Modeling the role of macrophage activation in fibrosis and fibroblast recovery from oxidative stress

Introduction: Cell-to-cell interactions impact risk of fibrosis following skin wounding. Macrophages promote tissue inflammation or tissue repair, depending on their activation state. An overabundance of anti-inflammatory (M2) macrophages relative to pro-inflammatory (M1) macrophages increases risk of fibrosis [1]. Tissue damage can be modeled in vitro using oxidative stress induced by hydrogen peroxide (H2O2) [2]. Hydrogel models offer a fruitful avenue for interrogating macrophage function during oxidative stress and capturing three-dimensional intercellular interactions important for tissue repair [3]. In this work, we leverage hydrogels to model the impact of macrophage activation on fibroblast response to oxidative stress.

To understand macrophage response in the context of oxidative stress, we encapsulated macrophages and dermal fibroblasts in polyethylene glycol (PEG) hydrogels wherein cells were exposed to H2O2. We hypothesize that following oxidative stress, M2 macrophages will facilitate increased fibroblast proliferation when compared to M1 macrophages. M2 macrophages will also promote expression of fibrosis-associated markers dependent on anti-inflammatory cytokine transforming growth factor-β (TGF-β). Our results serve as a proof-of-concept for use of a PEG hydrogel model to investigate how macrophage signaling impacts risk of fibrosis.

Materials and

Methods: Peptide conjugation: Arginine-glycine-aspartate-serine (RGDS) and the matrix-metalloproteinase-sensitive peptide GGGPQGIWGQGK (PQ) were separately conjugated to acrylate-PEG using amine substitution with acrylate-PEG-succinimidyl valerate. Cell culture: We cultured dermal fibroblasts and peripheral blood mononuclear cells (PBMCs) isolated from Caucasian females aged 18 to 35. PBMCs were treated with M-CSF to generate macrophages. Macrophages were skewed to an inflammatory phenotype using lipopolysaccharide and IFN-γ (M1 macrophages), towards an anti-inflammatory phenotype using IL-4 (M2), or not skewed (M0). All cell culture occurred in phenol-free media with charcoal-stripped serum. Encapsulation: Macrophages and dermal fibroblasts were encapsulated in 3.5mM PEG-RGDS and 6% PEG-PQ-PEG. Encapsulated cells were treated with white light to initiate crosslinking and hydrogel formation. Hydrogen peroxide treatment: Encapsulated cells were cultured with H2O2 for 24 hours. Cells for two-dimensional experiments seeded on 24-well plates were similarly treated with H2O2 for experiments outside of hydrogels. Cells in two-dimensional conditions or encapsulated in PEG hydrogels were then washed to remove H2O2, followed by culture for 3 or 7 days in the absence of H2O2. Imaging: Brightfield microscopy was performed on the Oxion Inverso microscope. Live and dead cells were detected using calcein AM and ethidium homodimer-1, respectively. Cell nuclei were stained with DAPI. Samples were stained with CD11b, CD90, alpha-smooth muscle actin, or collagen primary antibodies. Secondary antibodies were then applied. Imaging for immunocytochemistry was performed on the BC43 and FV3000 confocal microscopes. Secretomic analysis: Cytokine levels were evaluated using a Luminex bead assay, and an ELISA was performed targeting soluble collagen I and III.

Results, Conclusions, and Discussions: To mimic oxidative stress in a sterile wound environment, we first determined the level of H2O2 exposure sufficient for inducing moderate cell death and subsequent cell recovery in our model. Treatment with 250µM or 500µM of H2O2 for 24 hours did not significantly impact macrophage survival under two-dimensional conditions (data not shown). Dermal fibroblasts cultured outside of hydrogels did not recover from 250µM or 500µM of H2O2 exposure for 24 hours (figure 1). Co-encapsulation of fibroblasts and macrophages in hydrogels, however, led to persistent cell survival 3 days after 500µM of H2O2 exposure (figure 1). We therefore used a 500µM H2O2 treatment for 24 hours to induce oxidative stress in our experiments.

Addition of M1 macrophages increased levels of inflammatory cytokines such as IFN-γ, while addition of M2 macrophages increased levels of anti-inflammatory cytokines including TGF-β. M2 macrophages promoted fibroblast survival following oxidative stress, with M1 and M0 macrophages promoting survival to a lesser extent. M2 activation was also associated with increased collagen deposition and expression of alpha-smooth muscle actin, a marker of activated fibroblasts, along with increased secretion of collagen I and III.

These results suggest that M2 macrophages in our hydrogel system facilitate a fibrotic phenotype that serves as a model for fibrosis (figure 1). Future work will modulate cytokine signaling in this model to evaluate how cytokines such as TGF-β impact the fibrotic phenotype we observed. Prior research suggests fibrosis following skin wounding occurs more frequently in populations of African ancestry compared to those of European ancestry [4]. Our future work will therefore incorporate dermal fibroblasts and macrophages from individuals of African genetic ancestry and European ancestry to examine how ancestry contributes to fibrosis risk.

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