Immunostimulatory activity of gamma-irradiation-killed Streptococcus pneumoniae whole cell in human bronchial epithelial cells : 감마선 조사 사멸 폐렴구균에 의한 사람 기관지 상피세포의 면역활성

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자연과학대학 협동과정 유전공학전공
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서울대학교 대학원
학위논문 (석사)-- 서울대학교 대학원 : 자연과학대학 협동과정 유전공학전공, 2018. 8. 한승현.

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been introduced and its efficacy and immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immune responses, the present study investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cells by comparing with that of heat-inactivated S. pneumoniae (h-SP) or formalin-inactivated S. pneumoniae (f-SP).


To investigate the efficacy of inactivated whole-cell vaccines, nonencapsulated S. pneumoniae TIGR4 strain was generated and subsequently inactivated by gamma-irradiation, heat, or formalin. Human bronchial epithelial cells, BEAS-2B, were stimulated with r-SP, h-SP, or f-SP. The mRNA expression and protein secretion of interleukin (IL)-6 and IL-8 were determined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA expression of CCL5, CCL28, and CXCL10 was analyzed by real-time RT-PCR. Cell viability was determined by trypan blue staining and annexin V/PI staining. Toll-like receptor (TLR) 2 and TLR4 activation was determined using Chinese hamster ovary (CHO) cells overexpressing human CD14 and TLR2 (CHO/CD14/TLR2 cells) or TLR4 (CHO/CD14/TLR4 cells) by analyzing CD25 expression with flow cytometry. Phosphorylation of mitogen-activated protein (MAP) kinases and TLR2 induction were analyzed by western blot assay. To determine the intracellular signaling pathway involving MAP kinases, the cells were pretreated with U0126, SP600125, or SB203580 followed by stimulation with inactivated S. pneumoniae, and the expression of IL-6 and IL-8 was detected by ELISA. Each inactivated S. pneumoniae was separated into soluble and insoluble components by centrifugation. Soluble components separated from each inactivated S. pneumoniae were detected by silver staining and Coomassie blue staining.


When BEAS-2B cells were stimulated with inactivated S. pneumoniae, r-SP potently induced IL-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner compared with h-SP or f-SP, but not other chemokines, CCL5, CCL28, and CXCL10. Stimulation with inactivated S. pneumoniae followed by analyzing CD25 expression on CHO/CD14/TLR2 cells demonstrated that r-SP induced activation of TLR2, whereas h-SP or f-SP showed a minimal activation. In addition, CHO/CD14/TLR4 cells showed that only r-SP activated TLR4, while h-SP or f-SP did not. On the other hand, TLR2 synthesis was not induced by r-SP in BEAS-2B cells. Moreover, r-SP potently phosphorylated ERK, JNK, and p38 kinase, whereas h-SP and f-SP poorly phosphorylated them. The potent expression of IL-6 and IL-8 induced by r-SP was diminished by inhibitors for ERK, JNK, or p38 kinase, suggesting that r-SP induced IL-6 and IL-8 expression through the activation of MAP kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the expression of IL-6 and IL-8 and activation of TLR2 and TLR4. Of note, soluble components of r-SP potently induced IL-6 and IL-8 expression as much as whole r-SP compared with insoluble components of r-SP. r-SP potently released a large amount of different soluble components compared with h-SP or f-SP.


The present study demonstrated that r-SP potently stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8 via MAP kinase pathway by activating TLR2, which might influence the induction of adaptive immune responses and enhance vaccine efficacy.
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