No abstract

Medical devices and implants made of synthetic materials can induce an immune-mediated process when implanted in the body called the foreign body response, which results in formation of a fibrous capsule around the implant. To explore the immune and stromal connections underpinning the foreign body response, we analyzed fibrotic capsules surrounding surgically excised human breast implants from 12 individuals. We found increased numbers of interleukin 17 (IL17)–producing γδ+ T cells and CD4+ T helper 17 (TH17) cells as well as senescent stromal cells in the fibrotic capsules. Further analysis in a murine model demonstrated an early innate IL17 response to implanted synthetic material (polycaprolactone) particles that was mediated by innate lymphoid cells and γδ+ T cells. This was followed by a chronic adaptive CD4+ TH17 cell response that was antigen dependent. Synthetic materials with varying chemical and physical properties implanted either in injured muscle or subcutaneously induced similar IL17 responses in mice. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to implanted synthetic materials and the development of p16INK4a senescent cells. IL6 produced by senescent cells was sufficient for the induction of IL17 expression in T cells. Treatment with a senolytic agent (navitoclax) that killed senescent cells reduced IL17 expression and fibrosis in the mouse implant model. Discovery of a feed-forward loop between the TH17 immune response and the senescence response to implanted synthetic materials introduces new targets for therapeutic intervention in the foreign body response.

Biomaterials induce an immune response and mobilization of macrophages, yet identification and phenotypic characterization of functional macrophage subsets in vivo remain limited. We performed single-cell RNA sequencing analysis on macrophages sorted from either a biologic matrix [urinary bladder matrix (UBM)] or synthetic biomaterial [polycaprolactone (PCL)]. Implantation of UBM promotes tissue repair through generation of a tissue environment characterized by a T helper 2 (TH2)/interleukin (IL)–4 immune profile, whereas PCL induces a standard foreign body response characterized by TH17/IL-17 and fibrosis. Unbiased clustering and pseudotime analysis revealed distinct macrophage subsets responsible for antigen presentation, chemoattraction, and phagocytosis, as well as a small population with expression profiles of both dendritic cells and skeletal muscle after UBM implantation. In the PCL tissue environment, we identified a CD9hi+IL-36γ+ macrophage subset that expressed TH17-associated molecules. These macrophages were virtually absent in mice lacking the IL-17 receptor, suggesting that they might be involved in IL-17–dependent immune and autoimmune responses. Identification and comparison of the unique phenotypical and functional macrophage subsets in mouse and human tissue samples suggest broad relevance of the new classification. These distinct macrophage subsets demonstrate previously unrecognized myeloid phenotypes involved in different tissue responses and provide targets for potential therapeutic modulation in tissue repair and pathology.

Conflict of interest: DZ is an inventor of a pending patent application for BCL-xL inhibitors and targeted senolytic agents for age-related diseases (Compounds that induce degradation of anti-apoptotic BCL-2 family proteins and the uses thereof, no. US20190135801). DZ and JC are cofounders, advisors for, and shareholders of Unity Biotechnology, which develops senolytic agents. JHE is an equity holder of Unity Biotechnology, receives patent royalties through Johns Hopkins University School of Medicine (Unit dose of a cis-imidazoline for treating an osteoarthritic joint by removing senescent cells, no. US9849128B2), and is a founder and equity holder of Aegeria Soft Tissue (AST). DMP is a consultant for Aduro Biotech, Amgen, Astra Zeneca, Bayer, DNAtrix, Dynavax Technologies, Ervaxx, FLX Bio, Janssen, Merck, and Rock Springs Capital. DMP also provides research support and consulting to Compugen and Bristol Myers Squibb and receives patent royalties through Johns Hopkins University School of Medicine. DMP is on the scientific advisory board of Camden Nexus II; is on the board of directors of and owns stock in Dracen Pharmaceuticals, Five Prime Therapeutics, and WindMIL Therapeutics; is a consultant for Immunomic Therapeutics and Tizona Therapeutics; has equity and patent royalties through Johns Hopkins University School of Medicine (Use of multivalent chimeric peptide-loaded, MHC/lg molecules to detect, activate or suppress antigen-specific, T cell-dependent immune responses; no. PCT/US98/18909; Manipulation of regulatory T cells using LAG-3 antibodies, genes, agonists and antagonists, no. PCT/US04/006133; Microsatellite instability as a pharmacogenomic marker of therapeutic response to immune checkpoint inhibition, no. PCT/US2015/060331; Immune checkpoint chimeric antigen receptors (CAR) therapy, no. PCT/US2016/040010; Marrow infiltrating lymphocytes (MILs) as a source of T-cells for chimeric antigen receptor (CAR) therapy, no. PCT/US2016/041521; Combination of immunotherapy with local chemotherapy for the treatment of malignancies, no. PCT/US2016/028861; Use of SON-211 and Ontak as a regulatory T cell targeted cancer immunotherapy in combination with anti-PD-1 therapy for the treatment of cancer, no. PCT/US2019/020959); and has founder equity in Potenza Therapeutics and Trieza Therapeutics.

Biomaterials serve as the basis of implants, tissue engineering scaffolds, and multiple other biomedical therapeutics. New technologies, such as single cell RNA sequencing (scRNAseq), are enabling characterization of the biomaterial response to an unprecedented level of detail, facilitating new discoveries in the complex cellular environment surrounding materials. We performed scRNAseq and integrated data sets from multiple experiments to create a single cell atlas of the biomaterials response that contains 42,156 cells from biological extracellular matrix (ECM)-derived and synthetic polyester (polycaprolactone, PCL) scaffold biomaterials implanted in murine muscle wounds. We identified 18 clusters of cells, including natural killer (NK) cells, multiple subsets of fibroblasts, and myeloid cells, many of which were previously unknown in the biomaterial response. To determine intra and intercellular signaling occurring between the numerous cell subsets, including immune-stromal interactions in the biomaterial response, we developed Domino (github.com/chris-cherry/domino), a computational tool which allows for identification of condition specific intercellular signaling patterns connected to transcription factor activation from single cell data. The Domino networks self-assembled into signaling modules and cellular subsets involved in signaling independent of clustering, defining interactions between immune, fibroblast, and tissue-specific modules with biomaterials-specific communication patterns. Further compilation and integration of biomaterials single cell data sets will delineate the impact of materials chemical and physical properties and biological factors, such as anatomical placement, age, or systemic disease, that will direct biomaterials design.

AbstractSynthetic biomaterials and medical devices suffer to varying levels from fibrosis via the foreign body response (FBR). To explore mechanistic connections between the immune response and fibrosis from the FBR, we first analyzed fibrotic capsule surrounding human breast implants and found increased numbers of interleukin (IL)17-producing γδ+T cells and CD4+TH17 cells as well as senescent cells. Further analysis in a murine model demonstrated an early innate IL17 response to synthetic implants, mediated by innate lymphoid cells and γδ+T cells, was followed by a chronic adaptive antigen dependent CD4+TH17 cell response. Mice deficient in IL17 signaling established that IL17 was required for the fibrotic response to materials and the development of p16INK4asenescent cells. Treatment with a senolytic agent reduced IL17 expression and fibrosis. Discovery of a feed-forward loop between the TH17 and senescence response to synthetic materials introduces new targets for therapeutic intervention in the foreign body response.

Soft tissue reconstruction remains an intractable clinical challenge as current surgical options and synthetic implants may produce inadequate outcomes. Soft tissue deficits may be surgically reconstructed using autologous adipose tissue, but these procedures can lead to donor site morbidity, require multiple procedures, and have highly variable outcomes. To address this clinical need, we developed an “off-the-shelf” adipose extracellular matrix (ECM) biomaterial from allograft human tissue (Acellular Adipose Tissue, AAT). We applied physical and chemical processing methods to remove lipids and create an injectable matrix that mimicked the properties of lipoaspirate. Biological activity was assessed using cell migration and adipogenesis assays. Characterization of regenerative immune properties in a murine muscle injury model revealed that allograft and xenograft AAT induced pro-regenerative CD4+ T cells and macrophages with xenograft AAT additionally attracting eosinophils secreting interleukin 4 (Il4). In immunocompromised mice, AAT injections retained similar volumes as human fat grafts but lacked cysts and calcifications seen in the fat grafts. The combination of AAT with human adipose-derived stem cells (ASCs) resulted in lower implant volumes. However, tissue remodeling and adipogenesis increased significantly in combination with ASCs. Larger injected volumes of porcine-derived AAT demonstrated biocompatibility and greater retention when applied allogeneicly in Yorkshire cross pigs. AAT was implanted in healthy volunteers in abdominal tissue that was later removed by elective procedures. AAT implants were well tolerated in all human subjects. Implants removed between 1 and 18 weeks demonstrated increasing cellular infiltration and immune populations, suggesting continued tissue remodeling and the potential for long-term tissue replacement.

The avascular nature of cornea tissue limits its regenerative potential, which may lead to incomplete healing and formation of scars when damaged. Here, we applied micro- and ultrafine porcine urinary bladder matrix (UBM) particulate to promote type 2 immune responses in cornea wounds. Results demonstrated that UBM particulate substantially reduced corneal haze formation as compared to the saline-treated group. Flow cytometry and gene expression analysis showed that UBM particulate suppressed the differentiation of corneal stromal cells into α-smooth muscle actin–positive (αSMA+) myofibroblasts. UBM treatments up-regulated interleukin-4 (IL-4) produced primarily by eosinophils in the wounded corneas and CD4+ T cells in draining lymph nodes, suggesting a cross-talk between local and peripheral immunity. Gata1−/− mice lacking eosinophils did not respond to UBM treatment and had impaired wound healing. In summary, stimulating type 2 immune responses in the wounded cornea can promote proregenerative environments that lead to improved wound healing for vision restoration.