The Role Of Tumor Necrosis Factor Alpha In Immune Defense Of Secondary Otitis Media

ObjectiveOtitis Media?OM?is one of the most common infectious diseases in children,with high morbidity and high recurrence rate.Respiratory viruses are often followed by a high incidence of pneumonia and otitis media.Many children have a history of upper respiratory viral infection before suffering from otitis media.Influenza A virus?IAV?is one of the most important respiratory viruses causing OM and Streptococcus pneumoniae?S.pn?is one of the most important pathogens causing OM.Epidemiological investigations,clinical and experimental studies have confirmed that the previous IAV respiratory infections are highly correlated with the occurrence of OM.Therefore,establishing a mouse model of secondary otitis media by Streptococcus pneumoniae?S.pn?following Influenza A viruses?H1N1?has an important scientific significance to study the pathogenesis of otitis media and contribute to research novel influenza and bacterial vaccine.Methods1.C57BL/6 mice were infected intranasally with different dose of H1N1?PR8?.Five days later,mice were again inoculated with S.pn via transbullar injection.Weight change and survival rate were observed daily after PR8 infection.The middle ear?ME?tissues for histological examination as well as the middle ear lavage fluid?MELF?for total cells quantification and S.pn load determination were collected at different time points after S.pn inoculation Exploring appropriate doses of PR8 and S.pn by comparing the difference of S.pn clearance capacity and immune response between a group of S.pn infection alone and group of PR8+S.pn developed a mice model of secondary otitis media by streptococcus pneumoniae following influenza A viruses H1N1.2.WT and TNF-?^-/-mice were infected with dose of 550 TCID₅₀ PR8influenza virus firstly.Five days later,mice were again inoculated with S.pn?10⁶CFU/ear?via transbullar injection.Pro-inflammatory cytokines TNF-?,IL-1?,IL-6 and IFN-?in MELF were measurement by ELISA.Bactericidal substance NO also were collected at different time points after S.pn inoculation.Preliminary study the role of PR8 in secondary otitis media.3.Weight change and survival rate were observed daily in secondary otitis media from WT and TNF-?^-/-mice.The middle ear?ME?tissues for histological examination as well as the middle ear lavage fluid?MELF?for total cells quantification,S.pn load determination and cytokines measurement were collected at different time points after S.pn inoculation from WT and TNF-?^-/-mice to study the role of TNF-?in a mouse model of secondary otitis media with S.pn after influenza virus infection.Results1.We found that antecedent PR8 infection enhanced the mortality in a dose-dependent manner.We also found that the pathogenic effects of secondary AOM,including weight loss,inflammation cells infiltration,S.pn burden were more severe in the group of secondary otitis media by S.pn following PR8 than in the group of S.pn infection alone;the survival in secondary otitis media were 80%when dose of PR8 was 550 TCID₅₀ and S.pn?10⁶CFU/ear?,which had no significant difference comparing to simple infection otitis media.A model of Streptococcus pneumoniae otitis media following influenza A virus infection in C57BL/6 mice was successfully established.2.WT and TNF-?^-/-mice were infected with dose of 550 TCID₅₀ PR8influenza virus firstly.Five days later,mice were again inoculated with S.pn?10⁶CFU/ear?via transbullar injection.There were no significant differences in production of proinflammatory cytokine IL-6 and IFN-?among the group of secondary otitis media,whereas TNF-?and NO showed a remarkable increasing,but IL-1?decreased in the group of secondary otitis media than in the group of S.pn alone infection otitis media.S.pn infection promoted the production of NO which is mediated by TNF-?;compared to S.pn alone infection otitis media,over-production of TNF-?in secondary otitis media aggravated middle ear injury.3.Mortality rate of TNF-?deficiency mice was more increased than WT mice.TNF-?deficiency impaired the clearance of Streptococcus pneumoniae in middle ear and lung;TNF-?deficiency aggravated Middle ear and lung tissue injury in;TNF-?deficiency had no significant effect on the production of MPO in the middle ear but decreased in the lung tissue.ConclusionA model of Streptococcus pneumoniae otitis media following influenza A virus infection in C57BL/6 mice was successfully established.Although over-production of TNF-?aggravated middle ear tissue injury at early,TNF-?still had immunoprotection in entire process of otitis media.