Background Interferon (IFN)-α is a key contributor to susceptibility and severe manifestations in systemic lupus erythematosus (SLE). In SLE, the PRKG1 rs7897633 variant has been previously identified as the top hit in European ancestry patients with elevated circulating IFN-α compared to those with low IFN-α levels.
However, the mechanisms by which this PRKG1 variant impacts type I IFN production have not been studied. PRKG1 codes for the cGMP-dependent protein kinase I (PKGI). Activation of PKGI is thought to lead to the inhibition of Rho-associated kinases (ROCK). The RhoA/ROCK pathway plays critical roles in autoimmunity, at least in part by modulating type I IFN production. Accordingly, we aimed to evaluate whether the PRKG1 gene variant affected PRKG1 gene expression and ROCK activity level in immune cells. In addition, we assessed the effect of a PKGI agonist on type I IFN production.
Methods We used B lymphoblastoid cell lines (LCL; Coriell repositories) from healthy European ancestry subjects for all experiments. LCLs were categorized as homozygous (AA or CC) or heterozygous (AC) at the rs7897633 SNP. PRKG1 gene expression of LCLs (AA n=6, AC n=9, CC n=3) was assessed at baseline by RT-qPCR using gene-specific primers, normalized by GAPDH, and measured by the 2-ΔΔCT method. ROCK2 enzymatic activity was performed in LCLs (AA n=4, AC n=3, CC n=3) at baseline conditions by a colorimetric assay (Cell Biolabs). For type I IFN production assays, LCLs were treated with R848 +/- the PKGI agonist (PKGIa) 8-CPT-cGMP (n=7), and supernatants were collected after 18 hours. WISH cells were then incubated with the LCL supernatants for 6 hours, after which canonical type I IFN-induced gene expression (IFIT1, MX1, and PKR) was measured by RTqPCR, standardized to healthy controls, and summed to generate a type I IFN score. Statistically significant differences were determined by the Mann-Whitney U test, the Wilcoxon matched-pairs signed-rank test, or the sum-of-squares F test, as appropriate.
Results PRKG1 expression was lower in the homozygous AA and heterozygous genotypes compared to the homozygous CC genotype in LCLs (relative expression AA = 1.1 [IQR 4.2], AC= 0.7 [IQR 1.7], CC= 6.9 [IQR 47.3]; Mann-Whitney U test, AA vs. CC p=0.02, AC vs. CC p=0.009, AA vs AC=not significant).
Compared to LCLs with the homozygous AA variant of rs7897633, homozygous CC LCLs have lower baseline ROCK activity (OD for AA = 0.60 [IQR 0.1], AC = 0.56 [IQR 0.1], CC = 0.48 [IQR 0.1]; sum-of- squares F test, p=0.03). Pretreatment with the PKGI agonist 8-CPT-cGMP significantly reduced R848- induced type I interferon production by LCLs (IFN scores for R848 alone = 538.5 [IQR 1326.1] Vs. R848+PKGIa = 222.4 [IQR 947.4]; Wilcoxon matched-pairs test, p=0.016).
Conclusion PRKG1 rs7897633 AA and AC genotypes are associated with lower PRKG1 mRNA expression compared to the CC genotype, suggesting a recessive effect. The AA genotype is also associated with greater ROCK2 baseline activity, suggesting a potential role of genetic variation at PRKG1 in modulating the RhoA-ROCK pathway. In addition, direct stimulation of PKGI inhibits TLR7/8-induced type I IFN production by LCLs, a finding that could have therapeutic implications in type I IFN-driven autoimmune diseases such as SLE.
This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/ .
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.