MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice

Abstract

Background: Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.

Methods: Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.

Results: RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.

Conclusions: MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Figure 1

RSV infection results in an increase in BALF cellularity. Mice were infected with 1.5 × 10⁷ PFU of RSV and a group of animals were sacrificed at days 1, 3, 5, 7 and 9 post-infection. Total cells (A), macrophages (B), lymphocytes (C) and neutrophils (D) were all significantly increased following RSV challenge in both BALB/c mice and nude mice. Graphs are represented as the mean ± s.e.m. Data are representative of two independent experiments performed on 10 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the corresponding mice groups are connected by a line.

Figure 2

RSV infection results in significant lung histopathological lesions. Lungs were harvested at days 1, 3, 5, 7 and 9 post-RSV infection, stained with hematoxylin-and-eosin (HE), and scored for levels of inflammation as described in the Materials and Methods section. Representative HE staining of mouse lung tissue sections (original Magnification × 100) were shown. A: mock-infected BALB/c mice; B-F: RSV-infected BALB/c mice at days 1, 3, 5, 7 and 9 respectively. G: mock-infected nude mice; H-L: RSV-infected nude mice at days 1, 3, 5, 7 and 9 respectively. RSV induced a mass of inflammatory cells accumulation around the bronchiole, vascular and alveolar compartments, with goblet cell hyperplasia, mucus hypersecretion as well as partial destruction of regular tissue structures. M: Histological scores were remarkably elevated in the RSV-infected mice compared with the mock group. Graphs are representative of two independent experiments performed on 10 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the corresponding mice groups are connected by a line.

Figure 3

RSV infection results in enhanced AHR. Airway hyperresponsiveness to increasing doses of methacholine was assessed using plethysmography at days 1 (A), 3 (B), 5 (C), 7 (D) and 9 (E) post-RSV infection. Penh values were significantly increased at all indicated time points following RSV challenge in both BALB/c mice and nude mice. Graphs are represented as the mean ± s.e.m. Data were representative of two independent experiments performed on 10 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 for RSV-infected versus mock-treated BALB/c mice; ^, p < 0.05, ^^, p < 0.01, ^^^, p < 0.001 for RSV-infected versus mock-treated nude mice.

Figure 4

IFN-γ induced by RSV is predominantly produced by T cells. We confirmed that nude mice were deficient in CD3⁺ T cells (A) and found that IFN-γ was significantly increased at days 5 and 7 post-RSV infection in BALB/c mice, but was nearly negligible in nude mice (B). We further found that both CD3⁺CD4⁺IFN-γ⁺ Th1 cells and CD3⁺CD8⁺IFN-γ⁺ Tc1 cells were remarkably elevated at day 5 when IFN-γ levels peaked in BALB/c mice (C). Data are representative of two independent experiments performed on 6 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the corresponding mice groups are connected by a line.

Figure 5

MMP-12 contributes to RSV-induced resriratory symptoms. Mice were killed at days 1, 3, 5, 7 and 9 post-infection, and MMP-12 was detected using ELISA. A: MMP-12 levels in BALF. B: MMP-12 levels following MMP408, a specific inhibitor of MMP-12,administration. C: Inflammatory cells in BALB/c mice. D: Inflammatory cells in nude mice. E: AHR in BALB/c mice. F: AHR in nude mice. mock: mock-infected mice group; control: mock-infected and PBS treated mice group; RSV: RSV-infected and PBS treated mice group; RSV + MMP408: RSV-infected and MMP408 treated mice group, Disease parameters were assessed on day 9 and data are representative of two independent experiments performed on 6–10 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the control mice groups to other groups. ^, p < 0.05, ^^, p < 0.01, ^^^, p < 0.001 for RSV + PBS groups versus the RSV + MMP408 groups.

Figure 6

SARM-TRIF signaling pathway is involved in regulating MMP-12 expression. Nude mice were inoculated with RSV or mock infected with the culture supernatant. Resveratrol or PBS was injected intraperitoneally for 5 days consecutively. SARM siRNA 3–1 and the negative siRNA vector were transfected intranasally. The disease parameters were assessed at day 5 post-RSV infection. Nude mice were divided into five groups: control: mock-infected and PBS treated mice group; RSV: RSV-infected and PBS treated mice group; RSV + RES: RSV-infected and resveratrol treated mice group; RSV + RES + 3-1: RSV-infected, resveratrol treated and SARM siRNA 3–1 transfected mice group; RSV + RES + Negative: RSV-infected, resveratrol treated and negative siRNA vector administrated mice group. A: Inflammatory cells infiltrating into BALF. B: AHR in response to increasing doses of methacholine. C: Western blotting analysis of the expression of SARM and TRIF in the lung tissues. D: Semi-quantified expression of SARM and TRIF normalized to β-actin. E: MMP-12 protein levels in BALF. Data are representative of two independent experiments performed on 12 animals per group. In graph B, ***, p < 0.001 shown comparing the RSV group to the RSV + RES group; ^^^, p < 0.001 shown comparing the RSV + RES + 3-1 group to the RSV + RES + Negative group. In the other graphs, *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the corresponding mice groups are connected by a line.

Figure 7

IFN-γ suppresses MMP-12. To assess the effects of IFN-γ on MMP-12 production in the condition of RSV challenge, BALB/c mice were treated with IFN-γ neutralizing antibody and nude mice were treated recombinant murine IFN-γ on days 0, 1, and 3 respectively. Samples were collected on day 5. A: IFN-γ levels in BALB/c mice. B: IFN-γ levels in nude mice. C: MMP-12 levels in BALB/c mice. D: MMP-12 levels in nude mice. control: mock-infected and PBS treated mice group; RSV + anti-IFN-γ: RSV-infected and IFN-γ neutralizing antibody treated BALB/c mice group; RSV + r-IFN-γ: RSV-infected and recombinant murine IFN-γ treated nude mice group, RSV: RSV-infected and isotype control antibodies treated BALB/c mice or nude mice groups. Data are representative of two independent experiments performed on 6–10 animals per group. *, p < 0.05, **, p < 0.01, ***, p < 0.001 shown comparing the the corresponding mice groups are connected by a line.