(J-388) Antigen Presentation and Adaptive Immune Response to Muscle Injury and Biomaterial Implantation

Introduction:: Biomaterial implants are being developed as a treatment to potentially restore tissue function after an injury overwhelms the innate regenerative capacity of a tissue. However, more investigation is needed into characterizing tolerogenic or inflammatory immune responses to such materials after surgery and implantation. Previously, a subset of cross-presenting dendritic cells (DCs) has been identified as enriched in mice after receiving pro-regenerative materials such as decellularized ECM, suggesting a role for DCs in self-tolerance and tissue regeneration. It is known that cytokines produced by DCs, such as TGF-β induce self-tolerance, but little is known about the transcriptional landscape of DCs after injury and biomaterial implantation. Thus, it is important understand the differential gene expression of DCs and clonal diversity in T and B cells, which could lead to favorable outcomes such as collagen remodeling, tissue regeneration, and possibly restoration of tissue function. To accomplish this, single cell RNA sequencing was used to determine how biomaterials drive differential recruitment and the transcriptional landscape of various DC subsets in muscle tissue.

Materials and Methods:: Female C57BL/6 mice underwent volumetric muscle loss (VML) surgery, involving removing a 3mm piece of quadricep muscle bilaterally, and received 50µL of saline, 50µL decellularized extracellular matrix (ECM), or 50µl polyethylene (PE) particles in the wound. At 1-week post-surgery, muscles were removed and diced finely prior to digestion in 0.5 mg/ml Liberase TL with 0.2 mg/ml DNAse for 45 minutes in serum-free DMEM at 37C. Resultant material was mechanically pressed through a 70 µm cell strainer and centrifuged at 350 xg for 5 minutes. The samples were washed, stained with an antibody cocktail, and transferred to FACS tubes for sorting. Dendritic cells (Live CD45+ SiglecF- F4/80- Cd11blo CD11c+ MHCII+) in muscle tissue were sorted using a BD FACS Melody sorter. The sorted cells were then frozen under liquid nitrogen prior to single cell RNA sequencing (10xGenomics, Azenta Life Sciences). Raw sequencing data was then analyzed in R.

Results, Conclusions, and Discussions:: After clustering, co-expression of key genes specific to DC subsets confirmed the identities of the main populations, including plasmacytoid DCs (Siglech, Ccr9, Ly6d, Lys6a), cDC1s (Xcr1, Clec9a, Cadm1, Batf3), cDC2s (Clec10a, Sirpa, Itgam, Notch2), and mregDCs (Cd274, Pdcd1lg2, Cd200, Cd40, Ccr7, Il12b). Enrichment of the mregDC cluster did not change significantly between ECM and PE groups, but ECM and PE treatment groups displayed cDC1s with different phenotypes. ECM cDC1s, compared to those found in the PE treatment group, displayed upregulation of anti-inflammatory genes Tnip3 and Tnfaip8, and downregulation of pro-fibrotic Il1b as well as Irf7. Similarly, anti-inflammatory genes such as Arg1, Retnla, and Slpi were enriched in the ECM pDC population, while in the PE pDC population, Il1b and Cd14 were found to be upregulated. Additionally, a unique CD4+CD8+ pDC population was discovered in the ECM group which showed significant expression of Ccr9 compared to PE (p < 0.01). Also unique to the ECM treatment group was a myeloid macrophage-like DC population with large variety of scavenger receptors (as indicated by Cd14, Mrc1, Cd36, Clec7a). The ECM treatment group at large, compared to PE, displayed significant upregulation of the anti-inflammatory gene Ccl6, Clu (known to be involved in apoptotic cell efferocytosis and play a protective role in autoimmune diseases and inflammation), and Hbegf, which enables growth factor activity and heparin binding activity during wound healing (p ≤ 0.01). PE treatment resulted in upregulation of the pro-inflammatory gene Gpr84, Map4k3, which has been linked to induction of autoimmune responses, and Fstl1, a gene that enhances antigen presentation ability in DCs (p < 0.01). The behavior of these DC populations reveals a possible mechanism for inflammation mediation, which in the future may be harnessed to develop therapeutics or immunomodulatory materials for tissue regeneration and medical device implantation.

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