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Dry Mouth, Focal Lymphocytic Sialadenitis, and Oral Dysbiosis in IkB-z-Deficient Mice : IkB-z 결핍 쥐에서 구강건조증과 림프구성 타액선염의 발생 및 구강세균총의 변화

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Authors

이준호

Advisor
최영님
Issue Date
2019-08
Publisher
서울대학교 대학원
Keywords
Sjögren's syndrome (SS)focal lymphocytic sialadenitis (FLS)salivary flow rateoral microbiotaIkB-z-deficient mice
Description
학위논문(석사)--서울대학교 대학원 :치의학대학원 치의과학과,2019. 8. 최영님.
Abstract
1. 배경

쇼그렌 증후군은 전신 염증성 자가면역질환으로서 외분비 샘의 기능장애와 건조 증상이 특징이다. 발명의 원인은 잘 알려져 있지 않지만 다양한 유적적, 환경적 요인이 관여한다. IkB-z 단백질이 결여된 상피세포는 NF-kB 의 전사 활동이 다르게 일어나, caspase-3 활성화와 세포 사멸이 증가한다. 상피의 손상은 외부 환경으로부터 내부조직을 보호해주지 못하고, 세균 감염이 자가 면역을 유도 할 수도 있다. 전 연구에서 IkB-z 가 결여된 쥐에서 눈물 분비 감소, 눈물샘염 그리고 자가 항체 생성 같은 쇼그렌 증후군의 증상을 나타냈지만, 타액선에는 아무런 증상을 보이지 않았다. 그러나, 저희 실험동물실에서는 IkB-z 단백질이 결여된 쥐에서 타액 감소가 관찰되었기에 IkB-z 결핍에 따른 분비율 변화, 타액선염, 구강 세균의 관계를 알아보고자 한다.

2. 방법

실험에 사용된 IkB-z 단백질이 결여된 쥐는 서울대학교 연건 치의학대학원 특정 병원체 부재 동물실에서 번식 그리고 유지시켰다. Nfkbiz+/+, Nfkbiz+/-, Nfkbiz-/- 5 마리씩 24 주 동안 사육한 후에 타액선 그리고 구강 세균을 채취했다. 타액은 필로카폰을 주입하여 10 분 동안 채취했다. 타액선은 파라핀 포매 후 hematoxlin-eosin (H&E) 로 염색해 림프구성 타액선 염 발생을 관찰하였다. 타액선의 세균의 침투 여부는 in situ hybridization 으로 관찰하였다. 또한 타액선의 일부를 급속 냉각하여 얼린 조직을 만들고, CD3-z 와 B220 형광 염색 관찰하였다. 구강 세균은 멸균된 면봉으로 채취 후 Power Soil DNA Isolation Kit 를 사용해 genomic DNA 를 얻었다. 구강 세균은 high throughput sequencing 으로 분석했다.

3. 결과

필로카폰에 의해 유도된 타액을 채취하여 각 그룹들끼리 비교했다. Nfkbiz+/+ 쥐에 비해 Nfkbiz+/- 쥐와 Nfkbiz-/- 두 그룹에서 타액 분비량이 감소하였다. 헤마톡실-에오신에 염색된 타액을 각 그룹끼리 비교 했다. 그룹 간에 유의한 차이는 없었으나 Nfkbiz-/- 군 5마리 중 3마리에서 SS 진단 기준에 부합하는 1점 이상의 림프구성 타액선염이 관찰되었다. 형광 염색에서는 대부분의 T 세포가 타액선염 초기단계에 발견되었지만, 진행이 된 타액선염에서는 B 세포가 주로 관찰되었다. 그리고, in situ hybridization 결과에서 도관 세포를 통해 타액선으로 침투한 세균들이 모든 유전자형 쥐에서 관찰되었다. 구강 세균총 분석 결과에서는 Nfkbiz+/+ 에 비해 Nfkbiz-/- 쥐에서 세균총의 균일성과 다양성이 증가하였음을 보여주었다. 또한, 베타 다양성 결과에서 Nfkbiz+/+, Nfkbiz+/- 그룹에 비해 Nfkbiz-/- 가 따로 떨어져 있어, 매우 다른 구강세균총을 갖고 있음을 제시하였다. Streptococcus danieliae 는 Nfkbiz+/+, Nfkbiz+/- 에서 전체 구강 세균의 70% 이상을 차지하는데 반면 Nfkbiz-/- 쥐에는 크게 감소하였다. 그 대신, Nfkbiz-/- 쥐에서 Staphylococcus sciuri group, Escherichia coli group, Enterococcus faecium group, Corynebacterium mastitidis group 이 유의하게 증가하였다. Nfkbiz-/- 쥐에서 증가한 구강세균들은 FLS score of ≥ 1 그룹에서도 유의하게 증가하였다. Lactobacillus mudanjiangensis, Lactobacillus parabrevis group, Lactobacillus brevis, Lactobacillus plantarum group, Staphylococcus aureus group, Cupriavidu metallidurans and Lactobacillus_uc 세균종은 특이하게도 Nfkbiz+/- 쥐에서만 유의하게 증가하였다. 이 세균들은 salivary flow rate 평균 이하의 그룹에서도 유의하게 증가하였다.

4. 결론

Nfkbiz-/- 쥐에서 자발적으로 구강건조증, 림프구성 타액선염, 그리고 구강세균총의 변화가 일어났다. 구강세균 분석 결과, Staphylococcus sciuri group, Escherichia coli group, Enterococcus faecium group, Corynebacterium mastitidis group 이 FLS 와 관련이 있음을 제시하였다.
Background

Sjögren syndrome (SS) is a systemic autoimmune disease characterized by dryness of eyes and mouth. Etiopathogenetic mechanisms of SS remain elusive because numerous factors contribute to the progression of SS. It is believed that the cause of SS involves a combination of genetic and environmental factors. Mice lacking IkB-z, a protein encoded by Nfkbiz gene, would be an appropriate animal model for SS because genetic and environmental factors are involved in the model. IkB-z-deficient epithelial cells cause changes in the transcriptional activity of NF-kB, leading to increased caspase-3 processing and apoptosis. Damaged epithelia fail to protect from the external environment. It is more accessible to encounter the pathogens and their substances. Moreover, a previous study demonstrated that IkB-z-deficient mice spontaneously develop SS-like autoimmune disease, resulting in increased autoantibody production, reduced tear secretion and lymphocyte-infiltrated dacryoadenitis. Although the previous study reported no symptom in the salivary glands of IkB-z-deficient mice, reduced salivary rates and sialadenitis were observed in my animal facility.

Methods

IkB-z-deficient mice were bred and maintained under specific-pathogen-free condition in Laboratory Animal Facility at the School of Dentistry, Seoul National University. Five of each Nfkbiz+/+, Nfkbiz+/- and Nfkbiz-/- mice were used in the experiment. Salivary glands and oral microbiota were obtained at 24 weeks. Salivary flow rate was measured for 10 min after pilocarpine injection. Salivary glands were harvested, fixed with 10% neural formalin solution, and embedded with paraffin. They were subjected to staining with hematoxlin-eosin (H&E) or in-situ hybridization using a universal probe for bacterial 16s ribosomal RNA (rRNA). Focal lymphocytic sialadenitis (FLS) and bacterial invasion were examined under a microscope. The other pieces of salivary glands were embedded in OCT compound and then frozen in liquid nitrogen. The sections were stained with anti-mouse CD3-z and anti-mouse B220 antibodies. Immunofluorescence images were captured under a confocal microscope. Oral microbiota was collected with clean cotton swabs. Genomic DNA isolated from oral swab was subjected to microbiota analysis by high throughput sequencing.

Results

Both Nfkbiz+/- and Nfkbiz-/- mice produced less saliva than Nfkbiz+/+ mice in response to pilocarpine injection. FLS area and foci score tended to increase in Nfkbiz-/- mice compared to other genotypes, but statistical significance was not achieved. Importantly, three out of five IkB-z-deficient mice developed FLS with score ≥ 1. Immunohistofluorescent detection with anti-mouse CD3-z and anti-mouse B220 antibodies demonstrated that infiltrating lymphocytes were mostly T cells in the early stage. Afterwards, there was a large mass of B cells occupying salivary glands. In addition, in-situ hybridization using a universal probe for 16S rRNA revealed that bacterial invasion of ductal cells occurs in submandibular glands. Oral microbiota was analyzed in Nfkbiz+/+, Nfkbiz+/- and Nfkbiz-/- mice. In alpha diversity, Simpson (measure of evenness) and Shannon (measure of species richness and evenness) indexes showed significant differences between Nfkbiz+/+ mice and Nfkbiz-/- mice. Beta diversity revealed that oral bacterial communities in Nfkbiz+/+ mice and Nfkbiz+/- mice clustered closely; on the other hand, oral bacterial communities in Nfkbiz-/- mice were separated from those of other genotypes. While Streptococcus danieliae predominated the oral microbiota in Nfkbiz+/+ and Nfkbiz+/- mice, it was substantially decreased in Nfkbiz-/- mice. Instead, Staphylococcus sciuri group, Escherichia coli group, Enterococcus faecium group and Corynebacterium mastitidis group were significantly increased in Nfkbiz-/- mice compared to Nfkbiz+/+ mice. Surprisingly, those bacteria higher in Nfkbiz-/- mice significantly increased in FLS score of ≥ 1. Nfkbiz+/- mice had unique sets of oral bacteria, which were Lactobacillus mudanjiangensis, Lactobacillus parabrevis group, Lactobacillus brevis, Lactobacillus plantarum group, Staphylococcus aureus group, Cupriavidu metallidurans and Lactobacillus_uc. Those oral bacteria were also significantly higher in the groups that were below the salivary flow rate of 7.8 (mean).

Conclusion

In conclusion, Nfkbiz-/- mice spontaneously developed dry mouth, FLS, and dysbiosis of oral microbiota. Oral microbiota analysis demonstrated that the groups Staphylococcus sciuri, Escherichia coli, Enterococcus faecium, and Corynebacterium mastitidis may be associated with FLS. These findings may provide valuable information pertaining to the combined effects of host genotype and oral microbiota acting in conjunction with environmental factors to develop dry mouth and FLS.
Language
eng
URI
https://hdl.handle.net/10371/161684

http://dcollection.snu.ac.kr/common/orgView/000000158425
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