# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 3 Discussion
Membrane permeable 8-Br-cGMP (0.1 nM-10 uM) reduces LPS-induced NFkB activity in a dose-dependent manner in human embryonic kidney (HEK BLUE) cells not expressing IRAK3 [14], whereas cGMP effect is dose-independent in human monocytic THP-1 cells that natively express IRAK3 (Figure 1A). Collectively, these data imply that IRAK3 synergically acts within a nanoenvironment of cGMP to modulate inflammation. Although IRAK3 protein expression is unaffected by cGMP treatment (Supplemental Figures S3 and S4), cGMP can modulate IRAK3 actions without changing its expression level. As IRAK3 generates cGMP, it may create a localised nanoenvironment that can inhibit inflammation or alternatively mediate IRAK3 enzymatic activity or interaction with other IRAKs or MyD88 to control the TLRs/IL-1R pathway. Complementation with IRAK3 wild type in the knockdown cells causes decreases in TNF-\(\alpha\) and IL-6 production as expected, showing the knockdowns are effective (Figure 5 and Supplemental Figure S9). LPS-induced TNF-\(\alpha\) and IL-6 protein levels are not significantly altered by complementation with any of the IRAK3 mutants tested (Figure 5 and Supplemental Figure S9), implicating these residues in the death domain or GC centre in enabling IRAK3 actions. The IRAK3 R372L mutant is less successful in complementing the knockdown cells than wild type protein, and not significantly different at inducing cytokine production from the control untransfected cells (Figure 5 and Supplemental Figure S9). However, the IRAK3 R372L mutant was not statistically different from the complementation with the wild type (Figure 5 and Supplemental Figure S9). One reason may be that enough basal cGMP is present in monocytes for the mutant IRAK3 to function and this mutant partially responds to additional cGMP as shown previously [14]. The other two mutated versions of IRAK3 (R97A and W74) contain mutations introduced in the death domain that are required for IRAK3 to bind to other proteins, including other IRAKs and MyD88, thus IRAK3 action on inflammation is inhibited [7; 10]. Correspondingly, stably expressing either two death domain mutants in wild type THP-1 cells also raised IL-6 or TNF-\(\alpha\) protein levels compared to IRAK3 wild type--transduced cells [7]. Complementation with either W74A or R97A mutants confirmed the importance of IRAK3 interactions via the death domain to downregulate IL-6 and TNF-\(\alpha\) production (Figure 5 and Supplemental Figure S9). Despite not directly engaging IRAK3 interaction with myddosome complexes involved in NF\(\kappa\)B activation cascade, GC centre still influenced IRAK3 effects on expression of inflammatory cytokines, possibly via a cGMP nanoenvironment generated by GC centre itself. The second messenger cGMP formed by soluble GCs, that are activated by NO or deactivated by soluble GC inhibitors such as ODQ, downregulates inflammatory responses to endotoxin stimulation via IRAK3 molecules (Figure 6). Additionally, IRAK3 produces a low amount of cGMP potentially involved in regulating its functions (Figure 6). Together this suggests that the inflammatory responses can be modulated by regulating cellular cGMP levels. We show how modifying cellular cGMP levels using different approaches may contribute to the localised cGMP nanoenvironment generated by IRAK3 and thus modulating the degree of inflammation. Our results hint at the possibility of facilitating IRAK3 action by repurposing existing drugs (at low doses) that modify cGMP levels to complement other treatments such as glucocorticoids that are associated with many side effects. Our study confirms the effectiveness of very low changes in cellular cGMP levels in modulating TNF-\(\alpha\) and IL-6 cytokine production and NF\(\kappa\)B activity using an in vitro cell model.