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

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

PROVIDER: PRJNA1090473 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28411300.fastq.gz	Fastqsanger.gz
	SRR28411301_1.fastq.gz	Fastqsanger.gz
	SRR28411301_2.fastq.gz	Fastqsanger.gz
	SRR28411302_1.fastq.gz	Fastqsanger.gz
	SRR28411302_2.fastq.gz	Fastqsanger.gz

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Cardiopulmonary bypass (CPB) vs sham-operated controls

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

Project description: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.

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Chronic dysfunction of Store-operated calcium entry by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila

Project description:The purpose is to have a overview on how chronic dysfunction of store-operated calcium entry change transcription profiles of adult fly fat tissue. Methods:To achieve chronic dysfunction of store-operated calcium entry, we carried out the Temperature-caused RNAi Pulse Induction of Stim (29 celcius degree incubation, Stim-TRiPI, or Stim-TRIP in data file) in fat storage tissue of 6 days old adult male flies. At day 10 and 11 after Stim-TRiPI (Stim-TRIP), we isolated the fat tissues and carried out RNAseq analysis based gene-level read counts.

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Project description:Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2 and ORAI3 are known to comprise the CRAC channel, however the contributions of individual isoforms to neutrophil function is not well understood. Here we show that loss of ORAI1 partially decreases calcium influx while loss of both ORAI1 and ORAI2 completely abolishes store-operated calcium entry. In other immune cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2- and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions including phagocytosis, degranulation, leukotriene and ROS production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies novel subpopulations of neutrophils where cell membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for new mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

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