Mechanisms Of Acute And Persistent Airway Inflammation And Ahr Induced By RSV Infection In Nude Mice

PART ONE THE ESTABLISHMENT OF NUDE MOUSE MODEL WITH RSV INFECTION AND ITS PATHOLOGICAL CHARACTERISTICS ANALYSISObjective:To establish a nude mouse model with RSV infection; to observe the viral replication and pathological characteristics (including airway inflammatory cells infiltration, lung tissue damage and AHR) of RSV in the absence of T cells; and finally o lay the foundation for the further exploration of how innate immunity contributes to the pathogenesis of RSV infection.Methods:Female,6-8 weeks old nude mice (on a BALB/c background) and normal BALB/c mice were inoculated with cell culture supernatant (control groups) or RSV A2 (RSV groups) intranasally. The whole lungs were harvested on days 1 to 7 sequentially, and on days 9,12 and 14 to detect RSV viral load with plaque assay. Samples were collected on days 1,3,5,7,14,21,30 and 60, inflammatory cells in bronchoalveolar lavage fluid (BALF) were counted, left lung tissues were stained with HE and histopathological score (HPS) was performed. AHR was measured by whole-body plethysmography. The concentrations of the cytokines in BALF were determined with ELISA.Results:(1) RSV viral load reached the peak on day 3 in nude mice and BALB/c mice, with lower levels in nude mice within the first 4 days, but on days 9 and 12, viral replication was stilled detected in the lungs of nude mice but not in BALB/c mice. (2) RSV caused overt airway inflammation, lung tissue damage as well as AHR in both mice strains. These disorders were already significant on day 1, reached the peak on days 5 and 7, and then decreased over time, but did not return to normal till day 60. (3) Time dynamics of cytokines following RSV infection showed diphasic changes in both mice strains. Type I cytokiens were significantly increased on day 1, with a greater magnitude in nude mice. IFN-y was significantly increased on days 5 and 7, and returned to normal on day 14 in BALB/c mice but not increased throughout the disease in nude mice. Th2 cytokines (IL-4, IL-5, IL-13, IL-10, TSLP, IL-25) and Th17 cytokines (IL-17, IL-22) were significantly increased during the later phase of RSV infection, especially on days 21 and 30 in both mice strains.Conclusions:(1) Viral clearance is delayed in nude mice, indicating that although innate immunity can control RSV replication in the early stage, but the efficient elimination of virus relies on T cells. (2) The innate immunity can lead to RSV-associated acute and chronic airway inflammation and AHR independent of T cells. (3) Time dynamics of cytokines following RSV challenge show diphasic changes, suggesting that the underlying pathogenesis may change over time and the therapeutic strategies may be changed correspondingly. Moreover, the cytokine profiles in nude mice and BALB/c mice are not completely overlapped, implying that innate immune and T cells contribute to RSV pathogenesis via different mechanisms.PART TWO MMP-12 CONTRIBUTE TO ACUTE AIRWAY INFLAMMATION AND AHR POST RSV INFECTION INDEPENDENT OF IFN-γ IN NUDE MICEObjective:We have previously demonstrated that IFN-γ is markedly increased and contributes to the acute airway inflammation and AHR on day 5 post RSV infection in BALB/c mice. However, the primary cellular source of IFN-γ remains to be determined. In the first part, we found that RSV caused significant airway inflammation and AHR in nude mice, but unexpectedly, IFN-γ was not elevated, which indicated other pathogenesis independent of IFN-γ. The present study is to identify the predominant producers of IFN-γ, and further to explore other IFN-γ-independent culprits in the condition of RSV infection.Methods:Female,6-8 weeks old nude mice and BALB/c mice were infected with RSV intranasally. BALF was collected on days 1,3,5,7 and 9 post infection and differential cell analysis was done. T cells and NK cells in the lung tissue were detected by flow cytometry, and intracellular IFN-γ was analysed. Rabbit anti-mouse asialo GM-1 antibody was used to deplete NK cells. IFN-γ and MMP-12 were determined 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 si RNA was transfected to RSV-infected and resveratrol-treated mice to knock-down SARM expression. SARM and TRIF protein were semi-quantified by Western blot. AHR was measured by whole-body plethysmography.Results:(1) In the lung tissue of nude mice, T cells were almost undetectable. While in BALB/c mice, both CD3+CD4+IFN-γ+Th1 cells and CD3+CD8+IFN-γ+Tc1 cells were significantly increased on day 5 post RSV infection. Moreover, IFN-γ was not decreased by NK cells depletion. (2) MMP-12 was dramatically increased in nude mice on days 3 to 9. In BALB/c mic, MMP-12 began to increase on day 7 and was significantly increased on day 9. When MMP-12 was inhibited by MMP408, RSV-associated airway inflammation and AHR were alleviated. (3) MMP-12 was significantly increased in the IFN-y neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice. (4) SARM was significantly suppressed while TRIF and MMP-12 were significantly enhanced by RSV in nude mice. Following resveratrol administration, SARM inhibition was prevented, TRIF and MMP-12 were correspondingly down-regulated and 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.Conclusions:(1) IFN-γ induced by RSV mainly derived from T cells. (2) MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. (3) IFN-γ can suppress MMP-12 in the condition of RSV infection. (4) 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-TRIF on MMP-12. These findings help to elucidate the complicated network of TLRs and lung proteases triggered by RSV and further highlight the potential to target SARM-TRIF- MMP-12 signaling cascades in the treatment of RSV infection.PART THREE NK CELLS CONTRIBUTE TO PERSISTENT AIRWAY INFLAMMATION AND AHR INDUCED BY RSV INFECTION VIA PROMOTING TH2 AND TH17 CYTOKINES IN NUDE MICEObjective:RSV can lead to persistent airway inflammation and AHR, and is intimately associated with recurrent wheezing or asthma, but the underlying mechanisms remain largely unknown. Recently, mounting researches have focused the crucial roles of innate immune in the development of asthma, especially the glucocorticoid-resistant asthma phenotype. NK cells, an important component of innate immune, have been demonstrated to be involved in acute RSV infection and asthma. But whether and how NK cells contribute to the RSV-associated persistent airway diseases have not been reported. We have found in the first part that airway inflammation and AHR lasted at least for 60 days post RSV challenge in both nude mice and BALB/c mice. Moreover, during the later stage of infection, Th2 cytokines and Th17 cytokines were significantly increased in both mice strains. Based on these observations, we further used nude mouse model to determine whether NK cells can lead to RSV-associated persistent airway inflammation and AHR via promoting Th2 cytokines and Th17 cytokines in the absence of T cells.Methods:Female,6-8 weeks old nude mice and BALB/c mice were infected with RSV intranasally. BALF was collected on days 7,14,21,30 and 60 post infection and differential cell analysis was done. T cells and NK cells in the lung tissue were detected by flow cytometry. Rat anti-mouse CD8a and rabbit anti-mouse asialo GM-1 antibodies were used to deplete the CD3+CD8+T and NK cells respectively. Resveratrol was given intraperitoneally. AHR was measured by whole-body plethysmography. The concentrations of the cytokines were determined with ELISA. TRIF protein expression was detected using western blot.Results:(1) Lung tissue NK cells were significantly increased during the later phase of RSV infection in nude mice and BALB/c mice, with a greater magnitude in nude mice. Depletion of NK cells in both mice strains significantly decreased Th2 cytokines, Thl7 cytokines and the persistent airway disease without affecting T cells. (2) In contrast, after CD8+T cells were depleted in BALB/c mice, NK cells were significantly increased, Th2 cytokines, Th17 cytokines and the persistent airway disease were markedly exacerbated subsequently. (3) Moreover, TRIF inhibition with resveratrol significantly suppressed NK cells, Th2 cytokines, Th17 cytokines and the persistent airway disease.Conclusions:NK cells contribute to persistent airway inflammation and AHR in the later stage of RSV infection via promoting Th2 cytokines and Th17 cytokines independent of T cells. Moreover, the recruitment and/ or activation of NK cells may be associated with TLRs-TRIF pathway.