Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: Establishing an Inflammatory pain model and assessing the expression profiles of IL-27. (A) Course of mechanical hyperalgesia in the mouse pain model (n = 5), two-way ANOVA tested P values ( vs. NC) with Dunnett’s multiple comparisons test. (B–G) The dynamic concentration of IL-27 (IL-27p28 and Ebi3) in the brain, spinal cord (L3–L5), spleen, ipsilateral and contralateral DRG (tested by qPCR), and the ELISA method to measure IL-27p28 in the serum. N = 4, GAPDH was used as an internal reference. (H, I) The mRNA level of Wsx-1 in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. (J, K) The mRNA level of IL-12p35 (IL-35) in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. The P value ( vs. NC) was measured using one-way ANOVA with Dunnett’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ebi3: Epstein-Barr virus-induced gene 3 protein, Ips-DRG: ipsilateral-dorsal root ganglion, qPCR: quantitative PCR, λ-carr: λ-carrageenan.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Virus, Real-time Polymerase Chain Reaction

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Staining, Flow Cytometry, Labeling, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Cell Culture

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Knockdown, shRNA, Plasmid Preparation, Injection, Expressing

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27 induces the differentiation of AAM. (A) The picture shows the procedures of this part. (B–D) Quantitative real-time mRNA levels of M2 indicators, including Arg-1 (B), Chi3l3 (C), Retnla (D), when BMDMs were exposed to IL-27 (100 ng/mL for 24 hours) and IL-4 (20 ng/mL for 24 hours) (n = 3). GAPDH was regarded as a reference. The P value was compared with the IL-27-and IL-4-treated group, using two-way ANOVA followed by Tukey’s multiple comparisons test. (E–G) qRT-PCR levels of M1 indicators, including IL-1β, IL-6, TNF-α, when BMDMs were exposed to LPS (20 ng/mL for 24 hours), IL-27 (100 ng/mL for 24 hours), or LPS (20 ng/mL for 24 hours) + IFN-γ (20 ng/mL for 24 hours) (n = 3). (H, I) IL-27 (H) and IL-4 (I) promote the expression of Arg-1 at the protein level in BMDM with different patterns (n = 3). GAPDH was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. (J) IL-27 has no significant impact on the expression of Arg-1 when IL-27 stimulates microglia in vitro , which indicates that IL-27 doesn’t induce the phenotype switch of microglia (n = 3). ACTB was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± standard error of the mean. IL: interleukin, LPS: lipopolysaccharide, IFN: interferon, TNF: tumor necrosis factor, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Quantitative RT-PCR, Expressing, In Vitro, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: RNA Sequencing, Gene Expression, Knockdown, Expressing, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Two Tailed Test, Expressing, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27-Ucp2-mediated AAM and a subsequent activation of the transcription factor FoxO3. (A) A diagram of the experimental process of this section. (B) Volcano plot analysis of differentially expressed proteins (DEPs) comparing IL-27 vs. NC, IL-27 vs. si-Ucp2, and IL-27 vs. GNP-treated groups. The marked Acod1 was indicated. (C) IL-27 induced a higher expression of Irg1 in BMDM, n = 3, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (D) Compared to the WT group, IL-27-induced Arg-1 expression was inhibited in the si-Irg1 treated group. GAPDH was used as a reference, n = 3; the P values (si-Irg1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E) Transcription factor (TF) prediction for both DEGs and DEPs induced by IL-27-stimulated BMDM. (F) The binding motif of FoxO3 predicted by the JASPAR platform. (G) The mRNA level of FoxO3 in BMDM with either NC, IL-27 (100 ng/mL), and IL-4 (20 ng/mL) treatment. N = 4, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (H) Compared to the WT group, IL-27-induced FoxO3 expression was inhibited in si-Irg1/si-Ucp2/GNP-treated group. GAPDH was used as a reference, n = 4; two-way ANOVA tested the P values with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, GNP: Genipin explicitly inhibits the protein function of Ucp2, BMDM: bone marrow-derived macrophage, WT: wild type, DEGs: differential expression analysis of genes.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Activation Assay, Expressing, Two Tailed Test, Binding Assay, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: FoxO3 controls IL-27-induced AAM differentiation and mitigates inflammatory pain in mice. (A) Immunofluorescence (IF) microscopy of FoxO3 in the nucleus of BMDMs upon stimulation with either NC, IL-27 (100 ng/mL for 12 hours), and IL-4 (20 ng/mL for 24 hours). n = 3, The scale bar indicates 1cm, and (B) the P values were tested by a one-way ANOVA with Tukey’s multiple comparisons test. (C) By comparing the knockdown efficiencies of three candidate siRNAs targeting FoxO3, Si#1 was identified as the most effective and will use it in subsequent experiments. GAPDH was used as a reference, n = 3, and the P values ( vs. NC) were tested by a one-way ANOVA with Dunnett’s post hoc test. (D) Compared to the FoxO3 mRNA in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 4, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E–G) Three M2 markers (Arg-1, Chi3l3, Retnla) were assessed by qPCR (E) and ELISA methods (F, G) in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 3, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (H) Flowchart of the experimental process of the adoptive transfer strategy. (I, J) Course of mechanical hyperalgesia in mice with five different intervention groups. (I) The BMDM transfer time was indicated, n = 5, and P values (M vs. MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (J) The results of IL-27M-IL-27MSF3 groups. N = 5, and P values (IL-27M vs. IL-27MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Data are shown as mean ± standard error of the mean. FoxO3: forkhead box class O3, IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage, WT: wild type, M: macrophages, MSF3: macrophage + si-FoxO3, IL-27M: IL-27-primed macrophage, IL-27MSF3: IL-27-primed macrophage+si-FoxO3.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Immunofluorescence, Microscopy, Knockdown, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: Establishing an Inflammatory pain model and assessing the expression profiles of IL-27. (A) Course of mechanical hyperalgesia in the mouse pain model (n = 5), two-way ANOVA tested P values ( vs. NC) with Dunnett’s multiple comparisons test. (B–G) The dynamic concentration of IL-27 (IL-27p28 and Ebi3) in the brain, spinal cord (L3–L5), spleen, ipsilateral and contralateral DRG (tested by qPCR), and the ELISA method to measure IL-27p28 in the serum. N = 4, GAPDH was used as an internal reference. (H, I) The mRNA level of Wsx-1 in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. (J, K) The mRNA level of IL-12p35 (IL-35) in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. The P value ( vs. NC) was measured using one-way ANOVA with Dunnett’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ebi3: Epstein-Barr virus-induced gene 3 protein, Ips-DRG: ipsilateral-dorsal root ganglion, qPCR: quantitative PCR, λ-carr: λ-carrageenan.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Virus, Real-time Polymerase Chain Reaction

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Staining, Flow Cytometry, Labeling, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Cell Culture

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Knockdown, shRNA, Plasmid Preparation, Injection, Expressing

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27 induces the differentiation of AAM. (A) The picture shows the procedures of this part. (B–D) Quantitative real-time mRNA levels of M2 indicators, including Arg-1 (B), Chi3l3 (C), Retnla (D), when BMDMs were exposed to IL-27 (100 ng/mL for 24 hours) and IL-4 (20 ng/mL for 24 hours) (n = 3). GAPDH was regarded as a reference. The P value was compared with the IL-27-and IL-4-treated group, using two-way ANOVA followed by Tukey’s multiple comparisons test. (E–G) qRT-PCR levels of M1 indicators, including IL-1β, IL-6, TNF-α, when BMDMs were exposed to LPS (20 ng/mL for 24 hours), IL-27 (100 ng/mL for 24 hours), or LPS (20 ng/mL for 24 hours) + IFN-γ (20 ng/mL for 24 hours) (n = 3). (H, I) IL-27 (H) and IL-4 (I) promote the expression of Arg-1 at the protein level in BMDM with different patterns (n = 3). GAPDH was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. (J) IL-27 has no significant impact on the expression of Arg-1 when IL-27 stimulates microglia in vitro , which indicates that IL-27 doesn’t induce the phenotype switch of microglia (n = 3). ACTB was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± standard error of the mean. IL: interleukin, LPS: lipopolysaccharide, IFN: interferon, TNF: tumor necrosis factor, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Quantitative RT-PCR, Expressing, In Vitro, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: RNA Sequencing, Gene Expression, Knockdown, Expressing, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: 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.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Two Tailed Test, Expressing, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27-Ucp2-mediated AAM and a subsequent activation of the transcription factor FoxO3. (A) A diagram of the experimental process of this section. (B) Volcano plot analysis of differentially expressed proteins (DEPs) comparing IL-27 vs. NC, IL-27 vs. si-Ucp2, and IL-27 vs. GNP-treated groups. The marked Acod1 was indicated. (C) IL-27 induced a higher expression of Irg1 in BMDM, n = 3, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (D) Compared to the WT group, IL-27-induced Arg-1 expression was inhibited in the si-Irg1 treated group. GAPDH was used as a reference, n = 3; the P values (si-Irg1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E) Transcription factor (TF) prediction for both DEGs and DEPs induced by IL-27-stimulated BMDM. (F) The binding motif of FoxO3 predicted by the JASPAR platform. (G) The mRNA level of FoxO3 in BMDM with either NC, IL-27 (100 ng/mL), and IL-4 (20 ng/mL) treatment. N = 4, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (H) Compared to the WT group, IL-27-induced FoxO3 expression was inhibited in si-Irg1/si-Ucp2/GNP-treated group. GAPDH was used as a reference, n = 4; two-way ANOVA tested the P values with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, GNP: Genipin explicitly inhibits the protein function of Ucp2, BMDM: bone marrow-derived macrophage, WT: wild type, DEGs: differential expression analysis of genes.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Activation Assay, Expressing, Two Tailed Test, Binding Assay, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: FoxO3 controls IL-27-induced AAM differentiation and mitigates inflammatory pain in mice. (A) Immunofluorescence (IF) microscopy of FoxO3 in the nucleus of BMDMs upon stimulation with either NC, IL-27 (100 ng/mL for 12 hours), and IL-4 (20 ng/mL for 24 hours). n = 3, The scale bar indicates 1cm, and (B) the P values were tested by a one-way ANOVA with Tukey’s multiple comparisons test. (C) By comparing the knockdown efficiencies of three candidate siRNAs targeting FoxO3, Si#1 was identified as the most effective and will use it in subsequent experiments. GAPDH was used as a reference, n = 3, and the P values ( vs. NC) were tested by a one-way ANOVA with Dunnett’s post hoc test. (D) Compared to the FoxO3 mRNA in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 4, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E–G) Three M2 markers (Arg-1, Chi3l3, Retnla) were assessed by qPCR (E) and ELISA methods (F, G) in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 3, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (H) Flowchart of the experimental process of the adoptive transfer strategy. (I, J) Course of mechanical hyperalgesia in mice with five different intervention groups. (I) The BMDM transfer time was indicated, n = 5, and P values (M vs. MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (J) The results of IL-27M-IL-27MSF3 groups. N = 5, and P values (IL-27M vs. IL-27MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Data are shown as mean ± standard error of the mean. FoxO3: forkhead box class O3, IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage, WT: wild type, M: macrophages, MSF3: macrophage + si-FoxO3, IL-27M: IL-27-primed macrophage, IL-27MSF3: IL-27-primed macrophage+si-FoxO3.

Article Snippet: The forced elevation of IL-27 concentration in the mice was obtained by i.v. injection of recombinant mouse IL-27 cytokine (rIL-27, MCE) ( ).

Techniques: Immunofluorescence, Microscopy, Knockdown, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Derivative Assay

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 expression positively correlates with proinflammatory markers in M1-polarized macrophages. (A-C) PD-L1-overexpressing (PD-L1 HI ) and control (PD-L1 NC ) THP-1-M cells were stimulated with LPS (100 ng/ml) + IFN-γ (20 ng/ml) for 12 h followed by RNA sequencing. A Heatmap of inflammatory gene expression in PD-L1 HI vs. PD-L1 NC cells. B MA plot showing differential gene expression between PD-L1 HI and PD-L1 NC cells. C TPM expression values of IL-6, IL-27 and NOS2. D - E PD-L1 HI and PD-L1 NC cells were stimulated with LPS + IFN-γ for 24 h. ( n = 3 independent biological replicates) ( D ) qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). E ELISA for IL-6/IL-27 and Griess reagent assay for NO in supernatants ( n = 3 independent biological replicates). (F-G) PD-L1-knockout THP-1-M (PD-L1 KO /THP-1-M) and PD-L1 NC /THP-1-M were stimulated with LPS + IFN-γ for 24 h. F qRT-PCR analysis of IL-6, IL-27 and NOS2 mRNA levels ( n = 3 independent biological replicates). G ELISA and Griess reagent detection of IL-6/IL-27 proteins and NO in supernatants ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C, D, E, F and G was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Expressing, Control, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: Triple-verification strategy (gene silencing/antibody neutralization/site-directed mutagenesis) confirms regulatory hierarchy of PD-L1/AIM2/IL-18/STAT1 signaling axis in macrophage M1 polarization. A PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were pretreated with AIM2-targeting siRNA or scramble siRNA (scr siRNA) prior to 24 h LPS (100 ng/ml) + IFN-γ (20 ng/ml) co-stimulation, with subsequent Western blotting analysis of STAT1 phosphorylation. B - C Anti-IL-18 neutralizing antibody (1 µg/ml) and isotype control (1 µg/ml) were pretreated for 24 h followed by LPS + IFN-γ 24 h co-stimulation. B qRT-PCR quantification of IFN-γ mRNA ( n = 3 independent biological replicates). C STAT1 phosphorylation profiling by Western blotting. D Truncation mutant validation: LPS + IFN-γ-stimulated (24 h) PD-L1 Δ35–90 cells analyzed for STAT1 activation vs. full length type. E Following AIM2 silencing in PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells, qRT-PCR quantification of IL-6, IL-27 and NOS2 mRNA after 24 h LPS + IFN-γ stimulation ( n = 3 independent biological replicates). F Post IL-18 neutralization, mRNA levels of IL-6, IL-27 and NOS2 were assessed by qRT-PCR under LPS + IFN-γ 24 h stimulation ( n = 3 independent biological replicates). G STAT1-silenced cells were analyzed for IL-6/IL-27/NOS2 transcriptional changes post 24 h LPS + IFN-γ exposure ( n = 3 independent biological replicates). H Truncated PD-L1 Δ35–90 mutants were evaluated for IL-6, IL-27 and NOS2 mRNA expression following LPS + IFN-γ 24 h challenge ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B, E, F, G and H was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Neutralization, Mutagenesis, Western Blot, Phospho-proteomics, Control, Quantitative RT-PCR, Biomarker Discovery, Activation Assay, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: PD-L1 mediates M1 polarization of macrophages via the AIM2/IL-18/STAT1 signaling axis during sepsis

doi: 10.1186/s12964-025-02578-1

Figure Lengend Snippet: PD-L1 knockout reduces septic mortality dampens systemic inflammation and alleviates vital organ (cardiac/hepatic/renal) injury in murine models. A PD-L1 wild-type (WT) and knockout (KO) mice were subjected to CLP followed by survival monitoring, inflammatory marker assessment, and multi-organ functional evaluation. B Survival rates of PD-L1 WT and PD-L1 KO mice at indicated time points post-CLP ( n = 15 mice per group). Results were compared by log-rank test. C mRNA levels of IL-6, IL-27, and NOS2 in monocytes analyzed by qRT-PCR at 24 h post-CLP ( n = 3 mice per group). D Serum concentrations of IL-6 and IL-27 proteins measured by ELISA, and NO levels determined by Griess assay at 24 h post-CLP ( n = 3 mice per group). E Serum biomarkers of organ function including ALT and AST for hepatic injury, BUN for renal dysfunction, and cTnI for cardiac damage at 24 h post-CLP ( n = 3 mice per group). The data are presented as the mean ± SEM. Statistical analysis for C, D and E was performed by Student’s t-test. * p < 0.05

Article Snippet: IL-27, IL-6, IL-18, and IFN-γ concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm.

Techniques: Knock-Out, Marker, Functional Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Griess Assay