Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

Hinokitiol, a component of the essential oil isolated from Cupressaceae, possesses antibacterial and antifungal activities and has been used in oral care products. In this study, the antibacterial activities of hinokitiol toward various oral, nasal and nasopharyngeal pathogenic bacteria, including Streptococcus mutans, Streptococcus sobrinus, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Fusobacterium nucleatum, methicillin‐resistant and ‐susceptible Staphylococcus aureus, antibiotic‐resistant and ‐susceptible Streptococcus pneumoniae, and Streptococcus pyogenes were examined. Growth of all these bacterial strains was significantly inhibited by hinokitiol, minimal inhibitory concentrations of hinokitiol against S. mutans, S. sobrinus, P. gingivalis, P. intermedia, A. actinomycetemcomitans, F. nucleatum, methicillin‐resistant S. aureus, methicillin‐susceptible S. aureus, antibiotic‐resistant S. pneumoniae isolates, antibiotic‐susceptible S. pneumoniae, and S. pyogenes being 0.3, 1.0, 1.0, 30, 0.5, 50, 50, 30, 0.3–1.0, 0.5, and 0.3 μg/mL, respectively. Additionally, with the exception of P. gingivalis, hinokitiol exerted bactericidal effects against all bacterial strains 1 hr after exposure. Hinokitiol did not display any significant cytotoxicity toward the human gingival epithelial cell line Ca9‐22, pharyngeal epithelial cell line Detroit 562, human umbilical vein endothelial cells, or human gingival fibroblasts, with the exception of treatment with 500 μg/mL hinokitiol, which decreased numbers of viable Ca9‐22 cells and gingival fibroblasts by 13% and 12%, respectively. These results suggest that hinokitiol exhibits antibacterial activity against a broad spectrum of pathogenic bacteria and has low cytotoxicity towards human epithelial cells.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme‐linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix.

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