30 jan. 2026
Heieis et al. (BioRxiv)
Keywords
Alternative activation of macrophages
O-GlcNAcylation
Immunometabolism
Cell cycle regulation
Tissue-resident macrophages
Main Findings
Macrophages are an important innate immune cell type involved in tissue homeostasis and immune response during infection and cancer. Type II cytokines such as IL-4 and IL-13 polarize macrophages towards an ‘anti-inflammatory’ M2 phenotype, also known as alternative activation of macrophages (AAM). Previous studies showed that a hexosamine biosynthesis pathway (HBP) metabolite, Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) was enriched in AAM. Being at the crossroads of glycolysis, glutamine, fatty acid, and nucleotide metabolism, HBP functions as nutrient sensor. In addition, UDP-GlcNAc also serves as the donor substrate of a post-translational modification, O-GlcNAcylation. Previous literature implicated O-GlcNAcylation in modulating metabolism, cell cycle and various other processes. Mechanistically, O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). In this preprint by Heieis G.A., et al., the role of O-GlcNAcylation in AAM and in vivo tissue-macrophage homeostasis were investigated. The key findings are as follows:
OGT inhibition blocked AAM both in vitro and in vivo. Both pharmacological inhibition of OGT (OGTi) in wild-type primary macrophages in vitro and myeloid-specific genetic knockout of Ogt, Lyz2ΔOgt in vivo reduced O-GlcNAcylation. In parallel canonical M2 polarization marker expression was decreased. In vivo injections of IL-4 complex (IL-4c) in Ogt-deficient mice failed to induce proliferation of tissue-resident large cavity macrophages (LCM). Instead, macrophage populations were mainly composed of monocyte-derived small and converting cavity macrophages (SCM and CCM, respectively). During helminth infection, Lyz2ΔOgt mice failed to control parasite burden, showed a decrease in macrophage abundance and exhibited a similar skewing of macrophage populations. Even during the resolving phase of helminth infection, there was a lack of tissue-resident TIM4+ macrophages in Lyz2ΔOgt mice indicating a requirement for OGT in macrophage tissue residency.
O-GlcNAcylation regulated survival of macrophages during homeostasis. Under steady-state conditions, Lyz2ΔOgt mice exhibited loss of LCMs. Co-transfer experiments using a pre-labelled mixture of F4/80hi LCMs demonstrated selective loss of Ogt-deficient LCMs regardless of recipient genotype. Hence, cell-intrinsic mechanisms were implicated in the loss of Ogt-deficient LCMs.
Both pharmacological inhibition and genetic depletion of OGT in macrophages led to alteration in T-cell response. During helminth infection, Lyz2ΔOgt mice displayed a skewing of Th2 to Th1-biased immune responses. Macrophage-T-cell co-culture experiments revealed that OGTi increased costimulatory molecule expression on macrophages and relieved proliferation block of T-cells.
O-GlcNAcylation regulated metabolism and proliferation of LCMs. Transcriptomics, functional metabolic assays and flow cytometry analyses revealed that Ogt-deficient LCMsexhibited metabolic dysregulation. These cells accumulated in a G2/M phase instead of a quiescent G0 stage. Ogt-deficient LCMs acquired a “senescence associated secretory phenotype” characterized by larger cell size, increased senescent markers and secretion of inflammatory cytokines. These cells showed higher dependence on glycolysis, dysregulated oxidative phosphorylation, increased reactive oxygen species (ROS)and lipid accumulation. Ex vivo glucose depletion reduced ROS production, DNA damage markers and improved cell recovery of LCMs. These results linked OGT-dependent LCM differentiation to glucose metabolism.
O-GlcNAcylation was dynamically regulated during LCM differentiation. Monocyte-derived populations of tissue-resident macrophages are often found to be in an actively replicating state. Transcriptomics and flow cytometry-based analyses revealed that Ogt expression was negatively correlated with metabolic regulators. In the Lyz2ΔOgt mice, LCMs were incapable of downregulating metabolic protein expression and remained in a proliferative state. Hence, O-GlcNAcylation could be a cell cycle regulator during LCM differentiation, possibly by sensing nutrient availability.
Requirement of O-GlcNAcylation in tissue macrophages was highly variable but required for macrophage homeostasis. Beyond the peritoneal cavity, OGT depletion affected abundance and proliferation of select tissue-resident macrophages during AAM, including SiglecF+ alveolar macrophages in the lungs and Kupffer cells in the liver. In the small intestine, increased CD11c expression and reduced TIM4⁺ macrophages indicated impaired tissue-resident macrophage differentiation.
Limitations & Suggestions
Identification of direct O-GlcNAcylated protein targets, particularly metabolic enzymes and regulators, would provide deeper mechanistic insight in macrophage homeostasis.
Despite Lyz2-mediated Ogt deletion, residual O-GlcNAcylation persisted in different tissue macrophages highlighting the need to show activity of OGA.
Using physiologically relevant culture conditions, including pO2, tissue specific cytokines and recombinant MCSF may better reflect O-GlcNAcylation dynamics.
The authors observed a series of effects on the metabolism and cell cycle of the Ogt-deficient mice and showed a certain rescue with glucose depletion. However, it remains unclear how the Ogt-deficiency leads to cell death and the changes in metabolism and cell cycle. Future studies could test whether ROS scavenging, mitochondrial metabolism modulation, or genetic complementation of Ogt could reverse senescence of Ogt-deficient LCMs.
Multiplex immunofluorescence in infected tissues could further validate metabolic, cell cycle and senescence markers in situ.
Significance/Novelty
The study identified O-GlcNAcylation as a key regulator for macrophage metabolism, tissue residency, cell cycle and differentiation during homeostasis and type II immune responses. While centered on the peritoneal cavity, the work extends to other tissues such as lung, liver, intestine indicating tissue specific requirements for O-GlcNAcylation. OGT depletion led to a more Th1 skewed response impairing helminth control but enhanced anti-tumour immune responses. Given that Ogt is encoded on the X chromosome and some of the effects of Ogt deficiency were more pronounced in male mice, O-GlcNAcylation could provide interesting molecular insights in sex dependent immune differences. The current preprint lays a foundation for further metabolic and molecular insights of O-GlcNAcylation regulated fate and function of macrophages.
Credit
Reviewed by Amitava Sinha as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.
The author declares no conflict of interests in relation to their involvement in the review.