Fig 1: Pharmacological Inhibition of Set7 Dose-Dependently Attenuates the Pro-inflammatory Cytokine Response of Trained Immunity In Vitro(A) Western blot analysis of β-glucan-trained macrophages incubated with cyproheptadine (CPH) for 24 h. HSP90 was used as a loading control.(B) Graphical overview of in vitro training methods. Adherent monocytes (Mo) were stimulated with β-glucan or RPMI culture medium for 24 h in the presence of CPH or DMSO vehicle control, allowed to differentiate to macrophages (Mϕ), and restimulated for 24 h with LPS, Pam3Cys, or RPMI on day 6.(C and D) Production of (C) TNFα and (D) IL-6 by β-glucan-trained macrophages incubated with CPH or 5′-methylthioadenosine (MTA) for the first 24 h of in vitro training and restimulated with LPS (n = 7 healthy volunteers).(E) Production of TNFα by β-glucan-trained macrophages incubated with CPH for the first 24 h of in vitro training and restimulated with Pam3Cys (n = 3 healthy volunteers).(F) Expression of SETD7 mRNA on day 6 by cells trained with β-glucan in the presence of 100 μM CPH (n = 6 healthy volunteers; open bar represents DMSO vehicle control).(G) Expression of RPL29 mRNA at 24 h by cells trained with β-glucan in the presence of 100 μM CPH (n = 3 healthy volunteers; open bar represents DMSO vehicle control).(H) Analysis of lactate dehydrogenase (LDH) as a measure of cytotoxicity in cells incubated with 100 μM CPH for 24 h (n = 3 healthy volunteers).(I) Analysis of viability and apoptosis with Annexin V and PI staining in cells incubated with 100 μM CPH versus DMSO vehicle controls for 24 h (n = 3 healthy volunteers). Fold change difference between CPH and vehicle controls.(J and K) Production of (J) TNFα and (K) IL-6 by β-glucan-trained macrophages incubated with sinefungin for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers).(L) Production of TNFα and IL-6 by bacillus Calmette-Guérin (BCG)-trained macrophages incubated with 100 μM CPH for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers).(M) Production of TNFα and IL-6 by laminarin-trained macrophages incubated with 100 μM CPH for the first 24 h of in vitro training and restimulated with LPS (n = 6 healthy volunteers).Data are represented as mean ± SEM. ∗p < 0.05, Wilcoxon signed-rank test or t test where appropriate.See also Figures S1 and S2.