Autocrine Interferon Priming in Macrophages but Not Dendritic Cells Results in Enhanced Cytokine and Chemokine Production after Coronavirus Infection

Zhou, Haixia; Zhao, Jincun; Perlman, Stanley

Real-time quantitative PCR (qPCR) analysis. RNA was isolated from BMM, BMDC, or 17Cl-1 cells using Trizol (Invitrogen). The levels of mRNA of genes of interest were determined by real-time qPCR as previously described (43) . Primers are listed in Table 1 . Cycle threshold (C T ) values were normalized to the values for the housekeeping gene hypoxanthine phosphoribosyltransferase (HPRT) by the following equation: C T ϭ C T (gene of interest) Ϫ C T (HPRT) . All results are shown as a ratio to HPRT calculated as Ϫ2 C T . IFN bioassay. The levels of IFN were measured using a bioassay based on the inhibition of vesicular stomatitis virus (VSV) growth in L929 cells. Supernatants were harvested from infected BMM or BMDC, and infectious virus was inactivated by exposure to UV light. L929 cells infected with 10,000 PFU VSV were treated with dilutions of supernatants or defined amounts of recombinant murine IFN-␤ at 30 min p.i. The titers of VSV were determined on Vero cells. IFN levels were calculated on the basis of standard curves generated with recombinant IFN-␤.