천식에 대한 기존의 마우스 모델은 ovalbumin로 기도 염증이 유발된 것 입니다. 마우스는 여러번의 ovalbumin 복강 주사 투여 후, 에어로졸화 된 ovalbumin을 흡입하게 됩니다. 이 ovalbumin으로 유도된 마우스 모델에서는, IgE와 호산구의 농도 뿐만이 아니라, 기도의 점액과 염증성 림프구의 투과도 증가하게 됩니다. 바이오사이토젠은 강력하고 검증된 ovalbumin으로 유도된 천식 모델을 제공합니다. 바이오사이토젠 천식 모델의 가장 중요한 특징은, 마우스의 IL-4와 IL-4 수용체가 인간의 것으로 대체된 B-hIL4/IL4RA 인간화 마우스에서 ovalbumin을 사용하여 유도된 천식 모델이라는 것 입니다. 이 B-hIL4/IL4RA는 마우스에서 인간 IL-4 경로를 타겟으로 하는 천식 치료제들의 직접적인 평가를 가능하게 합니다.
B-IL4/IL4R mice 와 표현형 분석
천식 모델과 유효성 평가
Acute murine asthma model scheme
B-hIL4/hIL4RA mice between 5 and 8 weeks of age were acclimatized for at least 1 week before initiation of sensitization and challenge with ovalbumin (OVA) in the creation of the acute murine model of asthma.
OVA로 유도된B-hIL4/IL4RA 인간화 천식 마우스 모델에서의 BALF(Bronchoalveolar lavage fluid) 호산구 증가
Upon ovalbumin sensitization and challenge, B-hIL4/hIL4RA mice showed a considerable increase of infiltration of leukocytes including eosinophils and neutrophils. This represented a clear allergic phenotype at day 26 in BALF when compared with mice not sensitized or challenged.
Figure 1. BALF cell count
Mice (n=5/group) were sensitized and challenged with OVA. Twenty-four hours following the last challenge, BALF cells were collected and eosnophils were counted with flow cytometry.
천식 마우스 모델에서의 BALF(Bronchoalveolar lavage fluid) 호산구 증가
Ovalbumin sensitization and challenge induced a clear allergic phenotype at day 26 characterized by considerable increase of eosinophilia in BALF when compared with mice that were not sensitized or challenged.
천식 마우스 모델에서의 IgE 증가
Level of OVA-specific IgE antibody was approximately five-fold higher in OVA-challenged B-hIL4/IL4RA mice compared with sham-challenged B-hIL4/IL4RA mice.
천식 마우스 모델에서의 H&E 염색
Histology of lung of B-hIL4/hIL4RA mice sensitized and challenged with OVA or PBS. Examination of H&E stained lung tissue of B-hIL4/hIL4RA mice revealed patchy peribronchial inflammatory infiltrates, composed primarily of eosinophils and lymphocytes. In contrast, sham-challenged mice exposed to PBS showed normal lung histology.
B-hIL4/hIL4RA 마우스 모델을 이용한 천식 치료제의 효능 평가 예시
B-hIL4/hIL4RA 마우스 모델을 이용한 항 인간 IL-4 항체 (dupilumab)의 효능 평가
All drugs were administered i.p.
The experimental design for OVA challenge asthma model in B-hIL4/hIL4RA mice and treatment with or without dupilumab.G1: Mice were challenged with PBS and treated without any drug. G2: Mice were challenged with OVA and treated without any drug. G3: Mice were challenged with OVA and treated with dupilumab before challenged as a prophylactic treatment. G4: Mice were challenged with OVA and treated with dupilumab after challenged as a therapeutic treatment.
B-hIL4/hIL4RA 마우스에서 OVA로서 유도된 급성 천식 모델에서, Dupilumab은 BALF에서의 염증성 세포 투과를 줄였다.
BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The proportions of eosinophils subpopulations were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.
dupilumab투여로 BALF에서의 염증 세포 제거를 보여주는 FACS 분석 결과
BALF immune cells were isolated from homozygous B-hIL4/hIL4RA mice (n=7). The number of CD45+ cells and eosinophils were determined by flow cytometry in B-hIL4/IL4RA mice treated with or without dupilumab. Prophylactic treatment before OVA challenge (G3) with dupilumab almost completely abolished the infiltration of eosinophils as opposed to untreated group (G2). Therapeutic treatment after OVA challenge (G4) with dupilumab could also obviously reduce the infiltration of eosinophils.
Dupilumab는 천식 마우스 모델에서 OVA에 대항하여 IgE의 생성을 제거했다.
Serum were collected at the study endpoint. OVA-specific IgE antibody levels were analyzed. The results showed that IgE levels were dramatically decreased in the dupilumab treatment group compared with the untreated group.
B-hIL4/IL4RA에 dupilumab을 투여하였을 때의 염증 감소를 보여주는 H&E 염색 결과
Airways from B-hIL4/hIL4Ra mice exposed to PBS aerosols (G1)  did not show any inflammation. OVA exposure resulted in a significant increase in peribronchial and perivascular inflammation in B-hIL4/hIL4RA mice, as well as an increase in the level of mucus secretion (G2). A reduction in inflammatory infiltrates and mucus secretion was observed in mice treated with dupilumab (G3, G4).