Cardiopulmonary bypass activates classical monocytes via shear-mediated activation of Store-Operated Calcium Entry

Exposure to cardiopulmonary bypass (CPB) during cardiac surgery results in a significant inflammatory response that contributes to morbidity and mortality, which is especially prominent in neonatal patients. The molecular and cellular mechanisms that underpin this inflammatory process remain poorly understood. To gain deeper insight, we performed snRNA and snATAC sequencing on peripheral blood mononuclear cells (PBMCs) isolated from neonatal CPB patients prior to start of CPB, at the end of CPB, as well as 8 and 24 hours after CPB. The dramatic increase in the proportion of classic monocytes following bypass surgery indicates their essential role in inflammation associated with cardiopulmonary bypass (CPB). Immune dysregulation exhibited at activation of inflammatory genes in classical monocytes, as well as in other cell types, after CPB exposure. A series of in vitro experiments in non-adherent monocytic cells identified two novel genes SPTAN1 and RAF1 as effectors of hemodynamic stress. These two genes promoted STIM1 coupling to ORAI1 channel leading to calcium icon influx, thereby driving inflammation and cell death. snATAC-Seq revealed dynamically changing patterns of chromatin accessibility and transcription factors JUN and FOS binding motifs being highly enriched in classical monocytes after CPB exposure. Increased calcium in turn stimulates JUN binding to DNA, as suggested by CUT&RUN data derived from shear stressed non-adherent monocytic cells. Together, these data indicate that shear stress via a calcium dependent mechanism contributes to the CPB-associated activation of classic monocytes. These findings provide deeper insight not only in the pathogenesis of CPB-associated inflammation, but also have implications for the understanding of early stages of sterile inflammation and how non-adherent cells “sense” shear stress. We performed either snRNA-Seq or 10X Genomics Multiome snRNA/ATAC-Seq on samples collected from neonatal patients prior to start of CPB, end of CPB, 8 hours after the end of CPB, and 24 hours after the end of CPB (workflow outlined in Figure 1A) to answer questions about the cell types and gene expression in an unbiased and systematic manner. PBMCs were isolated since granulocytes such as neutrophils are technically challenging to study using the 10X Genomics Multiome snRNA/ATAC-Seq pipeline. Bulk RNA seq for sheared vs. static THP-1 cells Bulk ATAC seq for sheared vs. static THP-1 cells CUT&RUN for sheared vs. .static THP-1 cells