Multi-modal single-cell sequencing reveals network transition in circulating monocytes that aligns with faster recovery in trauma patients and favors a response to M-CSF

Previous transcriptomic studies to characterize the changes in circulating immune cells after major trauma in humans have demonstrated a dramatic early upregulation of inflammatory features and simultaneous downregulation of MHCII and IFN signaling pathways in monocytes; changes that gradually return to baseline. The magnitude and duration of these changes is associated with post-injury complications. To determine if unique immune cell subsets or features emerging in the subacute time frame associated with patient outcomes after severe injury, we applied DOGMA-seq (single-cell RNA + ATAC + ADT sequencing) on peripheral blood mononuclear cells isolated at day 3 after injury. Patients were segregated into slow or fast recovery from critical illness (8 patients/ group) and compared to age and sex matched healthy controls. An analysis that integrated all three single cell modes identified a subset of CD172a hi/MHCII hi, CD14+ monocytes overrepresented in patients that recovered faster and distinct from baseline monocytes. This monocyte subset appeared prior to recovery and increased over time and was associated with changes in several key gene co-expression networks (referred to as a red-to-green network transition). A green-shifted pattern (associated with chemotaxis and cell adhesion) was overrepresented in fast recovery patients, continuously deviated from baseline, and specifically associated with increased accessibility of AP1 family motifs. In contrast, the red-shifted pattern (mainly pro-inflammatory genes) was associated with slow recovery, increased accessibility of CEBPB, less accessibility of IRF motifs, and gradually returned to baseline. Subsequent in vitro cell culture demonstrated that, compared to red-shifted cells, green shifted cells favored higher response to M-CSF-stimulated macrophage differentiation that included the upregulation of the genes involved in cell motility, while not obviously influenced the response to GM-CSF stimulation. Our findings add a new information layer to the current human paradigm for the immune response to injury, that includes the trauma-inducible features augmented along recovery leading to post-traumatic monocytes continuously different from baseline. The changes in circulating monocytes may have a wide influence on peripheral tissues via differentiated macrophages.