SIRT4 Controls Macrophage Function and Wound Healing through Control of Protein Itaconylation in Mice

Abstract

Proper regulation of inflammatory responses is essential for organismal health. Dysregulation can lead to accelerated development of the diseases of aging and the aging process itself. Here, we identify a novel enzymatic activity of the mitochondrial sirtuin SIRT4 as a lysine deitaconylase that regulates macrophage inflammatory responses. Itaconate is a metabolite abundantly produced in activated macrophages. We find it forms a protein modification called lysine itaconylation. Using biochemical and proteomics approaches, we demonstrate that SIRT4 efficiently removes this modification from target proteins both in vitro and in vivo. In macrophages, elevated protein itaconylation increases upon LPS stimulation, coinciding with elevated SIRT4 expression. SIRT4-deficient macrophages exhibit significantly increased IL-1β production in response to LPS stimulation. This phenotype is intrinsic to macrophages, as demonstrated by both lentiviral over-expression and acute SIRT4 knockdown models. Mechanistically, we identify key enzymes in branched-chain amino acid (BCAA) metabolism as targets of hyperitaconylation in SIRT4-deficient macrophages. The BCKDH complex component dihydrolipoamide branched chain transacylase E2 (DBT) is hyperitaconylated and has reduced BCKDH activity in SIRT4KO macrophages. Physiologically, SIRT4-deficient mice exhibit significantly delayed wound healing, demonstrating a consequence of dysregulated macrophage function. Our data reveal a novel protein modification pathway in immune cells and establish SIRT4 as a critical regulator at the intersection of metabolism and inflammation. These findings have implications for understanding immune dysregulation in aging and metabolic disease.

Keywords: Immunometabolism; Itaconylation; Macrophage; SIRT4; Wound healing.

Figure 1:. SIRT4 possesses deitaconylase activity and removes lysine itaconylation

(A) Chemical structures of itaconate and other structurally similar acyl modifications targeted by SIRT4, including glutaryl, methylglutaryl (MG), and hydroxymethylglutaryl (HMG) groups. (B) SIRT4 deitaconylase activity demonstrated in vitro using NAD⁺ consumption assay. Recombinant SIRT4 was incubated with chemically itaconylated BSA in the presence of ³²P-NAD⁺. Formation of ³²P-O-acyl-ADPR (upper band) indicates active deacylation. (C) Validation of anti-itaconyl-lysine antibody specificity. Western blot showing antibody reactivity against unmodified BSA, succinylated BSA (suc-BSA), and itaconylated BSA (ita-BSA), demonstrating specificity for itaconyl-lysine over structurally similar succinyl-lysine. (D) Protein itaconylation increases in macrophages upon LPS stimulation. Western blot of whole-cell and mitochondrial lysates from wild-type and SIRT4KO bone marrow-derived macrophages (BMDMs) with or without LPS stimulation (10ng/ml, 24h), probed with anti-itaconyl-lysine antibody. Red arrows indicate where SIRT4KO samples show enhanced itaconylation signal at baseline, upon LPS stimulation, or both compared to wild-type in mitochondrial fractions.

Figure 2:. SIRT4 regulates macrophage inflammatory response by controlling IL-1β production

(A) Differential serum cytokine abundance between SIRT4KO and WT mice after intraperitoneal injection of LPS (2μg/g body weight). Serum was collected before and after LPS stimulation (2h time point shown) and analyzed for inflammatory cytokines using V-PLEX Mouse Cytokine 19-Plex Kit. Each point represents a cytokine, with the x-axis showing the log2 fold change (KO/WT) and the y-axis the −log10 of the adjusted p-value (limma). Cytokines that are significantly different between genotypes (adjusted p-value < 0.05 and absolute log2 fold change > 0.5, dotted lines) are highlighted in red. (B) Elevated serum IL-1β in SIRT4KO mice after intraperitoneal injection of LPS (2μg/g body weight). Serum was collected before and after LPS stimulation and analyzed for inflammatory cytokines using V-PLEX Mouse Cytokine 19-Plex Kit. Data are shown as mean ± SEM; *p<0.05. (C) Cytokine Functional Classification Heatmap. Cytokines grouped by immune function, with color intensity (p-values ranging from 0.002 to 0.967; red to gray) indicating statistical significance between KO and wild-type mice. Inflammatory mediators and T cell-related cytokines show the strongest differential abundance. (D) Western blotting of wild-type and SIRT4KO BMDM cell extracts showing elevated pro-IL-1β in KO samples (upper panel) and increased SIRT4 expression in WT samples following LPS stimulation (middle panel). OxPhos was used as a loading control (lower panel). (E) Increased IL-1β secretion from SIRT4KO BMDMs after LPS stimulation. BMDMs from wild-type and SIRT4KO mice were stimulated with LPS (10ng/ml) for 24h, and secreted IL-1β was quantified by ELISA and normalized to total protein. Data are shown as mean ± SEM; *p<0.05 (F & G) Macrophage-intrinsic SIRT4 directly controls inflammatory responses. BMDMs from WT and SIRT4KO mice were transduced with either empty pBABE vector (empty) or SIRT4-expressing pBABE vector (SIRT4) using a retroviral delivery system, then stimulated with LPS. Pro-IL-1β protein levels (Western blot, F) and secreted IL-1β (ELISA, G) demonstrate rescue of inflammatory phenotype upon SIRT4 reintroduction.

Figure 3:. SIRT4 regulates BCAA metabolism in macrophages through control of protein itaconylation

(A) Differential protein itaconylation changes in BMDMs in SIRT4KO mice compared to wild-type following LPS stimulation for 0h, 6h and 24h. Log2 fold change (KO/WT x-axis) vs. statistical significance (−log10 p-value, y-axis). Red: upregulated sites (p<0.05, log2FC>0.5); blue: downregulated sites (p<0.05, log2FC<−0.5); gray: non-significant changes. Dashed lines indicate significance threshold (p=0.05) and fold change thresholds (±0.5). (B) Itaconylation Sites Per Protein. Distribution of proteins based on number of distinct itaconylation sites. Single site: 97.3%; two sites: 2.0%; three or more sites: 0.7%. Most proteins contain only a single specific modification site. (C) List of high-confidence itaconylated peptides identified by mass spectrometry, with posterior error probability (PEP) scores ≤0.010 (1% local FDR). Proteins involved in BCAA metabolism (AUH and DBT) are highlighted in red. (D) Untargeted metabolomics from BMDMs in SIRT4KO mice compared to wild-type following LPS stimulation for 0h, 6h and 24h. The x-axis represents log2 fold change, with negative values indicating downregulation and positive values indicating upregulation. The y-axis shows −log10(p-value), where higher values indicate greater statistical significance. The plot highlights a notable metabolite: 4-methyl-5-oxopentanoate (increased) involved in BCAA metabolism. Horizontal and vertical dashed lines represent significance and fold change thresholds, respectively. (E) Analysis of itaconylated-dihydrolipoamide branched chain transacylase E2 (ita-DBT) peptide abundances indicates hyperitaconylation in LPS-stimulated BMDMs from SIRT4KO mice compared to wild-type at 6h and 24h post-stimulation. Data are shown as normalized peptide abundance from mass spectrometry analysis. P-values indicated from mass spectrometry analyses. (F) BCKDH complex activity is reduced in BMDMs from SIRT4KO mice. Oxygen consumption rate (OCR) was measured in permeabilized BMDMs using the XF24 Seahorse analyzer. NADH produced from either NAD⁺ alone (untreated, as negative control), α-ketoisocaproate (αKIC, as a substrate to test BCKDH), or pyruvate (as a test of PDH). Data are normalized to protein content and shown as mean ± SEM; *p<0.05; n=3 independent experiments. (G) Schematic showing DBT in the BCKDH complex

Figure 4:. SIRT4 deficiency impairs wound healing in vivo

(A) Representative images of excisional wounds in wild-type and SIRT4KO male mice at days 0, 3, 6, and 9 post-wounding. Full-thickness dorsal wounds were created using 5mm biopsy punches. (B-C) Quantification of wound closure in male (B) and female (C) mice. Wound diameters were measured using digital calipers over 9 days in wild-type and SIRT4KO mice (n=3 per genotype). Data are shown as mean ± SEM; *p<0.05 by t-test at individual time points; ANOVA revealed statistically significant difference between genotypes (p<0.05). (D) Proposed model illustrating SIRT4’s role in regulating macrophage function and wound healing.