Activation of dendritic cells by crosslinked collagen hydrogels (artificial corneas) varies with their composition

Abstract

Activated T cells are known to promote fibrosis, a major complication limiting the range of polymeric hydrogels as artificial corneal implants. As T cells are activated by dendritic cells (DC), minimally activating hydrogels would be optimal. In this study, we evaluated the ability of a series of engineered (manufactured/fabricated) and natural collagen matrices to either activate DC or conversely induce DC apoptosis in vitro. Bone marrow DC were cultured on a series of singly and doubly crosslinked hydrogels (made from recombinant human collagen III [RHCIII] or collagen mimetic peptide [CMP]) or on natural collagen‐containing matrices, MatrigelTM and de‐cellularised mouse corneal stroma. DC surface expression of major histocompatibility complex Class II and CD86 as well as apoptosis markers were examined. Natural matrices induced low levels of DC activation and maintained a “tolerogenic” phenotype. The same applied to singly crosslinked CMP‐PEG gels. RHCIII gels singly crosslinked using either N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide with the coinitiator N‐hydroxy succinimide (EDC‐NHS) or N‐cyclohexyl‐N‐(2‐morpholinoethyl)carbodiimide metho‐p‐toulenesulfonate with NHS (CMC‐NHS) induced varying levels of DC activation. In contrast, however, RHCIII hydrogels incorporating an additional polymeric network of 2‐methacryloyloxyethyl phosphorylcholine did not activate DC but instead induced DC apoptosis, a phenomenon observed in natural matrices. This correlated with increased DC expression of leukocyte‐associated immunoglobulin‐like receptor‐1. Despite low immunogenic potential, viable tolerogenic DC migrated into and through both natural and manufactured RHCIII gels. These data show that the immunogenic potential of RHCIII gels varies with the nature and composition of the gel. Preclinical evaluation of hydrogel immunogenic/fibrogenic potential is recommended.

TGF‐β1‐activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full‐thickness hydrogel transplantation

“…As expected, the effects were lost in the presence of TGF‐β1 neutralising antibody blocking downstream TGF‐β1 effects (Figure ), or pro‐inflammatory LPS, provoking classical DC activation thereby mimicking the “pro‐inflammatory EDC‐NHS hydrogel effect” discovered earlier (Figures and ). In agreement with those in vitro data presented in our related report (Mölzer et al, ), the above results emphasise the importance of hydrogel composition in graft outcome. Drawing from the evidence presented in this report, we suggest that CMC‐NHS gels provide a more tolerising environment to DC, causing less inflammation, hence supporting wound healing and graft integration.…”

“…We also report that the CMC-NHS gel was associated with RCM formation and wound healing, with markedly lower levels of tenascin c expression in the epithelial and subepithelial RCM stroma. We have shown in vitro CMC-NHS gels promote "tolerant" DC (tolDC) that express lower levels of costimulating molecules along with increased levels of apoptosis (Mölzer et al, 2019), and demonstrate here that CMC-NHS induced tolDC only in low numbers. Dendritic cells can directly impact fibroblast-mediated production of tenascin c. As this protein is secreted by keratinocytes and fibroblasts involved in tissue regeneration (Midwood & Orend, 2009;Pearson, Pearson, Shibahara, Hofsteenge, & Chiquet-Ehrismann, 1988), we are therefore proposing an association between DC and tissue fibrosis.…”

“…Both cross‐linked hydrogel types were manufactured to give comparable mechanical and optical properties and were functionally equivalent in mouse models as orthotopic grafts (Ahn et al, ), with CMC providing more controllable reaction kinetics. However, in companion studies examining the effects of the two differentially cross‐linked hydrogels on DC in vitro, we found that EDC‐cross‐linked hydrogels promoted maturation of immune competent DC, whereas CMC‐cross‐linked hydrogels promoted a tolerising DC phenotype (Mölzer et al, ).…”

“…In the context of corneal hydrogel implants, we propose in an accompanying report that differences in hydrogel properties may result in the differential recruitment of and/or recognition by immune cells including DC (Mölzer et al, ). This may in turn differentially impact DC‐directed responses of fibroblasts, epithelial cells, keratinocytes, and smooth muscle cells involved in tissue repair.…”

“…We previously examined the effects of EDC versus CMC on the activation or maturation status of corneal DC and suggested a possible role in the wound healing responses, in maintaining tissue homeostasis, and their possible involvement in directing tissue repair (Ahn et al, 2013). In corneal epithelial wound repair, bidirectional interactions between intraepithelial DC and epithelial cells lead to enhanced wound closure mediated by the chemokine CXCL10, thymic stromal lymphopoeitin, or interleukin-1β, whereas depleting corneal DC delayed this outcome (Gao, Yin, Yoon, Mi, & Yu, 2011) In the context of corneal hydrogel implants, we propose in an accompanying report that differences in hydrogel properties may result in the differential recruitment of and/or recognition by immune cells including DC (Mölzer et al, 2019). This may in turn differentially impact DC-directed responses of fibroblasts, epithelial cells, keratinocytes, and smooth muscle cells involved in tissue repair.…”