Anti-inflammatory macrophage activation and TGF-β signaling drive a fibrosis-like phenotype in response to oxidative stress in a hydrogel model

Introduction: : Fibrosis involves excessive deposition of extracellular matrix (ECM) following injury. Activated fibroblasts promote fibrosis by secreting ECM proteins [1]. Macrophage immune cells facilitate fibrosis by secreting anti-inflammatory cytokines that activate fibroblasts [2]. Greater understanding of fibrosis requires models that assess these cell-to-cell interactions and ECM deposition. In particular, hydrogels can provide a three-dimensional environment for ECM deposition and for cell-to-cell interactions. Hydrogel models also facilitate in vitro modulation and interrogation of signaling pathways important for fibrosis.

We employed a poly(ethylene glycol) (PEG)-based hydrogel to model fibrosis. We co-encapsulated fibroblasts and macrophage in our hydrogel to characterize the interactions between each cell type. Injury was then induced using oxidative stress following H2O2 treatment [3]. We then assessed fibrosis-related phenotypes, including fibroblast activation, proliferation, and production of ECM proteins. We also assessed the anti-inflammatory cytokine transforming growth factor beta (TGF-β). TGF-β signaling promotes fibroblast survival, deposition of ECM proteins, and anti-inflammatory macrophage activation. Oxidative stress induces TGF-β signaling pathways [3-5]. Macrophages are also an important source of TGF-β in several disease models [2]. We hypothesize that TGF-β signaling and anti-inflammatory macrophage activation promote fibroblast, activation, and ECM deposition following oxidative stress in our hydrogel model.

Materials and

Methods: : Macrophage activation: Human peripheral blood-derived monocytes (PBMCs) were cultured for 24 hours, rinsed, cultured for 48 hours, rinsed, and cultured for 72 hours in macrophage colony stimulating factor. Following an additional rinse, adherent macrophages were cultured for 48 hours +/- interleukin-4 (anti-inflammatory activation) or +/- LPS and IFN-γ (inflammatory activation).

Peptide conjugation: Acrylate-polyethylene-glycol-succinimidyl valerate was mixed with arginine-glycine-aspartate-serime peptide (RGDS) or GGGPQGIWGQGK (PQ) peptide. These two mixtures were wrapped in foil, left overnight at 4°C with agitation, dialyzed, and stored at -80°C overnight. The frozen PEG-RGDS and PEG-PQ-PEG mixtures were then lyophilized and stored at -80°C.

Cell encapsulation: 5x104 human dermal fibroblasts with or without 5x103 macrophages were encapsulated in 5µL hydrogels containing 3.5mM PEG-RGDS and 6% w/v PEG-PQ-PEG. Encapsulated cells were placed on a methacrylated coverslip, exposed to white light, and placed in culture.

Hydrogen peroxide and TGF-β treatment: Encapsulated cells were cultured in fibroblast media for 24 hours +/- 20ng/mL TGF-β, then cultured for 24 hours +/- TGF-β and +/- 125µM H2O2. The cells were then immunostained, or cultured without H2O2 and +/- TGF-β for another three or seven days before immunostaining.

Immunostaining and image analysis: Encapsulated cells were fixed in paraformaldehyde, permeabilized with Triton-X, and blocked overnight with donkey serum. Hydrogels were then incubated overnight with primary antibodies, washed, and incubated with fluorescently-labeled secondary antibodies overnight. Cells were washed, treated with DAPI, washed, and imaged using a BC43 benchtop confocal microscope. Images were then analyzed using Imaris software.

Results, Conclusions, and Discussions:: At seven days following 125μm H2O2 treatment, human dermal fibroblasts encapsulated in PEG hydrogels increased expression of Ki67, a proliferation marker elevated during abnormal fibrotic scarring (Fig. 1A). The proportion of Ki67+ cells significantly increased from 21%% to 44%. There was also a significant increase from 28% to 64% in the proportion of fibroblasts expressing p16, an inhibitor of reactive oxidative species accumulation whose expression may persist in profibrotic contexts. Treatment with 125μm H2O2 decreased fibroblast number by a factor two, with greater decreases at higher H2O2 concentrations (Fig. 1A). This suggested 125μm H2O2 as the appropriate concentration to induce oxidative stress without too sharply limiting cell survival.

We then sought to determine the impact of macrophage activation and TGF-β signaling on the fibroblast response to oxidative stress. At seven days following H2O2 treatment, TGF-β treatment significantly increased the proportion of Ki67+ fibroblasts by 12% (Fig. 1B). Fibroblast number was also elevated by two-fold (Fig. 1B). Next, macrophages were either left unactivated, treated with IL-4 (anti-inflammatory activation), or treated with LPS and IFN-γ (inflammatory activation), followed by encapsulation with fibroblasts. Co-encapsulation with anti-inflammatory macrophages increased the proportion of fibroblasts expressing alpha smooth muscle actin (α-SMA), a marker of activated fibroblasts or myofibroblasts. This represented a significant increase from 14% to 24% (Fig. 1C).

Taken together, these results suggest anti-inflammatory macrophage activation and TGF-β signaling promotes fibroblast survival and activation following oxidative stress. Our PEG-based hydrogel model is able to recapitulate markers and in vivo signaling seen in the fibroblast response to sterile injuries such as those from oxidative stress. Future work will investigate the role of TGF-β signaling and macrophages in fibroblast deposition of ECM proteins following oxidative stress. Modulating cytokine signaling and macrophage activation within this model may identify important targets for mitigating fibrosis risk.

Acknowledgements and/or References (Optional):: 1. Xu, X. et al. Burns & Trauma 8 (2020).
2. Wang, Z.-C. et al. Front. Immunol. 11 (2020).
3. Kizilay Mancini, O. et al. Rheumatology 61 (2020).
4. Kim, K. K.et al. Cold Spring Harb Perspect Biol 10 (2018).
5. Frangogiannis, N. G. J Exp Med 217 (2020).