MODULATING PROFIBROTIC MACROPHAGES FOR THE TREATMENT OF PULMONARY FIBROSIS FROM THERAPEUTIC TARGET IDENTIFICATION TO INHALABLE DRUG DELIVERY

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease marked by excessive extracellular matrix (ECM) deposition and lung scarring, leading to respiratory failure. Current therapies can decelerate disease progression but are associated with significant systemic side effects. Profibrotic M2-like macrophages, key drivers of lung fibrosis, promote fibroblast activation and ECM accumulation, sharing phenotypic traits with tumor-associated macrophages (TAMs). The heterogeneity of macrophage phenotypes and the lack of specific, targetable markers have limited the development of macrophage-focused therapies. This thesis addresses these challenges by identifying actionable targets such as Dectin-1 and leveraging yeast beta-glucan (YBG) microparticles to modulate profibrotic macrophages. By optimizing YBG for enhanced inhalability and preserved bioactivity, we aim to support localized therapies that mitigate fibrosis while minimizing systemic toxicity. To enable therapeutic targeting of these macrophages, we conducted integrative computational analyses of publicly available microarray-based transcriptomic datasets encompassing diverse macrophage phenotypes. This analysis produced a conserved 35-gene signature characteristic of profibrotic macrophages across human and murine models. Validation using the NanoString nCounter platform in THP-1-derived macrophages and primary murine bone marrow-derived macrophages (BMDMs) refined this to six consistently upregulated surface receptor genes, with CLEC7A (encoding Dectin-1) emerging as a lead candidate. Single-cell RNA sequencing from IPF patients and bleomycin-induced murine models confirmed elevated CLEC7A expression in monocytes and macrophages. Spatial transcriptomics using the NanoString CosMX™ platform in non-small cell lung cancer (NSCLC) samples showed that CLEC7A⁺ macrophages clustered in regions with high expression of fibrosis-related genes such as ACTA2 and profibrotic macrophage markers CD163, CCL18 and MRC1. Histological and immunohistochemical staining of serial sections from IPF and lung adenocarcinoma patients revealed enrichment of Dectin-1, αSMA, Masson’s Trichrome, and CD163 in anatomically overlapping regions, indicating regional convergence of fibrotic and immune activity. Functional studies in Clec7a knockout mice further demonstrated exacerbated fibrosis and impaired lung function, consistent with a protective role for Dectin-1 in pulmonary remodeling. We then examined YBG microparticles as immunomodulators to shift profibrotic M2-like macrophages toward an anti-fibrotic phenotype, targeting their role in disease progression. These microparticles engage Dectin-1, but their efficacy is highly dependent on fabrication conditions. To improve particle consistency and performance, we employed Pressurized Gas eXpanded (PGX) liquids technology to produce PGX-YBG microparticles with greater surface area, lower density, and more uniform size than conventional spray-dried YBGs. PGX-YBG showed enhanced Dectin-1 activation in vitro with minimal TLR2/4 signaling, reducing potential off-target effects. In both human and murine in vitro models, PGX-YBG modulated M2-like macrophages and reduced fibrosis-associated features. Ex vivo murine precision-cut lung slices (PCLS) confirmed PGX-YBG's capacity to modulate profibrotic macrophages, supporting its utility as a localized therapeutic. To further evaluate translational potential, we assessed the inhalability and immunomodulatory activity of PGX-YBG compared to in-house spray-dried YBG prepared from identical feedstock. PGX-YBG formed uniform blends with inhalation-grade lactose and exhibited aerodynamic diameters of 3–4 µm, with roughly double the fine particle fraction of the spray-dried counterpart. These properties improved deposition in distal lung regions and interaction with profibrotic macrophages. Both YBG types induced pro-inflammatory cytokine expression in vitro and in ex vivo PCLS. However, only PGX-YBG demonstrated enhanced uptake by macrophage, along with a significant reduction in arginase-1 and CD206. Additionally, PGX-YBG exhibited lower cytotoxicity, indicating a more favorable safety profile. Together, this work establishes a translational pipeline that integrates target discovery, biomaterial engineering, and inhalable delivery to modulate profibrotic macrophages in lung fibrosis. It highlights Dectin-1 as a viable therapeutic target and demonstrates the potential of PGX-YBG as a safe, effective, and clinically promising inhalable immunotherapy for IPF and related fibrotic diseases.