Poster O9 - Biomaterial tools to monitor and modulate inflammatory monocytes in idiopathic pulmonary fibrosis

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease of intractable fibrotic remodeling in the distal airways and alveoli that is driven by unknown etiology. Inflammatory monocytes, especially in the pulmonary interstitium, drive fibrosis through poorly understood mechanisms. No IPF therapy so far has successfully targeted profibrotic macrophages. Here, we develop two complimentary biomaterial tools, an immune-monitoring implant and a leukocyte-targeting nanoparticle therapy, to study and perturb IPF’s profibrotic interstitial macrophages in vivo.

The implant (hereafter, scaffold), is comprised of non-degradable, biocompatible poly(ε-caprolactone) polymer with >90% porosity that is implanted subcutaneously. Within days, the scaffold is colonized by endogenous vasculature, extracellular matrix, fibroblasts, and immune cells, which incites “tissue-resident” immune functions that emerge in tissues but are muted or completely absent in circulating, quiescent blood. In IPF, lung-recruited monocytes acquire significant profibrotic functions, especially in interstitial fibrotic foci, that may be reflected by the scaffold. We hypothesized that the scaffold would recapitulate aspects of pulmonary fibrosis including the accumulation of excess collagen and the infiltration of inflammatory monocytes (CD45+CD11b+Ly6C+).

The nanoparticles are approximately 500 nM in diameter and consist of high molecular weight 50:50 poly(lactide-co-glycolide) with a negative zeta potential. We previously demonstrated that these particles modulate circulating monocyte trafficking and phenotype in several acute inflammation models. Most notably, the particles reduced tumor size and metastatic burden in a breast cancer mouse model. We hypothesized that the nanoparticles would reduce pulmonary fibrosis burden by modulating the trafficking and polarization of circulating leukocytes.

Materials and

Methods: Oropharyngeal aspiration of bleomycin (0.5 U/kg in 50 uL sterile saline) induced fibrosis in male C57/BL6J mice aged 6-10 weeks. Animals were housed on a 12 hour light/dark cycle with ad libitum water and chow in accordance with institutional guidelines under an approved protocol. Collagen was quantified using the Quickzyme Total Collagen Assay (Quickzyme Biosciences) according to manufacturer instructions. For flow cytometry, lungs and scaffolds were finely minced and enzymatically dissociated, incubated in RBC lysis buffer, and incubated with live/dead (Zombie Violet) and Fc block (TruStain FcX Mouse). Samples were stained with pre-optimized antibody panels, run on the ZE5 Cell Analyzer (BioRAD), and processed in FlowJo.

Scaffolds were fabricated using a previously described protocol in our lab. Briefly, poly(ε-caprolactone) was melted and dispersed onto NaCl crystals, then compressed into a scaffold mold, heated to crosslink, and de-salted in water. Sterilized scaffolds were implanted subcutaneously and integrated for 7-12 days before bleomycin. Scaffolds were explanted on Days 9, 14, and 28 post-bleomycin.

Nanoparticles were fabricated using an oil-water emulsion method as previously described by our lab. Briefly, 50:50 poly(lactide-co-glycolide) was emulsified by sonication in a surfactant solution (water plus poly(ethyl methacrylate)) and allowed to mix overnight at room temperature. Particles were washed, lyophilized, and stored under vacuum until use. For intravenous delivery, particles were suspended in sterile saline and delivered through the lateral tail vein. Nanoparticles were administered on either Days 7, 9, 11, 14, 16, and 18 (early timepoint) or on Days 14, 15, 16, 17, and 18 (late timepoint).

Results, Conclusions, and Discussions: The scaffold recapitulated key features of pulmonary fibrosis in a remote, non-lung setting. Scaffolds from fibrotic mice accumulated collagen more quickly from day 9 to 28 compared to control (saline) mice. Additionally, the scaffolds represented the fibrotic lungs’ composition of inflammatory monocytes (CD45+CD11b+Ly6C+) and neutrophils (CD45+CD11b+Ly6G+) more closely than the clinically standard biopsy specimen, bronchoalveolar lavage (BALF). These results suggest that the scaffold reflects important aspects of pulmonary fibrosis in an extrapulmonary site. Future work will focus on how the scaffold’s resident leukocytes contribute to fibrosis, and whether these mechanisms mimic those known to occur in the lung. Ultimately, this tool may be translated for prognostication of individual patient outcomes by reflecting lung physiology in a longitudinally accessible biopsy site that reflects key aspects of monocyte phenotypes that are represented in the scaffold and important to pathophysiology.

The nanoparticles were protective from pulmonary fibrosis, with both early (starting Day 7) and late (starting Day 14) delivery reducing collagen in bleomycin-treated animals back to the level of the control animals. Additionally, few studies have reported effective attenuation of fibrosis with therapeutic intervention 14 days post-bleomycin. Typically, at this relatively late timepoint, the fibrotic process is well established and difficult to interrupt. Therefore, this preliminary result supports the importance of circulating inflammatory monocytes as drivers of established pulmonary fibrosis and suggests promise for clinical translation to human patients.

In sum, we developed two complimentary biomaterial-based approaches to understand the role of inflammatory monocytes in IPF. The implanted scaffold acts as surveillance of the lung interstitum in a remotely accessible site for longitudinal studies of individuals, which can be leveraged in the future for precision medicine. The nanoparticles target circulating leukocytes and protect against both emerging and established pulmonary fibrosis, underscoring the important role of monocytes in fibrosis perpetuation. Together, these tools address an unmet need for approaches to monitor and target profibrotic leukocytes in pulmonary fibrosis.

Acknowledgements (Optional):