Extended Data Fig. 7: Impairment of macrophage function in Snx25+/− mice.
(a) Confocal images of the hind paw skin (naïve and 7 days after formalin injection) and DRG (L4) (naïve and 7 days after formalin injection) immunolabeled for Iba1 (green) and CD206 (red) in WT and Snx25+/− mice. Scale bars, 100 μm. (b) Quantification of mean CD206 fluorescence intensity at 7 days after formalin injection in the hind paw skin and DRG of WT and Snx25+/− mice. Hind paw skin (WT: n = 8; Snx25+/−: n = 8, p = 0.061) or DRG (WT: n = 4; Snx25+/−: n = 6, p = 0.028) sections from at least 3 different mice. Note the reduced number of the DRG macrophages in the Snx25+/− mice that may play an additive role to that of dermal macrophages in dull response in the inflammation paradigm. (c) Immune-related genes were examined with a mini-microarray (QIAGEN, RT2 Profiler PCR Array, PAMM-077ZC). Relative gene expression patterns of hind paw skin at 3 days after formalin injection in the WT and Snx25+/− mice are color-coded (n = 3 mice per group). (d) Left, a representative immunoblot showing TGFβ receptor type I (TGFbRI) levels in the ipsilateral (Ipsi) injected side and the contralateral (contra) side of the hind paw skin of WT and Snx25+/− mice at 30 min after formalin injection. Right, a representative immunoblot showing TGFbRI levels in the macrophage cell line RAW264.7 treated with scramble siRNA (siCtr) or Snx25 siRNA (siSnx25). Note that TGFbRI is upregulated in the Snx25-decreased tissues and cells. (e) Confocal microscopic images of the hind paw skin and DRG (L4) (7 days after formalin injection) immunolabeled for CCR2 (green) in WT and Snx25+/− mice. Insets show magnified views of boxed areas and arrowheads indicate CCR2+ cells. Scale bar, 100 μm. (f) Quantification of mean CCR2 fluorescence intensity at 7 days after formalin injection in the hind paw skin and DRG of WT and Snx25+/− mice. Hind paw skin (WT: n = 7; Snx25+/−: n = 6, p = 0.09) or DRG (WT: n = 3; Snx25+/−: n = 3, p = 0.099) sections from at least 3 different mice. (g) Size distribution of DRG neuron diameters (L4) of Snx25fl/fl mice (TAM, 1551 cells from 3 mice, blue columns) and Snx25Cx3cr1-cKO mice (TAM, 1627 cells from 3 mice, red columns) are plotted. X-axis values indicated the maximum diameter in each 5-μm range. (h) Immune-related genes were examined with a mini-microarray as in (c). Relative gene expression patterns of hind paw skin at 3 days after formalin injection in Snx25Cx3cr1-cKO mice (comparison with Snx25fl/fl mice as a control) are color-coded (n = 3 mice per group). (i) Experimental schedule of flow cytometry using Snx25Cx3cr1-cKO mice. (j) Flow cytometry strategy using 7-AAD, CD45, F4/80, and CD11b marker expression. F4/80+/CD11b+ cells were collected as macrophages in the hind paw skin of Snx25Cx3cr1-cKO mice after 3 days of formalin injection. (k) Expression patterns of representative chemokines (No TAM: n = 3; TAM: n = 5). Ccl2, p = 0.045; Ccl3, p = 0.023; Ccl4, p = 0.083; Cxcl2, p = 0.034. (l) Proportion of myeloid population in the hind paw skin of Snx25Cx3cr1-cKO mice (No TAM: n = 3; TAM: n = 3). CD11b+ F4/80+, p = 0.0378; CD11b+F4/80-, p = 0.029. Results are represented as mean ± SEM. Significance was calculated using the two-tailed Student’s t-test. *p < 0.05, **p < 0.01. Representative of three independent experiments (a and e).
