Mitochondria complex III-generated superoxide is essential for IL-10 secretion in macrophages

Abstract

Mitochondrial electron transport chain (ETC) function modulates macrophage biology; however, mechanisms underlying mitochondria ETC control of macrophage immune responses are not fully understood. Here, we report that mutant mice with mitochondria ETC complex III (CIII)-deficient macrophages exhibit increased susceptibility to influenza A virus (IAV) and LPS-induced endotoxic shock. Cultured bone marrow-derived macrophages (BMDMs) isolated from these mitochondria CIII-deficient mice released less IL-10 than controls following TLR3 or TLR4 stimulation. Unexpectedly, 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 mitochondria CIII-deficient BMDMs following LPS stimulation. In addition, mitochondria CIII deficiency did not affect BMDM responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the essential role of mitochondria CIII-generated superoxide in the release of anti-inflammatory IL-10 in response to TLR stimulation.

Fig. 1.. Macrophage mitochondrial CIII function is required for recovery following influenza A virus infection.

(A) Survival curves following intratracheal instillation of 15 PFU IAV [A/WSN/H1N1/(1933)] into mice expressing CX₃CR1^{Cre-ERT2-YFP/WT} while on tamoxifen chow. (B) IAV PFU from lung homogenates taken at 7 DPI. QPC-HET (n = 7) and QPC-KO (n = 6). (C) Weight was tracked for 23 days post-instillation in mice QPC-WT (QPC^WT/WT) [n = 6 (M = 3, F = 3)], QPC-HET (QPC^WT/Fl) [n = 14 (M = 4, F = 10)], and QPC- KO (QPC^Fl/Fl) [n = 14 (M = 9, F = 5)]. Cross indicates dates of mouse death events. Flow cytometric analysis 7 DPI of % Tomato⁺ of (D) classical monocytes in blood (E) classical monocytes in the lungs and (F) lung MoAMs. Seven DPI numerical calculations based on flow and cell counts for (G) classical monocytes in blood (H) classical monocytes in the lungs and (I) lung MoAMs. For (A) Kaplan-Meier tests with Mantle-Cox log rank and Gehan-Breslow-Wilcoxon test, adding extra weight for early time points was used. In (C), unpaired t tests were used. Data represent means ± SEM.

Fig. 2.. Mitochondrial CIII function is required for LPS induction of IL-10 protein in macrophages.

(A) Survival curve of mice following intraperitoneal injection of 30 mg/kg to LPS QPC-HET (LysM^Cre/CreQPC^WT/FlRosa26^Ai14/Ai14) [n = 22 (M = 13, F = 9)], QPC-KO (LysM^Cre/CreQPC^Fl/FlRosa26^Ai14/Ai14) [n = 16 (M = 8 F = 8)]. (B) Plasma IL-10 levels 2 hours post-LPS injection in QPC-HET (n = 6) and QPC-KO (n = 6). BMDMs generated from QPC-HET (LysM^Cre/Cre QPC^WT/Fl Rosa26^Ai14/Ai14) or QPC-KO (LysM^Cre/CreQPC^Fl/FlRosa26^Ai14/Ai14) mice were examined via seahorse for (C) Basal respiration (n = 4 for each group) and (D) Amplex red assay for mitochondrial ROS production (n = 6 for each group). BMDMs treated with heavy-labeled U-¹³C-Glucose for 0, 1, or 2 hours to examine lactate labeling in (E) untreated and (F) LPS-stimulated BMDMs QPC-HET (100 ng/ml; n = 5) and QPC-KO (n = 3). IL-10 secretion from QPC-HET (n = 4) and QPC-KO (n = 4) BMDMs following 2-hour stimulation with (G) LPS (100 ng/ml) or (H) poly (I:C) (20 μg/ml). For data in (A) Kaplan-Meier tests with Mantle-Cox log rank and Gehan-Breslow-Wilcoxon test, adding extra weight for early time points was used. In (B) to (H), unpaired Student’s t tests were used. Data represent means ± SEM.

Fig. 3.. AOX restores mitochondrial CIII–dependent respiration.

(A) Schematic of mitochondrial ETC and mitochondrial function in QPC-HET, QPC-KO, and QPC-KO + AOX mice was created in https://BioRender.com. (B) Seahorse analysis of oxygen consumption of QPC-HET (n = 7), QPC-KO (n = 5), and QPC-KO + AOX (n = 5) BMDMs with injections of mitochondrial CIII inhibitor myxothiazol (100 μM), AOX inhibitor SHAM (16 mm), and combination antimycin (1 μM)/piericidin (1 μM). Calculations of (C) mitochondrial CIII–dependent and (D) AOX-dependent respiration. Calculations of (E) basal respiration QPC-HET (n = 8), QPC-KO (n = 8), and QPC-KO + AOX (n = 7) (F) coupled respiration of QPC-HET (n = 6), QPC-KO (n = 6), and QPC-KO + AOX (n = 5) and (G) basal glycolytic rate QPC-HET (n = 8), QPC-KO (n = 8), and QPC-KO + AOX (n = 7) (H) glycolytic capacity QPC-HET (n = 4), QPC-KO (n = 4), and QPC-KO + AOX (n = 4) of BMDMs with injections of mitochondrial CIII inhibitor oligomycin (2 μM), AOX inhibitor SHAM (16 mm), and combination antimycin (1 μM)/piericidin (1 μM). In (C) to (H), ordinary one-way analyses of variance (ANOVAs) with Holm-Sidak’s multiple comparisons test with a single pooled variance were used to compare levels between the three groups. Data represent means ± SEM.

Fig. 4.. Mitochondrial CIII, independent of enabling respiration, is required for LPS induction of IL-10 protein in macrophages.

(A) NAD⁺/NADH ratio in BMDMs ±2-hour LPS (100 ng/ml) stimulation (n = 4 all groups). (B) IL-10 secretion 2-hour post-LPS simulation QPC-HET (n = 8), QPC-KO (n = 10), and QPC-KO + AOX (n = 7) BMDMs. RNA-seq data for BMDMs ±2-hour LPS stimulation [QPC-HET (n = 5), QPC-KO (n = 5), and QPC-KO + AOX (n = 3)] examining summed gene module expression for (C) GO:0071222, (D) IL-10/STAT3 AIR (34), and a (E) heatmap of genes from gene module (GO:0071222). (F) Survival of QPC-KO and QPC-KO + AOX mice following injection of LPS (10 mg/kg) from three experiments QPC-KO (n = 32) and QPC-KO + AOX (n = 26). Plasma (G) IL-10 and (H) TNF-α levels 2-hour post-LPS injection (30 mg/kg) in QPC-HET (n = 11), QPC-KO (n = 13), and QPC-KO + AOX (n = 8) mice. (I) Intracellular IL-10 concentration in BMDM lysates following LPS stimulation ± Brefeldin A. (J) Secreted IL-10 in BMDMs following 2-hour stimulation with LPS (100 ng/ml) ± db-cAMP (100 μM) or H89 (1 μM). (QPC-HET, n = 7; QPC-KO, n = 11; db-cAMP, n = 4; LPS + db-cAMP+H89, n = 3). In (A) and (I), an ordinary two-way ANOVA was performed with Sidak’s multiple comparisons test. In (B), (G), and (H), an ANOVA with mixed effects analysis with Dunnett’s multiple comparisons test was performed. In (C) and (D), pairwise comparisons were performed using pairwise Mann-Whitney U tests with false discovery rate (FDR) correction. In (E), hierarchical clustering was performed using Ward’s method (D2) with a Euclidean distance metric. Gene expression values are z-normalized DESeq2 counts. In (F), Kaplan-Meier tests with Mantle-Cox log rank and Gehan-Breslow-Wilcoxon test were used. In (J), an ordinary one-way ANOVAs with Holm-Sidak’s multiple comparisons test were used to compare groups. Data represent means ± SEM.

Fig. 5.. Mitochondrial CIII function is not required for IL-4 stimulation of macrophages.

RNA-seq analysis of QPC-HET (n = 5) and QPC-KO (n = 5) BMDMs 18 hours post–IL-4 (10 ng/ml) stimulation or vehicle control by: (A) Principal components analysis (PCA), (B) heatmap of gene expression of the top 20 genes, and (C) summed module expression of the IL-4 response module as defined in (41). Frequency of (D) Arg-1⁺, (E) Relm-α⁺, and (F) PD-L2⁺ BMDMs 18 hours post–IL-4 (10 ng/ml) stimulation. Frequency of (G) Arg-1⁺ and (H) Relm-α⁺ peritoneal macrophages 4 days post-initial thioglycolate and IL-4c stimulation QPC-HET (unstimulated, n = 3; IL-4c stimulated, n = 10) and QPC-KO (unstimulated n = 3; IL-4c stimulated, n = 12). Data represent means ± SEM. In (A), PCA was performed using single value decomposition of the top 1000 genes by variance. In (B), hierarchical clustering was performed using Ward’s method (D2) with Euclidean distance as a distance metric. Gene expression values are z-normalized DESeq2 counts. In (C), pairwise comparisons were performed using pairwise Mann-Whitney U tests with FDR correction. In (D) to (H), ordinary one-way ANOVAs with Holm-Sidak’s multiple comparisons test with a single pooled variance were used to compare groups. Data represent means ± SEM.