Figure 4. NRR1 inhibition of Notch1 dampens the immune cell response after injury.

Punch-wounded wildtype mice injected i.p. with 5 mg kg⁻¹ NRR1 (a–e), NRR2 (e) or IgG isotype control (a–d) antibodies for 7 days before injury. (b) Wound openness was measured as a per cent of initial wound size at 2 and 5 dpw. NRR1-treated wounds were significantly more open 2 dpw (P=0.039; *) but not 5 dpw. (c–e) Dorsal back skin from punch-wounded NRR1 or control IgG treated mice was collected 2 or 5 dpw. Single cell suspensions of whole back skin were co-stained with antibodies against Lineage markers (Lin), CD45, CD3, CD11b, CD25, ST2, NK1.1, CD127, F4/80 and/or RORγt prior to analysis by flow cytometry. Cells were initially gated on the basis of CD45 expression and immune cell numbers quantified on the basis of expression of diagnostic surface markers: CD11b (polymorphonuclear neutrophils/monocytes/macrophages), NK1.1 (NK/NKT cells), F4/80⁻CD3⁻CD127⁺NK1.1^– (ILC1s), Lin⁻CD127⁺CD25⁺ST2⁺ (ILC2s), Lin⁻CD127⁺ RORγt⁺ (ILC3s). Graphs represent the average per cent (%) of total skin cells from three biological replicates. Student's t-test (b–d) between NRR1-treated and control-treated samples was used to determine significance marked with **; P-values included on graphs. (e) Sections of wounds 5 dpw from NRR1 or NRR2-treated mice stained with an antibody to RORγ (green). RORγ⁺ cells marked by white arrows and wound sites marked with white asterisks. Wounding recruits RORγ⁺ cells into the dermis; pre-treatment with NRR1, but not NRR2 receptor blocking antibodies inhibits RORγ⁺ cell infiltrate after wounding; scale bars equal 100 microns.