# Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3
## 3 Discussion  
There is still a great need to understand the mechanisms to fine tune inflammatory immune responses to add to the repertoire of existing anti-inflammatory drugs for treatments of inflammatory diseases such as allergy, asthma and ulcerative colitis. It is noteworthy that glucocorticoids which are a highly effective mainstay treatment for inflammatory conditions upregulate expression and activity of IRAK3 [4], thus demonstrating that modulating IRAK3 activity is a viable therapeutic target. In addition, cGMP regulates many biological processes [20], and GC inhibitors and activators (e.g., Nesirtilde, Sildenafil, Ricoiguat) have been developed as pharmaceutical therapies for pulmonary hypertension, cardiovascular diseases, angina pectoris or erectile dysfunction [47; 48]. Sildenafil, a phosphodiesteraseerase inhibitor used for the treatment of blood hypertension and erectile dysfunction, also influences the proliferation of regulatory T cells and pro-inflammatory cytokines [18]. Those data underlie the immunomodulatory effects and potential use of GC inhibitors and activators as therapeutic agents in immune diseases but clinical data in the human immune system are limited. Hence, it is necessary to gain further insights into mechanism(s) of cGMP regulation in human immune responses. Here, we explored the association of IRAK3--an immune checkpoint molecule--with cGMP, as IRAK3 has GC activity and is predicted to generate a cGMP nanoenvironment surrounding itself and interacting proteins (Figure 6) [14; 26]. We show that low levels of cGMP can reduce monocytic TNF-\(\alpha\) and IL-6 output and that this depends upon the abundance of IRAK3. The results highlight the need for IRAK3 protein to contain an active death domain for binding to protein complexes and to a lesser extent to contain an active GC centre. Moreover, the results point to the possibility of repurposing existing drugs (at low doses) that modify cGMP levels to facilitate IRAK3 action in suppressing cytokine production. Figure 5: Complementation of K6-3 THP-1 IRAK3\({}^{-/-}\) cells with wild type and mutant IRAK3 proteins. (**A**) Schematic of IRAK3 domain architecture indicating point mutations (purple stars) introduced in the death domain (W74A, R97A) and in the GC centre (R372L). (**B,C**) TNF-\(\alpha\) and IL-6 protein levels in K6-3 THP-1 IRAK3\({}^{-/-}\)-cells complemented with IRAK3 wild type (WT) or specific IRAK3 mutants. K6-3 THP-1 IRAK3\({}^{-/-}\) cells were transfected with vectors carrying wild type or mutated IRAK3 gene; untransfected K6-3 THP-1 IRAK3\({}^{-/-}\) cells were used as the negative control. After transfection, the cells were treated with 1 ug/mL LPS for 24 h, and cell supernatants were collected for quantification of TNF-\(\alpha\) and IL-6 protein levels using ELISA (mean \(\pm\) SEM, \(n\) = 4–9, One-way ANOVA followed by [PERSON]’s multiple comparisons test; * \(p\) < 0.05, ** \(p\) < 0.01, ns not significant, pink hash mark (#) in columns indicates the statistically significant difference between WT and IRAK3 mutants, # \(p\) < 0.05, # \(p\) < 0.01). The effects of cGMP across a large concentration range from femtomolar to micromolar were examined to fill a gap left by most prior studies that only investigated higher concentrations of cGMP in the micromolar to millimolar range [28; 29; 31; 32; 34; 39; 40; 41; 42; 43]. cGMP at sub-nanomolar (0.1 nM) levels is surprisingly effective at downregulating NFxB activity, and pro-inflammatory cytokine (TNF-\(\alpha\) and IL-6) levels induced by LPS challenge in monocytes (Figure 1). We also report that higher cGMP concentrations are effective and these findings agree with previous studies reporting the inhibitory effect of higher cGMP concentrations (1 \(\upmu\)M-1 mM) on inflammation in LPS-stimulated cells [28; 34; 42; 50].