study indicates that APN activates macrophage autophagy through the adenosine 5'-monophosphate-activated protein kinase pathway and suppresses Ang II-induced inflammatory responses, thereby reducing the extent of cardiac fibrosis.

Selective Inhibition of PI3K/Akt/mTOR Signaling Pathway Regulates Autophagy of Macrophage and Vulnerability of Atherosclerotic Plaque

Macrophage infiltration contributes to the instability of atherosclerotic plaques. In the present study, we investigated whether selective inhibition of PI3K/Akt/mTOR signaling pathway can enhance the stability of atherosclerotic plaques by activation of macrophage autophagy. In vitro study, selective inhibitors or siRNA of PI3K/Akt/mTOR pathways were used to treat the rabbit's peritoneal primary macrophage cells. Inflammation related cytokines secreted by macrophages were measured. Ultrastructure changes of macrophages were examined by transmission electron microscope. mRNA or protein expression levels of autophagy related gene Beclin 1, protein 1 light chain 3 II dots (LC3-II) or Atg5-Atg12 conjugation were assayed by quantitative RT-PCR or Western blot. In vivo study, vulnerable plaque models were established in 40 New Zealand White rabbits and then drugs or siRNA were given for 8 weeks to inhibit the PI3K/Akt/mTOR signaling pathway. Intravascular ultrasound (IVUS) was performed to observe the plaque imaging. The ultrastructure of the abdominal aortic atherosclerosis lesions were analyzed with histopathology. RT-PCR or Western blot methods were used to measure the expression levels of corresponding autophagy related molecules. We found that macrophage autophagy was induced in the presence of Akt inhibitor, mTOR inhibitor and mTOR-siRNA in vitro study, while PI3K inhibitor had the opposite role. In vivo study, we found that macrophage autophagy increased significantly and the rabbits had lower plaque rupture incidence, lower plaque burden and decreased vulnerability index in the inhibitors or siRNA treated groups. We made a conclusion that selective inhibition of the Akt/mTOR signal pathway can reduce macrophages and stabilize the vulnerable atherosclerotic plaques by promoting macrophage autophagy.