A privileged hunter: role of NLRP7 in immune privilege

Abstract

NOD-like receptors (NLRs) are a family of cytoplasmic receptors with members regulating apoptosis and inflammation. NLRP7 is a novel PYRIN domain containing NLR family member and its functions are still under investigation. NLRP7 is known to possess an oncogenic role in testicular seminomas and found to be overexpressed in cer-tain cancers. Interestingly, in addition to some other tissues, NLRP7 is expressed in brain, testis and placenta which are immune privilege sites in the body. Furthermore, mutations in NLRP7 can cause recurrent hydatidiform moles leading to stillbirths and abortions. One study suggested that NLRP7 down-regulates IL-1β and hence it is thought that NLRP7 may have an immuno-suppressive function. The aim of this project is to clarify the possible role of NLRP7 in immune privilege and also in inflammation. As a first step to the project, NLRP7 cDNA was cloned into several tagged (FLAG-, HA-, pcDNA3-MYC, RFP and EGFP) mammalian expression vectors for confocal anal-ysis for cellular localization and for detection of its interaction partners by co-immunoprecipitation. The effect of NLRP7 overexpression on NF-κB signaling regula-tion was measured by performing luciferase assay in HEK293FT cells. In order to eluci-date, in order to elucidate the possible role of NLRP7 in immune privilege, shifts in death receptor and HLA expressions were measured using real-time PCR and FACS analysis, respectively, in stable NLRP7 knock-down HEC-1-A cell line. Confocal analy-sis of RFP or EGFP fused NLRP7 reveals that NLRP7 is a cytosolic protein with minor co-localization to mitochondria. Furthermore, confocal co-localization studies show clear overlap between NLRP7 and caspase-1 and/or ASC. We also found that NLRP7 forms an inflammasome that activates IL-1β through binding to ASC and Caspase-1. NLRP7 appears likely to regulate IL-1β expression. Finally, NLRP7 down-regulation decreases the expression of TRAIL-R3, TNF-R2 and TWEAK and also HLA A, B, C and increases Fas expression.