1Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland
Background
Hypertension is a significant global health burden and is the major risk factor for cardiovascular disease (CVD). Current evidence points to the importance of inflammation and immunity in the pathophysiology of hypertension and CVD. In particular, the NLRP3 inflammasome and its key cytokines IL-1β and IL-18 have been highlighted. The activation of the NLRP3 inflammasome in immune cells is a two-step process. The priming step is induced by molecules (e.g. LPS) through TLR4/CD14 receptors, leading to the activation of the NFκB pathway. Recent evidence has shown that the recycling route and level of CD14 are controlled by sorting nexin (SNX) 1, 2 and 6, and SNXs can also affect the levels of TLR4. The activation step is a subsequent process that results in the maturation and release of pro-inflammatory cytokines, IL-1β and IL-18, and induction of pyroptosis. Previously, our group has identified Cysteine-rich protein 1 (CRIP1 ) in strong association with blood pressure (BP) and BP changes in a large-scale population-based transcriptomic analysis using human monocytes. In addition, Crip1 expression was shown to be significantly affected in circulating monocytes in an angiotensin II-induced mouse model of hypertension, suggesting a link between CRIP1 and the pathophysiology of hypertension via the immune system.
Objectives
This project aims to investigate the molecular mechanism by which CRIP1 interferes with hypertension-related inflammatory signaling pathways, focusing on the regulatory mechanism for priming of the NLRP3-inflammasome in monocytes.
Material and Methods
CRIP1 knockdown (>80%) was established in the monocyte-like cell line THP-1 using a vector-based shRNA system (shCRIP1). NLRP3 inflammasome priming was induced by lipopolysaccharide (LPS). Protein and gene expression levels were assessed by immunoblotting and gene expression analyses, respectively. Cell surface molecule expression was examined by flow cytometric analysis. Using transcriptomic data of human monocytes, linear mixed models were used to calculate associations between CRIP1 and SNXs mRNA expression in the Gutenberg Health Study (GHS) using transcriptome data. The potential interactions between SNXs and CRIP1 were verified by co-immunoprecipitation (Co-IP).
Results
CRIP1 downregulation significantly reduced the expression of the CD14/TLR4 receptors. In line with this, less phosphorylation of NFkB was observed in shCRIP1 cells, indicating a reduced activation of NFkB signaling after priming with LPS. Further investigation of regulation upstream of TLR4/CD14 receptors revealed that SNX1, 2, and 6 levels were all downregulated in shCRIP1 at both mRNA and protein levels. Analysis of the transcriptomic data indicated an association of CRIP1 with SNX1 mRNA expression (N=1527; r=-0.277; p=3e-28). Consistently, immunoblot analysis of immunoprecipitated CRIP1 confirmed SNX1 as a functional interaction partner.
Conclusion
Our results suggest that CRIP1 downregulation reduced NLRP3 inflammasome signaling in monocytes by affecting TLR4/CD14-NFkB signaling through a functional interaction with SNX1. CRIP1 might contribute through relevant mechanisms to the development of hypertension.