TRIF, and TRIF-interacting TLRs differentially modulate several adenovirus vector-induced immune responses

Abstract

The use of Adenovirus (Ad)-based vectors has proven to be a useful platform for the development of gene therapy and vaccine protocols. The immunological mechanisms underlying these properties need to be identified and understood to foster safer, more efficacious use of this important gene transfer platform. Our recent studies have confirmed an important role for MyD88 dependent toll-like receptor (TLR) pathways as mediators of these responses. In this study, we confirm that TLR3, TLR4 and TRIF (TIR-domain-containing adapter-inducing interferon-beta) can also have augmentative or inhibitory roles during Ad-induced immune responses. Importantly, our studies revealed that TLR4 acts to suppress several aspects of the Ad-induced innate immune response, a finding not previously reported for this TLR in any model system. In addition, using MyD88 and TRIF double knockout mice, we demonstrate that the MyD88 and TRIF adaptor proteins can play either additive or redundant roles in mediating certain aspects of Ad vector-induced innate and adaptive immune responses. Furthering this complexity, our model system strongly suggests that non-TLR based systems must not only exist, but also have a significant role to play during Ad vector-mediated induction of adaptive immune responses.

Fig. 1

Role of TRIF, TLR3 and TLR4 in Ad-vector induced Iκ Bα degradation and ERK phosphorylation. Wild-type C57BL/6, TRIF-KO (a, b), TLR3-KO (c, d), TLR4-KO (e, f) mice were injected intravenously with adenoviral vectors (1.5 × 10¹¹ viral particles per mouse). The livers were collected at the indicated time points, snap frozen and lysates extracted as described in ‘Materials and Methods’. IκBα and tubulin (a, c, e) and p-ERK1/2 and ERK2 (b, d, f) levels were determined by Western blot analysis using Licor's Odyssey. Quantification was performed after normalizing the p-ERK levels to ERK2 and IκBα levels to Tubulin, to control for loading. n = 3 for all groups of mice. For each analysis, quantification is shown on the top and a representative blot on the bottom. The bars represent mean ± SD. Statistics were completed using 1-way ANOVA followed by Tukey's post-hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. = not significant; NI = no injection.

Fig. 2

Dual roles for TRIF utilizing receptors in Ad stimulated induction of cytokines and chemokines. C57BL/6 (n = 18), TLR3-KO (n = 13), TRIF-KO (n = 18), C57BL/10 (n = 8) and TLR4-KO (n = 8) mice were intravenously injected with 7.5 × 10 10 v.p./mouse of Ad5-LacZ or mock injected with PBS (n = 3 for all). Plasma samples were isolated at 6 h.p.i. and analyzed using a custom multiplex bead assay from Bio-Rad. The bars represent the mean × SE. Statistical analyses consisted of ANOVA followed by Newman-Keuls post-hoc test for C57BL/6, TLR3-KO and TRIFKO comparisons. A homoscedastic 2-tailed t test was used to compare levels obtained in C57BL/10 mice compared to TLR4-KO animals. * p < 0.05; ** p < 0.01, vs. identically injected wildtype mice.

Fig. 3

Partial roles for TRIF-utilizing TLRs in antibody responses generated against Ad vectors and their encoded transgene (β-galactosidase). C57BL/6 (n = 5), TLR3-KO (n = 4), TRIF-KO (n = 5), C57BL/10 (n = 3) and TLR4-KO (n = 5) were intravenously injected with 7.5 × 10¹⁰ v.p./mouse of Ad5-LacZ. Anti-β-gal and anti-Ad-specific antibody titers were evaluated at the appropriate plasma dilutions. Bars represent mean ± SD. A homoscedastic 2-tailed t test was used to compare levels obtained in knockout animals with those in control wild-type animals at each time point. T = TRIF-KO; 3 = TLR3-KO. A single (p < 0.05) or double (p < 0.01) character indicates statistically different values compared to identically injected wild-type mice. * p < 0.05; ** p < 0.01, TLR4-KO vs. C57BL/10 wild-type mice.

Fig. 4

Ad-induced innate toxicities are partially mediated by both MyD88 and TRIF. C57BL/6 (n = 5), MyD88-KO (n = 4), TRIF-KO (n = 5), and DBL-KO (n = 4) were intravenously injected with 7.5 × 10 10 v.p./mouse of Ad5-LacZ. a Thrombocytopenia was evaluated at 24 h.p.i. by enumeration of blood platelet levels. Bars represent mean ± SD. b Plasma levels of cytokines and chemokines were measured at 6 h.p.i. using a custom multiplex bead assay from Bio-Rad. Bars represent the mean ± SE. For all comparisons, a homoscedastic 2-tailed t test was used to compare levels obtained in knockout animals compared to control wild-type animals. The dashed line indicates values obtained in TRIF-KO animals. * p < 0.05; ** p < 0.01, vs. identically injected wildtype mice.

Fig. 5

Both MyD88 and TRIF mediate the Ad- and transgene-specific humoral responses following systemic Ad-vector injection. C57BL/6 (n = 5), MyD88-KO (n = 4) and Dbl-KO (n = 5) were intravenously injected with 7.5 × 10 10 v.p./mouse of Ad5-LacZ. Anti-β-gal and anti-Ad-specific antibody titers were evaluated at the appropriate plasma dilutions. Bars represent mean ± SD. A homoscedastic 2-tailed t test was used to compare levels obtained in knockout animals compared to control wild-type animals at each time point. M = MyD88-KO; D = TRIF^−/− MyD88^−/− Dbl-KO mice. A single (p < 0.05) or double (p < 0.01) letter indicates statistically different values compared to identically injected wild-type mice.