John, S. V. | Search bioRxiv

bioRxiv2026

Multi-omic analysis reveals nitric oxide dependent remodeling in classically activated macrophages and identifies negative regulation mediated by AKR1A1

Nitric oxide (NO*) is an important signaling molecule in many biological processes, including immune response. During response to classical activation stimuli lipopolysaccharide (LPS) and interferon-{gamma} (IFN{gamma}), macrophages generate NO* via inducible nitric oxide synthase (iNOS). To comprehensively define the effects of NO*, we applied a multi-omic strategy integrating proteomics and transcriptomics to profile murine macrophages across conditions with or without LPS/IFN{gamma}-activation, with or without iNOS expression or exogenous NO* donor treatment. The results revealed NO* has broad, yet selected and controlled, regulatory effects, playing a key role in coordinating the systematic remodeling during macrophage classical activation. Among the proteins that are most suppressed in a NO*-dependent manner, electron transport chain (ETC) is the most enriched. NO* drives complex-specific remodeling of ETC, causing selected downregulation of complex I, II, and IV, through a different combination of transcriptional and post-transcriptional mechanisms for each complex. Functionally, we found NO* is required, but not sufficient, for the strong suppression of cellular respiration upon macrophage activation. Among the most consistently upregulated proteins are many enzymes involved in redox defense. AKR1A1 was identified as a top hit. We found Akr1a1 induction requires both NO* and LPS/IFN{gamma} stimulation. The S-nitroso-CoA reductase activity of AKR1A1 mitigates NO*-driven inhibition of pyruvate dehydrogenase complex by limiting the inhibitory modifications targeting its lipoyl cofactor. Knocking out Akr1a1 causes accelerated remodeling of TCA cycle, dysregulated immunoregulatory metabolite level, and altered functional gene expression and cytokine production at later stage of immune response. Thus, the NO*-dependent upregulation of AKR1A1 forms a negative regulatory loop to fine-tune NO*-mediated metabolic and functional remodeling during immune response. Together, this work provided a systems-level map of NO*-dependent regulation, revealed the crosstalk between NO* and immune signaling, and demonstrated mechanisms providing redox adaptation and precise control of NO*s effects.

immunology↗

bioRxiv2023

Macrophages undergo functionally significant reprograming of nucleotide metabolism upon classical activation

During an immune response, macrophages specifically rewire their metabolism to support functional changes. Using a multi-omics approach, we identified nucleotide metabolism as one of the most significantly rewired pathways across the metabolic network in classically activated macrophages. Further isotopic tracing studies revealed the substantial changes in nucleotide de novo synthesis, degradation, and salvage fluxes in stimulated macrophages, as well as the key reactions where metabolic regulation occurs: 1) de novo synthesis of purine nucleotides is shut down and particularly blocked at the last step of IMP synthesis catalyzed by ATIC; 2) de novo synthesis of pyrimidines is maintained up to UMP, but further synthesis of CTP (catalyzed by CTPS) and dTMP (catalyzed by TYMS) is greatly reduced; 3) Nucleotide degradation to nitrogenous bases is increased, but further oxidation of purine bases (catalyzed by XOR) is inhibited, causing a great accumulation of nucleosides and bases; and 4) cells switch to salvaging the nucleosides and bases as the primary means to maintain purine nucleotides. Mechanistically, we found these changes are driven by a combination of transcriptional regulation and enzyme inhibition. Nitric oxide (NO) was identified as a major regulator, driving the strong inhibition of ATIC and XOR, and the transcriptional downregulation of Tyms. To understand the functional impact of the activation-induced switch from purine de novo synthesis to salvage, we knocked out the purine salvage enzyme Hprt. Hprt knockout significantly alters functional gene expression in activated macrophages, suppresses macrophage migration, and increases pyroptosis. Furthermore, knocking out Hprt or Xor increases the proliferation of the intracellular parasite Toxoplasma gondii in macrophages. Together, these results comprehensively uncovered the dynamic rewiring of nucleotide metabolism in classically activated macrophages, elucidated the key regulatory mechanisms, and identified the functional significance of such rewiring.

cell biology↗