Comprehensive immune profiling and analysis of significance of S100A8-positive immune cells in breast cancer

우지원 서울대학교 대학원 2022 국내박사

Background: Immune microenvironment differs according to hormone receptor (HR) status or tumor progression stage. Its importance is ever-increasing as drugs targeting tumor immunity came up as a game changer in cancer therapeutics. Myeloid-derived suppressor cells (MDSCs) are one of the key players in tumor immunity but not well studied in human tissue because of its phenotypic complexity. They are known to promote tumor progression via various immune-mediated and non-immune-mediated mechanisms. Methods: Differential expression of immune-related genes was evaluated via comprehensive immune profiling according to unsupervised clustering and HR status, and a target gene with significant fold change, possibly MDSC-associated gene, was searched for further analysis. The difference in its expression was validated using tissue microarray of 700 cases of human breast cancer. Its clinicopathological significance and relationship between other tumor infiltrating lymphocytes (TILs) including CD4+, CD8+ and FOXP3+ TILs or PD-L1+ immune cells (ICs) were investigated. Results: In immune profiling analysis, S100A8, a known MDSC marker, showed the most striking fold change in the cluster 2 in unsupervised clustering and HR status dependent grouping. In immunohistochemistry (IHC), S100A8 was stained in both tumor cells and ICs in breast cancer samples. Infiltration of S100A8+ ICs was associated with aggressive clinicopathological features and poor clinical outcome. They were already present in pre-invasive stage of breast cancer and were associated with increased infiltration of CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs. Prognostic impact of S100A8+ ICs was stronger in HR-positive, PD-L1+ IC-negative, CD8+ TIL-low and FOXP3+ TIL-low subgroups. When stained with various IHC markers, S100A8+ ICs were mostly revealed as CD33+ CD15- CD163+ cells which are thought to be monocytic-MDSCs derived macrophages. Conclusion: S100A8 is a key molecule that differentiates distinct immune clusters and also highly expressed in HR-negative breast cancers. S100A8 is usually expressed in monocytic-MDSCs derived macrophages and contributes to breast cancer progression from pre-invasive stage. S100A8+ ICs were associated with increase in CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs but prognostic impact of S100A8+ ICs was stronger in less immunogenic condition. 서론: 유방암의 면역 미세환경은 호르몬 수용체 발현이나 종양의 진행 단계에 따라 다양한 모습을 보인다. 종양 면역을 대상으로 하는 면역치료제의 효과가 입증됨으로서 유방암 면역에 대한 연구의 중요성은 날로 커지고 있다. 골수유래 면역억제세포는 종양 면역에 중요한 기능을 담당하고 있으나 그 표현형이 복잡하기 때문에 인체 조직에서 연구가 어려운 분야이다. 이 세포들은 면역과 관련된 기전 뿐만 아니라 면역과 관련되지 않은 기전을 통하여서도 다양하게 암의 진행을 촉진시킨다. 방법: 포괄적인 면역 프로파일링을 이용하여 비지도 군집분석과 호르몬 수용체 발현에 따른 면역 관련 유전자들의 발현 차이를 평가하였다. 추가 분석을 위하여 유의미한 발현량 차이를 보이는 유전자로 나타난 골수유래 면역억제세포 관련 유전자를 선택하였다. 700명의 유방암 환자 조직으로 만들어진 조직 마이크로어레이에서 해당 단백질 발현의 차이를 확인하였다. 이 단백질 발현이 가지는 임상병리학적 중요성과 다른 종양 침윤 림프구 (CD4, CD8, FOXP3 양성 림프구), PD-L1 양성 면역세포와의 관계를 분석하였다. 결과: 면역 프로파일링 자료를 비지도 군집분석에서 확인된 ‘군집 2’와 다른 군집들의 비교 및 호르몬 수용체 발현에 따라서 분석한 결과 골수유래 면역억제세포 표지자로 알려진 S100A8이 가장 큰 발현량 차이를 보였다. 면역조직화학염색 결과 S100A8은 유방암 조직의 종양세포와 면역세포 모두에서 염색되었다. 특히 S100A8 양성 면역세포의 침윤은 나쁜 임상병리학적 특성, 나쁜 예후와 관련이 있었다. 이들은 유방암의 침윤 전 단계부터 존재하였고 CD4, CD8, FOXP3 양성 종양 침윤 림프구, PD-L1 양성 면역세포 침윤의 증가와 연관을 보였다. S100A8 양성 면역세포의 예후적 중요성은 호르몬 수용체 양성인 경우, PD-L1 양성 면역세포가 없는 경우, CD8, FOXP3 양성 림프구가 적은 경우에 더 두드러지게 나타났다. 다양한 면역조직화학검사 결과 S100A8 양성 면역세포는 대부분 CD33 양성, CD15 음성, CD163 양성 표현형을 보여 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포로 생각되었다. 결론: S100A8은 특정 면역 군집을 구분하는 중요한 단백이며 또한 호르몬 음성 유방암에서 증가하였다. 인간 유방암 조직에서 S100A8은 주로 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포에서 발현되며 침윤 전 단계부터 존재하여 유방암의 진행을 촉진하는 역할을 할 것으로 생각된다. S100A8 양성 면역세포는 CD4, CD8, FOXP3 양성 종양 침윤 림프구와 PD-L1 양성 면역세포의 증가와 연관이 있었으나 그 예후적 중요성은 면역성이 낮은 조건에서 더 두드러지게 나타났다.

S100A8 and S100A9 induction is involved in inflammation-associated hepatocarcinogenesis under hypoxic conditions via the JNK pathway

조혜기 서울대학교 대학원 2017 국내석사

Background: S100A8 and S100A9 are two heterodimeric members of the cytoplasmic S100 Ca2+ signaling protein family. They act as proinflammatory signals and play a role in tumorigenesis as they affect inflammation, proliferation, and invasion of tumor cells in various types of cancer, including hepatocellular carcinoma (HCC). Hypoxia plays an important role in inflammation-associated tumorigenesis, but the direct role of S100A8 and S100A9 in hepatocarcinogenesis has not yet been elucidated, particularly with regard to the impact of hypoxia. The present study investigated the role of S100A8 and S100A9 in HCC under hypoxic conditions. Methods: The expression of S100A8 and S100A9 was quantified using real-time polymerase chain reaction. Human HCC cells (SNU-761 and SNU-3058) were grown with either S100A8/A9 treatment or control, under either normoxic or hypoxic conditions. MTS and invasion assays were performed to evaluate the role of S100A8 and S100A9 in HCC. Immunoblot analyses were performed to investigate the signaling pathway that is involved in the activity of S100A8 and S100A9. Results: S100A8 and S100A9 mRNA expression increased under hypoxic conditions compared with normoxic conditions. S100A8 and S100A9 treatment significantly increased the invasion of HCC cells, especially under hypoxic conditions, whereas no significant changes in HCC cell proliferation were observed. S100A8 treatment significantly enhanced the expression of interleukin-8 (IL-8), an inflammatory cytokine that is involved in tumor progression, under hypoxic conditions. IL-8 siRNA transfection attenuated the S100A8-induced invasion of HCC cells. S100A8 treatment attenuated bile acid-induced HCC cell apoptosis under hypoxic conditions. Moreover, S100A8 increased the protein expression of mesenchymal markers, including vimentin, matrix metalloproteinase 9 (MMP-9), and MMP-2, which were attenuated by a c-Jun N-terminal kinase (JNK) inhibitor. Conclusion: Hypoxia-induced S100A8/A9 expression increased the invasion of HCC cells and attenuated bile acid-induced HCC apoptosis by increasing IL-8 under hypoxic conditions. Considering the enhanced expression of epithelial-mesenchymal transition (EMT) markers, the JNK pathway may be involved in controlling the activity of S100A8 and S100A9. These results indicate that S100A8 and S100A9 may serve as therapeutic targets for the treatment of HCC under hypoxic conditions.

S100A8/A9에 의한 1차 배양 미세아교세포 및 성상교세포에서 증가되는 S100A8의 TLR4/NF-κB/NLRP3 경로를 통한 pyroptosis 조절

김지은 건양대학교 대학원 2023 국내석사

염증 매개체 중 하나인 S100A8/A9는 알츠하이머병(AD) 또는 외상성 뇌손상(TBI)과 같은 여러 신경퇴행성 질환에서 증가하며 염증의 신뢰할 수 있는 마커로 간주된다. S100A8/A9는 위험 관련 분자 패턴(DAMPs)에 속하며 면역 반응을 유도할 수 있다. 미세아교세포와 성상교세포는 중추신경계(CNS)의 비신경 세포에 해당된다. 미세아교세포는 염증과 손상이 일어나는 동안 대식세포로 기능하며 중요한 식세포 역할을 한다. 또한 성상교세포는 여러 사이토카인의 분비 및 신경독성 물질의 방출을 담당한다. 염증과 관련된 세포 사멸 중 파이롭토시스(pyroptosis)는 유전적으로 암호화된 괴사의 경로 중 하나이며 비염증성 형태의 세포 사멸과는 다른 유형의 세포 사멸이다. 지금까지 다른 연구들에선 S100A8/A9를 사용한 암의 세포 사멸을 확인했지만 뇌 질환에서는 현재까지 밝혀진 바가 많지 않다. 본 연구에서는 S100A8/A9 처리에 의한 S100 A8 발현이 1차 미세아교세포 및 성상교세포의 파이롭토시스에 영향을 미치는지 조사하였고 그 결과, 1차 미세아교세포와 성상교세포에서 S100A8/A9에 의해 유발된 caspase-1의 활성화와 특정 사이토카인의 성숙은 TLR4/NFκB/ NLRP3 경로를 통해 파이롭토시스 세포 사멸을 유도함을 확인했다. 또한 S100 A8 short-hairpin RNA (shS100A8)는 ninjurin-1 (NINJ1) 및 GSDMD와 같은 파이롭토시스 관련 단백질의 발현을 억제하였다. 요약하면, 본 연구 결과는 1차 소교 세포 및 성상교세포에서 S100A8/A9에 의한 증가된 S100A8 발현이 파이롭토시스를 유도한다는 것을 밝히는 새로운 관점을 제안한다. One of the inflammatory mediators, S100A8/A9 is increased in several neurodegenerative diseases such as Alzheimer’s disease (AD) or traumatic brain injury (TBI) and is regarded as a reliable marker of inflammation. S100A8/A9 belongs to the danger -associated molecular patterns (DAMPs) and types of molecules that can elicit an immune response. Microglia and astrocytes are non-neuronal cells of the central nervous system (CNS). During inflammation and injury, microglia function as macrophages and play an important phagocytic role. Astrocytes take charge of secretion of multiple cytokines or release of neurotoxic amounts. Among the cell death associated with inflammation, pyroptosis is one of the genetically encoded pathways of necrosis and it is a different type of cell death from the non-inflammatory form of apoptosis. So far, other studies have confirmed cell death through S100A8/A9 in cancer, but not in brain disease. As a heretofore not revealed, I investigated whether S100A8 expression by each treatment of S100A8 and S100A9 affect the pyroptosis of primary microglia and astrocytes. The activation of caspase-1 and maturation of specific cytokines in the S100A8/A9-triggered primary microglia and astrocytes induced pyroptotic cell death through TLR4/NF-κB/ NLRP3 pathway. Furthermore, S100A8 short-hairpin RNA (sh S100A8) inhibited the expression of pyroptosis-related proteins like ninjurin-1 (NINJ1) and GSDMD. In summary, our findings suggest a novel perspective, revealing that the increased S100A8 expression by S100A8/A9 in primary microglia and astrocytes induce pyroptosis.

Effects of S100A8/S100A9 (calprotectin) on human respiratory mucosal epithelium and its regulatory mechanism

안상현 Graduate School, Yonsei University 2023 국내박사

Inflammation is a host's defense mechanism against external stimulants, and the immune response by the first frontline barrier epithelium and inflammatory cells is complexly generated. Chronic rhinosinusitis (CRS) is a chronic inflammatory disease that occurs in the paranasal sinuses. A number of substances are involved in the immune response of CRS, and various pathophysiological mechanisms have been suggested. In particular, calprotectin as a damage-associated molecular pattern is a heterodimer complex of S100A8 and S100A9 and performs various functions in epithelial and inflammatory cells. In addition, although S100A8 and S100A9 are known to have strong pro-inflammatory effects, studies on their effects on the human nasal epithelial (HNE) cells and pathogenesis of CRS are rare. Therefore, this study aimed to investigate the effects of S100A8, S100A9, and calprotectin on human nasal epithelium, CRS, and regulatory mechanisms. Chapter I explores the conditions under which S100A8 and S100A9 are elevated in the upper airway nasal epithelium and investigates the roles of S100A8 and S100A9 in HNE cells. S100A8 and S100A9 increased when pro-inflammatory cytokines, such as TNF-α and IL-1β, were treated. They were inhibited by anti-inflammatory cytokines, such as IL-4 and IL-13. In addition, they were overexpressed in the squamous epithelium, not in normal tissues, through immunohistochemical findings of CRS tissues. In order to check whether S100A8 and S100A9 affect squamous metaplasia, recombinant S100A8, S100A9, and calprotectin were applied to HNE cells. Matrix metalloproteinase 9 (MMP9), a marker for tissue remodeling, and Involucrin, a marker for squamous metaplasia, were then used to confirm the optimal experimental conditions. Furthermore, the effects of S100A8, S100A9, and calprotectin on HNE cells were investigated through bulk RNA sequencing analysis. It was particularly confirmed that S100A9 induces squamous metaplasia by being involved in retinoic acid metabolism, which is important for HNE cells differentiation. In Chapter II, the role of calprotectin, known as an antimicrobial peptide, on eosinophils and eosinophil extracellular traps (EETs) in the pathophysiology of CRS is investigated. Previously, calprotectin was known to be predominantly expressed in neutrophils and be mainly involved in neutrophil extracellular traps (NETs). However, CRS with nasal polyps (CRSwNP) insufficiently explained the presence of only calprotectin and neutrophil because eosinophils were known to be important type 2 inflammation cells in disease development. Therefore, the purpose of this study was to investigate the relationship between calprotectin and eosinophil and between calprotectin and EETs. To prove this hypothesis, using eosinophilic CRS mouse and human tissues, major basic protein (MPB) and myeloperoxidase (MPO), which are markers specific to eosinophils and neutrophils, respectively, were subjected to simultaneous immunofluorescence with calprotectin. As a result, calprotectin-positive cells were co-localized with MBP-positive cells, and calprotectin was involved in EETs as well as NETs. In addition, calprotectin-positive cells in the tissue showed a positive correlation with tissue and blood eosinophils. Additionally, calprotectin showed a correlation with olfactory dysfunction, with the Lund-Mackay CT score indicating the severity of CRS and the use of JECREC scores. Through this, it was demonstrated that the expression of calprotectin in CRS tissue can be a biomarker indicating disease severity. Summarizing these two studies, increased S100A8 and S1000A9 due to inflammation in HNE cells induces tissue remodeling, such as squamous metaplasia through the modulation of retinoic acid metabolism. In addition, calprotectin is secreted by neutrophils and eosinophils and has an important role in the innate immune response of CRSwNP, owing to its involvement in EETs. In conclusion, S100A8, S100A9, and calprotectin expressed in epithelial and inflammatory cells reflect the disease severity of patients with CRS. 염증은 외부 자극에 대한 숙주의 방어 기제로 상기도의 첫 번째 장벽인 비강 점막 상피세포와 점막 하층에 분포하는 염증세포에 의한 복합적인 면역반응으로 인해 발생한다. 비강과 부비동에 발생하는 염증성 질환인 만성 비부비동염은 점막 상피세포와 다수의 염증 관련 단백질, 그리고 다양한 면역세포가 관여하고 있으며 이들과 관련한 다양한 병태생리학적 면역 기전이 제시되어 있다. 특히, S100A8과 S100A9의 이종이량체 복합체인 calprotectin (칼프로텍틴)은 손상 관련 분자 패턴으로써 비강 점막 상피 세포와 염증 세포에서 다양한 기능을 수행하며 S100A8과 S100A9는 강력한 전염증 효과가 있는 것으로 알려져 있다. 하지만 이 단백질들이 비강 점막 상피세포에 미치는 영향과 만성 비부비동염의 발병 기전에 대한 연구는 아직까지 드물다. 따라서 본 연구에서는 S100A8, S100A9 및 칼프로텍틴이 비강 점막 상피세포와 비강의 염증성 질환인 만성 비부비동염에 미치는 영향과 조절 기전을 조사하였다. 1장에서는 S100A8과 S100A9이 비강 점막 상피세포에서 증가되는 조건과 그 역할을 조사하였다. S100A8과 S100A9는 비강 점막 상피세포에서 TNF-α 및 IL-1β와 같은 전염증성 사이토카인을 처리했을 때 증가했고, IL-4 및 IL-13과 같은 항염증성 사이토카인에 의해 억제되었다. 또한, 만성 비부비동염 환자의 조직을 가지고 면역조직화학염색을 시행하고 분석하였다. 그 결과는 S100A8과 S100A9이 정상 상피층이 아닌 편평 상피층에서 과발현되었다. 이를 통해 S100A8과 S100A9이 비강 점막 상피세포의 편평 상피 화생에 영향을 미치는지 알아보기 위한 실험을 진행하였다. 배양된 비강 점막 상피세포에 재조합 S100A8, S100A9, 칼프로텍틴을 각각 처리한 후 조직 리모델링 표지자인 MMP9와 편평 상피 화생의 표지자인 Involucrin의 발현을 확인하였다. S100A8과 S100A9이 편평 상피 화생에 영향을 주는 최적의 실험 조건을 확인하였고 bulk RNA 시퀀싱 분석을 통해 S100A8, S100A9, 칼프로텍틴이 비강 점막 상피세포에 미치는 영향을 확인하였다. 이 실험을 통해 S100A8과 S100A9, 그리고 칼프로텍틴이 비강 점막 상피세포에 미치는 공통점과 차이점을 확인할 수 있었다. 특히, S100A9는 비강 점막 상피세포 분화에 중요한 레티노산 대사에 관여한다는 것을 확인하였고, 편평 상피 화생과 조직 리모델링에 관련이 있는 것을 확인하였다. 2장에서는 만성 비부비동염의 병태생리학에서 중요한 염증세포로 알려진 호산구와 그와 관련한 호산구 세포외 트랩에 대하여 항균 펩타이드로 알려진 칼프로텍틴의 관련성을 조사하였다. 기존의 문헌들에 따르면 칼프로텍틴은 주로 호중구에서 발현되며, 선천적 염증 반응에 중요한 호중구 세포외 트랩에 관여하는 것으로 알려져 있었다. 하지만 비용종을 동반한 만성 비부비동염은 호산구가 질병 발달에 중요한 제2형 염증 질환으로 알려져 있어 칼프로텍틴과 호중구만을 설명하기에는 불충분하였다. 따라서 본 연구의 목적은 호산구에서 칼프로텍틴의 발현을 확인하고 칼프로텍틴과 호산구 세포외 트랩의 관계를 조사하였다. 이 가설을 증명하기 위해 호산구성 만성 비부비동염 마우스 모델과 만성 비부비동염 환자의 조직을 사용하여 각각 호산구 및 호중구에 특이적인 표지자인 major basic protein (MBP)와 myeloperoxidase (MPO), 그리고 칼프로텍틴 항체를 가지고 동시에 삼중 면역 형광 염색을 진행하였다. 칼프로텍틴 양성 세포는 MPO 양성 세포와 일치할 뿐만 아니라 MBP에 양성을 나타내는 세포도 일치하는 소견을 보였다. 이를 통해 칼프로텍틴이 호중구와 호산구에서 각각 발현되는 것을 확인할 수 있었다. 또한, 칼프로텍틴은 호중구 세포외 트랩뿐만 아니라 호산구 세포외 트랩에서도 발현되는 것을 확인하였다. 이 결과를 통해 조직 내 칼프로텍틴 양성 세포가 많을 수록 조직 및 혈액의 호산구가 비례하게 증가하는 임상적인 의미를 발견할 수 있었다. 뿐만 아니라 칼프로텍틴을 발현하는 염증세포는 후각장애, 만성 비부비동염의 중증도를 나타내는 Lund-Mackay CT 점수, JECREC 점수와 상관관계를 보였다. 결국, 이 연구를 통해 만성 비부비동염의 조직에서 칼프로텍틴의 발현이 만성 비부비동염의 중증도를 나타내는 바이오마커가 될 수 있음을 입증하였다. 이 두 연구를 요약하면, 비강 점막 상피세포에서 염증에 의해 증가된 S100A8과 S1000A9는 레티노이드 대사의 조절에 관여하고 편평 상피 화생과 같은 조직 리모델링을 유도하였다. 또한, 이들의 복합체인 칼프로텍틴은 항균 펩타이드로써 호중구와 호산구에서 각각 분비되며 호산구 세포외 트랩에서도 관여하기 때문에 비용종을 동반한 만성 비부비동염의 선천 면역 반응에 중요한 역할을 하였다. 결론적으로 본 연구를 통해 비강 상피 점막세포와 염증세포에서 발현되는 S100A8, S100A9, 칼프로텍틴은 만성 비부비동염 환자의 질병 중증도를 반영하는 중요한 생체 지표가 될 수 있음을 확인하였다.

The mechanisms of differentiation and migration of eosinophils induced by S100A8 and S100A9

Gu, Ayoung Graduate School, Eulji University 2017 국내석사

S100A8 and S100A9 function as essential factors in inflammatory response and they induce cell differentiation, chemotactic activities, adhesion, apoptosis. In this study, I investigated the contribution of S100A8 and S100A9 to differentiation and chemotactic activity in the human eosinophilic cell line EoL-1. It is performed that a proliferation assay, chemotaxis assay and Western blotting. S100A8 and S100A9 increased the percentage of vacuole cell, the number of vacuole per one cell and the protein expression of EPO and MBP, indicating the differentiation of EoL-1 cells. S100A8 and S100A9-induced differentiation was inhibited by rottlerin, an inhibitor of protein kinase C delta (PKCδ). S100A8 and S100A9 induced the migration of EoL-1 cells and the migration was inhibited by TLR4, an inhibitor of TLR4, Ly2294002, an inhibitor of PI-3 kinase, AKTi, an inhibitor of AKT, rottlerin, PD98059, an inhibitor of ERK, SB202190, an inhibitor of p38 MAPK, SP600125, an inhibitor of JNK and BAY-11-7085, an inhibitor of NF-κB. S100A8 and S100A9 induced phosphorylation PKCδ, AKT, ERK, p38 MAPK, JNK and NF-κB in a time-dependent manner. The phosphorylation of ERK, p38 MAPK, JNK and NF-κB were inhibitied by TLR4i, Ly294002, AKTi and rottlerin. These findings contribute to an understanding of S100A8 and S100A9 in eosinophil biology and to the development of novel therapies for the treatment of allergy.

Inflammatory Responses Induced by S100A8 and S100A9 in Human Keratinocytes

Kim, Mun Jeong Graduate School, Eulji University 2017 국내석사

Atopic dermatitis (AD) is an inflammatory skin disorder caused by genetic factors and immunological deficiency. Dryness, infectious agents such as bacteria, germs, and fungi, as well as environmental and psychological factors can contribute to AD, but the most direct causative factor has yet to be identified. In this study, I examined whether IL-6, IL-8, and MCP-1 protein expressions are increased by S100A8 and S100A9 treatment in HaCaT cells in time- and dose-dependent manners. Alterations of cytokine expression were examined by performing ELISA with various signal protein-specific inhibitors. Activation of the MAPK pathway and NF-κB was evaluated by using western blotting and NF- κB activity test. The expressions of IL-6, IL-8, and MCP-1 increased after treatment with S100A8 and S100A9, and the increase was significantly blocked by specific signal inhibitors such as TLR4i, rottlerin, PD98059, SB203580, and BAY-11-7085. Because these inhibitory effects indicate the association of ERK and p38 MAPK with S100A8 and S100A9 mechanisms, the activation of ERK and p38 MAPK was evaluated; both were activated in a time-dependent manner after treatment with S100A8 and S100A9. Phosphorylations of ERK and p38 MAPK were blocked by TLR4i and rottlerin. S100A8 and S100A9 induced translocation of NF- κB in a time-dependent manner, and the activation of NF- κB was inhibited by TLR4i, rottlerin, PD98059, SB203580, and BAY-11-7085. In addition, S100A8 and S100A9 decreased the expression of the skin barrier proteins filaggrin and loricrin. These results contribute to elucidating the pathogenic mechanism for AD and to developing strategies for controlling clinical features of AD. 아토피 피부염은 가장 흔한 만성 염증성 피부질환으로 유전적인 요소와 면역체계 이상으로 인한 염증성 피부질환이다. 그 외에 피부가 건조하거나 정상인에 비해 쉽게 피부 가려움증을 느끼는 증상이 있으며, 환경적 요인으로 세균·바이러스·곰팡이 등에 의한 감염과 정서적 요인이 복합적으로 작용하여 발병하는 것으로 알려져 있지만, 명확한 원인은 아직 밝혀지지 않았다. 본 연구에서는 사람 정상 각질세포주인HaCaT 세포에서 S100A8 및 S100A9 처리에 의해 IL-6, IL-8 및 MCP-1 이 시간 및 용량 의존적으로 증가하는지 여부를 조사하였다. HaCaT 세포주에서 S100A8 및 S100A9 처리한 후에 다양한 신호전달 억제제를 사용하여 ELISA를 이용한 사이토카인의 변화를 조사 하였다. Western blotting과 NF-κB 활성도 실험을 통해서 MAPK 경로와 NF-κB 활성화를 보았다. HaCaT 세포주에서 S100A8 및 S100A9 처리 후 IL-6, IL-8 및 MCP-1은 증가하였고, 증가한 IL-6, IL-8 및 MCP-1은 TLR4i, rottlerin, PD98059, SB203580 및 BAY-11-7085와 같은 특이적 신호전달 억제제에 의해 현저히 줄어들었다. 이러한 억제 효과가 ERK 및 p38 MAPK와 S100A8 및 S100A9의 연관성을 나타내기 때문에 ERK 및 p38 MAPK의 활성화를 알 수 있다. HaCaT 세포주에서 S100A8 및 S100A9두 가지 모두 처리한 후에 시간의존적으로 활성화되었다. 인산화된ERK와 p38 MAPK는 TLR4i와 rottlerin에 의해 억제되었다. HaCaT 세포주에서 S100A8 및 S100A9는 시간에 따라 NF-κB 의 활성화를 유도하였고 NF-κB 의 활성화는 TLR4i, PP2, rottlerin, PD98059 및 SB203580에 의해 억제되었다. 또한, HaCaT 세포에서 S100A8과 S100A9는 피부 장벽 단백질인 filaggrin과 loricrin의 발현을 감소시켰다. 이러한 결과는 AD의 병인기전을 밝히고 AD에 임상적으로 제어할 수 있게 기여한다.

The Pathogenic Roles of S100A8, S100A9 and Their Interaction with SFPQ in Parkinson’s Disease

Ayesha Kashif 을지대학교 대학원 2022 국내박사

Parkinson’s disease (PD) refers to a neurodegenerative disorder wherein dopaminergic neurons (DA) in the brain are insolvent or damaged selectively. The pathogenesis of PD is unknown but it is likely to include an intricate interaction between environmental and genetic variables. The EF-hand proteins, S100A8 and S100A9 act as pro-inflammatory mediators and amyloidogenic proteins. They are implicated in the etiology of several diseases and pivotally influence many cellular functions such as danger signaling and motility. This study investigates the pathogenic roles of S100A8 and S100A9 in the PD model. The recombinant S100A8, S100A9, and SFPQ proteins (splicing factor proline and glutamine-rich) were purified using conventional techniques. The consequences of S100A8 and S100A9 treatment, with or without 1-methyl-4-phenylpyridinium (MPP+), were evaluated by MTT assay, annexin V-FITC apoptosis staining, Western blot, and immunocytochemistry analysis. Interaction of S100A8 and S100A9 with SFPQ was identified by conducting pull-down assay and surface plasmon resonance (SPR). The prevalence of S100A8, S100A9, and SFPQ in normal and PD brain tissues was investigated by using Immunofluorescence and Western blot. The results uncovered the apoptotic roles of S100A8 and S100A9 in MPP+-induced SH- SY5Y cells. S100A8 and S100A9 impaired cell apoptosis by regulating JNK and p38-MAPK, increasing the expression of apoptosis-associated proteins, and caspase 9/3 in MPP+-induced SH-SY5Y cells. Besides, the combo also enhanced the expression of the proteins associated with PD, namely a- synuclein (a-syn), PINK1, and Parkin. Moreover, this study identified SFPQ as a functional interacting partner of S100A8 and S100A9 in PD pathophysiology. S100A8 and S100A9 were shown to directly interact with SFPQ with an apparent dissociation constant of 25.0 ± 0.1 nM and 20 ± 0.2 nM, respectively. Interestingly, S100A8 and S100A9 treatment promoted the cytoplasmic mislocalization of SFPQ, most likely impairing the nuclear regulatory functions of SPFQ. The siRNA experiments confirmed that SFPQ is not only interacting with S100A8 and S100A9 but also intricate in apoptosis of MPP+-induced SH-SY5Y cells, employing the same pathway. In line with the findings from SH-SY5Y cells, the primary neuron cells from rat brain validated that S100A8 aggravates apoptosis. S100A8 + MPP+ treated primary neuron cells showed marked dislocation of SFPQ to the cytoplasm and colocalization with S100A8. In the postmortem brain of PD patients, S100A8 and S100A9 were significantly upregulated compared with control samples, emphasizing their pathological consequences. In a nutshell, the findings from this study highlighted the apoptotic roles of S100A8 and S100A9 in association with SFPQ, as a critical pathway contributing to the progression of PD.

Pathogenic mechanism induced by S100A8 and S100A9, and neuroprotective effect of a novel herbmedicine, Hepad in Parkinson's disease : 파킨슨 질환에서 S100A8과 S100A9에 의한 병인기전과 Hepad의 신경보호효과

Baek, Seung Yeop Graduate School, Eulji University 2016 국내석사

S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are secreted and exert extracellular paracrine and autocrine functions. S100A8 and S100A9 affect cell death in 1-methyl-4-phenylpyridinium (MPP+) - treated dopaminergic neuronal cells. S100A8 and S100A9 induce cell death via activation of MAPK such as p38 MAPK, JNK and expression of pro-apoptotic proteins, and activation of caspase 9 and caspsase 3 in SH-SY5Y cells. S100A8 and S100A9 also trigger cell death through the Receptor for Advanced Gycation End products (RAGE) and Toll-like receptor 4 (TLR4). Injection of the 6-hydroxydopamine (6-OHDA) plus S100A8 or S100A9 causes more significant loss of tyrosine hydroxylase (TH) immunopositive cell bodies from the substantia and microgliosis than injection group of 6-OHDA in a rat model of Parkinson’s disease. These results indicate that S100A8 and S100A9 may induce neurodegenerative effect. Neuroprotective effects of a novel herb formula, Hepad was investigated on Parkinson’s disease. Dose-dependent treatment with MPP+ decreased the viability of SH-SY5Y cells, and Hepad inhibited the toxic effect of MPP+ and MPP+ plus S100A8 or S100A9. Hepad blocked the production of reactive oxygen species (ROS) induced by MPP+ in SH-SY5Y cells, and suppressed the activation of caspase 9 and caspase 3 due to MPP+. A rat model of Parkinson’s disease was generated by 6-OHDA injection into the left medial forebrain bundle (MFB) of SD rats. In amphetamine sulfate-induced rotational behavioral tests, Hepad administration attenuated circling behavior relative to the 6-OHDA-treated disease group. In addition, Hepad treatment significantly increased the TH- positive cells in the substantia nigra pars compacta (SNpc) that had decreased in response to 6-OHDA treatment. OX-6 expression, which indicates the presence of microglial cells, significantly decreased after treatment of Hepad in contrast to the 6-OHDA-treated disease group. These results indicate that Hepad may be a useful neuroprotective material for the treatment of neurodegenerative disorders such as Parkinson’s disease.

The role of S100A8 and S100A9 in interaction between bronchial epithelial cells and neutrophils

Kim, Da Hye Graduate School, Eulji University 2017 국내석사

Allergic asthma is characterized by bronchoconstriction, allergen-specific IgE and bronchial inflammation. S100A8 and S100A9 are important proteins in the pathogenesis of allergic asthma. In this study, I examined that the pathogenic mechanism of asthma is associated with interaction of cytokine release in bronchial epithelial cells with constitutive apoptosis of neutrophils. S100A8 and S100A9 increased the secretion of MCP-1, IL-6 and IL-8 in a time and dose dependent manners. TLR4 was constitutively expressed in BEAS-2B cells. This secretion was suppressed by TLR4i, an inhibitor of TLR4, LY294002, an inhibitor of PI3K, AKTi, an inhibitor of Akt, PD98059, an inhibitor of ERK, SB202190, an inhibitor of p38 MAPK, an inhibitor of JNK, SP600125 and BAY-11-7085, NF-κB inhibitor. S100A8 and S100A9 induced activations of Akt, ERK, p38 MAPK and JNK activations in a time-dependent manner. Supernatants collected from bronchial epithelial cells after S100A8 and S100A9 treatment suppressed the apoptosis of normal and asthmatic neutrophils. The supernatants inhibited the cleaved caspase 3 and caspase 9. The co-culture of human neutrophils with BEAS-2B cells inhibited more neutrophil apoptosis after S100A8 and S100A9 treatment than co-culture of human neutrophils with BEAS-2B cells without S100A8 and S100A9. These findings improve understanding of the role of S100A8 and S100A9 in interaction between bronchial epithelial cells and neutrophils in allergic asthma and will enable elucidation of asthma pathogenesis.

S100A8/A9, a DAMP molecule activated by IL-17 and house dust mite is upregulated in atopic dermatitis

김산 Graduate School, Yonsei University 2011 국내석사