chimeric receptor, efferocytosis 논문 (Morioka et al., Cell, 202212)

Highlights (Impact & Novelty)

efferocytosis 향상시키기 위한 efferocytosis(CHEF)용 chimeric receptors 설계.

CHEF 통한 efferocytosis 촉진은 in vivo에서 multuple inflammatory insults 약화.

protein folding 과 misfolded protein degradation은 efferocytosis의 rate-limiting step임.

CHEF expression은 진행중인 질병의 결과를 개선할 수 있음.

introduction

Seminars in Immunopathology   volume 38 ,  pages 409–423 ( 2016 )

efferocytosis : 식세포에 의해 apoptotic cell이 제거되는 과정 → 조직의 항상성 유지에 필수적.

efferocytosis이 망가져있으면 죽상동맥경화증, 궤양성 대장염 등의 질병이 증가하는 것에 기여요인으로 보고있음.

apoptotic cell이 축적될 경우, 이차적으로 괴사가 일어날 수 있고 차례로 염증 및 병리학적 측면을 악화시킬수 있음.

apoptotic cell을 인식하고 제거할 때, apoptotic cell의 phosphatidylserine(PtdSer)의 노출이 중요한 역할을 함.

apoptosis induction → PtdSer 외부로 전위 → phagocytes receptor가 직.간접 인식 통해 PtdSer과 결합.

Lemke, G. (2019).  How macrophages deal with death. Nature Reviews Immunology, 19(9), 539–549.

αvβ3 and αvβ5 , tyrosine kinase receptors 은 MFG-E8, Gas6/Protein 통해 indirectly apoptotic cell 인식.

TIM4, BAI1은 direct RtdSer binding receptors.

BAI1은 cytoplasmic tail domain을 통해 ELMO와 상호작용

Results

fused a C-terminal region of ELMO1 (amino acids 532–727) to the efferocytic receptor BAI1

BELMO

: deleting the natural ELMO1 binding site → directly fused this truncated BAI1 to ELMO1

 

BELMO expression → efferocytosis 5배 증가 , 더 많은 apoptotic cell 삼킴.

apoptotic cell 흡수는 강하게 촉진, live cell, necroptotic, perroptotic cell에서는 흡수 촉진하지 않음.

BELMO : StdSer dependence

Rac1 활성 억제시, BELMO-driven enhaced phagoxytosis 없어짐.

cytochalasinD에 의한 actin polumerization 방지 → BELMO-dependent efferocytosis 억제.

 

gut injury

generated BELMO transgenic mice : cell-type-specific Cre would induce expression of BELMO and the bicistronic eGFP marker

BELMO+복막 macrophages : more dfferocytosis, greater corpse-derived fluorescence on a per-cell basis than control macrophages ex vivo

 

To study BELMO in the context of gut injury

the Villin-cre BELMOTg mice: colonic crypt architecture와 colon length 유지.generated BELMO transgenic mice : cell-type-specific Cre would induce expression of BELMO and the bicistronic eGFP marker

BELMO+복막 macrophages : more dfferocytosis, greater corpse-derived fluorescence on a per-cell basis than control macrophages ex vivo

To study BELMO in the context of gut injury

the Villin-cre BELMOTg mice: colonic crypt architecture와 colon length 유지.

 

BAI1 형질변환보다 BELMO발현의 효과가 colon pathology줄이는데 더욱 효과적.

Villin-cre BELMOTg mice : 전염증성 사이토카인 발현 적고 IL-10 발현이 더 많음.

IECs에서 BELMO발현 → DSS-indecces injury model에서 보호효과 나타남.

 

hepatotoxicity model

control Alb-cre only mice에서만 TUNEL+ cell과 caspase-3 활성 증가.

DNase II-mediated DNA breaks (which occur in the phagolysosomes during the digestion of apoptotic cells) → Alb-cre BELMOTg mice에서 더 많은 efferocytic cell 관찰.

Plasma alanine aminotransferase (ALT)도 Alb-cre BELMOTg mice에서 현저히 낮게 관찰.

 

cisplatin-induced nephrotoxicity

cisplatin, a widely used cancer chemotherapeutic agent, induces nephrotoxicity with apoptosis of kidney tubular epithelial cells (TECs)

PEPCK-cre BELMOTg mice

TUNEL+ cells and cleaved caspase-3+ cells 적게 검출,

more efferocytic TECs

Plasma creatinine levels and the overall survival rate were significantly improved

three different inflammatory : BELMO expressions →

IECs, hepatocytes, and kidney TEC로 인한 efferocytosis 강화 → tissue injury and inflammation 완화할 수 있음.

BELMO-mediated efferocytosis and associated benefits occur locally

 

transcriptomic profiles of BELMO-expressing and control fibroblasts

gene ontology analysis of BELMO-dependent genes ; protein folding/endoplasmic reticulum (ER) function

thapsigargin (TG), an inhibitor of ER calcium transport, led to a strong reduction in efferocytosis in control phagocytes.

BELMO+ phagocytes were more resistant

BiP expression ↑

 

protein folding, degradation of unfolded protein, ER funtion 관련된 유전자 siRNA knock down

→ BELMO-mediated enhanced efferocytosis 약화.

 

acute kidney injury (AKI), linked to defective ER proteostasis

PEPCK-cre BELMOTg 마우스

Creatinine 수치 감소.

생존력 ↑↑, health/activity 개선

신장 대부분 보호

Bip 사향 조절.

 

ongoing/chronic disease model

BELMO size 너무 커서 TIM4에 ELMO1 부착한 TELMO 생성.

ELMO1 통한 efferocytosis 증가시키는 것 확인.

AAV9-TELMO 생성 → 왼쪽 신장 정맥에 주입.

 

TELMO-AAV kidneys

creatinine, BUN 상당히 낮춤.

콜라겐 침착, 섬유화 적음.

Discussion

BAI1 또는 TIM4의 extracellular domain 통해 RtdSer인식, ELMO1통한 intracellular signaling → strongly enhance efferocytosis

CHEF mediates the typical anti-inflammatory features of efferocytosis and promotes anti-inflammatory signaling.

CHEF expression → DSS-induced colitis in the gut, a hepatocyte-injury model in the liver, and cisplatin-induced nephrotoxicity 완화할 수 있었음.

BELMO increases the expression of proteostasis-promoting genes such as BiP, DNAJC3, ATP2A3, and VIMP

BELMO+ phagocytes may be more potent in dampening inflammation in at least two ways

: faster removal of the apoptotic cells & improved proteostasis in the BELMO+ phagocytes

CHEF → boosting efferocytosis → attenuates multiple inflammatory insults

 

 

https://doi.org/10.1016/j.cell.2022.11.029