SAECs are not commonly used in pulmonary research, and this is reflected in a lack of literature on both SAEC primary research and methodological reports. Primary SAECs are an important resource as they enable the study of the small airways, which are implicated in a variety of pulmonary diseases, including chronic obstructive pulmonary disease (COPD). The detailed protocol described here bridges the knowledge gap on how to successfully differentiate primary human SAECs at the ALI.

Evidence is presented that the multifunctional enzyme tissue transglutaminase (TG2) is a driver of fibrosis pathology in the lung. Here, we investigate a candidate therapeutic antibody that blocks the protein cross-linking function of TG2, in a novel rabbit model of silica-induced pulmonary fibrosis. Our results show that treatment with zampilimab can reduce accumulation of fibrotic tissue and hydroxyproline content by 50% compared with control, suggesting that zampilimab may represent a novel therapy for pulmonary fibrosis.

Thoracic radiation is an effective mainstay treatment for lung cancer, however patients risk developing an adverse side effect known as radiation-induced lung injury (RILI). RILI is dose-limiting, can be permanent, and may threaten normal lung function, but the underlying mechanism is not well characterized. RILI can include both inflammatory (radiation pneumonitis) and fibrotic (radiation-induced lung fibrosis) pathologies. Myofibroblasts are the main effector cells of fibrosis, and we and others have shown that ionizing radiation induces differentiation of normal lung fibroblasts to the myofibroblast phenotype (FMT, fibroblast to myofibroblast transdifferentiation). We previously reported that radiation induces production of excess lactate, which promotes an acidic microenvironment that activates the major profibrotic cytokine, transforming growth factor - β (TGFβ). TGFβ in turn upregulates production of lactate, creating a pro-fibrotic feed-forward loop. Here, we performed targeted metabolomics and metabolic tracer studies to determine how radiation alters cellular metabolism to promote fibrosis in cultured human lung fibroblasts and a mouse model of radiation induced lung fibrosis. Radiation upregulated both glycolysis and the pentose phosphate pathway (PPP), and we found that the PPP was a significant source of lactate production. Inhibition of glycolysis by targeting pyruvate kinase M2 prevented radiation-induced FMT and lactate production but did not affect fibronectin expression. However, when the gluconic shunt or the non-oxidative pentose phosphate pathway is blocked by targeting glucose-6-phosphate dehydrogenase, FMT, lactate production, and fibronectin are markedly reduced. Our data reveals that the PPP is an important compensatory mechanism and driver of lactate accumulation observed in RILI.

Hyperhomocysteinemia (hHcy) has been demonstrated to induce NLRP3 inflammasome activation and inflammatory exosome release in podocytes of mice, which can be enhanced by podocyte‐specific Smpd1 gene overexpression or blocked by global Smpd1 gene deletion. However, it remains unknown whether hHcy‐induced podocyte injury and glomerular inflammation are attributed to these exosomes secreted from podocytes during hHcy. In the present study, we found that exosomes extracted from urine of mice with hHcy induced by folate free (FF) diet induced podocyte injury when they were infused into the renal cortical interstitial space in both WT/WT and Smpd1‐/‐ mice, as shown by reduction of podocin and elevation of desmin in glomeruli. These exosomes from mice with hHcy also increased T‐cells in glomeruli, which was not blocked by global Smpd1 gene deletion. Functionally, proteinuria was detected in both WT/WT and Smpd1‐/‐ mice receiving these exosomes from mice with hHcy. However, infusion of urinary exosomes from mice on the normal diet (ND) had no effects on these podocyte injury and glomerular inflammatory changes. We also performed in vitro experiments with cultured podocytes. We treated murine podocytes with urinary exosomes from mice on the ND and FF diet. It was found that exosomes from mice on the FF diet produced no significant effects on podocytes compared with control exosomes if they were cultured without immune cells. If murine podocytes were co‐cultured with T‐cells, macrophages, or both, the exosomes from mice with hHcy caused significant podocyte damage as shown by reduction of podocin and elevation of desmin, but exosomes from mice on the ND failed to influence podocytes even in the presence of immune cells. These results suggest that hHcy increases urinary exosomes and these exosomes importantly contribute to podocyte injury and glomerular inflammation during hHcy.