# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 3 Discussion
cGMP has no effect on NFxB activity in unstimulated human monocytes (Supplemental Figure S1B), and this is consistent with previous results [41; 51], although other studies showed that cGMP at 10 \(\upmu\)M increased LPS-unstimulated NFxB activity and cytokine expression [28; 32; 39]. The reasons for these differences are unclear but may be attributed to the measured outcomes, NFxB--DNA binding activity [28; 39; 49] versus the NFxB SEAP reporter assay used in this study. Figure 6: Schematic of a proposed mechanism for the interaction of IRAK3 and cGMP to modulate inflammatory cytokine production in TLR4/IL1R signalling pathway. Activation of TLR4/IL1R by endotoxins such as LPS (indicated by stars) leads to the formation of myddosome complexes comprising MyD88, IRAK4 and IRAK1/IRAK2, then activates TRAF6, that transduces the signal to degrade IxB\(\alpha\) (an NFxB inhibitor) and trigger nuclear translocation of the transcription factor NFxB containing p50 and RelA subunits. NFxB induces the transcription of inflammatory genes (TNF-\(\alpha\) and IL-6), consequently leading to cytokine production. IRAK3 generates cGMP to create a nanoenvironment surrounding its protein interactome and inhibits the association of IRAK1 or 2 with TRAF6, thus suppressing the downstream cascade. Sub-nanomolar levels of cellular cGMP directly added via membrane permeable cGMP (mp-cGMP) or pharmacologically modulated using DEA NONOate (NO donor) or ODQ (a soluble GC inhibitor), lead to changes in NFxB activity and cytokine production. NO released from NO donor also stabilizes IxB\(\alpha\) by inhibiting its degradation from NFxB, causing reduced NFxB activity [49]. Figure drawn in Adobe Illustrator, red stop ampel man from Wiki commons image downloaded 3 June 2020. Pharmacologically increasing cellular cGMP levels by treatment with the NO donor (DEA NONOate, a soluble GC stimulator) also decreased LPS-induced NF\(\kappa\)B activity and pro-inflammatory cytokines (Figure 2A,B). Membrane permeable cGMP at 0.1 nM and 100 nM further decreased NF\(\kappa\)B activity lowered by NO in LPS-stimulated cells (Figure 2C). This agrees with previous reports showing NO or other GC activators such as ANP inhibit NF\(\kappa\)B activation and pro-inflammatory cytokine expression [28, 32, 52]. However, in unstimulated conditions, NO has biphasic effects, including stimulatory action on inflammatory responses at lower (\(\leq\)10 nM) and inhibitory actions at higher (>100 nM) concentrations [39] in a cGMP-dependent manner [32, 33]. NO is synthesised along with L-citrulline from L-arginine in a reaction catalysed by NO synthase [53]. Thus, diminished bioavailability of L-citrulline and L-arginine can indicate a reduction in NO production, in turn resulting in increased levels of inflammatory cytokines. Notably, IRAK3 is highly expressed in severe asthma and COVID-19 cohorts, where levels of L-citrulline and L-arginine are decreased in sera of COVID-19 and asthma patients [53, 54]. These changes may contribute to the cytokine storm occurring in COVID-19 patients and is consistent with our data showing increasing NO donor reduces LPS-induced levels of TNF-\(\alpha\) and NF\(\kappa\)B activity (Figure 2A,B). Although IRAK3 gene expression is raised in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial cells [53], treatment with SARS-CoV-2 spike protein downregulated IRAK3 expression in THP1-differentiated macrophages, human peripheral blood mononuclear cells and monocytes [55]. These findings indicate that the SARS-CoV-2 infection causes a dramatic production of inflammatory cytokines by decreasing IRAK3 expression and level of NO. NO downregulates NF\(\kappa\)B activity via differential actions that partly involve IRAK3/cGMP pathway(s) (Figure 6). NO enhances the expression of IRAK3 which in turn diminishes molecular interactions of IRAK1 or 2 with TRAF6, and downstream cascades [56] (Figure 6).