Figures and data
IL-4 increases CD11c+ microglia in the SDH and ameliorates pain hypersensitivity in the SpNT mice.
(A) Paw withdrawal threshold (PWT) of Itgax-Venus mice before (Pre) and after SpNT (n = 7–8 mice). IL-4 or PBS was intrathecally administrated from day 14 to day 16 post-SpNT (once a day for three days). ****P < 0.0001 versus the ipsilateral side of PBS- treated group, two-way ANOVA with post hoc Tukey multiple comparison test. (B) Venus fluorescence (yellow) and P2Y12R and IBA1 immunostaining (magenta and cyan, respectively) in the SDH of Itgax-Venus mice with SpNT 21 days after PBS or IL-4 treatment (from day 14 to 16). Scale bars, 200 µm (middle), 100 µm (right). (C) Colocalization of Venus, P2Y12R and IBA1. Scale bar, 20 µm. (D) Flow cytometric quantification for the number of CD11c+ (CD11b+CD45+Venus+) and total (CD11b+CD45+) microglia in the 3–4th lumbar SDH contralateral (C) and ipsilateral (I) to SpNT (n = 6–7 mice). One-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
IL-4-induced remission of pain hypersensitivity requires spinal CD11c+ microglia.
(A) Paw withdrawal threshold (PWT) of Itgax-DTR-EGFP mice before (Pre) and after SpNT (n = 6 –7 mice). IL-4 or PBS was intrathecally administrated from days 14 to 16 post-SpNT (once a day for three days). On day 17, DTX (0.5 ng/mouse) or PBS was intrathecally injected. ****P < 0.0001 versus the ipsilateral side of PBS-PBS group, ††††P < 0.0001 versus the ipsilateral side of IL-4-PBS group, two-way ANOVA with post hoc Tukey multiple comparison test. (B) Flow cytometric quantification for the number of CD11c+ microglia (CD11b+CD45+EGFP+) in the 3–4th lumbar SDH contralateral (C) and ipsilateral (I) to SpNT in Itgax-DTR-EGFP mice treated with PBS/PBS, IL-4/PBS, or IL- 4/DTX (n = 4–5 mice). One-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
IL-4 alleviates pain hypersensitivity in female mice.
Paw withdrawal threshold (PWT) of Itgax-DTR-EGFP mice before (Pre) and after SpNT (n = 6 female mice). IL-4 or PBS was intrathecally administrated from days 14 to 16 post-SpNT (once a day for three days). On day 17, DTX (0.5 ng/mouse) or PBS was intrathecally injected. ****P < 0.0001 versus the ipsilateral side of PBS-PBS group, †††P < 0.001 versus the ipsilateral side of IL-4-PBS group, two-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
CD11c+ cells in the DRG are involved in IL-4-induced remission of pain hypersensitivity.
(A and B) Immunohistochemical analyses of CD11c+ cells (EGFP+ IBA1+) in the DRG (A) and SDH (B) on day 18 post-SpNT of Itgax-DTR-EGFP mice treated intrathecally with IL-4 (from day 14 to 16) (n = 4–5 mice). DTX (2 ng/g) or PBS was intraperitoneally injected on day 17. The myeloid marker IBA1 was also stained in the SDH (B). Scale bars, 100 µm. Unpaired t-test. (C) Effect of DTX (2 ng/g) intraperitoneally injected (on day 17) on PWT of SpNT mice treated intrathecally with PBS or IL-4 (from day 14 to 16) (n = 5 mice). Data are shown as mean ± SEM.
Blunted appearance of CD11c+ microglia in the SDH after SNI.
(A) PWT of wild-type (WT) mice before (Pre) and after SpNT and SNI (n = 5 mice). (B) Venus (yellow) and IBA1 immunostaining (magenta) in the SDH of SpNT and SNI mice on day 14. Scale bar, 200 µm. (C) Flow cytometric quantification of the number of CD11c+ (CD11b+CD45+Venus+) and total (CD11b+CD45+) microglia in the 3–4th lumbar SDH of Itgax-Venus mice after SpNT and SNI (n = 3–5 mice). ****P < 0.0001 versus the ipsilateral side of SNI group, two-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
Expression of Igf1 is lower in the SNI model.
(A-C) Igf1 mRNA in total RNA extracted from sorted SDH microglia (A; n = 4 mice), from tissue homogenate of the upper half of the SDH (B; n = 3–4 mice), or from the sorted CD11chigh microglia (C; n = 5–6 mice) in Itgax-Venus mice was quantified by qPCR on day 36 post-SpNT or SNI. Cells and tissues were collected from the contralateral (C) and ipsilateral (I) side to the SpNT or SNI. Values represent the relative ratio of Igf1 mRNA (normalized to the value for Actb mRNA) to the ipsilateral (A and C) or contralateral (B) side of SpNT mice. One-way ANOVA with post hoc Tukey multiple comparison test (A and B). Unpaired t-test (C). Data are shown as mean ± SEM.
IL-4 increases CD11c+ microglia in the SDH and ameliorates pain hypersensitivity in the SNI mice.
(A) Venus fluorescence (yellow) and P2Y12R and IBA1 immunostaining (magenta and cyan, respectively) in the SDH of Itgax-Venus mice with SpNT after PBS or IL-4 treatment. Scale bar, 200 µm. (B) Colocalization of Venus, P2Y12R and IBA1. Scale bar, 20 µm. (C) Flow cytometric quantification for the number of CD11c+ (CD11b+CD45+Venus+) and total (CD11b+CD45+) microglia in the 3–4th lumbar SDH contralateral (C) and ipsilateral (I) to SNI (n = 7 mice). One-way ANOVA with post hoc Tukey multiple comparison test. (D) Paw withdrawal threshold (PWT) of Itgax-Venus mice before (Pre) and after SNI (n = 6–8 mice). IL-4 or PBS was intrathecally administrated from day 14 to 16 post-SNI (once a day for three days). *P < 0.05, ***P < 0.001 versus the ipsilateral side of PBS group, two-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
IL-4 alleviates SNI-induced pain hypersensitivity in CD11c+ microglia-and IGF1-dependent manners.
(A) Flow cytometric quantification for the number of CD11c+ microglia (CD11b+CD45+EGFP+) in the 3–4th lumbar SDH contralateral (C) and ipsilateral (I) to SNI in Itgax-DTR-EGFP mice treated with IL-4/PBS or IL-4/DTX (n = 6 mice). One-way ANOVA with post hoc Tukey multiple comparison test. (B) PWT of Itgax-DTR-EGFP mice before (Pre) and after SNI (n = 6 mice). IL-4 was intrathecally administrated from day 14 to 16 post-SNI (once a day for three days). On day 17, DTX (0.5 ng/mouse) or PBS was intrathecally injected. **P < 0.01 versus the ipsilateral side of IL-4-PBS group, two-way ANOVA with post hoc Tukey multiple comparison test. (C) PWT of Cx3cr1CreERT2/+;Igf1flox/flox mice before (Pre) and after SNI (n = 5–7 mice). Tamoxifen or vehicle were administered 4 weeks before SNI to induce recombination. IL-4 was intrathecally administrated from day 14 to 16 post-SNI (once a day for three days). ***P < 0.001 versus the ipsilateral side of Corn oil group, two-way ANOVA with post hoc Tukey multiple comparison test. Data are shown as mean ± SEM.
