Poster CC7 - Glycolytic macrophages as a promising target to combat medical implant fibrosis.

Introduction: Polymeric implantable medical devices (PIMDs) are integral to modern medicine but are frequently associated with severe fibrotic complications. The resulting requirement for surgical revisions represents a significant burden to patient wellbeing and healthcare spending. The foreign body response (FBR) is the chronic fibrotic reaction that underlies most implant-associated complications. It is defined by prolonged, macrophage-driven inflammation, and the consequent formation of a contracting collagenous capsule around the implant, leading to structural deformations and chronic pain.
Though the clinical manifestations of the FBR are well-documented, therapeutically targeting this reaction is challenged by mechanistic complexities. In particular, the failure of classification systems to comprehensively identify pathological cells in this environment is challenging for the development of novel therapeutic treatments. This limitation is particularly true for multinucleated giant cells (MNCs), fused macrophages believed to drive pathological FBR fibrosis.
Recent developments in immunology have showcased central carbon metabolism as a primary determinant of macrophage function. Glycolytic metabolism is believed to dictate inflammatory behaviors, and oxidative phosphorylation (OXPHOS) defines regenerative wound-healing. In this project, we applied this perspective to the FBR associated with PIMDs, to elucidate macrophage behaviors that lead to undesirable fibrotic outcomes. Using novel flow cytometric tools to characterize metabolic utilization, we characterized implant-associated macrophages and MNCs based on their temporal utilization of glycolysis or OXPHOS, appreciating the role of metabolism in the persistence of implant associated inflammation.

Materials and

Methods: Methodology: Clinically relevant polymeric material discs (4mm x 2mm); poly(dimethylsiloxane) (PDMS), polyethylene (PE), polypropylene (PP) were subcutaneously implanted in male wild-type mice (C57BL6). Implants and associated capsular tissues were excised one-, three-, and six-weeks post-implantation (WPI) for subsequent analysis. Material-specific and temporal fibrotic responses to implanted polymers were characterized with Masson’s Trichrome staining of capsular tissues obtained three- and six-WPI. Capsular thickness was then quantified using ImageJ. RT-qPCR of implant-associated fibrotic gene expression (col1a1, col3a1, fn-1) was used to validate histological assessments.

Characterization of the functional metabolic behavior of peri-implant macrophages associated with PP, PDMS, and PP implants was performed using flow cytometric approaches. Here, capsular tissues were enzymatically digested for single cell analyses. Metabolic utilization of central-carbon metabolism was analyzed using SCENITH, a novel flow cytometry-based tool for characterization of metabolic utilization (10.1016/j.cmet.2020.11.007). Metabolic utilization was further assessed in MNCs using a combination of SCENITH and imaging flow cytometry (ImageStream), using a multinuclearity masking system.

Metabolic utilization was correlated with metabolic enzyme expression profiles using METFLOW (10.1038/s42003-020-1027-9), a flow cytometry-based assessment of inflammation-associated glucose and lipid metabolic pathways. Spatial organization and glycolytic metabolic enzyme expression (GLUT1, LDHA) in capsular macrophages was further assessed using multiplexed OPAL immunofluorescent (IF) histology. Co-localization of glycolytic macrophages (GLUT1+, F4/80+) with pro-fibrotic processes was characterized through co-staining of fibroblast (α-Smooth muscle actin (α-SMA), Vimentin) markers.

Results, Conclusions, and Discussions: Clinically relevant PIMD materials (PP, PE and PDMS implants) were associated with a thick fibrotic capsule. No statistically significant differences in capsular thickness were observed three- or six-WPI. A lack of material-specificity was corroborated with comparable levels of col1a1, col3a1 and fn-1 gene expression 1-6 WPI. These findings support the growing body of evidence suggesting that implant fibrosis is material-independent.

Macrophages (F4/80+ CD11b+, CD45+, Ly6G-) made up the most significant portion of the immune cells across timepoints. Assessment of macrophage central-carbon metabolic utilization indicated the persistence of glycolytic macrophages in all materials over time (Figure 1. A), with capsular tissues assessed six-WPI showing increased rates of glycolysis when compared with earlier timepoints (Figure 1. A). These data point to glycolysis as the dominant metabolic phenotype in persistent capsular macrophages up to six-WPI, contrasting with the canonical metabolic dynamics of resolute wound-healing.

Metabolic enzyme capacity was significantly different from one- to six-WPI. Upregulations in the capacity for glucose transport (GLUT1) and lipid synthesis (ACC1) were noticeable in glycolytic cells. ACC1 upregulation is consistent with the increased requirement for inflammatory cells to expand membrane biosynthesis, and to increase Golgi availability for protein modification. Spatial characterization highlighted the localization of macrophages with high glycolytic capacity (GLUT1High) to the implant-tissue interface (Figure 1. B). Activated fibroblasts (α-SMA+, Vimentin+) co-localized with GLUT1High macrophages, suggesting their role in driving fibrotic outcomes.

MNCs are well established drivers of chronic FBR associated complications. Imaging flow cytometry-based analysis of capsular tissues indicated glycolytic macrophages were larger than oxidative macrophages at each timepoint (Figure 1. C), and isolated analysis of MNCs indicated a nearly exclusive reliance on glycolysis (Figure 1. D). This identifies glycolysis as the primary metabolic pathway employed by MNCs, and presents a new potential therapeutic target to chronic implant inflammation.

Ultimately, this study supports the notion that synthetic polymeric material implants provoke an unresolving immune-reaction, that persists for the duration of the implant-lifecycle. The presence of unresolving material-associated glycolytic macrophages presents a novel potential therapeutic target in modulating PIMD complications.

Acknowledgements (Optional):