Fig 1: IL-23 potentiates blue light-induced, intensity-dependent, and sex-dependent spontaneous pain in ChR2-TRPV1 reporter mice. (A, C) Schematic of experimental design. Mice of both sexes were treated either with blue light only (A) or with IL-23 injection followed by blue light stimulation (C). Light-induced spontaneous pain was assessed by duration of pain response. (B) Blue light-induced spontaneous pain in male and female mice, as assessed by time spent on licking and lifting behavior. Optogenetic stimulation was applied to the left hindpaw plantar surface for 20 seconds. Duration of pain response was timed in seconds. n = 9 (male) or 11 (female) mice/group. ****p < 0.0001, Two-way ANOVA followed by Bonferroni post-hoc test. (D) Effects of IL-23 on blue light-induced pain in both sexes. One hour prior to optogenetic stimulation, mice were given an intraplantar injection of either vehicle (PBS, 10 µL) or IL-23 (100 ng, 10 µL). Optogenetic stimulation was applied to the left hindpaw plantar surface for 20 seconds at 2 mW/mm2. n = 4-6 mice/group. **p < 0.01, Two-way ANOVA followed by Bonferroni posthoc test. Data shown as mean ± SEM. Illustrations in A and C were made with BioRender with license.
Fig 2: Comparison of the serum IL-17 and IL-23 levels between SLE patients versus healthy controls. (A) IL-17 serum levels in SLE patients vs healthy controls. (B) IL-23 serum levels in SLE patients vs healthy controls. The bars in each scatter plot represent the median and interquartile range.
Fig 3: 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 4: 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 5: 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.
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