# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 2 Results
### Effects of cGMP on Inflammatory Markers in Wild Type and IRAK3 Knockdown/Knockout THP-1 Cells  
IRAK3 inhibits NFxB activity and cytokine production [3] and also contains a cryptic GC centre that generates cGMP [14]. To elucidate the relationship between IRAK3 and cGMP on inflammatory cytokine production, we first investigated the effect of cGMP on IRAK3 protein expression in THP-1 cells. Membrane permeable cGMP over the concentration range of 1 pM-10 \(\upmu\)M (10\({}^{-12}\) to 10\({}^{-5}\) M) had no effect on IRAK3 protein expression in THP-1 cells in either unstimulated or LPS-stimulated conditions (\(n=5\); Supplemental Figures S3 and S4). This finding indicates that cGMP does not induce or inhibit IRAK3 expression under the conditions used. To examine if cGMP modulates IRAK3 effects on inflammation, the IRAK3 gene in THP cells was inactivated using a CRISPR-Cas9 approach. We designed sgRNA primers targeting two sites at exon 1 of IRAK3 gene (Supplemental Figure S5) and co-transfected these with Cas9-GFP protein into THP-1 cells. Two IRAK3 knockdown monoclonal cell lines designated K5-1 and K6-3 were obtained using the limited dilution cloning method. Analysis of PCR products showed the cleavage occurred only at one of two sites in IRAK3 knockdown K5-1 and K6-3 cell lines (Supplemental Figure S6A). Sanger sequencing showed deletion of one nucleotide at one allele, and deletion of seven nucleotides at the other allele in the K6-3 line (Supplemental Figure S6B), indicating K6-3 is a heterozygous IRAK3 mutant. These nucleotide deletions in K6-3 cells cause separate frameshift mutations and early introduction of a stop codon to terminate translation, predicted to result in two different versions of truncated proteins with no similarity to IRAK3 (Figure 4A). Sanger sequencing showed a nucleotide substitution at one allele and deletion of two nucleotides at the other allele in the K5-1 line (Supplemental Figure S6B), indicating K5-1 is a heterozygous IRAK3 mutant. The nucleotide substitution in one allele of IRAK3 gene of K5-1 led to one amino acid substitution (N4T) in IRAK3 protein mutant, and the deletion of two nucleotides causes a frameshift mutation and early introduction of a stop codon, resulting in a truncated protein with no similarity to IRAK3 (Supplemental Figure S7A). Figure 4: IRAK3 protein expression and cytokine production in THP-1 wild type (WT) cells versus IRAK3 knockdown/knockout THP-1 cells. (**A**) The full length IRAK3 protein containing 596 amino acids has a death domain, pseudokinase domain embedded (purple stars) with a GC centre and TRAF6 binding domain (C-terminal domain). Positions where the anti-IRAK3 antisera bind to the death domain (NSJ Bioreagent) or C-terminal domain (Cell Signalling Technology—CST) are indicated on the full length IRAK3 protein schematic. The predicted knockouts of IRAK3 in K6-3 cell line contain one allele with 128 amino acids and a second allele with 130 amino acids producing unique truncated proteins with no similarity to wild type IRAK3 with 596 amino acids. (**B**) IRAK3 protein expression in the IRAK3\({}^{+/-}\) K5-1 or IRAK3\({}^{-/-}\) K6-3 THP-1 cells is reduced compared to wild type IRAK3\({}^{+/+}\) THP-1 cells. Cells were un-stimulated or mock PBS-treated (annotated as U in the blots), or stimulated with 1 ug/mL LPS (annotated as S in the blots) for 16 h, upon which cell lysates were collected for immunoblot experiments. IRAK3 was detected with the CST (upper panel) or NSJ (lower panel) antisera; \(\beta\)-tubulin was used as the loading control. Full-length immunoblots are shown in Supplemental Figure S8. (**C**,**E**) LPS-induced TNF-\(\alpha\) and IL-6 levels were significantly increased in K6-3 THP-1 IRAK3\({}^{-/-}\) cells compared to THP-1 IRAK3\({}^{+/+}\) wild type cells.