Single-Cell Analysis of Microglia and Monocyte Dynamics Uncover Distinct TNF-α-driven Neuroimmune Signatures after...

Innate immune cells contribute to both secondary brain injury and repair following intracerebral hemorrhage (ICH). However, the specific signaling pathways that govern initial inflammatory and subsequent reparative myeloid programs in living patients remain poorly understood. To better characterize mononuclear phagocyte cell changes over time, we generated a single-cell transcriptomic dataset of paired hematoma clot evacuates and peripheral blood samples from 10 patients following ICH (5 - 290 hours). We identified distinct populations of activated and TNF-low microglia, as well as a unique, highly activated population of CD14⁺ monocytes in the hematoma. Perturbation analysis identified TNF signaling as the primary driver of hematoma monocyte activation. Custom temporal trajectory analysis using single-cell foundation model embeddings found that this TNF response in monocytes was transient, peaking early after hemorrhage and decreasing over the following 48 hours as monocytes shifted to reparative transcriptional programs. Transiently activated microglia emerged as the likely acute source of TNF, signaling through monocyte TNFR2. Surprisingly, acute TNF signaling in CD14⁺ monocytes was also associated with better severity-adjusted neurological outcomes both in our cohort and an independent validation cohort. These findings suggest acute TNF signaling between activated microglia and hematoma-associated monocytes, particularly through TNFR2, may contribute to recovery following ICH.