Activation of MAPK/ERK signalling in Merlin-null Schwann cells leads to increased and sustained immune cell infiltration in the peripheral nervous system
Abstract Aims Previous work has shown that increased numbers of macrophages are associated with more rapid schwannoma tumour growth and we are interested in signals that control entry of macrophages and other immune cells into these tumours. Activation of the Raf-kinase domain and the Raf/MEK/ERK pathway within Schwann cells has been observed to induce an inflammatory response in peripheral nerves in the absence of injury. Activation of an inducible Raf-kinase transgene in Schwann cells allows modelling of acute demyelination of peripheral nerves without nerve injury. This Raf-oestrogen receptor fusion protein (Raf-TR) is activated by the oestrogen analogue Tamoxifen and so allows targeted, controlled activation of the Raf/MEK/ERK pathway within the Schwann cells. Here, in order to understand drivers of tumour formation, we assess the effect of MAPK activation in Merlin-null Schwann cells upon immune cell infiltration within the PNS. Method RafTR-P0CRE-NF2fl/fl mice of 4-6 weeks age were injected daily (IP) with 2mg of 4-hydroxy-tamoxifen or vehicle (corn oil) control for 5 consecutive days. RafTR was activated on either a Merlin (NF2) wild-type (NF2 fl/fl, P0-CRE-) or NF2 null (NF2 fl/fl, P0-CRE+) background and effects on immune cell infiltration studied in each condition. Immunofluorescence was performed in the dorsal root ganglia (DRGs) and sciatic nerves of mice to identify various immune cell infiltrates at various timepoints. These will include neutrophils, mast cells, T-Cells and macrophages using the cell markers Csf3r, C-kit, CD3 and IBA1 respectively. Results At 21 days post treatment, a significantly increased infiltration of macrophages within the sciatic nerve and dorsal root ganglia was observed in mice treated with Tamoxifen when compared to vehicle controls. Loss of NF2 led to a massive increase in the number of macrophages recruited to peripheral nerves in tamoxifen-treated mice compared to Cre- mice and Cre+ treated with vehicle alone. Further assessment of other immune cell infiltration including neutrophils, mast cells and T cells are ongoing. Conclusion Raf/MEK/ERK signalling, in the absence of tumour suppressor Merlin, significantly increases the infiltration of inflammatory cells such as macrophages into peripheral nerves even in the absence of injury. As this effect is enhanced in NF2 null mice, this suggests that Merlin plays an important role in inhibiting the inflammatory response in peripheral nerves. It also suggests that Merlin could be involved in maintaining the blood nerve barrier (BNB), as in its absence the greater influx of immune cells into the nerves and DRGs suggests a more complete loss of BNB function than just activation of the Raf/MEK/ERK cascade alone.
