CircShock4 is specifically regulated in cardiogenic shock and controls the increase in anti-inflammatory CD16+ monocytes

Luis Victor Baum (Leipzig)1, K. E. Kokot (Leipzig)1, S. Erbe (Leipzig)1, J. M. Kneuer (Leipzig)1, P. Büttner (Leipzig)2, H. Thiele (Leipzig)2, U. Laufs (Leipzig)1, J.-N. Boeckel (Leipzig)1

1Universitätsklinikum Leipzig Klinik und Poliklinik für Kardiologie Leipzig, Deutschland; 2Herzzentrum Leipzig - Universität Leipzig Klinik für Innere Medizin/Kardiologie Leipzig, Deutschland


Background: The mortality rate of cardiogenic shock (CS) is currently still around 50%. CS leads to a strong activation of the immune system. The class of non-coding RNAs includes circular RNAs (circRNAs). Their function is are largely unknown in CS. The aim of this study was to analyze the immune cell populations and the immune cell-specific regulation of circRNAs in CS at time of admission and after revascularization.

Methods and Results: Both bulk and single cell sequencing (scRNA-seq.) data of peripheral blood mononuclear cells (PBMC) from patients with CS after acute myocardial infarction (AMI) at admission and 24 h, 48 h and 72 h after revascularization were analyzed and compared with controls. Analyses of scRNA-seq. data show a 2.05-fold increase in monocytes at the immune cell level 24 h after revascularization of CS patients, with significantly more anti-inflammatory CD16+ monocytes (2.16-fold), this increase decreases until 72 h after revascularization.
Analyses by two independent bioinformatic algorithms revealed the expression of 19,182 circRNAs in immune cells, 170 of which were significantly regulated in CS. The circRNA CircShock4 was among the most upregulated. We validated the characteristics of CircShock4 by RNAse-R digestion, oligo-dT selection and Sanger sequencing of the back-splice junction (BSJ) and detected a predominant expression in monocytes. The observed regulation of CircShock4 was confirmed in a larger patient cohort of CS after AMI (n=52) compared to controls (n=38, +10.9-fold, p<0.0001). Interestingly, patients with AMI without CS (n=37) did not show an increase in CircShock4 compared to controls (n=31, p=0.2), indicating a possible CS-specific regulation of CircShock4 independent of AMI. CircShock4 levels identified patients with CS both compared to patients with AMI (AUC=0.9454, p<0.0001) and compared to controls (AUC=0.9378, p<0.0001).
ScRNA-seq. after treatment of monocytes with the anti-inflammatory cytokine IL10 showed a 5-fold increase in CD16+ monocytes (p<0.0001). Knockdown of CircShock4 in monocytes concomitant with IL10 treatment abolished the IL10-mediated increase in anti-inflammatory CD16+ monocytes by 90%. At the gene expression level, knockdown of CircShock4 in monocytes also showed an increase in a variety of inflammatory cytokines, including TNFA (14-fold, p<0.0001), CCL2 (48-fold, p<0.0001), CCL3 (77-fold, p<0.0001) and IL1B (486-fold, p<0.0001), which may indicate a cellular and expressional anti-inflammatory function of CircShock4.

Conclusion: There is a temporary increase in anti-inflammatory CD16+ monocytes in the blood of patients with CS after AMI following revascularization. Monocyte-specific CircShock4 showed CS-specific upregulation that appeared to be independent on AMI. After knockdown of CircShock4 in monocytes, the IL10-mediated increase in CD16+ monocytes is abolished and an increase in several inflammatory cytokines is observed. These data demonstrate for the first time an immune cell-specific circRNA regulation in CS, which may be involved in the monocyte subpopulation switch to anti-inflammatory CD16-positive monocytes in the course of CS after revascularization.
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