T Cells Contribute To Peripheral Nerve Regeneration Through Acellular Nerve Allografts Via Regulation Of Il-4
Deng Pan, Daniel Hunter, Lauren Schellhardt, Anja Fuchs, PhD, Haiying Zhou, PhD, Sally Jo, Katherine Santosa, MD, Alison Snyder-Warwick, MD, Mikhail Berezin, PhD, Susan Mackinnon, MD, Matt Wood, PhD.
Washington University in St Louis, Saint Louis, MO, USA.
Purpose: Peripheral nerve injury remains a significant public health issue. Traumatic nerve injuries often necessitate surgical repair with nerve grafts. While autologous nerve grafts are the clinical standard, acellular nerve allograft (ANAs) have been increasingly used. ANAs are prepared from nerve obtained from deceased donors treated with detergents to remove cellular debris and antigenic components. While it has the advantage of being available off-the-shelf, its ability to promote axon regeneration across a long nerve gap is limited. In this study, we evaluate why nerve regeneration across long ANAs is limited.
Method: For both rats and mice, we utilized a sciatic nerve transection with ANA graft as model of nerve repair. Two cm (short) and 4 cm (long) ANAs were used in rats. Grafts were analyzed using immunohistochemistry, RT-PCR, histomorphometry, and flow cytometry. For mice 1 cm grafts were used in all repairs.
Results: At 8 weeks, rats that were repaired using 2 cm ANAs regenerated significantly more axons than those that received 4 cm ANAs (1a). At 4 weeks, long ANAs contained fewer Schwann cells, and those Schwann cells were less proliferative (1b). Interestingly, T cells within long ANAs were significantly fewer than those in short ANAs (1d), and angiogenesis was also reduced (1c). To test if T cells impacts regeneration, we utilized RNU rats, which are T cell deficient. Eight weeks after nerve repair using short ANAs, we found that the RNU+/- rats (T cell sufficient) had significantly more regenerated axons than the RNU-/- rats (1e). In mice deficient in T cells (Rag1-/-), we observed significant fewer myelinated axons compared to wildtype (WT) control (1f). We also observed reduced Schwann cell accumulation and blood vessels in the Rag1-/- mice compared to WT (1g), with no significant alterations to macrophages (data not shown). T cell related cytokines, including IL-4 were significantly down-regulated in ANAs from Rag1-/- mice compared to WT (1h). Using IL-4GFP mice, in which cells secreting IL-4 also express GFP, we found that appearance of GFP+ cells on day 14 post surgery correlates with increase in T cells (1i). However, majority of IL-4+ cells were eosinophils (SiglecF+), suggesting T cell's role in regulating rather than secreting IL-4 (1j). Finally, utilizing an IL-4 knockout mice, we found that loss of IL-4 resulted in reduced myelinated axons, as well as reduced angiogenesis (1k), suggesting an IL-4 action on macrophages (no changes in macrophages were observed, data not shown). As therapeutic strategy, we demonstrate that IL-4 incorporated within ANAs can be sustained for release for more than 7 days (1l). Incorporation of IL-4 within ANAs resulted in increased angiogenesis and nerve regeneration (1m).
Conclusion: Our study uncovered the importance of T cells in promoting nerve regeneration. We showed that T cells regulate IL-4 via recruitment of eosinophils, and that loss of IL-4 impact regeneration due to loss of angiogenesis.
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