# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 2 Results
### Pharmacologically Altering cGMP Levels Changes Inflammatory Responses  
To test if changes in intracellular cGMP altered LPS-induced NF\(\kappa\)B activity or cytokine production, we employed pharmacological modulators of soluble GCs. As NO activates cGMP generation by soluble GCs [20], we used different concentrations of the NO donor (DEA NONOate) to determine if endogenous cGMP affects inflammatory responses. TNF-\(\alpha\) and IL-6 protein levels are significantly downregulated at micromolar concentrations of the NO donor in LPS-stimulated THP-1 cells (Figure 2A,B). To confirm whether the effect of the NO donor on inflammatory signals responds to additional cGMP, monocytes were treated with the NO donor (at 1 uM), simultaneously with 8-Br-cGMP and LPS for 18-24 h. NF\(\kappa\)B activity reduced by NO in LPS-stimulated cells is further decreased by membrane permeable cGMP at 0.1 nM (\(10^{-10}\) M) and 100 nM (\(10^{-7}\) M) (Figure 2C). Figure 1: Effects of cGMP on inflammatory markers (NF\(\kappa\)B activity, TNF-\(\alpha\) and IL-6 production) in monocyte cell lines. (**A**) 8-Br-cGMP affects LPS-induced NF\(\kappa\)B activity in THP-1-BLUE cells. THP-1-BLUE cells were treated with 1 ug/mL LPS and 8-Br-cGMP at concentrations from 0.1 fm to 10 uM (\(10^{-16}\) to \(10^{-5}\) M) for 24 h, and SEAP activity correlating to NF\(\kappa\)B activity was measured and normalised to control (Con) cells treated with LPS only (mean \(\pm\) SEM, \(n=6\), One-way ANOVA followed by [PERSON]’s multiple comparisons, *\(p<0.05\), **\(p<0.01\), ***\(p<0.001\), ***\(p<0.001\), ns: not significant). (**B,C**) 8-Br-cGMP affects LPS-induced TNF-\(\alpha\) (**B**) and IL-6 (**C**) protein production in THP-1 cells. THP-1 cells were treated with 1 ug/mL LPS and 8-Br-cGMP at 100 fm (\(10^{-13}\) M), 100 pM (\(10^{-10}\) M) and 100 nM (\(10^{-7}\) M), and 24 h post-treatment cell supernatants were collected for quantification of TNF-\(\alpha\) and IL-6 protein levels using ELISA. Control (Con) cells were treated with LPS only (mean \(\pm\) SEM, \(n=3\)–5, One-way ANOVA followed by [PERSON]’s multiple comparisons test, **\(p<0.01\), ***\(p<0.001\), ns: not significant). To determine whether downregulation of cellular cGMP levels influences inflammation, the effect of ODQ, an inhibitor of soluble GC, on NF\(\kappa\)B activity and cytokine level was examined. ODQ at 100 nM (10\({}^{-7}\) M) and 1 uM (10\({}^{-6}\) M) enhanced LPS-induced NF\(\kappa\)B activity (Figure 3A), while 10-100 nM (10\({}^{-8}\)-10\({}^{-7}\) M) ODQ resulted in significant increases in LPS-mediated TNF-\(\alpha\) protein production (Figure 3B). However, the effect of ODQ at higher concentrations (1 or 10 uM) tends to be weaker, which may be due to impairment of cell viability by ODQ (approximately 15% reduction in cell viability between untreated cells and cells treated with 10 uM ODQ; Supplemental Figure S2). To check if the effect of ODQ on inflammation is cGMP dependent, cells were treated simultaneously with ODQ and 8-Br-cGMP. A concentration of ODQ of 100 nM was selected as it did not affect cell viability (Supplemental Figure S2) but significantly enhanced NF\(\kappa\)B activity and TNF-\(\alpha\) levels (Figure 3A,B). The influence of 100 nM ODQ on LPS-stimulated NF\(\kappa\)B activation was reversed by the addition of at least 0.1 nM (10\({}^{-10}\) M) 8-Br-cGMP (Figure 3C). Figure 3: Effects of ODQ (a soluble GC inhibitor) on LPS-induced NF\(\kappa\)B activity and TNF-\(\alpha\) production. (**A**) ODQ stimulates LPS-induced NF\(\kappa\)B activity. THP-1 BLUE cells were treated with 1 ug/mL  
Figure 2: Effects of NO donor (DEA NONOate) on cytokine production and NF\(\kappa\)B activity. (**A**) and (**B**) NO donor decreases LPS-induced TNF-\(\alpha\) (**A**) and IL-6 (**B**) production. THP-1 cells were treated with 1 ug/mL LPS without (control; Con) or with DEA NONOate at indicated concentrations, and 24 h post-treatment cell supernatants were collected to quantify TNF-\(\alpha\) and IL-6 protein levels using ELISA.