Interleukin-1beta Participates in the Development of Pneumococcal Acute Lung Injury and Death by Promoting Alveolar Microvascular Leakage

Streptococcus pneumoniae (S. pneumoniae, also known as pneumococcus) infections are major causes of death worldwide. Despite the development and use of effective antibiotics, high, early mortality due to pneumococcal infections has not been decreased for the last few decades. Recent study found a deadly hemorrhagic acute lung injury (ALI) as a major cause of death at the early stage of severe pneumococcal infections. Interleukin (IL)-1beta was known to play critical roles not only for the development of ALI but also resolution of it. The role of IL-1beta on the pathogenesis of pneumococcal ALI, however, has not been well understood yet. This study aims to investigate the role of IL-1beta on the development of pneumococcal ALI and subsequent death. IL-1beta expression was upregulated in the lungs of pneumococcal ALI in wild-type (WT) mice, but not in the plasma. Despite an increased expression of pulmonary IL-1beta, no inflammatory cell infiltration into airway has been observed. Upregulation of IL-1beta expression was indeed dependent on pneumococcal cytoplasmic toxin pneumolysin and its cell surface receptor Toll-like receptor 4. Deficiency of IL-1R1, a cell surface receptor of IL-1beta, resulted in a markedly reduced hemorrhagic pulmonary edema and early death in pneumococcal ALI. Finally, IL-1beta neutralization in WT mice protects against pulmonary hemorrhagic edema and death. These data suggest that pulmonary expression of IL-1beta exacerbates pneumolysin-induced ALI and death by promoting alveolar hemorrhagic edema.

Dynamic levels and significance of the triggering receptor expressed on myeloid cells-1 expression in injury brain tissues of rat caused by pneumolysin / 中华实用儿科临床杂志

Objective To investigate the dynamic level changes and significance of triggering receptor expressed on myeloid cells-1 (TREM-1) in the injury brain tissues of rats caused by pneumolysin (PLY).Methods Sixty-four SD rats were randomly and equally divided into PLY group and control group,0.1 mL PLY and isopyknic normal saline was given through left internal carotid artery respectively.Brain tissue gross and histological changes were observed at different time(4 h,6 h,12 h,24 h),meanwhile the expression levels of neurocyte damage marker glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) protein were detected by immunohistochemistry;and the expression levels of TREM-1,tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were detected respectively by enzyme-linked immunosorbent assay.Results The observation of brain tissue gross and histological changes indicated the existence of brain injury,and the expression levels of GFAP,NSE,TNF-α and IL-6 protein increased from 4 h after PLY were injected and augmented dynamically as time went on,compared with the control group at corresponding time points,the differences were statistically significant (all P ＜ 0.05).The level of TREM-1 in the PLY group reached a peak at the 4 h time point,but decreased somewhat at the 6 h time point,the level of TREM-1 was still higher than that in control group,the differences were statistically significant(all P ＜ 0.05).However,the level of TREM-1 in the PLY group declined obviously at 12 h and 24 h time points,compared with that in control group,there were no significant differences (all P ＞ 0.05).Conclusions The expression levels of TREM-1 up-regulated obviously in the early stage of brain damage induced by PLY,which might be involved in the pathological process of brain damage by promoting the expression of TNF-α and IL-6.

Construction of ply gene-deletion mutant of Streptococcus pneumoniae and research of its virulence change / 中华微生物学和免疫学杂志

Objective To lay the foundation for further exploration on parasitifer's defence reaction to pneumolysin through constructing ply gene-deletion strain of Streptococcus pneumoniae and researching on its virulence change. Methods A linker fragment with erm gene in middle and homologous upstream and downstream fragment of ply gene at both sides was prepared by long flanking homology-polymerase chain reaction(LFH-PCR). The linker fragment was transformed into Streptococcus pneumoniae. ply-deficient strain was then screened out from blood plate which contains erythromycin and identified by PCR. ply-deficient strain growth in vitro was observed and virulence change was observed through infecting mouse model. Results PCR results showed that ply gene was replaced completely by erm gene. The ply deficient strain was successfully constructed. The growth of single strain culture medium showed that ply genetic defect made no influence on bacterial's external growth. While in the mice nasal cavity infecting experiment, deficient strain enter into blood after 6 h from infecting which obviously slower than that did wild-type(2 h). And the number of bacteria at each point was much smaller than that of wild-type(P ＜0. 01 ). The mice peritonaeum infecting experiment showed that median lethal time of wild-type was 3 d, while that of deficient strain was 18 d(P＜0. 01). Conclusion It is a good way to completely substitute ply gene using LFH-PCR. ply deletion made no influence on baterial's growth in vitro, but it resulting in reduction of bacterial virulence in vivo.

The study on the immunogenicity of Streptococcus pneumoniae pneumolysin DNA vaccine in Rhesus macaques / 中华微生物学和免疫学杂志

Objective To study the immunogenicity of Streptococcus pneumoniae pneumolysin DNA vaccine in Rhesus macaques. Methods The deletion of the gene sequence encoding for the 11 amino acids at the carboxyl terminal of pneumolysin (PN) from its wild type gene (pn) by PCR resulted in a mu-tant pneumolysin gene (pnd). The wild type pn gene encoding PN and the mutant gene (pnd) encoding PND were cloned into pVAX1 vector respectively and then tested as Ppn and Ppnd DNA vaccines. The PN and PND proteins were purified from recombinant E. coli. Rhesus macaques were immunized by intramuscu-larly (i.m.) injection of Ppn or Ppnd DNA vaccine with electroporation (EP). Results Because of the deletion of the gene sequence encoding for the eleven amino acids at the carboxyl terminal of the PN from pn gene, the recombinant PND antigen lost its hemolytic activity while its antigenicity was remained. The spe-cific humoral immunity against pneumolysin was induced by injecting monkey with 500 μg DNA followed by EP. Boosting the Ppn or Ppnd DNA/EP primed animals with corresponding recombinant protein, PN or PND, evoked strong immune response at about 4 fold increase in the antibody titer. Conclusion Specific antibody responses were induced in the monkeys by DNA vaccination and electroporation. The immunogenic-ity of the DNA vaccines were significantly enhanced when PN or PND protein boost was applied 10 d after third DNA vaccination.