Hypertension is a major global health issue, with the underlying molecular mechanisms remaining unknown. Current evidence point to the importance of inflammation and immunity in hypertension. Immune cell accumulation in blood vessels, kidneys, and hearts promotes a chronic inflammatory response that disrupts the regulation of blood pressure (BP) in these organs. Especially, the NLRP3 inflammasome and its key cytokines IL-1β and IL-18 have been highlighted. In a large-scale population-based transcriptomic analysis, our group identified cysteine-rich protein 1 (CRIP1) in human monocytes as being strongly associated with BP. CRIP1 is highly expressed in immune cells like in monocytes suggesting a correlation between CRIP1 and the pathophysiology of BP through the immune system. 

We aimed to investigate the influence of CRIP1 on hypertension-related inflammatory signaling pathways, focusing on priming and activation of the NLRP3-inflammasome. 

Knockdown of CRIP1 was established in the monocyte-like cell line THP-1 using a vector-based shRNA system. Inflammation was induced by addition of LPS/ATP. Differentiation of THP-1 monocytes into macrophages was induced by PMA. Gene expression was assessed using immunoblotting techniques and/or gene expression analyses. The concentration of secreted IL-1β and IL-18 was measured via a luminescence-based commercial kit or an ELISA kit. 

Stable downregulation of CRIP1 (>70%) was observed in CRIP1 shRNA transfected THP-1 cells (shCRIP1). Analysis of the components of NLRP3 inflammasome signaling pathway showed that expression of IL18 and NLRP3 were lower in shCRIP1 monocytes compared to control (shCtrl), whereas IL1B, CASP1, and NFKB showed no difference. Upon LPS stimulation/priming, the expression of IL1B, CASP1, and NFKB was significantly increased in shCtrl cells, while the effects were significantly decreased in shCRIP1 cells. Subsequently, ATP-induced NLRP3 activation, mature IL-1β and IL-18 protein were secreted into supernatant. The IL-18 secretion was lower in shCRIP1 cells, while IL-1β secretion showed no difference. In THP-1 macrophages, IL18 and NFKB expression were lower after CRIP1 knockdown and priming, while IL1B and CASP1 showed an increased expression. Interestingly, after induced NLRP3 activation,  IL-1β secretion was particularly lower in shCRIP1 macrophages.

The role of CRIP1 in the NLRP3 signaling pathway was further investigated through key receptors for NLRP3 priming (TLR4/CD14) and activation (P2RX7), as well as activation of NFKB-signaling. TLR4 and CD14 expression in both monocytes and macrophages were significantly reduced after CRIP1 knockdown. P2RX7 expression showed reduction only in macrophages. Following priming in monocytes, less IKBa (NFkB inhibitory regulator) protein level, with increased phosphorylation of IKBa and NFkB were observed in shCRIP1 cells, indicating a greater activation of NFKB signaling. Upon priming in macrophages, no activation of NFKB signaling was observed. Different CRIP1-related inflammatory mechanisms in monocytes and macrophages were revealed.

Our results suggest that low levels of CRIP1 may avoid inflammasome activation by reducing IL-1β/-18 protein secretion, and vice versa, if CRIP1 is present at normal/high levels, it will contribute to the activation of the inflammasome. CRIP1 might contribute through these mechanisms to the development of hypertension.