Figure 4: Dermal macrophage response to TRPV1 activation is CGRP dependent.
(A) Quantification of TUNEL+ F4/80+ cell number per field of view from dorsal skin of TRPV1 activated mice and controls 3.5 hours after a single intradermal CNO treatment. Data are combined from 6 to 7 fields of view per mouse, taken from 7 mice per group (****p<0.0001).
(B) Representative immunofluorescent images of 20μm skin sections from mice treated as in A. Sections stained with anti-F4/80 (red), TUNEL (green) and DAPI (blue). White arrowheads indicate F4/80+ TUNEL+ cells. Scale bar 50μm.
(C) Percentage of Annexin V+ dermal macrophages from dorsal skin of TRPV1 activated mice and controls 45 minutes after a single CNO intradermal injection (***p=0.0002).
(D) Mice back skin intradermally injected with CGRP on one side and vehicle on the other. (Left) Samples collected 45 minutes later and stained for annexin V as in C (*p-paired=0.017). (Right) samples collected 6 hours later and dermal macrophage frequency assessed (*p-paired=0.015).
(E) Representative immunofluorescent images of 150μm dorsal skin sections from TRPV1 activated mice and controls pretreated with CGRP8–37 or vehicle followed by single intradermal CNO injection. Samples collected 6 hours later and stained with anti-F4/80 (red), CD207 (green), and DAPI (blue). Dashed rectangles indicate the lower follicle and peri-follicular area. Scale bar 100μm.
(F) Quantification of F4/80 normalized MFI signal in the peri-follicular area from mice treated as in E. Data are combined from 6–8 ROIs per mouse taken from 5–8 mice per group (****p<0.0001).
(G) Percentage of dermal macrophages from TRPV1 activated mice and controls treated as in E (*p=0.048,0.012,****p<0.0001).
(H) Dermal macrophages frequency in dorsal skin of TRPV1 activated mice and controls reconstituted with Ramp1 KO or wild type BM. Chimeric mice given a single CNO injection and data collected 6 hours later (*p=0.04,***p=0.0009).
(I) Dermal macrophages frequency in dorsal skin of TRPV1 activated mice and controls reconstituted with CD64CreRamp1+/+ or CD64CreRamp1fl/fl BM. Mice treated with CNO as in H (*p=0.043,***p=0.0001).