Figure 4. β2integrin ligation by fibrinogen induces USP18, a MAPK-dependent negative regulator of IFN-α signaling.

Primary human macrophages were plated onto control wells or fibrinogen (Fb) coated wells.

A, Cumulative data from macrophages plated for 4 h. mRNA expression was measured by qPCR and normalized relative to GAPDH mRNA. Results are presented as mean ± SEM of 6 independent donors. Statistical analysis was performed using the 2-tailed, paired Student t test, p = 0.0198.

B, Macrophages were pretreated with either the vehicle control DMSO or two MAPK inhibitors: p38 inhibitor SB203580 (15 μM) and MEK inhibitor U0126 (15 μM) for 30 min. Macrophages were plated for 4 h, and IFN-α (1000 U/mL) was added for 15 min. Whole cell lysates were immunoblotted with Abs against USP18 and p38α. Data are representative of 2 independent experiments.

C, Macrophages were pretreated with either the vehicle control DMSO or Syk inhibitor piceatannol (80 μM) for 30 min. Macrophages were plated for 4 h, and IFN-α (1000 U/mL) was added for 15 min. Whole cell lysates were immunoblotted with Abs against USP18 and p38α. Data are representative of at least 3 independent experiments.

D – E, Macrophages were nucleofected with non-specific (control) or USP18-specific short interfering RNAs (siRNA), and 3 days later were plated onto control or Fb-coated wells and stimulated with IFN-α for 2 hrs. D, Immunoblot shows knockdown efficiency. E, mRNA expression was measured by qPCR and normalized relative to GAPDH mRNA. Error bars represent SD of triplicate wells, and data are representative of 3 independent experiments. F, Cumulative data from 3 donors is presented as mean ± SEM. Transcript expression in IFN-α-stimulated control cells was set as 100%, and expression in all other conditions was set relative to that control. * p = 0.0175, paired Student t test; NS = not significant (p = 0.1963).