Deficiency of surfactant protein-C (SPC) increases susceptibility to lung infections and injury, and suppressed expression of SPC has been associated with the severity of acute respiratory distress syndrome (ARDS). Alveolar type 2 epithelial cells (AT2) are critical for maintenance and repair of the lung. However, the role of the SPC in the regulation of AT2 cell lineage and the underlying mechanisms are not completely understood.
This study aimed to investigate the mechanisms by which SPC regulates AT2 lineages. Sftpc-/- mice were used to model the SPC deficiency in ARDS patients. We utilized three-dimensional (3D) organoids to compare AT2 lineage characteristics between wild type (WT) and Sftpc-/- mice by analyzing AT2 proliferation, alveolar type 1 cells (AT1) differentiation and CD74 expression, using colony-formation assay, immunofluorescence, flow cytometry, and immunoblots.
The results showed that Sftpc-/- mice demonstrated a reduced AT2 cell population. Influenza A virus subtype H1N1 (H1N1) infected Sftpc-/- mice demonstrated reduced AT2 proliferation and AT1 differentiation. Western blot indicated elevated levels of CD74 protein in AT2 cells of Sftpc-/- mice. Colony-forming efficiency was significantly attenuated in AT2 cells isolated from Sftpc-/- mice compared to the WT controls. Podoplanin (PDPN, a marker of AT1 cells) expression and transient cell count significantly increased in Sftpc-/- organoids. Moreover, siRNA-mediated gene silencing of CD74 in AT2 cells significantly increased AT2 proliferation and AT1 differentiation in Sftpc-/- organoids.
This study suggests that SPC regulates AT2 lineage in vitro and in vivo. The SPC might influence AT2 lineage during the lung epithelium repair by activating signaling mechanism involving CD74 receptor.

Alveolar type 2 epithelial (AT2) cells synthesize surfactant protein C (SPC) and repair an injured alveolar epithelium. A mutated surfactant protein C gene (SftpcL184Q, Gene ID: 6440) in newborns has been associated with respiratory distress syndrome and pulmonary fibrosis. However, the underlying mechanisms causing Sftpc gene mutations to regulate AT2 lineage remain unclear. We utilized three-dimensional (3D) feeder-free AT2 organoids in vitro to simulate the alveolar epithelium and compared AT2 lineage characteristics between WT (C57BL/6) and SftpcL184Q mutant mice using colony formation assays, immunofluorescence, flow cytometry, qRT-PCR, and Western blot assays. The AT2 numbers were reduced significantly in SftpcL184Q mice. Organoid numbers and colony-forming efficiency were significantly attenuated in the 3D cultures of primary SftpcL184Q AT2 cells compared to those of WT mice. Podoplanin (PDPN, Alveolar type 1 cell (AT1) marker) expression and transient cell count was significantly increased in SftpcL184Q organoids compared to in the WT mice. The expression levels of CD74, heat shock protein 90 (HSP90), and ribosomal protein S3A1 (RPS3A1) were not significantly different between WT and SftpcL184Q AT2 cells. This study demonstrated that humanized SftpcL184Q mutation regulates AT2 lineage intrinsically. This regulation is independent of CD74, HSP90, and RPS3A1 pathways.

Abstract The severity of allergic asthma is driven by the balance between allergen-specific T regulatory (Treg) and T helper (Th)2 cells. However, it is unclear whether specific subsets of conventional dendritic cells (cDCs) promote the differentiation of these two T cell lineaeges. We have identified a subset of lung resident type 2 cDCs (cDC2s) that display high levels of CD301b and have potent Treg-inducing activity ex vivo. Single cell RNA sequencing and adoptive transfer experiments show that during allergic sensitization, many CD301b+ cDC2s transition in a stepwise manner to CD200+ cDC2s that selectively promote Th2 differentiation. GM-CSF augments the development and maintenance of CD301b+ cDC2s in vivo, and also selectively expands Treg-inducing CD301b+ cDC2s derived from bone marrow. Upon their adoptive transfer to recipient mice, lung-derived CD301b+ cDC2s confer immunological tolerance to inhaled allergens. Thus, GM-CSF maintains lung homeostasis by increasing numbers of Treg-inducing CD301b+ cDC2s.

Organoid models have become an integral part of the research methodology in the lung field. These systems allow for the study of progenitor and stem cell self-renewal, self-organization, and differentiation. Distinct models of lung organoids mimicking various anatomical regions of mature lungs have emerged in parallel to the increased gain of knowledge regarding epithelial stem and progenitor cell populations and the corresponding mesenchymal cells that populate the in vivo niche. In the distal lung, type 2 alveolar epithelial cells (AEC2s) represent a stem cell population that is engaged in regenerative mechanisms in response to various insults. These cells self-renew and give rise to AEC1s that carry out gas exchange. Multiple experimental protocols allowing the generation of alveolar organoids, or alveolospheres, from murine lungs have been described. Among the drawbacks have been the requirement of transgenic mice allowing the isolation of AEC2s with high viability and purity, and the occasional emergence of bronchiolar and bronchioalveolar organoids. Here, we provide a refined gating strategy and an optimized protocol for the generation of alveolospheres from wild-type mice. Our approach not only overcomes the need for transgenic mice to generate such organoids, but also yields a pure culture of alveolospheres that is devoid of bronchiolar and bronchioalveolar organoids. Our protocol contributes to the standardization of this important research tool.

Pulmonary fibrosis, especially idiopathic pulmonary fibrosis (IPF), portends significant morbidity and mortality, and current therapeutic options are suboptimal. We have previously shown that type I collagen signaling through discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase expressed by fibroblasts, is critical for the regulation of fibroblast apoptosis and progressive fibrosis. However, the downstream signaling pathways for DDR2 remain poorly defined and could also be attractive potential targets for therapy. A recent phosphoproteomic approach indicated that PIK3C2α, a poorly studied member of the PI3 kinase family, could be a downstream mediator of DDR2 signaling. We hypothesized that collagen I/DDR2 signaling through PIK3C2α regulates fibroblast activity during progressive fibrosis. To test this hypothesis, we found that primary murine fibroblasts and IPF-derived fibroblasts stimulated with endogenous or exogenous type I collagen led to the formation of a DDR2/PIK3C2α complex, resulting in phosphorylation of PIK3C2α. Fibroblasts treated with an inhibitor of PIK3C2α or with deletion of PIK3C2α had fewer markers of activation after stimulation with TGFβ and more apoptosis after stimulation with a Fas-activating antibody. Finally, mice with fibroblast-specific deletion of PIK3C2α had less fibrosis after bleomycin treatment than did littermate control mice with intact expression of PIK3Cα. Collectively, these data support the notion that collagen/DDR2/PIK3C2α signaling is critical for fibroblast function during progressive fibrosis, making this pathway a potential target for antifibrotic therapy. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.