CRIP1 as a Novel Regulator of NLRP3 Inflammasome Activation in Hypertension

Hypertension is a major global health issue and significantly elevates the risk of cardiovascular disease (CVD). Emerging evidence links inflammation and immunity with hypertension pathophysiology, with the NLRP3 inflammasome and its key cytokines IL-1b and IL-18 drawing particular interest. Clinical trials, including CANTOS, COLCOT, and LoDoCo2, have provided evidence that inhibiting inflammation driven by NLRP3/IL-1b/IL-6/IL-18-mediated signaling significantly reduces the risk of cardiovascular events. The activation of the NLRP3 inflammasome in immune cells is a two-step process. The classic priming step is triggered by molecules like LPS through TLR4/CD14 receptors, leading to the activation of the NFkB. The activation step is a subsequent process that results in the maturation and release of pro-inflammatory cytokines IL-1b and IL-18, and induction of pyroptosis. Our group has previously identified Cysteine-rich protein 1 (CRIP1) as strongly associated with blood pressure in a large-scale population-based transcriptomic analysis of human monocytes. Moreover, Crip1 expression was significantly affected in circulating monocytes in an angiotensin II-induced hypertensive mouse model, suggesting a link between CRIP1 and the pathophysiology of hypertension via immune pathways.

This study investigates how CRIP1 impacts hypertension-associated inflammatory pathways, focusing on NLRP3 inflammasome priming and activation.

CRIP1 knockdown (>80%) was achieved in the monocyte-like cell line THP-1 via shRNA. To validate findings, bone marrow-derived monocytes (BMDMs, >90% marker-positive) from WT and Crip1-deficient mice were analyzed. In both models, NLRP3 inflammasome was primed with LPS and activated with ATP. Protein and gene expressions were measured by immunoblotting and qPCR; flow cytometry assessed cell surface molecule expression. Caspase-1 activity was analyzed using an immunofluorescence kit. ELISA measured IL-1β and IL-18 secretion, and potential protein interactions were evaluated by co-immunoprecipitation and colocalization analysis.

CRIP1 knockdown in THP-1 cells significantly reduced IL-1b and IL-18 protein expression and secretion. mRNA and surface expression levels of CD14/TLR4 receptors also decreased, with less NFkB phosphorylation observed in CRIP1 knockdown cells, indicating suppressed NFkB signaling. Co-IP and colocalization confirmed NFkB as a CRIP1 functional interaction partner, consistent with findings in cancer cell models. Caspase-1 activity remained unchanged.  The findings were validated in Crip1-deficient BMDMs. A rescue experiment is ongoing to further underline these results.

Our results suggested that CRIP1 knockdown/knockout reduced NLRP3 inflammasome signaling in monocytes by reduced IL-1b and IL-18 protein expression and secretion. The effect is possibly mediated by a functional interaction with NFkB and a feedback regulation upstream of the signaling pathway. CRIP1 may contribute to hypertension and CVD development through its role in relevant signaling pathways, underscoring its potential as a biomarker and therapeutic target for inflammatory diseases.