Plasmacytoid dendritic cells promote host defense against acute pneumovirus infection via the TLR7-MyD88-dependent signaling pathway

Abstract

Human respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants. In human infants, plasmacytoid dendritic cells (pDC) are recruited to the nasal compartment during infection and initiate host defense through the secretion of type I IFN, IL-12, and IL-6. However, RSV-infected pDC are refractory to TLR7-mediated activation. In this study, we used the rodent-specific pathogen, pneumonia virus of mice (PVM), to determine the contribution of pDC and TLR7 signaling to the development of the innate inflammatory and early adaptive immune response. In wild-type, but not TLR7- or MyD88-deficient mice, PVM inoculation led to a marked infiltration of pDC and increased expression of type I, II, and III IFNs. The delayed induction of IFNs in the absence of TLR7 or MyD88 was associated with a diminished innate inflammatory response and augmented virus recovery from lung tissue. In the absence of TLR7, PVM-specific CD8(+) T cell cytokine production was abrogated. The adoptive transfer of TLR7-sufficient, but not TLR7-deficient pDC to TLR7 gene-deleted mice recapitulated the antiviral responses observed in wild-type mice and promoted virus clearance. In summary, TLR7-mediated signaling by pDC is required for appropriate innate responses to acute pneumovirus infection. It is conceivable that as-yet-unidentified defects in the TLR7 signaling pathway may be associated with elevated levels of RSV-associated morbidity and mortality among otherwise healthy human infants.

Figure 1. The absence of TLR7-MyD88 signaling delays weight loss, clinical symptoms and virus recovery

Open symbols/bars represent vehicle-inoculated mice; closed symbols/bars, PVM-inoculated mice. WT mice are represented by circles; TLR7−/− mice by triangles; and MyD88−/− mice by diamonds. (A) Virus recovery (copies of PVM SH gene per 10⁷ copies HPRT) in the lungs of WT, TLR7−/− and MyD88−/− mice was measured by qRT-PCR at 1, 3, 5 and 7 days post inoculation (dpi). (B) WT, (C) TLR7−/− and (D) MyD88−/−mice were weighed at 7 days of age immediately prior to inoculation (day 0) with vehicle or PVM. Mice were weighed every 24 hours thereafter until euthanasia, and weight expressed relative to day 0. Data are mean +/− s.e.m, 4–13 mice in each group and * p<0.05, ** p<0.01, *** p<0.001.

Figure 2. PVM inoculation of WT but not TLR7- or MyD88-deficient neonatal (7 day old) BALB/c mice induces an innate inflammatory response

Open symbols represent vehicle-inoculated mice; closed symbols, PVM-inoculated mice. WT mice are represented by circles; TLR7−/− mice by triangles; and MyD88−/− mice by diamonds. The left lung lobe was mechanically dispersed and (A) FSc^lowGr-1^highCD11b⁺ CD11c⁻ neutrophils and (B) CD3-CD49b⁺ natural killer (NK) cells identified by flow cytometry. (C) CCL3 protein expression in right lung lobe homogenates was measured by ELISA. Data are mean +/-− s.e.m, 4–16 mice in each group and * p<0.05, ** p<0.01, *** p<0.001. * WT compared to TLR7-deficient mice, # WT compared to MyD88-deficient mice.

Figure 3. pDC recruitment and PVM-induced IFN and pro-inflammatory cytokine production requires TLR7-MyD88

Open symbols/bars represent vehicle-inoculated mice; closed symbols/bars, PVM-inoculated mice. WT mice are represented by circles; TLR7−/− mice by triangles; and MyD88−/− mice by diamonds. The key to symbols is shown in (A) and is applicable to panels (C) and (D). (A) The left lung lobe was mechanically dispersed and pDC (Siglec-H+, CD11c^lo, B220+) expressed as a percent of lung cells. pDC from vehicle-inoculated TLR7- and MyD88-deficient mice are shown at day 1 only for clarity. (B) Percentage of MHC class II-positive pDC in the lung at 5 days post inoculation (DPI) in response to vehicle or PVM. (C) qRT-PCR analysis of IFN–α4, IFN–β, IFN-λ2, and IRF-7 mRNA expression in whole lung from wild-type, TLR7−/− and MyD88−/− mice (normalised to lungs from vehicle-inoculated WT mice) at 5 dpi. (D) Right lung homogenates were analysed at 1, 3, 5 and 7 days post inoculation (DPI) for IL-12p40, IL-6 and TNF–α protein expression by ELISA. Data are represented as mean +/− s.e.m, 4–8 mice in each group, or shown as individual mice, and * p<0.05, ** p<0.01, *** p<0.001. * WT compared to TLR7-deficient mice, # WT compared to MyD88-deficient mice.

Figure 4. Activation of the TLR7-MyD88 pathway is essential for T cell infiltration, activation and production of IFN–γ

Open symbols/bars represent vehicle-inoculated; closed symbols/bars, PVM-inoculated. WT mice are represented by circles; TLR7−/− by triangles; and MyD88−/− by diamonds. (A–B) The percent of (A) CD4+ and (B) CD8+ T cells expressing stem cell antigen-1 (Sca-1, also known as LY6A, a surrogate marker of IFN-activation), in the lungs of wild-type, TLR7−/− and MyD88−/− mice were quantitated by flow cytometry at 7 days post inoculation (DPI). (C and D) IFN–γ gene and protein expression was measured in whole lung by (C) qRT-PCR and (D) ELISA at 1, 3, 5 and 7 dpi. Data are mean +/− s.e.m, 5 mice in each group and * p<0.05, ** p<0.01, *** p<0.001. Data in A and B, were compared by unpaired t test and in C and D by ANOVA and Bonferroni Post Hoc Test. * p<0.05, *** p<0.001. * WT compared to TLR7-deficient mice, # WT compared to MyD88-deficient mice.

Figure 5. PVM-specific CD8+ T cell responses and the innate IFN response are delayed in the absence of TLR7

WT (circles) and TLR7-deficient (triangles) mice were inoculated with PVM (closed) or vehicle (open) and sacrificed 10 days later. (A) The percent of LN cells that were CD3+CD8+ T cells at 10 days post infection (DPI). (B) Mediastinal lymph nodes were harvested, seeded at 0.3 × 10⁶/well and stimulated with the PVM-specific peptide (P261) or media alone. After a 3 day culture, cell-free supernatants were quantified for IFN–γ and TNF–α protein expression. (C) IFNα4, CCL3, Mx-1, RIG-I, NOD2 and NLRP3 gene expression in whole lung extracts was measured by qRT-PCR. Data are mean +/− s.e.m, 5–6 mice in each group. Data in (B) were compared by a two-tailed Mann-Whitney and in (A) and (C) by unpaired t test. * p<0.05, ** p<0.01.

Figure 6. PVM-induced upregulation of IFN–α expression by BM-derived pDC requires TLR7

(A) Bone marrow cells were harvested and cultured in Flt3L to generate a pDC-rich cell population (~20%). After a 10 day culture, cells were cultured overnight with PVM at an MOI of 1 in the presence of brefeldin A. IFN–α was detected by intracellular cytokine staining and localised to Siglec-H⁺, B220⁺ cells. (B) The percentage of Siglec⁺IFN–α⁺ cells as a fraction of total Flt3L-treated WT and TLR7−/− bone marrow cells cultured with or without PVM. Data are mean +/− s.e.m, 3–4 mice in each group and ** p<0.01.

Figure 7. TLR7 but not phosphorylated IRF7 is widely distributed

(A) Lung biopsies from naive neonatal WT (left panels) and TLR7-deficient (right panels) mice were probed for TLR7. Scale bar equals 20μm, top panels (×400 magnification); and 10μm, (×1000 magnification) lower panels. (B) Enumeration of phosphorylated IRF7-immunoreactive cells in WT mice at 1 dpi following inoculation with vehicle or PVM +/− anti-pDC antibody, and photomicrograph demonstrating phosphorylated IRF7-immunoreactive cells in PVM-inoculated WT mice. Scale bar equals 20 μm, top panel (×400 magnification); and 10 μm, (×1000 magnification) lower panel.

Figure 8. Reconstitution of TLR7−/− mice with TLR7+/+ but not TLR7−/− BM-derived pDC recapitulates host defense and accelerates viral clearance

Purified Flt3L-expanded pDC from TLR7+/+ and TLR7−/− bone marrow cells were adoptively transferred to TLR7−/−recipients. Mice were inoculated with PVM 2 h later and endpoints measured at 7 dpi (A and B). The left lung lobe was mechanically dispersed and (A) neutrophils (FSc^lowGr-1^highCD11b⁺ CD11c⁻) and NK cells (CD3⁻CD49b⁺) identified by flow cytometry. (C and D) CCL3 and TNF-α protein expression in right lung lobe homogenates was measured by ELISA. (E) Representative histogram of lung CD3+CD4+Sca-1+ T cells following transfer of TLR7−/−(top panel) and TLR7+/+ (bottom panel) pDC to TLR7−/− mice. Graphs represent the percent CD3+CD4+ and CD3+CD8+ T cells expressing Sca-1 as detected by flow cytometry. (F) Recipient TLR7−/− mice were weighed at 7 days of age immediately prior to inoculation with PVM (day 0). Mice were weighed every 24 hours thereafter until euthanasia, and weight expressed relative to day 0. (G) Virus recovery in the lungs was measured by qRT-PCR.