# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 1 Introduction
Sophisticated interplay or integration of several messenger molecules such as cGMP or cyclic adenosine monophosphate (cAMP) with calcium ions and strict intracellular compartmentalization of these messengers is important in mediating cyclic nucleotide signalling [24; 25]. Generating ordered spatial and temporal and stimulus-specific intracellular messages requires confining messengers (cGMP or cAMP) within defined cytoplasmic areas or nanoenvironment(s) involving the accurate intracellular placement of enzyme and scaffold protein components of signal cascades [24; 26; 27]. cGMP has different effects on inflammation under stimulated versus unstimulated conditions [28]. In unstimulated conditions, treatment with GC activators (e.g., NO donor, ANP) enhances expression of inflammatory mediators including NF*B activity and pro-inflammatory cytokines [28; 29; 30; 31; 32]. Whereas these cGMP modulating treatments attenuate inflammatory responses in LPS- or TNF-\(\alpha\)-stimulated conditions [28; 31; 32; 33; 34; 35; 36; 37; 38; 39; 40; 41; 42]. Decreasing GC activity and thus cGMP formation by using GC inhibitors suppresses cGMP effects on inflammation in both unstimulated and LPS-stimulated conditions [29; 30; 39]. However, there were controversial results; for instance in LPS stimulated conditions cGMP treatment increases TNF-\(\alpha\) protein levels [43] and downregulating cellular cGMP with GC inhibitors reduced LPS-induced TNF-\(\alpha\) production [29; 30]. Notably, the impact of NO on inflammation appears to be more complicated. NO is produced by NO synthases from stimulated innate immune cells and metabolised to other reactive nitrogen species that have cytocidal effects on pathogenic microbes [44]. NO also links innate and adaptive immunity via regulating T-cell activation and functions [44, 45]. NO has biphasic actions as it increases NF\(\kappa\)B and IL-6 mRNA and protein levels at lower concentrations (\(\leq\)10 \(\upmu\)M) and decreases these inflammatory markers at higher concentrations (\(>\)100 \(\upmu\)M) [39]. The GC activity of IRAK3 is similar to those reported in several plant receptor kinases with cryptic GC activity [24, 26]. A mutation in the IRAK3 GC centre reduces cGMP production and disables the inhibitory impact of IRAK3 on LPS-induced NF\(\kappa\)B activity [14], indicating the importance of this GC centre in regulating IRAK3 function. In a study by [PERSON] et al. [14], the addition of sub-nanomolar levels of membrane permeable cGMP reversed the impairment of LPS-induced NF\(\kappa\)B activity seen with the IRAK3 GC centre mutant. Thus, it is feasible that there is a direct interaction between cGMP and IRAK3 on inflammation. We hypothesise that low amounts of cGMP generated by IRAK3 may form a nanoenvironment surrounding IRAK3 and contribute to its actions. As there is a shortage of data on the inflammatory effects of cGMP at low (sub-nanomolar) levels, our study investigates how different levels of cGMP influence expression of inflammatory markers (NF\(\kappa\)B activity, IL-6 and TNF-\(\alpha\)) in monocytes where IRAK3 is endogenously expressed. Cellular cGMP levels were altered by direct addition of membrane permeable cGMP (8-Br-cGMP), or by treatment with pharmacological agents. To elucidate the role of IRAK3 and cGMP, we generated IRAK3 knockdown THP-1 monocyte cell lines and undertook complementation studies with wild type and loss-of-function IRAK3 mutants to further examine the mechanisms involved in modulating cytokine production.