Presentation_1.PDF

<p>Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, is able to efficiently manipulate the host immune system establishing chronic infection, yet the underlying mechanisms of immune evasion are not fully understood. Evidence suggests that this pathogen interferes with host cell lipid metabolism to ensure its persistence. Fatty acid metabolism is regulated by acetyl-CoA carboxylase (ACC) 1 and 2; both isoforms catalyze the conversion of acetyl-CoA into malonyl-CoA, but have distinct roles. ACC1 is located in the cytosol, where it regulates de novo fatty acid synthesis (FAS), while ACC2 is associated with the outer mitochondrial membrane, regulating fatty acid oxidation (FAO). In macrophages, mycobacteria induce metabolic changes that lead to the cytosolic accumulation of lipids. This reprogramming impairs macrophage activation and contributes to chronic infection. In dendritic cells (DCs), FAS has been suggested to underlie optimal cytokine production and antigen presentation, but little is known about the metabolic changes occurring in DCs upon mycobacterial infection and how they affect the outcome of the immune response. We therefore determined the role of fatty acid metabolism in myeloid cells and T cells during Mycobacterium bovis BCG or Mtb infection, using novel genetic mouse models that allow cell-specific deletion of ACC1 and ACC2 in DCs, macrophages, or T cells. Our results demonstrate that de novo FAS is induced in DCs and macrophages upon M. bovis BCG infection. However, ACC1 expression in DCs and macrophages is not required to control mycobacteria. Similarly, absence of ACC2 did not influence the ability of DCs and macrophages to cope with infection. Furthermore, deletion of ACC1 in DCs or macrophages had no effect on systemic pro-inflammatory cytokine production or T cell priming, suggesting that FAS is dispensable for an intact innate response against mycobacteria. In contrast, mice with a deletion of ACC1 specifically in T cells fail to generate efficient T helper 1 responses and succumb early to Mtb infection. In summary, our results reveal ACC1-dependent FAS as a crucial mechanism in T cells, but not DCs or macrophages, to fight against mycobacterial infection.</p

Inhibiting choline metabolism impairs mitochondrial structure and function.

A) Heatmap of z-scores for select mitochondrially-encoded genes. B) Western Blot of electron transport chain complexes I to V in macrophages treated with vehicle (DMSO), HC3 (250 μM), or RSM (1 or 5 μM). Total protein by trichloroethanol (TCE) staining shown for normalization. Representative blots of n = 3. C-I) Mito Stress Test assay of extracellular flux with sequential treatments of 1.5 μM oligomycin, 14 μM BAM15, and 1 μM rotenone/1 μM antimycin A/Hoechst 33342. Oxygen consumption rate (OCR) of M[0] (C) or M[IL-4] (E) treated with DMSO (Veh), HC3 (250 μM), or different concentrations of RSM. Extracellular acidification rate (ECAR) in M[0] (D) or M[IL-4] (E). Derived parameters of basal respiration (G), spare respiratory capacity (H), or non-mitochondrial respiration (I) from (C) and (E). Measurements (n = 3 in triplicate) were normalized to an arbitrary cell factor determined by Hoechst 33342 nuclei counts. One-way ANOVA with Šídák’s test for multiple comparisons (*** p OXPHOS) or glycolysis (ATPGlyco). Measurements (n = 3 in triplicate) were normalized per 103 cells. Mixed-effects analysis with Tukey’s test for multiple comparisons (ATPOXPHOS: ## p Glyco: * p < 0.05, *** p < 0.001, **** p < 0.0001). K) Transmission electron micrographs of macrophages treated with vehicle (DMSO) or RSM (5 μM) in M[IL-4]. Scale bars represent 2 μM (left) or 0.5 μM (right insets). White arrows indicate healthy mitochondria with intact cristae, orange arrows indicate mitochondria with aberrant membrane structure or absent cristae. Representative images from n = 3 and at least 10 cells imaged per condition. No cells with intact mitochondria were observed with RSM treatment.</p

<i>In vivo</i> choline kinase inhibition during secondary intestinal helminth infection alters peritoneal cell populations and macrophage alternative activation.

A-D) Enumeration of A) B-1 cells, B) monocytes, C) neutrophils, D) peritoneal macrophages among live PECs. n = 4–5. Unpaired t test (** p (TIF)</p

Reactome pathway analysis.

A-D) Reactome pathway analysis of genes up-regulated by HC3 (A) or RSM (C) in M[IL-4] or down-regulated by HC3 (B) or RSM (D), sorted by gene count enrichment for each Reactome pathway. (TIF)</p

<i>In vivo</i> consequence of inhibiting choline metabolism.

A) Gating strategies for peritoneal cells. B) Schematic of 3-day in vivo choline kinase inhibition. Mice were treated intraperitoneally with vehicle (40% DMSO in PBS) or RSM-932a (3 mg/kg) on day 0 and 2 and sacrificed on day 3. n = 3–4. C) Intracellular RELMα expression in live CD11b+ F480hiMHCIIlo, F480loMHCII+, F480−, Ly6G+ PMN, and CD11blo peritoneal cells. Two-way ANOVA with Šídák’s test for multiple comparisons (*** p Emr1 (Adgre1/F4/80), Retnla, Chil3, or Mrc1 in WAT. Unpaired t test (* p (TIF)</p

Choline uptake and phosphorylation is required for normal IL-4 signaling and M[IL-4] phenotype.

A-D) Macrophages were treated with vehicle (DMSO) or HC3 (250 μM) for 24 h, washed, then treated with IL-4 (20 ng/mL) for 24 h. Relative expression of M[IL-4] hallmark genes Arg1, Mrc1, Chil3, or Retnla normalized to Actb and compared to M[0]. n = 3–4, representative of 3–5 experiments. Unpaired t test (*** p Retnla normalized to Actb and compared to M[0]. n = 3, representative of 1 experiment. Two-way ANOVA with Tukey’s test for multiple comparisons (* p + macrophages. n = 3, representative of >4 experiments. One-way ANOVA with Tukey’s test for multiple comparisons (* p < 0.05, ** p < 0.01). I) Macrophages were treated with vehicle (DMSO), HC3 (250 μM), or RSM-932a (5 μM) for 24 h, washed, then treated with IL-4 (20 ng/mL) for 24 h. Detection of supernatant RELMα by ELISA. n = 5 (vehicle, HC3, IL-4) or n = 2 (RSM). Unpaired t test (** p < 0.01). Schematics were created using BioRender.</p

<i>In vivo</i> choline kinase inhibition in naïve mice and primary intestinal helminth infection alters peritoneal cell populations and macrophage alternative activation.

A) UMAP plots of peritoneal cells (PECs) from naïve and H. polygyrus-infected mice treated with vehicle or RSM-932a. See S6A Fig for population gating. B-G) Enumeration of B) total PECs, C) eosinophils, D) monocytes, E) B-1 cells, F) neutrophils, G) peritoneal macrophages among live PECs. n = 3–4 (naïve) or n = 5 (infected). Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). H-J) CD206, I) CD86, or J) PD-L1 expression (gMFI) on peritoneal macrophages. n = 3–4 (naïve) or n = 5 (infected). Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05), representative of 2 experiments.</p

Mitochondrial changes induced by inhibiting choline metabolism.

A) Densitometry of complex III and I from Fig 4B. Two-way ANOVA with Dunnett’s test for multiple comparisons. B) Bioenergetics analysis of ATP produced through oxidative phosphorylation (ATPOXPHOS) or glycolysis (ATPGlyco). Macrophages were treated for 1 h or 5 min with RSM (0.2, 1, or 5 μM) prior to Mito Stress Test assay as in Fig 4C–4F. Measurements (n = 3 in triplicate) were normalized per 103 cells. Mixed-effects analysis with Tukey’s test for multiple comparisons (ATPOXPHOS: # p Glyco: ** p (TIF)</p

IL-4 up-regulates choline metabolism in macrophages.

A) Saturation curves showing rate of 3H-choline uptake at increasing concentrations of unlabeled choline. n = 8. Michaelis-Menten least squares fit regression (**** p 3H-choline uptake by HC3. n = 4 (M[0]) or 5 (M[IL-4]). Four parameter log(inhibitor) vs. response regression F test (** p (TIF)</p

Graphical abstract.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.</div