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Background and objectives Immune checkpoint inhibitors (ICIs) bring cancer patients tumor control and survival benefits, yet they also trigger immune-related adverse effects (irAEs), notably checkpoint inhibitor-related pneumonitis (CIP), affecting about 5% of patients among whom 1–2% experiencing severe grade 3 or higher pneumonitis. Current research points to potential links with T cell subset dysfunction and autoantibody increase, but the specific mechanisms underlying different grades of CIP are understudied. Methods Herein, we employed single-cell RNA sequencing (scRNA-seq) on bronchoalveolar lavage fluid (BALF) from CIP patients across varying severity levels, aiming to elucidate underlying immune environment and mechanisms of CIP progression at cellular and molecular levels. Findings Totally, 121,409 high qualified cells from BALF of 11 patients were annotated and categorized into five major cell types. Severe CIP (CIP-S) cases have a significant increase in the percentage of unreported epithelial cells in their bronchoalveolar lavage fluid compared with mild CIP (CIP-M) cases. These cells were defined as aberrant basaloid cells. They upregulated SOX9, increased the expression of CXCL3/5, recruited neutrophils, and activated the immune system. Additionally, macrophages in the CIP-S group had stronger antigen-presenting abilities and resulted in more CD8 + effective T cells infiltrated. Conclusions Utilizing single-cell sequencing of BALF, we discovered an enriched population of aberrant basaloid cells in CIP-S patients, which had not been previously reported. Aberrant basaloid cells may upregulate SOX9 via CXCL3/5-CXCR2 to recruit and activate neutrophils, and further activate the immune system, resulting in CIP-S. This finding could identify new targets for stratified treatment of CIP patients, holding promise of a novel approach for clinical guidance.

Pulmonary fibrosis (PF) is a severe lung disease characterized by the destruction of lung architecture resulting from chronic epithelial injury. The PF microenvironment induces PF-specific epithelial cells, such as aberrant basaloid cells (ABCs). However, limited experimental models capable of inducing and activating PF-specific epithelial cells hinder the understanding of their roles. To address the lack of experimental models, in this study, we developed an ex vivo murine lung-organoid model designed to induce and activate ABCs. The organoids were subjected to bleomycin (BLM) stimulation. Dose-dependent reductions in number and size, structural disorganization, and transcriptomic changes were assessed following stimulation. Single-cell RNA-sequencing (scRNA-seq) analysis was performed to identify ABC subsets. Cell-cell interaction analysis was also conducted. Following BLM stimulation, the organoids displayed dose-dependent reductions in number and size, along with structural disorganization and transcriptomic changes that were similar to those observed in the in vivo murine fibrosis model. scRNA-seq analysis identified two ABC subsets: Krt5 Tp63 Krt17 ABCs_1, found in patients with idiopathic pulmonary fibrosis (IPF), and Krt5 Tp63 Krt17 ABCs_2, which have been observed in cultured tissues from patients with IPF but not in traditional murine models. BLM stimulation led to the induction of transforming growth factor beta (TGF-β2) expression in ABCs. Cell-cell interaction analysis suggested that BLM-damaged type 2 alveolar epithelial cells (AT2s) enhanced their direct and indirect interactions with ABCs_2 via ephrin-A signaling. In line with this observation, stimulation experiments of BLM-damaged organoids revealed that Ephrin A4 induced ABC cell differentiation-related gene expression changes, whereas Ephrin A3 enhanced epithelial proliferation-related gene expression changes and suppressed fibroblast activation-related gene expression changes. The developed organoid model serves as a novel platform for studying the roles and responses of PF-specific ABCs. This model may contribute to advancing the understanding of PF pathogenesis and facilitate the development of ABC-targeted therapies.

In idiopathic pulmonary fibrosis (IPF), keratin (KRT)17+/KRT5+ basal and KRT17+/KRT5− aberrant basaloid cells are atypically present within the alveolar space. We previously described the fibrosis-enriched outgrowth of alveolar basal cells from peripheral fibrotic lung tissue. Using single cell RNA sequencing (scRNA-seq), we here characterize the transcriptome of these cultured alveolar basal cells under different culture conditions. Methods: Fibrotic peripheral lung tissue pieces were placed in DMEM growth medium. Outgrown cells were analysed by scRNA-seq, TaqMan-PCR or immunofluorescence (IF) either directly or after medium change to an epithelial cell specific medium (Cnt-PR-A). Results: A fraction of alveolar basal cells cultured in DMEM growth medium showed close transcriptomic similarities to IPF basal cells. However, although they expressed KRT5, the transcriptome of the majority of cells matched best to the transcriptome of recently described KRT17+/KRT5− aberrant basaloid cells, co-expressing the canonical basal cell marker KRT17 and mesenchymal cell marker (VIM, FN1). A smaller fraction of cells matched best to secretory epithelial cells. Two differentiation gradients from basal to aberrant basaloid-like cells and basal to secretory epithelial-like cells were apparent. Interestingly, these differentiation paths seemed reversed when the cell culture medium was changed to Cnt-PR-A. Conclusions: Our results suggest that cultured alveolar basal cells have the capacity to differentiate towards secretory epithelial-like cells and to aberrant basaloid-like cells. However, due to the persistent expression of KRT5, a complete differentiation towards aberrant basaloid cells did not seem to be achieved in our culture conditions. Importantly, differentiation seemed reversible by changing the cells microenvironment. Determining specific factors influencing these differentiation paths may help to define novel drug targets for IPF therapy.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo , to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo . Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro . Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro . The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

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.