Fig 1: The knockdown of IL-27 intensified mechanical allodynia in mouse pain models. (A) Schematic diagram showing construction of pAAV2/9-U6-shRNA (IL-27p28)-CMV-EGFP vector. (B) The Sh-1 presents the most effective knockdown of IL-27 at the mRNA level in mouse DRG (L3–L5) tissue and was selected for use in the next operation (n = 3). One-way ANOVA with Tukey’s multiple comparisons test was applied. (C) The experiment procedures for i.t. injection of pAAV2/9-U6-shRNA (IL-27p28) to knock down IL-27 and behavior test in mice. (D) The timelines of mechanical hyperalgesia in WT, λ-carr, and sh-IL-27 mice after tail-vein injection of recombination mouse IL-27 in a hind paw (n = 5). P values (vs. λ-carr) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E, F) The timelines of mechanical hyperalgesia in mice with WT, sh-IL-27, IL-27 forced expression (IL-27 FE)-treated groups. Mice received the rIL-27 agent (100 ng/kg) at the indicated time (n = 5). P values (vs. λ-carr) were tested by two-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, DRG: dorsal root ganglion, i.t.: intrathecal injection, WT: wild type, λ-carr: λ-carrageenan.
Fig 2: IL-27 distinctly induces the polarization of AAM from IL-4. (A) The picture of the study pipeline in this part. (B, C, E) Bulk RNA-seq comparing the BMDM treated by NC, IL-27 (100 ng/mL for 12 hours), and IL-4 (20 ng/mL for 24 hours). Data containing the heatmap presenting differential gene expression (B), PCA (C), and a Venn plot to show the shared and non-shared DEGs (E). (D) The knockdown effect of the three candidates’ siRNA was targeted at Wsx-1 at the protein level (GAPDH as a reference). (F) The decreased expression of Arg-1 in the si-Wsx-1 group when BMDM was insulted by IL-27, compared to the IL-4-treated and WT group (n = 3). β-actin as internal reference, P values (WT vs. si-Wsx-1 specifically under IL-27 stimulation) were tested by two-way ANOVA with Sidak’s multiple comparisons test. (G) The mRNA of Arg-1 was inhibited in the IL-27-si-Wsx-1 group, compared to the IL-4-treated and WT group (n = 4). GAPDH was regarded as a reference. (H, I) Compared to the WT and NC groups, the expression level of Chi3l3 induced by IL-27 decreased after si-Wsx-1 treatment (H), whereas the IL-4-treated group (I) remained unaffected (n = 4). The P values (si-Wsx-1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test, n = 4, GAPDH as internal reference. ***P < 0.001, ****P < 0.0001. Data are shown as mean ± standard error of the mean. IL: interleukin, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage, PCA: principal component analysis, DEGs: differential expression analysis of genes, WT: wild type.
Fig 3: The identification of the source of IL-27. (A–D) The intracellular co-label staining by flow cytometry was applied to determine the source of IL-27. The four APCs were labeled by dendritic cell (CD11b, CD11c, B), neutrophil (CD11b, Ly6G, C), monocyte (CD11b, Ly6C, A), and macrophage (CD11b, F4/80, D), respectively. IL-27p28 antibody labeled the IL-27. The results indicated that neutrophil/monocyte-derived IL-27 was highly expressed in serum and spleen. IL: interleukin, APCs: antigen-presenting cells, SSC-A: side scatter area.
Fig 4: IL-27-Ucp2 signaling pathway mediates AAM. (A) The schematic illustration of this part. GNP: Genipin explicitly inhibits the protein function of Ucp2. (B) ECAR of glycolysis stress test of BMDMs, either NC or treated with IL-27 (100 ng/mL for 6 hours). (C) The Glycolytic capacity was compared with the NC and IL-27-stimulated group (n = 3), and an unpaired, two-tailed t-test was used. (D) OCR of Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours), n = 3. (E) Proton leak measured from Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours) and IL-4 (20 ng/mL for 6 hours), n = 3, one-way ANOVA followed by Dunnett post-test. (F) OCR of BMDM in IL-27 (100 ng/mL for 6 hours) and IL-4 (20 ng/mL for 6 hours)-treated group. (G) The mRNA analysis of Ucp proteins. GAPDH was used as a reference, n = 4, and a unpaired, two-tailed t -test was used. (H) The mRNA level of Ucp2 was dampened in the si-Wsx-1 group when treated with IL-27, GAPDH was used as a reference, and n = 4, one-way ANOVA followed by Tukey’s multiple comparisons test. (I) OCR of BMDMs either NC or treated with IL-27 (100 ng/mL for 6 hours) and GNP (100 mM for 12 hours). (J) Proton leak measured from Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours) and GNP (100 mM for 12 hours), n = 3, one-way ANOVA followed by Tukey’s multiple comparisons test. (K) Compared with the WT group, si-Ucp2 hampered the IL-27-induced Arg-1 expression at the mRNA level. N = 4, The P values (si-Wsx-1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test, n = 4, GAPDH as internal reference. *P < 0.05, **P < 0.01, ****P < 0.0001, ns: not significant. Data are shown as mean ± standard error of the mean. IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, ECAR: extracellular acidification rate, BMDM: bone marrow-derived macrophage, OCR: oxygen consumption rate, WT: wild type.
Fig 5: The source of IL-27 in the spinal cord is microglia. (A, B) The cultured primary microglia secret loads of IL-27 in response to LPS (100 ng/mL) insult for 12 hours, while cultured primary astrocytes released minimal IL-27 in the same condition (n = 3). (C, D) IFN-γ-stimulated (20 ng/mL) primary microglia were the main source of IL-27, compared with the astrocytes group (n = 3), which suggests IL-27 was primarily secreted from microglia in the spinal cord. IL: interleukin, LPS: lipopolysaccharide, IFN: interferon.
Supplier Page from Abcam for Mouse IL-27 p28 ELISA Kit