Therapeutic inhibition of glycolysis preferentially targets pathogenic monocyte subsets and attenuates CNS inflammation in flavivirus encephalitis

Infiltrating monocytes play a dual role in central nervous system (CNS) diseases, both driving and attenuating inflammation. However, it is unclear how metabolic pathways preferentially fuel protective or pathogenic processes and whether these can be therapeutically targeted to enhance or inhibit these opposing functions. Here, we employed single-cell RNA-sequencing and metabolic protein flow analysis of brain and bone marrow (BM) to map the metabolic signatures of monocyte-derived cells (MCs) to their functions during lethal West Nile virus encephalitis. Using trajectory analysis, we showed progression of BM monocytes through 3 metabolic profiles before their migration to the brain where they differentiated into metabolically distinct MC populations. These included a single pro-inflammatory HIF1-α MC cluster that diverged into two disparate populations: an inducible nitric oxide synthase-positive (iNOS ⁺ ) M1-like MC, with high glycolysis and amino acid metabolic scores, and a glycolytically quiescent, MHC-II ⁺ antigen-presenting MC. Daily in vivo glycolysis inhibition with 2-deoxy-D-glucose significantly reduced CNS leukocyte numbers, reducing neuroinflammation and disease signs without increasing viral load. Reduced leukocyte numbers were not due to decreased myelopoiesis, but a preferential decrease in iNOS ⁺ , compared to antigen-presenting MC, highlighting different glycolytic dependencies between these subsets. Importantly, HIF1-a was independent of glycolysis, enabling continued antigen-presenting MC differentiation, while glycolysis inhibition did not impair generation of an effective antiviral response by cervical node T cells. Together, this integrative approach unveils the tight coupling of MC function and metabolism in viral CNS disease, highlighting novel metabolic therapeutic intervention points, potentially with anti-viral therapy, during severe or uncontrolled inflammation.