Myeloid ATP Citrate Lyase Regulates Macrophage Inflammatory Responses In Vitro Without Altering Inflammatory Disease Outcomes

Abstract

Macrophages are highly plastic, key regulators of inflammation. Deregulation of macrophage activation can lead to excessive inflammation as seen in inflammatory disorders like atherosclerosis, obesity, multiple sclerosis and sepsis. Targeting intracellular metabolism is considered as an approach to reshape deranged macrophage activation and to dampen the progression of inflammatory disorders. ATP citrate lyase (Acly) is a key metabolic enzyme and an important regulator of macrophage activation. Using a macrophage-specific Acly-deficient mouse model, we investigated the role of Acly in macrophages during acute and chronic inflammatory disorders. First, we performed RNA sequencing to demonstrate that Acly-deficient macrophages showed hyperinflammatory gene signatures in response to acute LPS stimulation in vitro. Next, we assessed endotoxin-induced peritonitis in myeloid-specific Acly-deficient mice and show that, apart from increased splenic Il6 expression, systemic and local inflammation were not affected by Acly deficiency. Also during obesity, both chronic low-grade inflammation and whole-body metabolic homeostasis remained largely unaltered in mice with Acly-deficient myeloid cells. Lastly, we show that macrophage-specific Acly deletion did not affect the severity of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. These results indicate that, despite increasing inflammatory responses in vitro, macrophage Acly deficiency does not worsen acute and chronic inflammatory responses in vivo. Collectively, our results indicate that caution is warranted in prospective long-term treatments of inflammatory disorders with macrophage-specific Acly inhibitors. Together with our earlier observation that myeloid Acly deletion stabilizes atherosclerotic lesions, our findings highlight that therapeutic targeting of macrophage Acly can be beneficial in some, but not all, inflammatory disorders.

Keywords: ATP citrate lyase; immunometabolism; inflammation; macrophage; obesity; peritonitis.

Figure 1

Macrophage Acly deficiency upregulates LPS-induced inflammatory gene expression in vitro. (A) Control and Acly^M-KO BMDMs were stimulated with LPS for 3 or 24 h and analysed by RNA-sequencing. (B) Volcano plot of differentially expressed genes between control and Acly-deficient BMDMs after 3 or 24 hour LPS stimulation highlighting the top 5 most significant up- and down-regulated genes. (C) Venn diagram showing overlap in deregulated genes by Acly deficiency after 3 and 24 hours LPS-activation (D) Deregulated pathways in Acly-deficient macrophages as determined by Reactome, KEGG and GO pathways. (E, F) Expression in control versus Acly-deficient BMDMs of genes that are most highly LPS-induced after 3h (E) or 24h (F) LPS stimulation in control BMDMs. (n=3 per group).

Figure 2

Myeloid Acly deficiency barely alters systemic and local immune responses in acute endotoxin-induced peritonitis. (A) Control and Acly^M-KO mice received an intraperitoneal injection of LPS or vehicle control. After 2 hours, spleens, blood and peritoneal fluid were collected. (B) Splenic gene expression of inflammatory cytokines. (C) Peritoneal and plasma cytokine levels in vehicle control and LPS-treated control and Acly^M-KO mice. (D) Peritoneal exudate cell counts. (E) Relative distribution of peritoneal exudate cell levels as assessed by flow cytometry. (F) Relative distribution of white blood cells as assessed by flow cytometry. (G) Abundance of blood monocyte subsets as defined by Ly6C expression. Values represent mean ± SEM [n=3/3/10/10 (Ctrl vehicle/KO vehicle/Ctrl LPS/KO LPS)]. *P<0.05 by ordinary one-way ANOVA with Sidak’s post hoc test for multiple comparisons.

Figure 3

Obesity-related chronic low-grade inflammation remains unaffected by myeloid Acly deficiency. (A) Control mice received a control low-fat diet (LFD) or a high-fat diet (HFD) for 16 weeks, after which macrophages were sorted from white adipose tissue (WAT). (B) Acly expression in WAT macrophages after 16 weeks of LFD or HFD. [n=6/8 (LFD/HFD)]. (C) Control and Acly^M-KO received a LFD or HFD for 16 weeks, after which blood, liver and WAT were collected. (D) Body weight during the course of 16 weeks. (E) Blood glucose levels during glucose tolerance test (GTT) and derived area under the curve (AUC) in control and Acly^M-KO mice after LFD or HFD. (F) Blood glucose levels during insulin tolerance test (ITT) and AUC in control and Acly^M-KO mice after LFD or HFD [n=3/3/6/7 (Ctrl LFD/KO LFD/Ctrl HFD/KO HFD)]. (G) Plasma cytokine levels at 12 weeks of diet intervention [n=3/3/10/10 (Ctrl LFD/KO LFD/Ctrl HFD/KO HFD)]. (H) Circulating monocyte levels. (I) WAT myeloid cell abundance, (J) percentage of CD11c⁺ macrophages in WAT. (K) Liver myeloid cell abundance, (L) percentage of CD11c⁺ Kupffer cells. (M) Gene expression levels of indicated genes in liver and WAT [n=3/3/6/7 (Ctrl LFD/KO LFD/Ctrl HFD/KO HFD)]. Values represent mean ± SEM. *P<0.05, **P<0.01 by ordinary one-way ANOVA with Sidak’s post hoc for multiple comparisons.