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: iScience

Article Title: Interferon-γ and IL-27 positively regulate type 1 regulatory T cell development during adaptive tolerance

doi: 10.1016/j.isci.2025.112308

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Mouse IL-27 p28 (clone MM27.7B1) , BioXCell , Cat #BE0326; RRID:AB_2819053.

Techniques: Recombinant, Saline, Staining, Red Blood Cell Lysis, Sequencing, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 1. DNA vaccination-based anti-IL-27 Abs are highly specific. The Western blot shows that our DNA vaccination-based anti-IL-27 Ab binds mouse IL-27 p28 (lane 1; 27 kDa), but not recombinant mouse IL-18, IL-12, or TNF- (lanes 2, 3, and 4, respectively). A, Coomassie Blue staining verifies the appearance of each cytokine on the loaded gel. B, Western blot showing that of these cytokines, our DNA vaccination-based anti-IL-27 Ab binds only mouse IL-27 p28. These Ab also bound natural mouse IL-27 (verified by sequencing) from supernatant of activated MOGp35–55-specific cultured primary draining lymph node cells (not shown).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Western Blot, Recombinant, Staining, Sequencing, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 2. Anti-p28 Abs suppresses ongoing severe EAE. A, Four groups of 10 mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g/mouse of anti-p28 Ab (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. The experiment summarized in Fig. 2 shows the results of one of three experiments performed under similar experimental conditions, with similar results. The mean maximal score SE represents six mice per group. The other four mice were killed on day 30 and subjected to histological evaluation (see Fig. 3). B, Five groups of six mice each were subjected to MOGp35–55-induced EAE. Beginning at the onset of disease (day 17), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), anti-IL-18 Ab (F), anti-IL-1 Ab (), IgG obtained from Lewis rats previously subjected to an empty plasmid administration (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as the mean maximal score SE of six mice per group. C, Three groups of six mice each were subjected to induction of transferred EAE. Beginning at the onset of disease (day 5), these mice were repeatedly (every other day) administered 100 g of anti-p28 Ab/mouse (f), IgG obtained from naive Lewis rats (Œ), or PBS (E). An observer blind to the experimental procedure scored EAE daily. Results are shown as mean maximal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 4. The beneficial effect of anti-IL-27 is dependent on the con- tinuing administration of protective Abs. Three groups of six mice each were subjected to active induction of EAE. Beginning 1 day after the onset of disease (day 17), these mice were treated with either a single dose of anti-p28 Ab (100 g/mouse; E) or with repeated administration (every other day) of this Ab (Œ) or PBS (f). An observer blind to the experi- mental procedure scored EAE daily. Results are shown as the mean max- imal score SE of six mice per group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 3. Anti-IL-27 therapy reduces the histological score of EAE. Histological evaluation was conducted 30 days after disease induction. Lumbar spinal cord samples from naive mice or from EAE mice treated with PBS, IgG from naive mice, or anti-IL-27 p28 Abs were subjected to histological analysis (nine sections each group). The arrowheads point to the parenchymal mononuclear cell infiltration. The scale for mononuclear cell infiltration used was: 0, no mononuclear cell infiltration; 1, one to five perivascular lesions per section with minimal parenchymal infiltration; 2, five to 10 perivascular lesions per section with parenchymal infiltration; and 3, 10 perivascular lesions per section with extensive parenchymal infiltration. The mean histological score SE was calculated for each group.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 5. Protective administration of anti-IL-27 Abs decreases in vivo polarization of CD4 T cells into Th1 and suppresses IFN- production by Ag-specific T cells. C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 12, 14, and 16) of anti-IL-27 p28 Abs (100 g), PBS, or normal rat IgG. On day 17 cervical lymph node cells (that drain the autoimmune site) were subjected to intracellular staining of IL-4 and IFN-. A, FACS analysis of CD4 T cells in this experiment. This experiment represents results obtained in three different independent experiments with very similar data. Subsequently, cervical lymph node T cells from these mice were cultured in the presence of 100 M MOGp35–55. After 72 h of stimulation, supernatants were assayed for the protein level of IFN- (B) and IL-4 (not shown). This experiment represents results obtained in three different independent experiments with very similar data.

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: In Vivo, Staining, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Suppression of ongoing experimental autoimmune encephalomyelitis by neutralizing the function of the p28 subunit of IL-27.

doi: 10.4049/jimmunol.173.10.6465

Figure Lengend Snippet: FIGURE 6. Neutralizing the function of IL-27 reduces IFN- produc- tion by IFN--producing T cells. A, C57BL/6 mice (three per group) were subjected to active induction of EAE and then to repeated administration (days 3 and 6) of 100 g of anti-IL-27 p28 Abs (group 3), PBS (group 2), or normal rat IgG (group 1). On day 9, spleen cells were subjected to spot ELISA as previously described (38). A, Relative number of positive spots per 107 cultured cells. The average size of positive spots was analyzed. B, The MOGp33–55-specific CD4 T cell line was cultured with or without 100 M MOGp33–55. Cultured cells were supplemented with anti-IL-27 Abs at a final concentration of 10 g/ml (), normal rat IgG (f), or PBS (E). After 60 h of incubation, cells were plates in spot ELISA plates for an additional 24 h for the detection of IFN--positive spots (38). Number of positive spots (y-axis) and spot sizes (x-axis; logarithmic scale) determined as previously described (46).

Article Snippet: Our recombinant mouse IL-27 p28, produced as described above, and commercially available recombinant mouse IL-18, IL-12, and TNF- (R&D Systems) were each subjected to Western blot analysis according to the protocol described in detail previously (29, 32), with the minor modification of using a 12% (rather than 8%) running gel.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay, Incubation

Journal: Arthritis Research & Therapy

Article Title: Gene therapy using IL-27 ameliorates Sjögren's syndrome-like autoimmune exocrinopathy

doi: 10.1186/ar3925

Figure Lengend Snippet: Generation of functional IL-27-expressing vector (rAAV2- IL27 ) . (A) IL-27 p28 expression levels in the cell lysates or culture media of mock transfection which contain transfection reagents only (white bar) or control empty AAV2 plasmid (grey bar) or rAAV2-IL27 (black bar) transfected HEK293 cells at 48 hours after transfection. (B) Biological effects of IL-27 were generated from rAAV2-IL27 on C57BL/6 splenocytes by using media only or (a) mock, (b) IL-17 differentiation cocktail containing IL-6 and transforming growth factor-beta, or (c) IL-17 differentiation cocktail with supernatant IL-27 from rAAV2-IL27 transfected HEK293 cells (sIL-27). The experiment was done in triplicate and repeated three times for consistency. Values in bar graphs are mean. ** P < 0.01, rAAV2- IL27 group versus mock or rAAV2 transfected by one-way analysis-of-variance test. IFN-γ, interferon-gamma; IL, interleukin.

Article Snippet: Ebi3 was determined by Western blotting by using anti-mouse Ebi3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and p28 was detected by enzyme-linked immunosorbent assay (ELISA) by using a Quantikine kit for mouse IL-27 p28 (R&D Systems, Minneapolis, MN, USA).

Techniques: Functional Assay, Expressing, Plasmid Preparation, Transfection, Control, Generated