Acute otitis media (AOM) is one of the most common bacterial infectious diseases in children aged 2 to 7 years worldwide. We previously demonstrated that interleukin-17A (IL-17A) promotes an acute inflammatory response characterized by the influx of neutrophils into the middle ear cavity during Streptococcus pneumoniae-induced AOM. In general, the inflammatory response is viewed as an effector that frequently causes local tissue damage. However, little is known about the pathogenic effects of IL-17A in AOM. Here, we investigated the pathogenic effects of IL-17A by using wild-type (WT) and IL-17A knockout (KO) mouse models. The results showed that the pathogenic effects of AOM, including weight loss, histopathological changes, and proinflammatory cytokine production, were more severe in WT mice than in IL-17A KO mice, suggesting that IL-17A aggravates tissue damage in AOM. Furthermore, these pathogenic effects were found to be dependent on p38 mitogen-activated protein kinase (MAPK) and could be reversed in the presence of a p38 MAPK-specific inhibitor. It was also demonstrated that IL-17A promoted the production of neutrophil myeloperoxidase (MPO) through the p38 MAPK signaling pathway, which was responsible for the middle ear tissue injury. These data support the conclusion that IL-17A contributes to middle ear injury through the p38 MAPK signaling pathway.

目的 建立肺炎链球菌性急性中耳炎小鼠模型,研究白细胞介素17A(interleukin 17A,IL-17A)促进中耳上皮细胞凋亡以及凋亡促进中耳组织损伤的作用.方法 鼓膜穿刺术建立C57BL/6小鼠和IL-17A缺陷鼠急性中耳炎模型.收集中耳灌洗液,ELISA检测灌洗液中炎症因子和损伤标志物的表达.中耳组织石蜡切片经TUNEL染色和HE染色,观察小鼠中耳上皮细胞的凋亡和中耳组织损伤程度.采用广谱的凋亡抑制剂Z-VAD-FNK抑制凋亡后,观察中耳组织损伤的变化.结果 IL-17A显著上调白细胞介素6(IL-6)、肿瘤坏死因子α(TNF—α)和髓过氧化物酶(MPO)表达,加重了中耳炎症.IL-17A还促进了上皮细胞的凋亡和黏膜损伤.抑制凋亡后,显著降低了IL-17A介导的炎症反应,减轻了中耳组织损伤.结论 IL-17A可促进上皮细胞凋亡,加重中耳组织损伤.

We have recently reported that neutrophils play a pivotal role in innate defense against Streptococcus pneumoniae (Spn) during mouse acute otitis media (AOM). However, the underlying mechanism remains unclear. By constructing models of pneumococcal AOM in C57BL/6 mice and using a specific inhibitor in vivo, we investigated the role of myeloperoxidase (MPO), one of the most important protein components of neutrophils. Experiment results showed a significant increase in MPO production of the recruited neutrophils in Spn-infected mice. Neutrophils killed Spn in a MPO-dependent manner. MPO facilitated the generation of reactive oxygen species (ROS), and consequently promoted Spn clearance at an early stage and exacerbated tissue damage. Moreover, MPO induced neutrophil apoptosis and necrosis, which, in turn, worsened tissue damage. In summary, our study demonstrates that neutrophil MPO plays a paradoxical role in bacterial clearance and tissue damage in pneumococcal AOM.

To investigate the effect of Streptococcus pneumoniae (S.pneumoniae) on the formation of neutrophil extracellular traps (NETs),neutrophils were isolated from mouse bone marrow by magnetic activated cell sorting,and then stimulated by S.pneumoniae for 180 minutes.NETs structure was observed by immunofluorescence staining extracellular free DNA was detected by Sytox Green staining to quantify NETs.To further understand the roles of deoxyribonuclease (DNase) and reactive oxygen species (ROS) in NETs formation,DNase and NADPH oxidase inhibitor diphenylene iodonium (DPI) which inhibits production of ROS were used.S.pn was found to be a potent inducer of NETs production and the formation of NETs was significantly decreased after being treated with DNase or DPI,which indicating that ROS was involved in the formation of NETs induced by S.pn.In conclusion,S.pn can initiate the release of NETs.DNase and DPI could be inhibitors to balance formation of NETs.

Background: The natural course of otitis media (OM) in most children is acute and self-limiting; however, approximately 10–20% of children can experience persistent or recurrent OM. Determining the host factors that influence outcome of OM will help us design better therapies. This study focused on the role of Toll-like receptor 2 (TLR2) in a pneumococcal OM mouse model. Methods: The middle ears (MEs) of wild-type (WT) and TLR2 -/- mice were inoculated with Streptococcus pneumoniae ( Spn ) serotype 19F via transbullar injection. ME TLR2 expression in WT mice was determined by qRT-PCR and immunofluorescence. ME pathological manifestations, inflammatory response, and pneumococcal clearance between WT and TLR2 -/- mice were compared after Spn inoculation. Results: TLR2 expression in ME mucosa was markedly enhanced following infection with Spn in WT mice. In contrast to WT mice, TLR2 -/- mice exhibited unaffected early ME inflammatory response. During late stage of ME infection, however, the absence of TLR2 can lead to reduced macrophage recruitment, impaired Spn clearance, and prolonged ME inflammation. Conclusion: Our results demonstrate that TLR2 signaling is critical for bacterial clearance and timely resolution of inflammation in OM induced by Spn .

目的:研究肺炎链球菌(Streptococcus pneumoniae,S.pn)荚膜多糖(capsular polysaccharide,CPS)对C57BL/6小鼠急性中耳炎的炎症应答和组织损伤的作用.方法:首先通过同源重组的方式构建TIGR4△cps菌株,然后经鼓膜穿刺,分别接种5μl/耳的TIGR4和TIGR4△cps于C57BL/6小鼠的双侧中耳腔,对照组采用相同方法接种等体积无菌磷酸盐缓冲液(phosphate buffered saline,PBS).观察小鼠发病情况,分别于感染后1、3、5d制备中耳组织切片,HE染色观察中耳组织损伤及炎性细胞浸润变化,同时收集中耳灌洗液(middle ear lavage fluids,MELF),检测MELF中细菌载量、炎性细胞数量和细胞因子含量.结果:成功构建了TIGR4△cps菌株.与TIGR4组相比,TIGR4△cps组小鼠中耳上皮的损伤更轻,中性粒细胞募集减少(第1天时P=0.004;第3天时P=0.000;第5天时P=0.000),MELF中细胞因子TNF-α和IL-6水平更低(第1天时P=0.076、P=0.000;第3天时P=0.002、P=-0.003;第5天时P=0.000、P=0.000),但细菌清除更快(第1天时t=2.659,P=0.029;第3天时t=3.717,P=0.006).结论:在S.pn TIGR4致小鼠急性中耳炎过程中,CPS具有加重炎症应答和组织损伤,并导致宿主对S.pn的清除减慢的作用.

OBJECTIVE To develop a mouse model for acute otitis media (AOM) via transbullar injection method and evaluate its feasibility and practicability. METHODS The middle ears (ME) of C57BL/6 mice were inoculated via transbullar injection method with 5 µl streptococcus pneumoniae (S.pn) 19F suspension (1×10(7) CFU/ml), and the control group was inoculated equivalent phosphate buffered solution (PBS). Behavior changes were observed daily following inoculation. The ME tissues for histological examination and the middle ear lavage fluid (MELF) for total cells quantification, S.pn load determination and cytokines measurement were collected at 12 h, day 1, 2, 3, 5, 7 after inoculation, respectively. RESULTS Within 24 hours after instillation, the density of S.pn and the level of acute inflammatory cytokines in ME cavity increased rapidly, some mucosal hyperplasia was evident and leukocytic infiltration (primarily neutrophils) began. The level of ME inflammatory response reached maximal at 2-3 days after inoculation, with extensive effusion, leukocytic infiltration and mucosal thickening. Meanwhile, the density of S.pn decreased gradually. Bacterial clearance was completed by day 5 with extensive resolution of ME inflammation, although mucosal hyperplasia did not resolute until day 7. CONCLUSION A mouse model for AOM is successfully established via transbullar injection method, laying foundation for future study of AOM.

ABSTRACT Streptococcus pneumoniae is a Gram-positive and human-restricted pathogen colonizing the nasopharynx with an absence of clinical symptoms as well as a major pathogen causing otitis media (OM), one of the most common childhood infections. Upon bacterial infection, neutrophils are rapidly activated and recruited to the infected site, acting as the frontline defender against emerging microbial pathogens via different ways. Evidence shows that interleukin 17A (IL-17A), a neutrophil-inducing factor, plays important roles in the immune responses in several diseases. However, its function in response to S. pneumoniae OM remains unclear. In this study, the function of IL-17A in response to S. pneumoniae OM was examined using an in vivo model. We developed a model of acute OM (AOM) in C57BL/6 mice and found that neutrophils were the dominant immune cells that infiltrated to the middle ear cavity (MEC) and contributed to bacterial clearance. Using IL-17A knockout (KO) mice, we found that IL-17A boosted neutrophil recruitment to the MEC and afterwards induced apoptosis, which was identified to be conducive to bacterial clearance. In addition, our observation suggested that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in the recruitment and apoptosis of neutrophils mediated by IL-17A. These data support the conclusion that IL-17A contributes to the host immune response against S. pneumoniae by promoting neutrophil recruitment and apoptosis through the p38 MAPK signaling pathway.

OBJECTIVE:To compare the sensitivities of two different serotypes of Streptococcus pneumoniae in acute otitis media of C57BL/6 mice.METHODS:The middle ear cavity of C57BL/6 mice were inoculated via intratympanic injection of Streptococcus pneumoniae TIGR4 or 6B 1×10(8) CFU, and the control group were inoculated equivalent phosphate buffered solution (PBS). The incidence of mice, development of otitis media and the middle ear lavage fluid pathological changes by HE staining, as well as cell counts and cytokine levels were investigated.RESULTS:After inoculated with Streptococcus pneumoniae TIGR4, 6B and PBS, the survival rate of 6B group was significantly less than TIGR4 group and PBS control group (P < 0.01). Inflammatory cells in the middle ear cavity were mainly neutrophils, and the inflammatory cells recruitment in TIGR4 group were more than 6B group; The levels of IL-6 and TNF-α in the middle ear lavage fluid in TIGR4 group and 6B group were significantly increased compared with PBS control group, while the TIGR4 group were significantly increased compared with 6B group;6B group had delayed bacterial clearance in the middle ear.CONCLUSION:The study implied that Streptococcus pneumoniae 6B had higher pathogenicity for acute otitis media in C57BL/6 mice than TIGR4.

Objective:To establish a mouse model of acute otitis media and explore its pathological mechanism.Methods A total of 97 healthy,female C57BL/6 mice were used.Under anesthetized,5μl Streptococcus pneumoniae TIGR4 suspension(104,106 and 108 CFU) and equivalent phosphate buffer were puncture inoculation in the middle ear cavity through tympanic membrane.After inoculation,the mouse weight and the incidence were observed daily.Four mice from each group were taken heart blood and middle ear lavage fluid(MELF) at 1,3,5 days after inoculation.Cell count,cell classification,bacteria loads and cytokines were determinated.HE staining of tissue sections with 106CFU treated mouse was monitored at 1,3,5,7 days after inoculation.Results After inoculated with Streptococcus pneumoniae,mouse weight lost in a dose-dependent manner.Along with time,middle ear mucosa epithelial cell necrosis,mucosal thickening,inflammatory cell infiltration in the middle ear cavity and middle ear bone wall bone destruction were seen.Neutrophil recruitment phase was related with bacterial clearance phase.IL-6 and TNF-α were significantly elevated in the MELF.Conclusions In this study,mice with acute infection otitis media model were successfully established and supplied experimental basis for further study of the pathogenesis of acute otitis media.