The Role of M2 Macrophages and their Product, HB-EGF, as Regulators of Lung Fibrosis
Hult, Elissa
2022
Abstract
Idiopathic Pulmonary Fibrosis (IPF) is a highly debilitating lung disease with no known cure1–3. Previous work has shown that monocytes and macrophages are critical in the development and progression of pulmonary fibrosis in animal models4–7. As such, the overarching theme of this dissertation is to further explore myeloid cell regulation of lung fibrosis and specifically, to better understand how macrophages and macrophage-associated factors may be regulating fibrosis. This dissertation is comprised of two distinct but interrelated projects. The first set out to characterize the soluble products of proinflammatory (M1-like) and profibrotic (M2-like) macrophages to investigate how these factors mediate fibrosis and influence structural cells of the lung. Using M1- and M2-polarized bone marrow-derived macrophages (BMDMs) in an in vitro model, the data showed that M1-like and M2-like BMDMs have different gene expression profiles based on RNAseq analyses, with M2-like BMDMs containing gene clusters with higher enrichment for cell processing as well as increased expression for secreted proteins. While M2 supernatant increased profibrotic characteristics of fibroblasts and alveolar epithelial cells (AECs) compared to cells treated with M1 supernatant, all effects could be attributed to the lingering presence of IL-4 and IL-13, which was required to polarize the BMDMs to an M2 phenotype. The presence of a M2 protease(s) able to yield a false positive in an assay of AEC apoptosis by direct cleavage of the Caspase-Glo 3/7 substrate was also noted. Together, this work provides a novel M1-M2 transcriptome database, confirms profibrotic effects noted by M2 supernatant in pulmonary structural cells in vitro, and provides new evidence that profibrotic effects associated with M2 macrophages are likely related to polarization cytokines found in the fibrotic milieu. In the second project, the aim was to characterize the role of a known M2-associated growth factor, heparin-binding epidermal growth factor-like growth factor (HB-EGF) to determine if it acts as a major myeloid-derived profibrotic effector. The findings demonstrated that faster-progressing IPF patients had higher levels of both HB-EGF and its receptor, epidermal growth factor receptor, using SOMAmer technology, and also showed that HB-EGF expression is increased in lung macrophages of fibrotic mice. In addition, the results proved that mice lacking HB-EGF from the myeloid compartment (Hbegff/f;Lyz2Cre mice) are protected from bleomycin-induced fibrosis as evidenced by decreased levels of hydroxyproline and profibrotic gene expression. This protection is likely multifactorial, caused by genotypic differences in CCL2-dependent monocyte migration, decreased fibroblast migration, and decreased contribution of HB-EGF from AEC sources when HB-EGF is removed under the Lyz2Cre promoter. In summary, this work has explored the profibrotic impacts of M2-polarized macrophages and demonstrated a profibrotic effect of M2-derived HB-EGF on myeloid cell accumulation and fibroblast migration in lung fibrosis.Deep Blue DOI
Subjects
Lung Fibrosis Macrophages HB-EGF RNAsequencing
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