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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262146 | GEO | 2025/03/19

REPOSITORIES: GEO

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Project description:Cardiopulmonary bypass (CPB), the mechanical support required during the majority of cardiac surgeries, causes significant systemic inflammation and multi-organ dysfunction that is especially severe in neonatal patients. Currently, the limited understanding of the molecular mechanisms that underlie CPB-associated inflammation presents a significant barrier to the development of therapeutic interventions. To address this knowledge gap, we performed mRNA sequencing of total circulating leukocytes from neonatal patients undergoing CPB. The transcriptional signatures across 7 time points ranging from pre-CPB, during CPB, to 24 hours post-CPB were found to be dominated by myeloid cells, particularly monocytes. INTERLEUKIN-8 (IL8) and TUMOR NECROSIS FACTOR-α (TNFα) were two inflammatory cytokines specifically and robustly upregulated in the leukocytes from both CPB patients and piglets exposed to CPB. To delineate the mechanism how CPB activates cytokine production, we exposed THP-1 cells, a human monocyte cell line, in vitro to CPB-like conditions including artificial surfaces, high shear stress and cooling/rewarming. The in vitro CPB data recapitulated the in vivo CPB condition in that IL8 and TNFα were strongly upregulated. Shear stress was the major driver of cytokine expression as compared to the temperature or plastic exposure. Extracellular calcium influx was found essential for the shear-stress mediated upregulation of cytokines via the MEK/ERK/AP-1 and Calcineurin/NFAT pathways. After being exposed to CPB conditions, a subpopulation of THP-1 cells died by apoptosis and TNFα-mediated necroptosis, the latter form of which potentially contribute to the post-CPB tissue injuries. Together, our study is the first to elucidate the shear-stress modulated molecular mechanisms leading to systemic inflammation in pediatric CPB and the link between inflammation and organ damage. Calcium signaling pathways and TNFα signaling are novel targets for therapeutic development to improve clinical outcomes. To our knowledge, these are also the first data to implicate necroptosis in CPB and show that shear stress can activate necroptosis.

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

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