HHV-6B induces IFN-lambda1 responses in cord plasmacytoid dendritic cells through TLR9

Abstract

Human herpesvirus type 6B (HHV-6B) is a strong inducer of IFN-alpha and has the capacity to promote Th1 responses and block Th2 responses in vitro. In this study we addressed whether inactivated HHV-6B can also induce IFN lambda responses and to what extent interferons alpha and lambda affect Th1/Th2 polarization. We show that inactivated HHV-6B induced IFN-lambda1 (IL-29) but not IFN-lambda2 (IL-28A) responses in plasmacytoid DC and that this induction was mediated through TLR9. We have previously shown that HHV-6B promotes Th1 responses and blocks Th2 responses in both humans and mice. We now show that neutralization of IFN-alpha but not IFN-lambda1 blocked the HHV-6B-induced enhancement of Th1 responses in MLR, but did not affect the HHV-6-induced dampening of Th2 responses. Similarly, blockage of TLR9 counteracted HHV-6Bs effects on the Th1/Th2 balance. In addition, IFN-alpha but not IFN-lambda1 promoted IFN-gamma production and blocked IL-5 and IL-13 production in purified CD4+ T-cells. The lack of effect of IFN-lambda1 correlated with the absence of the IFN-lambda receptor IL-28Ralfa chain on the cell surface of both resting and activated CD4+ T-cells. We conclude that inactivated HHV-6B is a strong inducer of IFN-lambda1 in plasmacytoid DC and that this induction is TLR9-dependent. However, human CD4+ T-cells do not express the IFN-lambda receptor and are refractory to IFN-lambda1 treatment. The HHV-6B-induced alterations in the Th1/Th2 balance are instead mediated mainly through TLR9 and IFN-alpha.

Figure 1. HHV-6B induces the production of IFN-alpha, IFN-lambda1 but not IFN-lambda2 in pDC in a TLR9-dependent fashion.

Purified cord pDC were exposed to inactivated HHV-6B in the presence or absence of G-ODN, a TLR9-specific inhibitor. The levels of IFN-alpha (A; n = 20), IFN-lambda1 (B; n = 15) and IFN-lambda2 (C; n = 5) were analyzed in 24h culture supernatants. Data are expressed as mean + SEM. **  =  p<0.01, ***  =  p<0.001 using ANOVA with Bonferroni's multiple comparison test. The levels of IFN-lambda1 and IFN-lambda2 were also assessed over a 72 h time-period (D; n = 3).

Figure 2. Neutralization of IFN-alpha but not IFN-lambda1 blocks the HHV-6B-induced promotion of IFN-gamma responses in cord mixed lymphocyte reactions.

Cord blood pDC were incubated with allogeneic cord-blood CD4+ T-cells in the presence or absence of inactivated HHV-6B and neutralizing antibodies to IFN-alpha or IFN-lambda1. Supernatants were collected after 48 h and analyzed for IFN-gamma (A), IL-5 (B) and IL-13 (C) content. Data are expressed as medians and the 25% and 75% percentile (the boxes) with the minimum and maximum responses for n = 9. *  =  p<0.05 using ANOVA with Bonferronís multiple comparison test.

Figure 3. HHV-6B affects the IFN-gamma and IL-13 responses in cord mixed lymphocyte reactions via TLR9 signaling.

Cord blood pDC were incubated with allogeneic cord-blood CD4+ T-cells and inactivated HHV-6B in the presence or absence of G-ODN. Supernatants were collected after 48 h and analyzed for IFN-gamma (A), IL-5 (B) and IL-13 (C) content. Data are expressed as medians and the 25% and 75% percentile (the boxes) with the minimum and maximum responses for n = 5. *  =  p<0.05 using student's t-test.

Figure 4. IFN-alpha but not IFN-lambda1 promote Th1 responses and block Th2 responses in activated cord CD4+ T-cells.

Cord blood CD4+ T-cells were activated with anti-CD3 (A–B) or anti-CD3 and anti-CD28 (C) in the presence or absence of recombinant human IFN-alpha or IFN-lambda1. Supernatants were collected after 48 h and analyzed for IFN-gamma (A), IL-5 (B) and IL-13 (C) content. Data are expressed as medians and the 25% and 75% percentile (the boxes) with the minimum and maximum responses for n = 5. *  =  p<0.05 using ANOVA with Bonferroni's multiple comparison test.

Figure 5. CD4+ T-cells do not express IL-28Ralpha.

Resting or activated cord blood mononuclear cells were analyzed for IL-28Ralpha expression using FACS. IL-28Ralpha expression (histograms) on pDC (A) or CD4+ T-cells obtained directly upon isolation (B) or after 24 h incubation with anti-CD3 (C) or recombinant human IFN-alpha (D). Filled red areas represent isotype-PE control and blue lines represent IL-28Ralpha-PE.