Th17 cytokines are critical for respiratory syncytial virus-associated airway hyperreponsiveness through regulation by complement C3a and tachykinins

Abstract

Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. In this paper, we show RSV-infected wild-type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production also has been implicated in RSV pathophysiology, and tachykinin receptor-null mice were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in mice deficient in C3aR, whereas C3a levels were unchanged in tachykinin receptor-null animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.

Figures

Figure 1. C3aR1 deficient mice are protected from RSV associated airway pathophysiology

A. Airway hyperresponsiveness in RSV infected C3aR1−/− and wild type Balb/c mice assessed as changes in pulmonary resistance as a function of methacholine concentration relative to PBS control. Sham infected mice were administered UV-killed RSV under identical conditions. ■ RSV infected wild type mice, ▲ RSV infected C3aR1−/− mice, □ sham infected wild type mice, △ sham infected C3aR1−/− mice. *P<0.001 relative to RSV infected C3aR1−/− mice. B. Total CD45+ cells isolated from lungs of RSV or sham infected wild type and C3aR1−/− mice. C. Differential analysis of inflammatory cells isolated from lungs of RSV or sham infected wild type and C3aR1−/− mice. D. Expression analysis of the gob5 gene, mclca3 as a function of time after infection with 106 pfu of human RSV-A2 or UV-killed (sham) virus. Data normalized to GAPDH; significance of differences is indicated. E. Assessment of the lung burden of RSV as determined by quantitative real-time PCR of RSV matrix protein RNA at 4 and 7 days following infection normalized to GAPDH. Results are representative of 3 independent experiments (mean ± SEM for 4–5 animals per group).

Figure 2. Adoptive transfer of wild type bone marrow cells to C3aR1−/− mice restores wild type AHR associated with RSV infection

Airway responsiveness as a function of methacholine concentration in RSV infected C3aR1−/− and wild type mice after bone marrow transplantation as indicated. Sham-infected mice were administered UV-killed RSV under identical conditions. Results are expressed as the pulmonary resistance normalized to PBS control. ● C3aR1−/− mice transplanted with wild type bone marrow and infected with RSV, ■ wild type mice transplanted with C3aR1−/− bone marrow and infected with RSV, ○ C3aR1−/− mice transplanted with wild type bone marrow and sham infected, □ wild type mice transplanted with C3aR1−/− bone marrow and sham infected. *P<0.001 for RSV infected C3aR1−/− mice transplanted with wild type bone marrow relative to RSV infected wild type mice transplanted with cells from C3aR1−/− animals. Results are the mean ± SEM for 6–8 animals per group.

Figure 3. TACR−/−1 mice are protected from RSV associated airway pathophysiology

A. Airway hyperresponsiveness in RSV infected TACR1−/− and wild type Balb/c mice assessed as changes in pulmonary resistance as a function of methacholine concentration relative to PBS control. Sham infected mice were administered UV-killed RSV under identical conditions. ● RSV infected wild type mice, ■ RSV infected TACR1−/− mice, ○ sham infected wild type mice, □ sham infected TACR1−/− mice. *P<0.001 for RSV infected wild type mice relative to RSV infected TACR1−/− animals; **P<0.01 for RSV-infected TACR1−/− mice relative to sham-infected wild type or NK1R−/− animals. B. Expression analysis of the gob5 gene, mclca3 as a function of time after infection with 106 pfu of human RSV-A2 or UV-killed (sham) virus. Data normalized to GAPDH; significance of differences is indicated. C. Total CD45+ cells isolated from RSV or sham-infected TACR1−/− or wild type mice assessed by flow cytometry. D. Differential analysis of inflammatory cells isolated from lungs of RSV or sham infected wild type and TACR1−/− mice. Results are expressed as percent of total cells. E. Assessment of the lung burden of RSV as determined by quantitative real-time PCR of RSV matrix protein RNA at 4 and 7 days following infection. Results are the mean ± SEM for 5 animals per group. Significance of differences is indicated.

Figure 4. RSV-induced expression of tachykinins is reduced in C3aR1−/− mice with no concomitant alteration in C3a generation

A&B. Expression analysis of TAC1 (substance P; A) and TAC4 (hemokinin-1; B) mRNAs in the lungs of RSV and sham infected wild type and C3aR1−/− mice. Results are normalized to GAPDH; mean ± SEM for 4–5 mice per group. Significance of differences is indicated. C. ELISA determination of C3a levels in the lungs of RSV and sham infected wild type, C3aR1−/− and TACR1−/− mice. *P<0.05 for day 4-post RSV relative to sham infected mice. **P<0.001 for day 7-post RSV relative to sham infected mice; mean ± SEM for 5–9 mice per group. D. C3aR1 mRNA in the lungs of RSV and sham infected wild type and TACR1−/− mice. Results are normalized to GAPDH; mean ± SEM for 4–5 mice per group. Significance of differences is indicated.

Figure 5. Analysis of Th1 and Th2 cytokines in the lungs of RSV and sham infected wild type, C3aR1−/− and TACR1−/− mice

A&B. IFN-γ levels in wild type and C3aR1−/− (A) or TACR1−/− (B) mice following RSV or sham infection. C&D. TNF-α in lung homogenates of RSV or sham infected C3aR1−/− (C) or TACR1−/− (D) mice determined by ELISA. E&F. IL-4 (E) and IL-5 (F) levels in wild type and C3aR1−/− mice following RSV or sham infection. G. IL-4 levels in wild type and TACR1−/− mice following RSV or sham infection. Results are the mean ± SEM for 5 mice per group. Significance of differences is indicated.

Figure 6. Mice deficient in the C3aR1 or TACR1 fail to produce Th17 cytokines following RSV infection

A&B. IL-17A C&D. IL-6 E&F. IL-21 in lung homogenates of RSV or sham infected C3aR1−/− (A, C, E) or TACR1−/− (B, D, F) mice determined by ELISA. G&H. RORγt I&J. IL-23p19 mRNA levels in lungs of RSV or sham infected C3aR1−/− (G&I) or TACR1−/− (H&J) mice determined by quantitative real-time PCR. K&L IL-1β in lung homogenates of RSV or sham infected C3aR1−/− (K) or TACR1−/− (L) mice determined by ELISA. Results are the mean ± SEM for 5 mice per group. Significance of differences is indicated.

Figure 7. Flow cytometric identification of cells producing IL-17A, IL-6, and substance P-like immunoreactive material in the lungs following RSV infection

A&B. Intracellular cytokine staining for IL-17A in lung cells from wild type, C3aR1−/− (A) and TACR1−/− (B) mice. C&D. Intracellular staining for IL-6 in lung cells from wild type, C3aR1−/− (A) and TACR1−/− (D) mice. E. Intracellular staining for substance P-like immunoreactivity in lung cells from wild type and C3aR1−/− mice. Representative of 2 independent experiments. Results are the mean ± SEM for 3–5 mice per group. RU, relative units.