2024 Theses Doctoral

Treg cell–derived amphiregulin in heparan sulfate–mediated repair of viral damage in the lung and characterization of reparative Treg cells in lung injury models

Loffredo, Lucas Fedele

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.