# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 3 Discussion
Additionally, the induction and stabilization of the NF\(\kappa\)B inhibitor, alpha (I\(\kappa\)B\(\alpha\)) is mediated by NO, thereby attenuating NF\(\kappa\)B activity [49] (Figure 6). NO also suppresses the activity of caspase-1 enzyme which mediates processing of premature IL-1\(\beta\) and IL-18 cytokines, causing decreased IL-1\(\beta\) and IL-18 production [57, 58]. Therefore, there is a correlation between IRAK3, cGMP and NO effects to downregulate inflammatory cytokine production. The potential for altering cGMP levels to influence systemic inflammatory responses has been raised previously. In a polymicrobial sepsis-induced mouse model where exogeneous cGMP was administered to increase cGMP bioavailability, plasma levels of IL-6 and TNF-\(\alpha\) are substantially reduced, and ODQ (an inhibitor of soluble GCs) showed the opposite effect [16]. Our study confirmed that pharmacologically decreasing cGMP levels by ODQ promotes LPS-induced NF\(\kappa\)B activity and TNF-\(\alpha\) expression (Figure 3A,B). The effect of ODQ on LPS-induced NF\(\kappa\)B activity was reversed by simultaneously adding membrane permeable cGMP (Figure 3C), confirming the inhibitory effect of cGMP signalling on inflammation. However, there are contradictory findings about the influence of cGMP or soluble GC inhibitors on inflammation [29, 30, 43]. Such examples are: zaprinast (a selective inhibitor of cGMP-specific phosphodiesterase) enhanced LPS-stimulated secretion of TNF-\(\alpha\) and IL-1\(\beta\) in rat microglial cells [43]; soluble GC inhibitors such as methylene blue suppressed LPS-induced secretion of inflammatory cytokines in human pulmonary macrophages [29, 30]; and ODQ at 50 nM inhibited LPS-induced production of TNF-\(\alpha\) and IL-1\(\beta\) in rat microglial cells [43]. We found that ODQ at 0.1-1 nM raised LPS-induced NF\(\kappa\)B activity (Figure 3A), at 10-100 nM ODQ increased LPS-induced TNF-\(\alpha\) level (Figure 3B). However, at higher concentrations (10 nM), ODQ decreased the viability of THP-1 human monocytes (Supplemental Figure S2), that can lead to lower expression of inflammatory markers potentially explaining the decreased effect of 10 nM ODQ (Figure 3A,B). cGMP regulates cellular protein turnover, proteasome activity enhancing the elimination of misfolded or mutated proteins [59]. Hence, treatment with higher ODQ concentration can cause impaired proteosome function or defects in protein homeostasis and directly affects cell viability. Put together, soluble GC inhibitors may influence cytokine production in concentration dependent ways that are influenced by the cell types and effects on cell viability and mechanisms of action. A GC centre encapsulated in the pseudokinase domain of IRAK3 is responsible for its cGMP generating activity [14; 15]. The R372L mutation in the GC centre predicted to affect the affinity of GTP to IRAK3 protein decreases in vitro cGMP production by IRAK3 protein and importantly, impairs the suppressive functions of IRAK3 on LPS-induced NF\(\kappa\)B activation [14]. The inhibitory action of IRAK3 R372L mutant on NF\(\kappa\)B activity is recovered by supplementing cells with sub-nanomolar membrane permeable cGMP and not further improved when using higher amounts of cGMP [14]. Such low levels of cGMP correlate with the relatively low GC activity of IRAK3 (at least in vitro) and it has been suggested that IRAK3 may generate a cGMP-enriched nanodomain surrounding itself and interacting proteins [26]. Sub-nanomolar cGMP levels were confirmed to suppress inflammatory markers in LPS stimulated cells, but have no effect in the IRAK3 knockdown/knockout cells, suggesting cGMP-mediated inhibition of inflammatory cytokine production requires the normal degree of expression of wild type IRAK3 proteins in monocytes (Figures 1 and 4, and Supplemental Figure S7C,E). It is possible that immunosuppressive signal is amplified in the presence of cGMP via IRAK3 acting as a checkpoint molecule upon LPS stimulation.