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Interferon-γ (IFN-γ) inhibition of human keratinocyte migration in vitro and pediatric-foreskin Langerhans Cell polarization of T-cells away from IFN-γ may influence wound healing
Aviva C. Berkowitz, BA1, Jie Chen, MD1, Hideki Fujita, MD, PhD2, Hiroshi Mitsui, MD, PhD3, James G. Krueger, MD, PhD3, John A. Carucci, MD, PhD2, Diane Felsen, PhD1, Dix P. Poppas, MD1.
1Weill Cornell Medical College, New York, NY, USA, 2NYU Langone Medical Center, New York, NY, USA, 3Rockefeller University, New York, NY, USA.

BACKGROUND: It is well-known that pediatric wounds from urologic surgery heal better and faster than those of adults. Understanding the differences in molecular events associated with adult and pediatric wound healing could lead to novel interventions in wound healing. IFN-γ has been shown to inhibit wound healing by decreasing collagen deposition by fibroblasts. Interleukin-22 (IL-22) is known to enhance keratinocyte migration and proliferation. We sought to compare the direct effects of these cytokines on both pediatric and adult keratinocytes. METHODS: Using pediatric foreskins and normal adult human skin from surgical discard, we isolated epidermal Langerhans Cells (LCs) from acutely wounded samples and measured the degree of T-cell polarization toward IFN-γ producing CD8+ T-cells with flow cytometry. We examined the effects of IFN-γ and IL-22 on migration of an adult human keratinocyte cell line [HaCaT; n=4] and on primary pooled neonatal keratinocytes [HEKn; n=3] using an in vitro scratch assay, and examined receptor expression for IFN-γ and IL-22 on both cell types with RT-PCR and Western Blot. We also examined the effect of IFN-γ on HaCaT proliferation and apoptosis. RESULTS: We found that LCs from pediatric tissue polarized a smaller percentage of T-cells toward IFN-γ producing CD8+ T-cells compared to those from adult skin (Pediatric: 6.95% of T-cells present; Adult: 59.9%). Using HaCaT and HEKn cells, we found that IFN-γ caused a marked decrease in migration in both cell types [all rates reported as pixels/hour: HaCaT at 40 hours post-scratch (T40): Control-28,075±931; IFN-γ 20ng/mL-10,158±2,749 (p<0.0001). HEKn at T32: Control-28,367±3,555; IFN-γ 20ng/mL-11,236±335 (p<0.05)]. Similar numbers of control and IFN-γ treated live HaCaTs were found immediately post-scratch (T0) (T0 Control: 1.83x 106; T0 IFN-γ 20ng/mL 1.80 x 106) and at T40 (3.29 x 106 vs. 3.11 x 106). Similar TUNEL staining was found in control and IFN-γ treated HaCaTs at T40. Conversely, IL-22 was observed to enhance migration [Rate, HaCaT, T24: Control-28,983±4,779; IL-22 20ng/ml-36,656±3,165 (p<0.05). Rate, HEKn, T32: Control-14,942±2,721; IL-22 20ng/ml-22,075±4,600 (p<0.05)]. Expression of IFN-γ and IL-22 receptor subunits was similar in both cell types. CONCLUSIONS: IFN-γ is known to affect wound healing by inhibiting collagen deposition; we demonstrate herein that IFN-γ may directly affect wound healing by inhibiting keratinocyte migration. IFN-γ does not induce apoptosis or proliferation in HaCaTs. We also highlight a potential role for IL-22 in enhancing wound healing via increased keratinocyte migration. While the receptors of both cytokines are present in neonatal and adult cells and the observed effects of these cytokines were similar for both cell types, the presence of fewer IFN-γ producing T-cells (due to decreased polarization by LCs in pediatric skin) may contribute to better pediatric wound healing. Expansion of these studies to in vitro skin organ culture or an in vivo human skin transplant model [Petratos et al, PlastReconstrSurg 111:1988-97, 2003] can provide more direct evidence as to the role of these cytokines in both pediatric and adult wound healing, with the goal of translation into clinically useful treatments for dysfunctional wound healing.

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