Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment

Abstract

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Fig. 1. ECM scaffold promotes early immune cell infiltration with or without mast cells.

Representative images of toluidine blue staining of ECM scaffold (green outlined with asterisk where material is present) and neighboring dermal tissue in wild-type (a) and mast cell-deficient (b) mice at day 1 post-injection. Mast cells can be found as early as day 1, showing a lack of or minimal degranulation (black arrow). Representative fluorescent images of injected biomaterial (white outline with asterisk denotes where material is present) with Ly-6G/Ly-6C⁺ neutrophil (c) and F4/80⁺ pan-macrophage staining (d) in green and Hoechst nuclei counterstain in blue. Quantification of total nuclei (e, f), neutrophil (g, h) and macrophage (i, j) density at 1 and 3 days post-injection for male and female wild-type (green) and mast cell-deficient (red) mice. n = 7–9 per group for total nuclei, n = 4–5 per group for immune cell quantification. Data displayed as mean ± SD. Scale bars of 50 µm in toluidine blue (a, b) and fluorescent images (c, d). (*p < 0.05, **p < 0.01, and ***p < 0.001).

Fig. 2. Transition from a pro-inflammatory to pro-remodeling macrophage is dysregulated in mast cell-deficient mice in a sex-specific manner.

a Representative contour plots for assessing macrophage polarization by CD206-PE versus CD86-BV786 labeling. Red values indicate the M2/M1 ratio per plot, and contour lines separate 5% of the events, each with outliers as individual black dots. Ratios of CD206⁺ M2 versus CD86⁺ M1 macrophage counts were assessed at 3, 7, and 11 days post-injection between wild-type (green), mast cell-deficient (red), and mast cell-engrafted (blue) male (b) and female mice (c). n = 4–9 per group. Box plots display a median with a 25–75% percentile distribution. d Median M2/M1 values between male (circles with solid lines) and female (squares with dashed lines) over time with statistical analysis between sexes at the same timepoint. Quantified results were pooled from 2–3 independent experiments (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig. 3. Comparison of mast cell influence in response to non-decellularized and decellularized ECM scaffolds.

Representative hematoxylin and eosin staining in mast cell-deficient mice following dorsal subcutaneous injection of a non-decellularized and b decellularized ECM scaffold at 7 days post-injection (material outlined in green with an asterisk on side where material is present). Scale bar is 100 µm. Gene expression ratio at day 7 post-injection of M2/M1 macrophage markers Arg1 relative to Nos2 in c male and d female mice. Decellularized ECM scaffold in wild-type (green), mast cell-deficient (red), and mast cell-engrafted (blue) groups were compared by relative gene expression with 2^−ΔΔct normalized to non-decellularized (NDM) control per group. n = 6–10 per group. Data displayed as geometric mean ± geometric SD with results pooled from 2–3 independent experiments. (Value displayed for trend p ≤ 0.1, *p < 0.05, ***p < 0.001 and ****p < 0.0001).

Fig. 4. Immune marker profiling and hormone receptor gene expression demonstrating differentiation expression based on mast cell presence.

qRT-PCR gene expression in deficient and engrafted normalized to wild-type expression by 2^−ΔΔct at day 11 in male mice for a Il1b and b Il33 relative to the housekeeping gene, Gapdh. c Gene expression ratio of receptor antagonist, Il1rn, relative to Il1b. d Gene expression of Cd206 relative to the housekeeping gene, Gapdh. qRT-PCR gene expression at day 3 in female mice measured for e Nos2, f Esr1, and g Esr2 relative to Gapdh. n = 11–16 per group for male samples, n = 6–8 per group for female samples. Data displayed as geometric mean ± geometric SD with results pooled from 2–3 independent experiments. (*p < 0.05, **p < 0.01, and ***p < 0.001).

Fig. 5. Mast cell-deficient male mice maintain late pro-inflammatory gene expression profile in response to ECM biomaterial implantation.

Volcano plots labeled with the top 25 differentially expressed genes a in a multi-group comparison between mast cell-deficient versus both wild-type and mast cell-engrafted groups, and b in a pairwise comparison between engrafted versus wild-type samples. Dot colors indicate q < 0.05 (blue, red), absolute fold change greater than 2 (green, red) or nonsignificant (black). Dashed lines indicating different significance thresholds (*q < 0.05, **q < 0.01, ***q < 0.001 and ****q < 0.0001). c Bubble plot showing significant differentially expressed genes consistent across at least two pairwise analyses among the wild-type, deficient, and engrafted groups. The red to blue color scale displays log2 fold change that was up- or downregulated, respectively, and the circle size indicates the significance of the negative log₁₀ transformed q-value (1: q = 0.1, 2: q = 0.01, 3: q = 0.001, 4: q = 0.0001). n = 8 per group pooled from 2–3 independent experiments.

Fig. 6. Mast cell-deficient male mice maintain late pro-inflammatory response to biomaterial implantation while mast cell engraftment restores physiological cellular responses to biomaterial stimuli.

Bubble plot from gene set enrichment analysis for enriched pathways a consistent or b independently enriched among different group comparisons. Assessed databases included simplified biological process GO terms (“G_…”), KEGG pathways (“K_…”), Reactome pathways (“R_…”), and MSigDB hallmark gene sets (“H_…”). The red to blue color scale of each point represents the magnitude and directionality of up- and downregulated pathways based on normalized enrichment score (NES). Point size represents significance based on the negative of the log₁₀ transformed q-value (1: q = 0.1, 2: q = 0.01, 3: q = 0.001, 4: q = 0.0001). n = 8 per group pooled from 2–3 independent experiments.