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Part 1: Amphiregulin (Areg), a growth factor produced by regulatory T (Treg) cells to facilitate tissue repair/regeneration, contains a heparan sulfate (HS) binding domain. How HS, a highly sulfated glycan subtype that alters growth factor signaling, influences Areg repair/regeneration functions is unclear. Here we report that inhibition of HS in various cell lines and primary lung mesenchymal cells (LMC) qualitatively alters downstream signaling and highlights the existence of HS-dependent vs. independent Areg transcriptional signatures. Utilizing a panel of cell lines with targeted deletions in HS synthesis–related genes, we found that the presence of the glypican family of HS proteoglycans is critical for Areg signaling and confirmed this dependency in primary LMC by siRNA-mediated knockdown. Furthermore, in the context of influenza A virus (IAV) infection in vivo, we found that an Areg-responsive subset of reparative LMC upregulate glypican-4 and HS. Conditional deletion of HS primarily within this LMC subset resulted in reduced blood oxygen saturation following infection with IAV, with no changes in viral load. Finally, we found that co-culture of HS-knockout LMC with IAV-induced Treg cells results in reduced LMC responses. Collectively, this study reveals the essentiality of HS on a specific lung mesenchymal population as a mediator of Treg cell–derived Areg reparative signaling during IAV infection. Part 2: Regulatory T (Treg) cells are known to play critical roles in tissue repair via provision of growth factors such as amphiregulin (Areg). Areg-producing Treg cells have previously been difficult to study because of an inability to isolate live Areg-producing cells. In this report, we created a novel reporter mouse to detect Areg expression in live cells (AregThy1.1). We employed influenza A and bleomycin models of lung damage to sort Areg-producing and -non-producing Treg cells for transcriptomic analyses. Single cell RNA-seq revealed distinct subpopulations of Treg cells and allowed transcriptomic comparisons of damage-induced populations. Single cell TCR sequencing showed that Treg cell clonal expansion is biased towards Areg-producing Treg cells, and largely occurs within damage-induced subgroups. Gene module analysis revealed functional divergence of Treg cells into immunosuppression-oriented and tissue repair–oriented groups, leading to identification of candidate receptors for induction of repair activity in Treg cells. We tested these using an ex vivo assay for Treg cell–mediated tissue repair, identifying 4-1BB agonism as a novel mechanism for reparative activity induction. Overall, we demonstrate that the AregThy1.1 mouse is a promising tool for investigating tissue repair activity in leukocytes.

Chronic Obstructive Pulmonary Disease (COPD) is a progressive respiratory disease characterized by airway inflammation and abnormal alveolar enlargement. It is the third leading cause of death around the world. Although extensive research efforts have been made, there is still no curable treatment available for lung tissue damage in patients with COPD. Therefore, it is of great significance to elucidate the mechanisms of tissue damage and repair in COPD. As the first barrier against environmental insults and pathogens, pulmonary epithelial cells play an essential role in regulating injury response and repair. However, how pulmonary epithelial cells contribute to irreversible alveolar destruction in COPD is not well understood. In this study, we elucidated the mechanisms of epithelial cell damage in both cigarette smoke-induced COPD and alpha1 antitrypsin deficiency (AATD)-associated genetic COPD. To investigate alveolar epithelial cell damage and repair in cigarette smoke-induced emphysema, a lineage tracing model was utilized to fluorescently label and chase alveolar type II (AT2) epithelial cells, the adult progenitor cells in the alveolar epithelium. An assessment of cigarette smoke-induced changes in cellular composition and regenerative capacity of the alveolar epithelial cells was performed. Cigarette smoke was found to impede the AT2-directed alveolar epithelial regeneration and repair process, and this impaired progenitor cell function was not restored after smoke cessation. Moreover, comparison analysis between stains that are sensitive and resistant to smoke-induced damage revealed that deficiency in lipid metabolism may contribute to the dysregulation of alveolar epithelial repair by AT2 cells. Restoring alveolar progenitor functions through lipid metabolism may serve as a novel therapeutic for alveolar destruction in smoke-induced COPD. To explore the mechanism of epithelial damage in AATD-associated genetic COPD, we utilized a PiZ (p.Glu342Lys) transgenic mouse model expressing human ZAAT protein. Morphometric analysis of PiZ lungs suggests that the accumulation of ZAAT polymers in the lung directly leads to the spontaneous development of emphysema. To investigate epithelial damage induced by zAAT accumulation, we isolated the epithelial cell population from the lung of PiZ mice. We identified epithelial-specific expression of cleaved caspase 3, indicating a direct cytotoxic effect of ZAAT in impairing epithelial function and inducing epithelial cell death. Future therapeutics could directly target the cytotoxicity of pulmonary epithelial cells in AATD to reduce lung tissue damage. Overall, our findings suggest that pulmonary epithelial damage plays an essential role in the pathogenesis of lung tissue damage in COPD. Future epithelial cell-based therapies may contribute to pulmonary re-epithelialization and tissue repair in both cigarette smoke-induced and AATD-associated COPD.