TLR4 regulates ROS and autophagy to control neutrophil extracellular traps formation against Streptococcus pneumoniae in acute otitis media.

BACKGROUND: Otitis media (OM), a prevalent pediatric infectious disease, is mainly caused by Streptococcus pneumoniae (S.pn). Neutrophil extracellular traps (NETs), a novel antimicrobial strategy, were reported in 2004. We found that NETs formed in the middle ear with acute otitis media (AOM) induced by S.pn. However, the mechanisms of NETs formation are not entirely clear. METHODS: We stimulated neutrophils isolated from mouse bone marrow with S.pn clinical stain 19F in vitro, and established mouse model of AOM via transbullar injection with S.pn. NETs formation, reactive oxygen species (ROS) production, autophagy activation and bacterial load were analyzed in TLR4-/- and wild-type neutrophils stimulated in vitro with S.pn and in vivo during AOM. RESULTS: We found that autophagy and ROS were required for S.pn-induced NETs formation. Moreover, TLR4 partly mediated NETs formation in response to S.pn in vitro and in vivo during AOM. We also showed that attenuated NETs formation in TLR4-/- neutrophils correlated with an impaired ROS production and autophagy activation in vitro and in vivo. In addition, both the in vivo and in vitro-produced NETs were able to engulf and kill S.pn. CONCLUSIONS: TLR4 regulates ROS and autophagy to control NETs formation against S.pn in the course of AOM. IMPACT: S.pn can induce NETs formation in vitro and in vivo; TLR4 regulates NETs formation by ROS and autophagy; NETs contribute to the clearance of bacteria in acute otitis media. In this study, we firstly found that autophagy and ROS were required for S.pn-induced NETs formation in the model of acute otitis media (AOM). And to some extent, TLR4 mediated NETs formation during AOM. Our research might provide a potential strategy for the treatment of otitis media.

Interleukin-17A Aggravates Middle Ear Injury Induced by Streptococcus pneumoniae through the p38 Mitogen-Activated Protein Kinase Signaling Pathway.

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.

TLR2 promotes macrophage recruitment and Streptococcus pneumoniae clearance during mouse otitis media.

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.

The critical role of myeloperoxidase in Streptococcus pneumoniae clearance and tissue damage during mouse acute otitis media.

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.

Selective phenotyping for increased efficiency in genetic mapping studies.

The power of a genetic mapping study depends on the heritability of the trait, the number of individuals included in the analysis, and the genetic dissimilarity among them. In experiments that involve microarrays or other complex physiological assays, phenotyping can be expensive and time-consuming and may impose limits on the sample size. A random selection of individuals may not provide sufficient power to detect linkage until a large sample size is reached. We present an algorithm for selecting a subset of individuals solely on the basis of genotype data that can achieve substantial improvements in sensitivity compared to a random sample of the same size. The selective phenotyping method involves preferentially selecting individuals to maximize their genotypic dissimilarity. Selective phenotyping is most effective when prior knowledge of genetic architecture allows us to focus on specific genetic regions. However, it can also provide modest improvements in efficiency when applied on a whole-genome basis. Importantly, selective phenotyping does not reduce the efficiency of mapping as compared to a random sample in regions that are not considered in the selection process. In contrast to selective genotyping, inferences based solely on a selectively phenotyped population of individuals are representative of the whole population. The substantial improvement introduced by selective phenotyping is particularly useful when phenotyping is difficult or costly and thus limits the sample size in a genetic mapping study.

[A mouse model for acute otitis media via transbullar injection].

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.