Discovery Of Unique Immune Cell Subsets During The Development Of Capsular Fibrosis
Britta A. Kuehlmann, M.D., PhD, Melanie Rodrigues, PhD, Geoffrey C. Gurtner, M.D., FACS.
Stanford University School of Medicine, Palo Alto, CA, USA.
PURPOSE: Capsular fibrosis is the most common long-term complication after breast implant-based augmentation. The cellular alterations that underlie capsular fibrosis are still barely understood. The identification of cell types in the implant area can provide clues in determining the pathophysiology of the fibrotic changes to find a medical cure. Here, using an in vivo mouse model with silicone implants we analyze the cell types present in the capsule over time. Next, we use single cell analysis to characterize these cells to determine their involvement in fibrotic capsule development. METHODS: Capsular fibrosis was induced by inserting a customized silicone implant (same shape and filling as a human silicone breast implant) in C57/BL6 mice. A paravertebral incision was performed on the dorsum of the mice and the implant was placed in a subcutaneous pocket. Explantation was done on day 15, day 30 and day 90 by resecting the implant with the surrounding capsule en-block for SEM and TEM. The capsule itself was digested and cells were immunostained with CD45, EpCam, CD31, CD11b, F4/80 and Col I. Cells were subjected to further analysis by fluorescence-activated cell scanning (FACS). Single cells were sorted and subjected to single cell transcriptional analysis. H&E, Trichrome and Picosirius Red as well as immuno-staining were performed on the capsules.
RESULTS: FACS analysis revealed that the major cell types in the fibrotic capsule across all time-points were immune cells, not fibroblasts or endothelial cells. The immune cells could be classified as: (i)CD45+/CD11b+/F4/80+ (macrophages), (ii)CD45+/CD11b+/F480- (myeloid cells, including dendritic cells) and (iii)CD45+/CD11b-/F4/80- cells. On day 15, the maximum number of cells was CD45+/CD11b+/F4/80+ (mean:76.61%, SEM:5.13%) and there were no cells that were CD45+/CD11b-/F4/80-. On day 30 the CD45+/CD11b+/F4/80+ cells were reduced and the CD45+/CD11b+/F4/80- cells were the statistically significant population (mean:57.35%, SEM:8.78%). The most interesting finding was that CD45+/CD11b-/F4/80- cells that were not found at earlier time-points were seen on day 90 (mean: 27.2%, SEM:3.67%) and there was a further reduction in the macrophage population. The macrophages were found to deposit Col1 both by FACS and immunostaining of sections. Scanning electron microscopy and Transition electron microscopy were used to confirm the presence of these immune cells and collagen deposition. We are currently using single cell analysis to understand the identity of the CD45+/CD11b-/F4/80- cells that arise in the capsule at day 90.
CONCLUSIONS: For the first time, our results reveal that the most prominent cells at every time-point in the capsule are immune cells. Macrophages comprise the largest subset of immune cells at day 15 but the macrophage numbers decrease subsequently. At day 30 and day 90 CD45+/CD11b+/F4/80- myeloid cells, most likely dendritic cells, comprise the largest immune subset. Importantly on day 90, CD45+/CD11b-/F4/80-, most likely T cells arise in the capsule. We are currently conducting single cell and functional studies on these immune subsets to understand their identity and role in capsule formation over the course of time. Our findings have promising therapeutic implications for the treatment of capsular fibrosis.
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