Fig 2: IL-23 is expressed by macrophages in mouse DRG of both sexes. (A) Double staining shows co-localization of IL-23 with F4/80. Immunohistochemistry was performed with IL-23 and F4/80 staining on DRG sections of mice to assess IL-23 expression in macrophages. Results were imaged with a Confocal microscope. Columns from left to right represent IL-23 staining (green), F4/80 staining (red), merged images (yellow). The boxes in the third columns are magnified in rightmost images. DRG sections were counter stained with DAPI to label all the nuclei. Scale bars, 75 μm. (B) Number of IL-23 and F4/80 double-labeled macrophages in DRG sections of both sexes. Colocalization of IL-23 and F4/80 staining was quantified with Image J and quantification results were used to calculate the number of IL-23/F4/80-positive macrophages per mm2. Data were then analyzed in GraphPad Prism using an unpaired t-test. n = 4 mice per sex, and 4 DRG sections were included for each animal. Data shown as mean ± SEM. n.s., not significant.

Fig 3: IL-23 potentiates capsaicin-induced spontaneous pain in females. (A) Schematic of experimental design. Mice of either sex were given an intrathecal injection of either vehicle (PBS) or IL-23 (100 ng, 10 µL) 1 hour prior to intraplantar injection of capsaicin (500 ng, 10 µL). Pain was assessed by duration of pain response (in seconds) over five minutes following capsaicin injection. (B, C) Effects of IL-23 on capsaicin-induced spontaneous pain in males, as shown by time course (B) and accumulated value (C). n = 5 male mice/group. n.s., not significant. (D, E) Effects of IL-23 on capsaicin-induced spontaneous pain in females, as shown by time course (D) and accumulated value (E). n = 5 female mice/group. *p < 0.05, Mann-Whitney test (E). Data shown as mean ± SEM.

Fig 4: Effects of intrathecal IL-23 treatment on p38 phosphorylation in DRG neurons in male and female mice. (A–D) Immunohistochemistry was performed to identify p-p38-positive neurons in the DRG of female mice (A, C) and male mice (B, D), without IL-23 treatment (A, B) and with IL-23 treatment (C, D). Animals were sacrificed 30 min after intrathecal (I.T.) IL-23 (100 ng) treatment. Scale bar, 100 μm. (E) High magnification image of the white box in (C). White arrows indicate activation of many small neurons (presumably nociceptors). Yellow and blue arrows indicate activation of some satellite glial cells and immune cells, respectively. (F) Quantification of the percentage of p-p38-positive neurons in mouse DRGs using Image J. Data were then analyzed with GraphPad Prism. n = 5 mice per sex. *p < 0.05, ****p < 0.0001, Two-way ANOVA followed by Bonferroni’s posthoc test. Data shown as mean ± SEM.

Fig 5: THP-1 macrophages release IL-23 and IL-17A following estrogen and paclitaxel administration. ELISA tests were performed to measure IL-23 (A) and IL-17A (B) levels produced by THP-1 cells (500,000 in 400 μl), stimulated with estradiol (1 ng/ml, 24 h), paclitaxel (PTX, 1 μg/ml, 24 h), or estradiol plus paclitaxel (A, B), or estradiol and IL-23 [10 ng/ml, 24 h, (C)]. n = 9 cultures from two separate experiments. *p < 0.05, One-way ANOVA followed by Bonferroni’s posthoc test. Data are shown as mean ± SEM.