Three Unique Macrophage Subsets Drive Development of Capsular Fibrosis Through Collagen Deposition
Britta A. Kuehlmann, MD, PhD, Clark A. Bonham, BS, Geoffrey C. Gurtner, MD.
Stanford, Stanford, CA, USA.
Purpose- Biomedical implants are used to restore, maintain and monitor bodily function in response to a wide variety of diseases and injuries. The body's immune system attempts to destroy these foreign bodies through the foreign body response (FBR). As cells incapable of eliminating implants, a prolonged inflammatory response ensues, causing an irregular tissue healing response in the skin. A fibrotic, disorganized capsule forms around the implant in a process known as capsular fibrosis (CF). These capsules cause complications and often require corrective surgery.
Methods- We have attempted to discover the major cellular constituents that drive fibrotic development in CF. Using a novel murine model, we placed small silicone implants subcutaneously in the dorsa of Bl6-mice. After 90 days, we harvested the capsular tissue surrounding the implants and isolated the cells. Cells were then subjected to fluorescence-activated cell sorting (FACS), where they were sorted into single cells. Single cells were subjected to quantitative polymerase chain reaction (qPCR) and single cell RNA sequencing to determine their genetic profiles and function.
Results- FACS analysis yielded distinct cell populations within the fibrotic capsule, with CD45+/ CD11b+ macrophages being among the most prevalent. Notably, these cells appeared to be depositing collagen. To further investigate, we subjected the entire capsule at day 90 to collagenase digestion and subjected all cells to single cell RNA-sequencing without FACS separation. Again, macrophages were found to be the predominant cell type in the capsule, with three macrophage subsets identified. All subsets expressed the hematopoietic marker Ptprc (Cd45), the myeloid marker Itgam (Cd11b) and the monocyte marker Cd14. The first macrophage subset (Subgroup 1) uniquely expressed the surface marker and phagocytic receptor Cd36 known to promote fibrogenic pathways in other forms of organ fibrosis. Subgroup 1 is characterized by a highly inflammatory genetic profile, regulating cell signal production associated with acute inflammation. The second macrophage subset (Subgroup 2) distinctly expressed the surface marker Cd209, which is essential to recognizing foreign bodies, and inciting an inflammatory response. Macrophages in group 2 display an array of genes that contribute to acute inflammation. Subgroups 1 and 2 both expressed Adgre1, a gene that encodes F4/80, a well-known marker of murine macrophage populations. The third macrophage subset (Subgroup 3) maintained a genetic profile based on its unique expression of Ccr2. Further analysis of the highly expressed genes in the 3 subgroups displayed that each group contributes to the induction of inflammation through pro-fibrotic and phagocytic activity and signaling.
Conclusions- According to current literature, fibroblasts are the source of collagen deposition in the FBR. We have found three subgroups that drive fibrosis and deposit collagen and ECM themselves. We are currently conducting immunofluorescence stains to
verify the cells' colocalization with deposited collagen and determine each group's function. Our findings have promising therapeutic implications for the treatment of skin fibrosis.
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