Influenza Virus Infection And Mucosal Immune Injury

Under normal physiological activities of the body, the primary function of the lung is responsible for the exchange of the air. The function of the lung determines the structure of the lung that opens to the natural environments, which make the lung under the persistent challenge by foreign substances. Considering the large surface areas of the respiratory epithelium and the volumes of air inspired on a daily basis, it is surprising that there is so little inflammation in normal conditions.Similar to the intestinal tract, the upper respiratory tract of human is also colonized by large number of bacteria which is beneficial for the host commonly. Some studies have suggested that commensal intestinal bacteria are critical for the production of the intestinal immune system and the maintenance of the intestinal homeostasis. The components of commensal bacteria stimulate the Toll-like receptors on gut epithelium and protect it against the infection of harmful pathogens. Do the commensal bacteria in the respiratory tract have the similar function to the commensal intestinal bacteria, which can protect the respiratory tract against the infection of harmful pathogens and maintain the homeostasis of the lung? This is the first purpose of our study.In normal conditions, the immune system in respiratory tract is under strict control to prevent inflammation to innocuous antigens or commensal bacteria. However, when harmful pathogens enter into the respiratory tract and infection the lung, the immune system of the respiratory tract will be activated and eliminate the pathogens. But the bad news is that the immune response also leads to immune injury of the tissue. Influenza is a respiratory tract disease caused by influenza virus, causing regional and seasonal epidemics that result in severe morbidity and mortality involving3to5million people annually all over the world. Some studies have suggested that Immune-mediated pathology is responsible for influenza-caused death.The most common symptoms of influenza are cough, fever, headache and weakness. In addition, influenza is often accompanied with other clinical manifestations such as abdominal pain, nausea, vomiting and diarrhea, particularly in children, which are common symptoms in the gastroenteritis. Although these gastroenteritis-like symptoms have been found in many cases of influenza patients, the immune mechanisms underlying this clinical manifestation have not been investigated. So, this is the second purpose of our study.This paper contains two parts of results:Ⅰ. Airway microbiota counteract influenza virus by dampening lethal inflammation. Ⅱ. Lung influenza virus infection induces intestine immune injury via Th17cell-dependent inflammation.I. Airway microbiota counteract influenza virus by dampening lethal inflammationIn this study, we built a mouse model of S. aureus commensally colonized in upper respiratory tract and then study the effect of S. aureus priming on the influenza virus infection. We built a mouse model of influenza virus PR8infection. H&E staining was used to assess the histopathology of lung; ELISA was used to detect the cytokines in BALF; RT-PCR was used to determine the viral titer; Flow cytometry and RT-PCR were used to detect the expression of M2cells-related markers in alveolar macrophage; Adoptive transfer and in vitro proliferation experiments were used to detect the function of M2cells. The major results of this study are shown as follows:1. S. aureus priming protects mice against influenza virus infection-caused death.We treated mice i.n. with1×107CFU of S. aureus to build mouse model of S. aureus commensally colonized in upper respiratory tract, and then infected S. aureus priming mice i.n. with a lethal dose of PR8. We found that S. aureus priming protect mice against PR8-caused death, and this protective effect depends on down-regulation of inflammation.2.S. aureus priming-induced protective effect depends on the activation of TLR2.In Tlr2-/-mice, S. aureus priming can not protect mice against PR8-caused death. According to the results of Immunopathological analysis and cytokine detection, we found that S. aureus priming can not attenuate the immune injury induced by PR8infection. So, S. aureus priming-induced protective effect depends on the activation of TLR2.3. S. aureus priming-induced protective effect does not depend on the decrease of the viral titer.Compared the PR8titer in the lung between control mice and S. aureus primed mice, we found that S. aureus priming can reduce the viral titer in the lung during PR8infection. However, similar to the S. aureus primed WT mice, S. aureus primed Tlr2-/-mice also can reduce the viral titer in the lung during PR8infection. So, S. aureus priming-induced protective effect does not depend on inhibiting the proliferation of influenza virus.4, S. aureus priming-induced protective effect depends on alveolar macrophages (AMs).5. aureus priming increase the expression of TLR2on AMs and the number of AMs in the alveoli. We treat mice i.n. with CL^MDP-lip to deplete AMs and found that CL2MDP-lip treated mice can not protect mice against PR8-caused death. By adoptive transfer experiments, we found that the AMs from S. aureus primed WT mice can restore the ability of CL2MDP-liP treated mice to prevent PR8-caused death. So, S. aureus priming-induced protective effect depends on AMs.5. TLR2activation-mounted alveolar environment promote the polarization of AMs to M2cells.5. aureus priming increases the expression of M2cell-related marker Argl, Fizzl, IL-10and TGF-β in AMs. Furthermore, S. aureus priming promote the production of IL-13in the alveoli in WT mice but not in Tlr2-/-mice. So, TLR2activation-mounted alveolar environment promote the polarization of AMs to M2cells.6. M2cells inhibit the recruitment and proliferation of inflammatory cells during influenza virus infection.Analysed the constitution of inflammatory cells in BALF, we found that S. aureus priming can remarkable decrease the numbers of NK cells and neutrophils in BALF during PR8infection. By in vitro experiments, we found that the AMs from5. aureus primed mice can inhibit the proliferation of T cells stimulated by Con A.In conclusion, by building the mouse model of S. aureus commensally colonized in upper respiratory tract, we found that S. aureus priming protects mice against influenza virus infection-caused death, and this protective effect depends on down-regulation of acute inflammation rather than inhibition of influenza virus amplification. During S. aureus priming, TLR2activation-mounted alveolar environment promote the polarization of AMs to M2cells which inhibit the recruitment and proliferation of inflammatory cells during influenza virus infection.Ⅱ. Lung influenza virus infection induces intestine immune injury via Thl7cell-dependent inflammationIn this study, we built a mouse model of influenza virus PR8infection. H&E staining was used to assess the histopathology; ALT and BUN detection were used to determine the injury of liver and kidney, respectively; CBA was used to detect the cytokines in serum; PCR was used to detect the existence of virus; Flow cytometry was used to detect Th17cells; RT-PCR was used to detect the expression levels of IL-17A, IFN-γ, IL-6,TGF-β and CCL25in the small intestine; Antibiotics were used to deplete commensal bacteria; Neutralizing antibodies were used to block the function of IL-17A and CCL25. The major results of this study are shown as follows:1. Influenza virus infection induces intestine immune injury.In the mouse model of influenza virus PR8infection, we found that PR8infection not only induces lung immune injury but also causes small intestinal immune injury, but there are not liver and kidney immune injury. However, we can detect the PR8in the lung but not in the small intestine. So, Influenza virus lung infection induces intestine immune injury, and this small intestinal injury is not induced by PR8infection directly.2. Th17cells are involved in intestine injury caused by influenza virus infection.The result of microarray analysis showed that the expression of RORyt was increased in the small intestine during PR8infection. Furthermore, we found that Thl7cells are increased in intraepithelial lymphocyte (IEL) and Lamina proprial lymphocyte (LPL) but not in blood, mediastinal lymph node (LN) and mesenteric LN during PR8infection. At the same time, PR8infection promotes the expression of IL-17A and down-regulate the expression of IFN-y in the small intestine, and anti-IL-17A antibody treatment can inhibit the small intestinal injury induced by PR8infection. So, influenza virus infection-induced small intestinal injury depends on Th17cells.3. The depletion of commensal intestinal bacteria attenuates the small intestinal injury induced by influenza virus infection.By culture and detection, we found that PR8infection can not change the total number of bacteria in the intestinal tract. After depleting the gut bacteria by antibiotics, we found that PR8infection can not induce the small intestinal injury, and there are not Thl7cells in the IEL and LPL. So, commensal intestinal bacteria are involved in the small intestinal injury induced by influenza virus infection.4. Commensal intestinal bacteria promote the recruitment of CD4+T cells and the polarization of Th17cells.The result of microarray analysis showed that PR8infection increases the expression of CCL25in the small intestine. At the same time, PR8infection increases the numbers of CCR9+CD4+T cells in the mediastinal LN and the small intestine. Furthermore, the depletion of gut bacteria decreases the expression of IL-6, TGF-β and CCL25in the small intestine. So, commensal intestinal bacteria may promote the expression of CCL25to recruit the CCR9+CD4+T cells and maintain the expression of IL-6and TGF-β to promote the polarization of Th17cells.In conclusion, this study found that influenza virus infection can induce the small intestinal immune injury. During influenza virus infection, the number of Th17cells and the expression of IL-17A in the small intestine are increased. Depletion of commensal intestinal bacteria by antibiotics protect the mice against the immune injury induced by influenza virus infection, the mechanism in which may be that commensal intestinal bacteria-mounted special environment can promote the recruitment of T cells and the polarization of Th17cells.