Mitochondria complex III-generated superoxide is essential for IL-10 secretion in macrophages
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ABSTRACT: Mitochondrial electron transport chain (ETC) function modulates macrophage biology, however, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. Here we report that mutant mice with mitochondrial ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondrial CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Surprisingly, restoring mitochondrial respiration without generating superoxide using alternative oxidase (AOX) was not sufficient to reverse LPS-induced endotoxic shock susceptibility or restore IL-10 release. However, activation of protein kinase A (PKA) rescued IL-10 release in mitochondrial CIII-deficient BMDMs following LPS stimulation. Additionally, mitochondrial CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondrial CIII generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation
Project description:Electron transport chain (ETC) biogenesis is tightly coupled to energy levels and availability of ETC subunits. Coenzyme Q: cytochrome c oxidoreductase (complex III or CIII) occupies a central position in the ETC, receiving electrons from diverse fuel sources to control the ubiquinol:ubiquinone (CoQH2/CoQ) ratio. As such, CIII is an attractive node for controlling ETC biogenesis during metabolic stress. Here, we report the discovery of mammalian CoOrdinator of Mitochondrial CYTB or “COM” complexes that regulate CIII biogenesis in a step-wise fashion in response to nutrient and nuclear-encoded ETC subunit availability. The COMA complex, consisting of UQCC1/2 and the membrane anchor C16ORF91 (UQCC4), facilitates the translation of CIII enzymatic core subunit CYTB. Subsequently, microproteins SMIM4 and BRAWNIN, together with COMA subunits form the COMB complex that stabilizes nascent CYTB. Finally, UQCC3-containing COMC promotes CTYB maturation and association with downstream CIII subunits. This stepwise assembly enables cells to adapt to metabolic stress by increasing CIII biogenesis and inducing an integrated stress response when challenged. Furthermore, when nuclear CIII subunits are unavailable for assembly, COMB is required to chaperone nascent CYTB to prevent OXPHOS collapse. Our studies highlight CYTB synthesis as a key regulatory node of ETC biogenesis, and uncover the roles of mito-SEPs in mitochondrial homeostasis during energy stress.
Project description:We report the genome-wide RNA sequencing analysis in Il10-/- bone marrow-derived macrophages (BMDMs) stimulated by lipopolysaccharide (LPS) where IL-10 effect in macrophage inflammatory response was examined in IL-10-deficient BMDMs upon LPS stimulation with addition of exogenous IL-10.
Project description:IL-10 limits the magnitude of inflammatory gene expression following microbial stimuli and is essential to prevent inflammatory disease, however, the molecular basis for IL-10 mediated inhibition remains elusive. Using a genome-wide approach we demonstrate that inhibition of transcription is the primary mechanism for IL-10-mediated suppression in LPS-stimulated macrophages, and that inhibited genes can be divided into two clusters. Genes in the first cluster are inhibited only if IL-10 is included early in the course of LPS stimulation and is strongly enriched for interferon-inducible genes. Genes in the second cluster can be rapidly suppressed by IL-10 even after transcription is initiated, and this is associated with suppression of LPS-induced enhancer activation. Interestingly, the ability of IL-10 to rapidly suppress active transcription exhibits a delay following LPS stimulation. Thus, a key pathway for IL-10 mediated suppression involves rapid inhibition of enhancer function during the secondary phase of the response to LPS.
Project description:IL-10 limits the magnitude of inflammatory gene expression following microbial stimuli and is essential to prevent inflammatory disease, however, the molecular basis for IL-10 mediated inhibition remains elusive. Using a genome-wide approach we demonstrate that inhibition of transcription is the primary mechanism for IL-10-mediated suppression in LPS-stimulated macrophages, and that inhibited genes can be divided into two clusters. Genes in the first cluster are inhibited only if IL-10 is included early in the course of LPS stimulation and is strongly enriched for interferon-inducible genes. Genes in the second cluster can be rapidly suppressed by IL-10 even after transcription is initiated, and this is associated with suppression of LPS-induced enhancer activation. Interestingly, the ability of IL-10 to rapidly suppress active transcription exhibits a delay following LPS stimulation. Thus, a key pathway for IL-10 mediated suppression involves rapid inhibition of enhancer function during the secondary phase of the response to LPS.
Project description:IL-10 limits the magnitude of inflammatory gene expression following microbial stimuli and is essential to prevent inflammatory disease, however, the molecular basis for IL-10 mediated inhibition remains elusive. Using a genome-wide approach we demonstrate that inhibition of transcription is the primary mechanism for IL-10-mediated suppression in LPS-stimulated macrophages, and that inhibited genes can be divided into two clusters. Genes in the first cluster are inhibited only if IL-10 is included early in the course of LPS stimulation and is strongly enriched for interferon-inducible genes. Genes in the second cluster can be rapidly suppressed by IL-10 even after transcription is initiated, and this is associated with suppression of LPS-induced enhancer activation. Interestingly, the ability of IL-10 to rapidly suppress active transcription exhibits a delay following LPS stimulation. Thus, a key pathway for IL-10 mediated suppression involves rapid inhibition of enhancer function during the secondary phase of the response to LPS.
Project description:According to the fluidity model, complexes of the electron transport chain (ETC) in the inner mitochondrial membrane partition between free complexes and supercomplexes. However, conclusive proof of the physiological requirement of supercomplex formation is lacking, and the mechanisms regulating their formation remain enigmatic. Here, we show that genetic perturbations affecting the biogenesis or maturation of ETC Complex III (CIII) stimulates the formation of a specialized extra-large supercomplex (SC-XL) with a predicted stoichiometry of CI2+CIII2. SC-XL formation increases mitochondrial cristae density and sustains normal ETC output despite a 70% reduction in electron flow through CIII, effectively rescuing mild to moderate CIII deficiency. Increasing the SC-XL:free CIII2 ratio significantly reduced CIII ROS production and propensity for CI ROS triggered by reverse electron transport, leading to enhanced ETC efficiency. Furthermore, higher SC-XL:free CIII2 ratio reprogrammed mitochondria towards fatty acid oxidation, enhancing endurance exercise capacity and protection against ischemic heart failure in mice. Our study reveals an unanticipated plasticity in the mammalian ETC to buttress against intrinsic perturbations via structural adaptations, and suggests that ETC reprogramming via controlled regulation of SC-XL formation is a potential therapeutic strategy for remediating diseases characterized by a decline in ETC bioenergetics and oxidative damage.
Project description:IL-10 or IL-6 stimulation of control 129xC57BL/6 murine bone marrow derived macrophages in the presence of LPS. We used microarrays to detail the global programme of gene expression changes in response to IL-6 or IL-10 stimulation in the presence of lipopolysaccharide. BMDMs were isolated from control, IL-6-/-, and IL-10-/- mice on a 129XBL/6 mixed background mice and differentiated in the presence of CSF-1 for 6-7 days. Cells were scraped and plated in 6 well plates at 2x10e6/well. Cells were washed with complete DMEM and rested for 1-2 hr before stimulation with combinations of IL-10 (10 ng/ml), IL-6 (2 ng/ml) or LPS (100 ng/ml) for 45 min or 180 mins. Complete biological replicates were performed. Keywords: time course
Project description:IL-10 or IL-6 stimulation of control 129xC57BL/6 murine bone marrow derived macrophages in the presence of LPS. We used microarrays to detail the global programme of gene expression changes in response to IL-6 or IL-10 stimulation in the presence of lipopolysaccharide. BMDMs were isolated from control, IL-6-/-, and IL-10-/- mice on a 129XBL/6 mixed background mice and differentiated in the presence of CSF-1 for 6-7 days. Cells were scraped and plated in 6 well plates at 2x10e6/well. Cells were washed with complete DMEM and rested for 1-2 hr before stimulation with combinations of IL-10 (10 ng/ml), IL-6 (2 ng/ml) or LPS (100 ng/ml) for 45 min or 180 mins. Complete biological replicates were performed. Experiment Overall Design: Data sets from wild-type, IL-10-/- and IL-6-/- BMDMs treated with IL-6 or IL-10 in the presence of LPS over time
Project description:To analyze the age-associated changes in the nuclear encoded genes of the mitochondrial ETC following treatment with a Superoxide dismutase mimetic GC4419 using the mitochondrial energy metabolism profiler array from Qiagen.
Project description:We aimed to understand the role of cyclophilin D (CypD)-dependent mitochondrial permeability transition (mPT) in the immunosuppressive phase of lipopolysaccharide (LPS)-induced endotoxic shock. The liver plays an important role in immunity and organ dysfunction; therefore, on liver RNA sequencing (RNAseq) data, Ingenuity® Pathway Analysis (IPA ®) was used to investigate the complex role of mPT formation in inflammatory reprogramming and disease progression. LPS induced significant changes in the expression of 2715 genes, affecting 179 pathways related to mitochondrial dysfunction, defective oxidative phosphorylation, nitric oxide (NO) and reactive oxygen species (ROS) accumulation, nuclear factor, erythroid 2 like 2 (Nrf2), Toll-like receptors (TLRs), and tumor necrosis factor α receptors (TNFRs) mediated processes in wild-type mice. The disruption of CypD reduced the LPS-induced alterations in gene expression and pathways involving TNFRs and TLRs, in addition to improving survival and attenuating oxidative liver damage and the related NO- and ROS-producing pathways. CypD deficiency diminished the suppressive effect of LPS on mitochondrial function, nuclear- and mitochondrial-encoded genes, and mitochondrial DNA (mtDNA) quantity, which could be critical in improving survival. Our data propose that CypD-dependent mPT is an amplifier in inflammatory reprogramming and promotes disease progression. The mortality in human sepsis and shock is associated with mitochondrial dysfunction. Prevention of mPT by CypD disruption reduces inflammatory reprogramming, mitochondrial dysfunction, and lethality; therefore, CypD can be a novel drug target in endotoxic shock and related inflammatory diseases.