Roles of retinoic acid-inducible gene-I-like receptors (RLRs), Toll-like receptor (TLR) 3 and 2'-5' oligoadenylate synthetase as viral recognition receptors on human mast cells in response to viral infection

Abstract

To investigate the anti-viral responses of human mast cells, we performed PCR array analysis of these cells after infection with vesicular stomatitis virus (VSV). PCR array analysis revealed that human mast cells up-regulated several anti-viral genes, including melanoma differentiation-associated gene 5, retinoic acid-inducible gene-I, and Toll-like receptor 3, together with type I interferons and chemokines, upon VSV infection. Additionally, we found that 2'-5' oligoadenylate synthetase, which also works as a virus recognition receptor by activating the latent form of RNase L, leading to viral RNA degradation, was up-regulated in human mast cells upon VSV infection. Moreover, small interfering RNA analysis to identify the receptors responsible for mast cell activation by VSV revealed that these receptors reciprocally cooperate to produce anti-viral cytokines and chemokines, inhibiting VSV replication. Our findings suggest that human mast cells produce cytokines and chemokines using several viral recognition receptors, leading to the inhibition of viral replication. These data provide novel information that improves our understanding of the roles of human mast cells in immune responses against viruses.

Fig. 1

Human mast cell line LAD2 cells are responsive to virus infection by inducing anti-viral type I interferons. LAD2 cells were stimulated with the indicated concentrations of VSV for 24 h (a) or with VSV (MOI = 100) for the indicated times (b). The levels of mRNA for IFN-α and IFN-β were determined by quantitative PCR. The columns present the mean ± SD of 3 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with un-stimulated cells

Fig. 2

Viral infection directly causes anti-viral chemokine production in a human mast cell line. LAD2 cells were stimulated with various concentrations of VSV for 24 h (a) or with VSV (MOI = 100) for the indicated times (b). The levels of mRNA for CCL5, CXCL10, CXCL11 and IL-15 were determined by quantitative PCR. The columns present the mean ± SD of 3–5 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with un-stimulated cells

Fig. 3

Viral infection causes antiviral cytokine production at the protein level by human mast cell line LAD2 cells. The production of IFN-α, IFN-β, CCL5, CXCL10, CXCL11 and IL-15 upon stimulation with the indicated concentrations of VSV for 24 h was measured by ELISA as described in the “Materials and methods” section. The columns present the mean ± SD of 3 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with un-stimulated cells

Fig. 4

LAD2 cells do not degranulate upon VSV infection. LAD2 cells were stimulated with the indicated concentrations of VSV for 1 to 24 h. β-hexosaminidase release was measured as described in the “Materials and methods” section. PMA/ionomycin was used as a positive control. The columns present the mean ± SD of 3 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with un-stimulated cells

Fig. 5

LAD2 cells express several OAS proteins, in addition to RLRs, and their expression levels are up-regulated by viral infection. The mRNA levels of MDA5, RIG-I, and TLR3 in (a) un-stimulated LAD2 or (b) LAD2 cells stimulated with VSV (MOI = 100) for 24 h were quantified by real-time PCR and expressed as relative expression levels based on the β-actin levels. The mRNA levels of OAS1–3 in un-stimulated LAD2 cells (c) or LAD2 cells stimulated with VSV (MOI = 100) for 24 h (d) were quantified by real-time PCR and expressed as relative expression levels based on the β-actin levels. (e) The protein levels of these receptors were analyzed by Western blotting. Equal protein loading in each lane was confirmed by reprobing the same membrane with anti-β-actin. The columns present the mean ± SD of 3 experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the un-stimulated cells

Fig. 6

Several virus recognition receptors reciprocally contribute to anti-viral cytokines and chemokines and to VSV replication. Small interfering RNAs were used to knockdown endogenous MDA5, RIG-I and TLR3 or OAS 1-3 as described in the “Materials and methods” section. The cells were stimulated with VSV 24 h after the corresponding siRNA transfection. The levels of IFN-α, IFN-β, CCL5, CXCL10, CXCL11 and IL-15 were determined by real-time PCR 24 h after VSV infection (a). Virus replication was examined using Vero cells infected with the supernatants of LAD2 cells that were stimulated with VSV after the corresponding knockdown of receptors as described in the “Materials and methods” section (b, c, d). The columns present the mean ± SD of 3 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared with the control siRNA-treated cells