MAVS and MyD88 are essential for innate immunity but not cytotoxic T lymphocyte response against respiratory syncytial virus

Abstract

Infection by RNA viruses is detected by the host through Toll-like receptors or RIG-I-like receptors. Toll-like receptors and RIG-I-like receptors signal through the adaptors MyD88 and MAVS, respectively, to induce type I IFNs (IFN-I) and other antiviral molecules, which are thought to be essential for activating the adaptive immune system. We investigated the role of these adaptors in innate and adaptive immune responses against respiratory syncytial virus (RSV), a common human pathogen. Deletion of Mavs abolished the induction of IFN-I and other proinflammatory cytokines by RSV. Genome-wide expression profiling in the lung showed that the vast majority of RSV-induced genes depended on MAVS. Although Myd88 deficiency did not affect most RSV-induced genes, mice lacking both adaptors harbored a higher and more prolonged viral load and exhibited more severe pulmonary disease than those lacking either adaptor alone. Surprisingly, Myd88(-/-)Mavs(-/-) mice were able to activate a subset of pulmonary dendritic cells that traffic to the draining lymph node in response to RSV. These mice subsequently mounted a normal cytotoxic T-lymphocyte response and demonstrated delayed but effective viral clearance. These results provide an example of a normal and effective adaptive immune response in the absence of innate immunity mediated by MAVS and MyD88.

Fig. 1.

In vitro and in vivo cytokine responses to RSV infection are lost in the absence of MAVS. Adult lung fibroblasts (A), bone marrow-derived macrophages (BMDM; B and C), and cDCs (D and E) from WT^+/+ and Mavs^−/− mice were infected with SeV or RSV for 24 h before culture supernatants were analyzed for IFN-β or -α by ELISA. Data are represented as mean ± SD. (F) Mice of the indicated genotypes were intranasally infected with RSV (10⁷ pfu) for 24 h, and then BALF was harvested for cytokine measurements by ELISA (for IFN-α, IFN-β, and IL-1β) or Cytometric Bead Array (for IL-6, TNF-α, and MCP-1) (*, P < 0.001; **, P < 0.01; ***, P < 0.05; ANOVA, Tukey's test) (n = 4 per group). n.d., not detected. Data are represented as mean ± SEM.

Fig. 2.

Global gene expression analysis of lung RNA after infection with RSV reveals profound defects in mice lacking MAVS. For each genotype, two mice were mock treated and three were inoculated with RSV (10⁷ pfu) for 24 hours before lungs were harvested for total RNA extraction, which was analyzed by microarray. (A–D) Mean relative expression of genes known to be involved in immune responses, including interferon-related (A), chemokine or cytokine receptors (B), cytokines (C), and chemokines (D). (E) Induction of selected genes was confirmed by qPCR. Data are represented as mean ± SEM.

Fig. 3.

Mavs^−/− and DKO mice are able to clear RSV and resolve pulmonary disease. (A and B) WT, Myd88^−/−, Mavs^−/−, and Myd88^−/−Mavs^−/− (DKO) mice were infected with RSV (10⁷ pfu) and BALF was harvested at the indicated times to measure viral loads by plaque assay. (A: *, mavs^−/− vs. myd88^−/−, P < 0.001; **, mavs^−/− vs. DKO and mavs^−/− vs. myd88^−/−, P < 0.001; B: *, mavs^−/− vs. DKO and mavs^−/− vs. myd88^−/−, P < 0.01; ANOVA) (n = 3–5). (C) Lungs from infected mice and mock controls were analyzed by immunohistochemistry using a polyclonal anti-RSV antibody. (D) Mice infected with RSV were assessed by whole-body plethysmography to measure airway obstruction (*, P < 0.0001; **, P < 0.03; ***, P < 0.04; ANOVA). n.d., not detected; Penh, enhanced pause. Data are represented as mean ± SEM.

Fig. 4.

Adaptive immune responses in mice lacking Mavs and Myd88. (A) RSV-specific antibody subtypes, IgG, IgG1, and IgG2a, were measured by ELISA using sera taken on the indicated day after RSV infection (IgG; *, WT vs. Myd88^−/−; P < 0.01, Myd88^−/− vs. DKO, P < 0.05; ** and ***, WT vs. Myd88^−/−, P < 0.001) (IgG1; *, Mavs^−/− vs. DKO, P < 0.01; ** and ***, WT vs. Myd88^−/− and Myd88^−/− vs. DKO, P < 0.05) (IgG2a; *, WT vs. Myd88^−/− and Mavs^−/− vs. DKO, P < 0.01; **, WT vs. Mavs^−/−, P < 0.001; ***, WT vs. Myd88^−/−, P < 0.001) (ANOVA, Tukey's test) (n = 7–10 per group). (B) Mice were mock-infected or infected with RSV for 8 days before lung cells were harvested for stimulation with an RSV- or SeV (control)-derived peptide. Six hours after stimulation, intracellular IFN-γ levels in CD3⁺CD8⁺ cells were measured by FACS. The percentage of IFN-γ⁺CD3⁺CD8⁺ cells in RSV-peptide treated cultures minus that from SeV-peptide cultures is plotted for each genotype (P = 0.35; ANOVA) (n = 5 per group). (C) Bronchoalveolar lavage fluid IFN-γ was measured in WT and DKO mice at the indicated times after RSV infection (n = 3 per group). (D) Lung cells (effectors) from day 8 RSV- or mock-infected mice were incubated with EL4 target cells loaded with a peptide (RSV peptide or SeV control peptide). The target cells were differentially labeled with CFSE and incubated with the effector cells at the indicated effector:target (E:T) ratios for 4 hours. Cells were then analyzed by flow- cytometry and the specific killing of RSV peptide-loaded targets was calculated (n = 3 per group). (E) Mice were either depleted of CD8⁺ T cells (with antibody 2.43) or mock depleted (IgG) and infected with RSV. BALF was extracted on day 8 and IFN-γ was measured by ELISA (n = 3 per group). (F) BALF viral titers were measured for mice described in E. Data are represented as mean ± SEM.

Fig. 5.

A subset of pulmonary DCs is activated by RSV in mice lacking Mavs. (A) Mice of the indicated genotypes were infected with RSV or mock treated for 24 hours before CD86 levels were assessed on pulmonary DCs and alveolar macrophages by flow cytometry. Shaded histograms represent isotype-control antibody staining, blue histograms represent DCs from mock-infected mice and red histograms represent RSV infected mice. (B) Mediastinal lymph nodes (MLNs) were taken from mice described in A and surface expression of CD86 was assessed on dendritic cells by flow cytometry. Representative mice are shown for each genotype (n = 3 per group).