Age and sex alter the immune response to synthetic materials

Introduction: Fibrosis affects a wide range of clinical disorders including chronic wounds, the foreign body response (FBR) to implants, and diseases such as uterine fibroids. Analysis of the 2019 Global Burden of Disease study found that mortality from fibrotic diseases has steadily increased up to 17.8% since the 1990s. Aging is a significant contributor of fibrotic disease. With aging, immunologic changes are known to occur including increased susceptibility to inflammatory age-related diseases, reduced responses to infections and vaccines, and poor wound healing. Chronic inflammation is one of the 12 hallmarks of aging [1]. Our goal is to understand how biologic factors (e.g. age, sex) alter the immune response in chronic fibrosis to develop more precise immunomodulatory systems designed to reduce fibrosis and promote tissue regeneration. The overarching hypothesis is that age-related immune skewing towards an effector state compounds the adverse reaction to synthetic materials. The goal of this work is to use a chronic non-healing wound injury model with various material implants that induce the FBR to determine how sex and age alter the immune microenvironment.

Materials and

Methods: All procedures were done in accordance with approved ACUC protocols at JHU. To assess the immune response to synthetic material implants, young (2-month) and aged (18 month), male and female, C57BL/6 mice sourced from the NIA aging colony were given a bilateral critically sized defect to their quadricep femoris. Quadricep defects were subsequently treated with saline (injury control), polycaprolactone (PCL; < 600m, MW 50,000), or polyethylene (PE; 125µm, ultra-high molecular weight) powder to induce fibrosis. Tissues were harvested 6-weeks post injury. For histological evaluation, quadriceps were formalin fixed and paraffin-embedded before Masson’s trichrome staining. For flow cytometry, quadriceps with implants were enzymatic and mechanically digested into a single cell suspension, then antibody stained to identify eosinophils, neutrophils, monocytes, macrophages with activation markers, B, NK, and T cells (CD4, CD8, gamma delta). Data was collected on a CyTEK Aurora, analyzed using FlowJo, and statistical analysis was done using a 2-way ANOVA with Sidak posthoc for multiple comparisons with significance determined at a p-value of < 0.05 in GraphPad Prism.

Results, Conclusions, and Discussions: Young (2 months) and aged (18 month), male and female mice were given a chronic non-healing wound using the volumetric muscle loss model. Wounds were treated either with saline (injury control) or a synthetic material (PCL or PE), Figure 1A. 6-weeks post injury there is fibrosis around the material implants visualized by Masson’s trichrome staining, representative Figure 1B. Flow cytometry of the immune cell fraction showed that while each treatment has different numbers of CD45+ infiltrating immune cells there is no significant difference in overall immune cell counts within each group by either age or sex, Figure 1C. Within the T cell fraction, CD4+ T cells decrease and CD8+ T cell increase with age and there is a significant difference between sex in the aged mice (data not shown) regardless of treatment condition. In aging literature, there is a consistent observed loss of naïve T cells and shift towards more effector function with an increase in CD8+ T cells relative to CD4+ [2]. Within each of these injury conditions, this trend appears to hold.
Within the myeloid compartment, a subset of macrophages identified by CD86+CD206+ has some material-specific differences by age and sex in PCL that are not seen in saline or PE, Figure 1D. Within this population the median fluorescence intensity (MFI) of MHCII, CD206 and CD86 were analyzed. CD86 MFI shown in Figure 1E, has no significant difference in saline, but both an age and sex difference in PCL and PE, Figure 1E. This demonstrates that there are age and sex differences in the myeloid response to synthetic materials that must be considered when designing materials intended to skew CD206 or CD86 expression.
This finding of age- and sex-related differences in the immune response to foreign material highlights the importance of these factors when designing immunomodulatory biomaterials. Future work to characterize the mechanism behind these differences, as well as spatial information on these immune populations, can be used to better design synthetic implants.

Acknowledgements (Optional): [1] PMID: 36599349 [2] PMID: 18469829
The authors would like to acknowledge funding from Morton Goldberg Chair, Bloomberg Kimmel Institute for Cancer Immunotherapy, NIH Pioneer (DP1AR076959), and NIBIB R01 (R01EB028796) to JHE. JCM is funded by K99AG081564.