肺纤维化大鼠模型中异常基底样细胞的研究。

Department of Neuroscience, Psychology, Drug Area and Child Health, University of Florence, 50139 Florence, Italy.

Target Innovation Department, Global Research and Preclinical Development, Chiesi Farmaceutici S.p.A., 43122 Parma, Italy.

Front Biosci (Landmark Ed). 2024 Aug 22;29(8):305. doi: 10.31083/j.fbl2908305.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs.

METHODS

Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers.

RESULTS

Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial-mesenchymal crosstalk.

CONCLUSION

Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF.

背景

特发性肺纤维化（IPF）是一种慢性进行性肺间质疾病（ILD），其病因和发病机制尚不清楚。到目前为止，没有一种肺纤维化的动物模型能够成功重现所有的特发性肺纤维化特征，因此，使用不同的啮齿动物模型对于评估和开发新的有效药物治疗方法至关重要。最近，肺泡上皮功能障碍在特发性肺纤维化的发病机制中得到了强调。值得注意的是，一种异常的基底样细胞类型的作用，主要在人类中发现，并在小鼠中得到证实，似乎在疾病/模型的建立和进展中起着至关重要的作用。我们的工作旨在首次在博来霉素（BLM）双重给药诱导的大鼠肺纤维化模型中对该细胞群体进行特征描述，证明了该模型的转化价值及其在测试新的有效药物方面的潜在用途。

方法

大鼠在第 0 天和第 4 天接受气管内 BLM 给药。动物在 BLM 后 21 天和 28 天被处死。纤维化的评估通过组织学（Ashcroft 评分和自动图像分析）和免疫酶分析进行。免疫荧光用于异常基底样细胞标志物的特征描述。

结果

肺组织学显示，严重程度的 Ashcroft 评分和纤维化面积增加，导致胶原沉积在两个分析时间点均增加。免疫荧光染色显示，来自对照组（生理盐水，SAL）和 BLM 处理动物的支气管上皮细胞中存在 KRT8+细胞。有趣的是，KRT8+细胞仅存在于 BLM 处理动物的纤维化实质中（通过α-平滑肌肌动蛋白（α-SMA）染色对肌成纤维细胞进行确认）。此外，KRT8+细胞共表达 Prosurfactant protein C（Pro-SPC）和波形蛋白等标志物，提示其为肺泡 II 型（AT2）细胞的中间状态，并参与异常上皮-间充质细胞的相互作用。

结论

先前的临床前研究表明，在肺纤维化的小鼠模型中存在 KRT8+异常基底样细胞。本研究在博来霉素（BLM）双重给药诱导的不同啮齿动物（大鼠）肺纤维化模型中研究了相同的细胞群体。我们的结果进一步证实，在大鼠中，气管内给予 BLM 诱导出现了一群细胞，与先前在小鼠中描述的 KRT8+肺泡分化中间（ADI）细胞相吻合。这项工作加强了先前的证据，并进一步支持使用 BLM 诱导的大鼠模型来评估特发性肺纤维化新的临床候选药物。