Background: Hypertension is a major global health burden and a leading risk factor for cardiovascular disease (CVD). Increasing evidence implicates inflammation and immunity in its pathophysiology, with particular attention being given to the NLRP3 inflammasome and its cytokines IL-1β and IL-18. Clinical trials such as CANTOS, COLCOT, and LoDoCo2 have demonstrated that inhibiting NLRP3/IL-1β/IL-6/IL-18–mediated inflammation can significantly reduce cardiovascular events. NLRP3 inflammasome activation occurs in two steps: priming by lipopolysaccharide (LPS) via TLR4/CD14, activating NFκB; and a subsequent activation phase that triggers IL-1β/IL-18 maturation and pyroptosis. Recent studies suggest that the recycling and abundance of CD14 are regulated by sorting nexins (SNX) 1, 2, and 6, which also influence TLR4 levels. Recently, our group identified Cysteine-rich protein 1 (CRIP1) as strongly associated with blood pressure in human monocyte transcriptomes. CRIP1 expression was also altered in monocytes from angiotensin II–induced hypertensive mice, suggesting a link between CRIP1 and hypertension via immune pathways.
Objectives: To investigate how CRIP1 modulates hypertension-related inflammatory signaling, focusing on NLRP3 inflammasome priming and activation.
Materials and methods: CRIP1 knockdown (>80%) was achieved in THP-1 monocyte-like cells using shRNA. NLRP3 inflammasome priming and activation were induced by LPS and ATP. Protein and mRNA were quantified by immunoblotting and qPCR; surface molecules by flow cytometry. Caspase-1 activity was assessed by immunofluorescence, and IL-1β/IL-18 secretion by ELISA. Protein interactions were examined by co-immunoprecipitation and colocalization. For validation, CRIP1 knockdown was performed in primary monocytes from healthy donors using siRNA. Correlations between CRIP1 and inflammasome mediators were analyzed in the Gutenberg Health Study (GHS) and Cardiogenics Transcriptome Study (CTS) cohorts.
Results: CRIP1 knockdown in THP-1 cells significantly reduced IL-1β and IL-18 expression and secretion after inflammasome activation, with decreased NFκB phosphorylation indicating suppressed priming. However, Caspase-1 activity was unaffected, suggesting selective upstream inhibition. Upstream CD14 and TLR4 expression, as well as SNX1, 2, and 6 levels, were reduced in shCRIP1 cells. Co-immunoprecipitation confirmed a physical interaction between CRIP1 and SNX1. Transcriptomic data supported this link (N=1527; p=3×10⁻²⁸), and SNX1 overexpression partially rescued NLRP3 signaling in CRIP1-deficient THP-1 cells. In human primary monocytes, CRIP1 knockdown similarly decreased TLR4/CD14 and IL-1β expression. GHS and CTS analyses confirmed correlations between CRIP1 and inflammatory markers.
Conclusion: CRIP1 deficiency diminishes NLRP3 inflammasome signaling in monocytes by reducing IL-1β and IL-18 expression and secretion, potentially through SNX1-dependent regulation of TLR4/CD14 and NFκB activation. These findings suggest that CRIP1 contributes to hypertension-related inflammation and may serve as a potential biomarker and therapeutic target for inflammatory cardiovascular diseases.
