N. Nanda1,2, V. Akondy1, P. Bommekal1, A. Degala1, S. Keswani1, I. Huddle1, S. Sinha1, H. Li1, L. Yu1, M. Guerra1, S. Keswani1, S. Balaji1 1Baylor College Of Medicine, Department Of Pediatric Surgery, Houston, TX, USA 2Rice University, Wiess College Of Natural Sciences, Houston, TX, USA
Introduction: Scarring outcomes differ vastly among patients after similar skin injuries, and the underlying cause of this heterogeneity is not yet understood. We have shown a role for CD4+ T lymphocytes in regulating fibrosis and scarring, with CD4+CD44high+ Tr1 overexpression inducing regenerative wound healing. T cells depend on IL-2 for activation and function. Specifically, we have shown that regulatory T cells use their heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) for activation and function in vivo. However, the role of IL-2-HS-dependent activation of Tr1 in patient scarring heterogeneity is unknown. We hypothesize that differential cytokine repositories in wound milieu regulate CD4+ T cells, thereby affecting scarring outcomes.
Methods: We tested two models of variable scarring. Model 1- Skin sections from patients with clinically stratified scarring, i.e. high vs. low scarring properties based on Vancouver Scar Scoring >6 or <3, respectively, from our biobank. Model 2 – Small (2mm) and large (6mm) dorsal excisional stented skin wounds in 8-10 wk C57B/6J mice that heal with low vs. higher scarring, respectively, were tested at days 3 and 7 post-wounding. 5um sections were colabelled with HS and IL-2. Heparinase (I and III) treatment followed by staining determined colocalization of HS and IL-2. Quantitative analysis using ImageJ was completed to count double-positive T cells for HPSE and CD4. n=3 wounds per group.
Results: High scarring patient tissues had greater expression of HS, specifically in the epidermis and localized around blood vessels in the dermis. Both HS and IL2 were expressed in greater quantities in the basement membrane, compared to low scar patient tissues. Treatment with heparinase to enzymatically digest HS reduced the expression of both HS (as expected) and also IL-2, suggesting that IL-2 is co-localized to HS in the ECM. Hyaluronidase control did not reduce HS or IL-2. High scar patient tissues had increased CD4+ T cells. In murine wounds, qualitative assessment of staining suggested greater expression of HS and IL2 in larger 6mm wounds compared to 2mm, and there was increased expression on day 7 compared to day 3. Quantitative analysis of HPSE and CD4 double positive cells showed more numbers in 6mm wounds compared to 2mm wounds at day 3. 2mm wounds showed increased cell counts from day 3 to 7, whereas the 6mm wounds reached that level by day 3.
Conclusion: This data shows first-time evidence that cytokine repository of IL-2 sequestration by HS in the wound milieu has a direct correlation with CD4+ lymphocyte activation and function and scarring outcomes. These data can be leveraged to develop novel anti-scarring therapeutic strategies.