(476c) Utilizing Variable Substrate Stiffness to Investigate Macrophage Response in Healthy and Fibrotic Pulmonary Microenvironment.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic disease thought to be initiated by repeated micro-injuries to the alveolar epithelium, resulting in deposition and accumulation of scar tissue and increased tissue stiffness¹. Upregulation of Th2 cytokines (such as IL-13) is commonly observed in the fibrotic lungs, which is hypothesized to aid in fibrosis progression by activating macrophages (M2 phenotype)². While previous studies have shown the role of stiffness and profibrotic microenvironment in activating fibroblasts³, little is known about the role of substrate stiffness and profibrotic stimuli such as IL-13 in tuning macrophage phenotype and aiding in fibrosis progression. Here, we present an approach to investigate macrophage response in healthy and fibrotic conditions by utilizing well-defined hydrogel-based synthetic matrices.

Experimental Methods: Hydrogels with bioinspired mechanical properties and biochemical content were created utilizing photoinitiated thiol-ene click chemistry. Briefly, monomer solutions containing norbornene functionalized 4-arm PEG (2.5 wt% to 15 wt%), a non-degradable dithiol linker, monothiol integrin binding peptides, and the photoinitiator Lithium acylphosphinate (LAP) were irradiated (10 mW/cm² at 365 nm). Incorporation of integrin binding peptide in the hydrogel matrix was assessed by using a fluorescently tagged peptide and confocal microscopy. Alveolar macrophages (MH-S cells) were cultured on these substrates in comparison to controls (tissue culture polystyrene) and the role of both substrate stiffness and IL-13 stimulation in the polarization of alveolar macrophage cells was assessed with flow cytometry and immunostaining, including expression of pro-inflammatory marker (CD86) and anti-inflammatory marker (CD206).

Results and conclusions: Hydrogels with moduli in the range of healthy (Young’s modulus (E) ~ 1.5 kPa) to fibrotic lung tissue (E~20 kPa) were prepared by varying crosslinking density, and the equilibrium swollen modulus was measured using rheometry. Relevant conditions were established for independent tuning of matrix mechanical properties and biochemical content and promoting consistent macrophage attachment to these bioinspired substrates. Finally, the effect of IL-13 on MH-S cell polarization was observed, with a significant increase in CD206 expression and a significant decrease in CD86 expression with large stiffness values, suggesting the role of IL-13 in upregulating anti-inflammatory effects by macrophages that could aid in fibrosis progression. Further engineering and application of these hydrogel systems provides exciting opportunities to investigate macrophage response in both the initiation and progression of fibrosis.

References

1. Richeldi, L. Lancet, 2017; 389: 1941–1952
2. Blirando, K. EC Pulmonology and Respiratory Medicine, 2018; 3: 98-120.
3. Smithmyer, M.E, ACS Biomat. Sci. and Eng, 2018; 4: 3304-3316.