(B-53) Age and sex differences in the immune response to synthetic materials in a volumetric muscle loss injury model

Introduction:: Synthetic polymers are widely used in biomedical applications but suffer from fibrosis induced by the foreign body response (FBR). The FBR is a chronic inflammatory and fibrotic microenvironment that results in fibrous encapsulation of the implanted material. The adaptive immune response, specifically T cells, are required for proper tissue healing and for the maintenance of the FBR in both mice and humans. This response is dependent on the material as well as the tissue location and underlying pathophysiology (patient’s demographic background and health) of the implant site. A hallmark of aging is the immunological changes that increase susceptibility of the population to inflammatory age-related diseases and lower regenerative capacity. There is an established diversity in both the immune and stromal microenvironments with age and sex in mice and humans. There is increased granularity in neutrophils and increased macrophage recruitment in aged mice during inflammation and studies in aged acute muscle injury showed impaired wound healing mediated by T regulatory cells (1-3). The overarching hypothesis is that the age-related immune dysregulation towards an effector state compounds the adverse reaction to synthetic materials. The goal of this work is to understand the changes that occur in the FBR with age and sex in a mouse implant model to inform future design synthetic systems towards modulating these different responses.


Materials and Methods:: All mouse procedures were done in accordance with approved ACUC protocols at JHU. To assess the immune response to synthetic material implants a volumetric muscle loss (VML) injury model was used. C57BL6 mice were given a bilateral critically sized defect to their quadricep femoris that was then treated with saline (injury control) or with polycaprolactone (PCL; < 600mm, MW 50,000) to induce fibrosis. At 6-weeks post injury tissue were harvested. For gene expression studies, whole tissues were homogenized in Trizol Reagent followed by chloroform-ethanol RNA extraction, cDNA synthesis and RT-qPCR using TaqMan primers. For flow cytometry quadriceps with implants were enzymatic and mechanically digested into a single cell suspension, antibody stained, and data collected on a CyTEK Aurora. For T cell characterization the quadriceps were first processed with a percoll density gradient. Significance was determined using two-way ANOVA with Tukey’s posthoc (p < 0.05).


Results, Conclusions, and Discussions:: Flow cytometry analysis of 6-week young (6-8 week old) VML-PCL or VML-Saline (control) male and female muscle shows significant increases in the immune (CD45+) infiltrates in the female fibrosis (PCL) as well as an increase in the percentage of monocytes and dendritic cells while the male samples had a significant increase in macrophages, Figure Panel A. RT-PCR for established genes for fibrosis (Col3a1, S100a4, Acta2), senescence associated secretory proteins and inflammation (Il6, Tgfb1, Il1b, Nlrp3) were all significantly increased in the female FBR. Notably these differences were only observed in the FBR (PCL group) and not within the saline injury control for both flow and gene expression which suggests a material-responsive difference.

With the emerging importance of T cells contributions to the foreign body response as well as the established changes with age, we further characterized T cells in 6-week VML-PCL in both young (6-8 week old) and aged (72-74 week old) male and female mice using flow cytometry. In the young FBR there was a significant increase in CD3 infiltrates in female mice; with age there was no difference by sex but no significant change in number of infiltrating CD3s with age relative to the young. T regulatory cells compose a significantly greater proportion of the CD4 T cell compartment with age and there is also a significant increase in CD8 T cells in the aged male mice, Figure Panel B.

This finding of sex- and age-related differences in the immune response to foreign material highlights the importance of these factors when designing immunomodulatory biomaterials. Future work to characterize the broad immune cell landscape of the FBR across an organisms’ lifespan to determine consistent age and sex related mechanisms, as well as spatial information on these immune populations, can be used to better design biomaterial implants.


Acknowledgements (Optional): : This work was funded in part the Bloomberg ~ Kimmel Institute, the Morton Goldberg Professorship, and by the NIH awards DP1AR076959 and R01EB028796,to JHE and K99AG081564 to JCM

References (Optional): : 1. R. J. Lu et al., Multi-omic profiling of primary mouse neutrophils predicts a pattern of sex and age-related functional regulation. Nat Aging 1, 715-733 (2021).

2. C. C. Bain et al., Rate of replenishment and microenvironment contribute to the sexually dimorphic phenotype and function of peritoneal macrophages. Sci Immunol 5, (2020).

3. W. Kuswanto et al., Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33-Dependent Accumulation of Regulatory T Cells. Immunity 44, 355-367 (2016).