il 27  (R&D Systems)

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 impairs early B-cell development in the bone marrow. (A) Experimental scheme for AAV-mediated IL-27 delivery and analysis of splenic and BM B-cell compartments. (B–D) Splenic B-cell analysis 10 weeks after AAV-Ctrl or AAV-IL-27 treatment (n = 2 per group). (B) Frequency of total splenic B cells. (C) CIBERSORTx-based deconvolution of splenic B-cell subsets, including clustering and pseudotime analysis (left) and relative subset distribution (right). (D) Cd93 expression (TPM) in splenic B cells. (E–G) Analysis of splenic and BM B-cell compartments 3 weeks after treatment (n = 4 per group). (E) Representative flow cytometry plots. (F) Frequencies of B220 + CD19 − and B220 − CD19 + populations in the BM and spleen. (G) Relative distribution of B-cell developmental subsets in the BM. (H–I) Kinetics of B-cell populations following IL-27 treatment (n = 2 per group at each time point). (H) Frequency and absolute number of BM B220 + B cells. (I) Proportions of B-cell subsets within the B220 + compartment at the indicated time points. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Cell Analysis, Expressing, Flow Cytometry

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 inhibits CLP formation in the bone marrow. (A) qPCR analysis of B-lineage- and myeloid-associated transcription factors in BM cells 5 days after AAV-Ctrl or AAV-IL-27 administration (n = 3 per group). (B) qPCR analysis of CEBPA expression in Reh cells treated with hIL-27 and DAC for 3 days (n = 3 per group). (C–D) Frequencies and absolute numbers of CLPs in WT and CD19 Cre ;IL-27R fl/fl mice (n = 3 per group). (E–F) BM B-cell frequency (E) and proportion of B220 + CD43 hi IgM − B cells (F) in WT and CD19 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for WT Ctrl group; n = 3 for WT IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 4 for CD19 Cre IL-27 group). (G–H) BM B-cell frequency (G) and proportion of B220 + CD43 hi IgM − B cells (H) in CD4 Cre ;IL-27R fl/fl and Lyz2 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for CD4 Cre Ctrl group; n = 3 for CD4 Cre IL-27 group; n = 5 for Lyz2 Cre Ctrl group; n = 6 for Lyz2 Cre IL-27 group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 reshapes immune reconstitution after bone marrow transplantation. (A) Experimental scheme. WT mice underwent BM transplantation followed by AAV-Ctrl or AAV-IL-27 treatment. Splenic and BM immune reconstitution was analysed at 10 weeks after treatment (n = 4 mice for the Ctrl group; n = 5 mice for the IL-27 group). (B) Percentages and absolute numbers of CD45 + splenocytes. (C–F) Frequencies of major immune cell populations within CD45 + splenocytes: myeloid cells (C), macrophages (D), T and B cells (E), and NK cells (F). (G) Frequency of CD4 + T cells among splenic T cells. (H) Proportion of Tregs among CD4 + T cells. (I–J) BM B-cell reconstitution. Frequency of BM B220 + B cells (I) and distribution of B220 + B-cell subsets (J). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 inhibits B-cell development through both direct and indirect mechanisms. (A) Schematic of the BM chimera model. WT (WTR) or IL-27R −/− (KOR) recipients were treated with AAV-Ctrl or AAV-IL-27. Donor BM cells were a 1:1 mix of WT (CD45.1 + ) and IL-27R −/− (CD45.1 − ) cells. Analyses were performed at 5 weeks (B–E; n = 3 per group) or 10 weeks (F–I; n = 5 per group) post-transplantation. (B) Percentage of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (C) Absolute numbers of total CD19 + B cells in spleen and BM of WTR and KOR mice. (D) Absolute numbers of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (E) Percentage of B220 + CD19 − B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in BM of WTR and KOR mice. (F) Absolute numbers of B220 + CD19 - B cells from CD45.1 + or CD45.1 - (IL-27R −/− ) donors in BM of WTR and KOR mice. (G) Frequencies and absolute numbers of BM B220 + B cells derived from CD45.1 + or IL-27R −/− donors in WTR mice. (H) Proportion of CD43 hi IgM − subsets from CD45.1 + or IL-27R −/− donors in BM B220 + B cells in WTR mice. (I–J) Frequencies of CLPs (I) and their donor-specific contributions (J) in BM chimeras. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay, Derivative Assay

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 indirectly inhibits BM B-ALL cells. (A) Experimental design. GFP + B-ALL cells were transferred into WT mice, followed by AAV-Ctrl or AAV-IL-27 administration. BM and splenic B-ALL cells were analysed 9–10 days later (n = 3 per group). (B–C) Representative flow cytometry plots and quantification of GFP + B-ALL and GFP − B cells. (D) GSVA analysis of transcriptional changes in BM B-ALL cells isolated from mice in (A). (E) Gene Ontology (GO) enrichment analysis of DEGs between Ctrl and IL-27 groups in (A) (adjusted P < 0.05, |log 2 fold change| > 1). The outer ring indicates enriched GO terms, the middle ring represents relative gene expression changes in the IL-27 group, and the inner ring shows background gene counts, with colour intensity reflecting enrichment significance. Enriched pathways include interferon-γ-mediated signalling, interferon-β responses, adhesion-related processes, protozoan defence responses, calmodulin-dependent kinase signalling, and negative regulation of STAT tyrosine phosphorylation. (F–G) Expression of apoptosis- and cell cycle-related genes in BM B-ALL cells from (A). (H) Experimental design. WT and IL-27R −/− mice transplanted with GFP + B-ALL cells were treated with AAV-Ctrl or AAV-IL-27 (n = 4 for WT Ctrl group; n = 4 for WT IL-27 group; n = 5 for IL-27R −/− Ctrl group; n = 5 for IL-27R −/− IL-27 group). (I–J) Representative flow plots and quantification of BM B-ALL cells. (K–L) Flow cytometry plots and quantification of BM B-ALL cells in CD4 Cre ;IL-27R fl/fl , CD19 Cre ;IL-27R fl/fl , and Lyz2 Cre ;IL-27R fl/fl mice (n = 2 for CD4 Cre Ctrl group; n = 2 for CD4 Cre IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 3 for CD19 Cre IL-27 group; n = 3 for Lyz2 Cre Ctrl group; n = 3 for Lyz2 Cre IL-27 group). (M) Flow cytometry plots and quantification of BM B-ALL cells in Rag1 −/− mice (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Flow Cytometry, Isolation, Gene Expression, Phospho-proteomics, Expressing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 reshapes the BM niche, reducing B-cell support and leukaemogenesis. (A) Pseudotime analysis of BM B-cell subsets (C0–C10). (B) Expression of representative marker genes across B-cell clusters in (A). (C–G) BM cells were harvested 8 days after AAV-Ctrl or AAV-IL-27 administration for RNA-seq analysis (n = 3 per group). (C) CIBERSORTx analysis showing increased pre-pro-B and cycling pro-B subsets after IL-27 treatment. (D) KEGG pathway enrichment analysis highlighting alterations in adhesion-related pathways. (E–F) Heatmaps of transcription factors (E) and adhesion molecules (F). (G) RNA-seq analysis of genes shown in the figure in BM cells. (H) RNA-seq analysis of genes shown in the figure in human MSCs with or without hIL-27 treatment (n = 2 per group). (I) qPCR analysis of genes shown in the figure in murine MSCs with or without IL-27 (n = 3 per group). (J) RNA-seq analysis of genes shown in the figure in BM Gr-1 + cells 3 weeks after AAV-Ctrl or AAV-IL-27 administration (n = 2 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing, Marker, RNA Sequencing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 exhibits therapeutic potential while modulating B-cell compartments. (A–B) Experimental design of combination therapy in WT (A) and Rag1 −/− (B) mice, with corresponding Kaplan–Meier survival analysis. (C–D) Experimental design of IL-27R −/− CAR-T cell therapy combined with AAV-Ctrl or AAV-IL-27 in B-ALL-bearing mice, with Kaplan–Meier survival curves. B-ALL cells were transplanted either prior to chemotherapy (C) or after chemotherapy (D). (E–F) NSG mice transplanted with human G-PBSCs were injected intramuscularly with AAV-Ctrl or hIL-27. 10 days later, frequencies and absolute numbers of B cells in the BM and spleen were analysed (n = 5 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; log-rank test (A–D) and unpaired Student's t-test (E–F).

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Injection

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 impairs early B-cell development in the bone marrow. (A) Experimental scheme for AAV-mediated IL-27 delivery and analysis of splenic and BM B-cell compartments. (B–D) Splenic B-cell analysis 10 weeks after AAV-Ctrl or AAV-IL-27 treatment (n = 2 per group). (B) Frequency of total splenic B cells. (C) CIBERSORTx-based deconvolution of splenic B-cell subsets, including clustering and pseudotime analysis (left) and relative subset distribution (right). (D) Cd93 expression (TPM) in splenic B cells. (E–G) Analysis of splenic and BM B-cell compartments 3 weeks after treatment (n = 4 per group). (E) Representative flow cytometry plots. (F) Frequencies of B220 + CD19 − and B220 − CD19 + populations in the BM and spleen. (G) Relative distribution of B-cell developmental subsets in the BM. (H–I) Kinetics of B-cell populations following IL-27 treatment (n = 2 per group at each time point). (H) Frequency and absolute number of BM B220 + B cells. (I) Proportions of B-cell subsets within the B220 + compartment at the indicated time points. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Cell Analysis, Expressing, Flow Cytometry

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 inhibits CLP formation in the bone marrow. (A) qPCR analysis of B-lineage- and myeloid-associated transcription factors in BM cells 5 days after AAV-Ctrl or AAV-IL-27 administration (n = 3 per group). (B) qPCR analysis of CEBPA expression in Reh cells treated with hIL-27 and DAC for 3 days (n = 3 per group). (C–D) Frequencies and absolute numbers of CLPs in WT and CD19 Cre ;IL-27R fl/fl mice (n = 3 per group). (E–F) BM B-cell frequency (E) and proportion of B220 + CD43 hi IgM − B cells (F) in WT and CD19 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for WT Ctrl group; n = 3 for WT IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 4 for CD19 Cre IL-27 group). (G–H) BM B-cell frequency (G) and proportion of B220 + CD43 hi IgM − B cells (H) in CD4 Cre ;IL-27R fl/fl and Lyz2 Cre ;IL-27R fl/fl mice 2 weeks after AAV treatment (n = 3 for CD4 Cre Ctrl group; n = 3 for CD4 Cre IL-27 group; n = 5 for Lyz2 Cre Ctrl group; n = 6 for Lyz2 Cre IL-27 group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 reshapes immune reconstitution after bone marrow transplantation. (A) Experimental scheme. WT mice underwent BM transplantation followed by AAV-Ctrl or AAV-IL-27 treatment. Splenic and BM immune reconstitution was analysed at 10 weeks after treatment (n = 4 mice for the Ctrl group; n = 5 mice for the IL-27 group). (B) Percentages and absolute numbers of CD45 + splenocytes. (C–F) Frequencies of major immune cell populations within CD45 + splenocytes: myeloid cells (C), macrophages (D), T and B cells (E), and NK cells (F). (G) Frequency of CD4 + T cells among splenic T cells. (H) Proportion of Tregs among CD4 + T cells. (I–J) BM B-cell reconstitution. Frequency of BM B220 + B cells (I) and distribution of B220 + B-cell subsets (J). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 inhibits B-cell development through both direct and indirect mechanisms. (A) Schematic of the BM chimera model. WT (WTR) or IL-27R −/− (KOR) recipients were treated with AAV-Ctrl or AAV-IL-27. Donor BM cells were a 1:1 mix of WT (CD45.1 + ) and IL-27R −/− (CD45.1 − ) cells. Analyses were performed at 5 weeks (B–E; n = 3 per group) or 10 weeks (F–I; n = 5 per group) post-transplantation. (B) Percentage of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (C) Absolute numbers of total CD19 + B cells in spleen and BM of WTR and KOR mice. (D) Absolute numbers of CD19 + B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in spleen and BM of WTR and KOR mice. (E) Percentage of B220 + CD19 − B cells from CD45.1 + or CD45.1 − (IL-27R −/− ) donors in BM of WTR and KOR mice. (F) Absolute numbers of B220 + CD19 - B cells from CD45.1 + or CD45.1 - (IL-27R −/− ) donors in BM of WTR and KOR mice. (G) Frequencies and absolute numbers of BM B220 + B cells derived from CD45.1 + or IL-27R −/− donors in WTR mice. (H) Proportion of CD43 hi IgM − subsets from CD45.1 + or IL-27R −/− donors in BM B220 + B cells in WTR mice. (I–J) Frequencies of CLPs (I) and their donor-specific contributions (J) in BM chimeras. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Transplantation Assay, Derivative Assay

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: Exogenous IL-27 indirectly inhibits BM B-ALL cells. (A) Experimental design. GFP + B-ALL cells were transferred into WT mice, followed by AAV-Ctrl or AAV-IL-27 administration. BM and splenic B-ALL cells were analysed 9–10 days later (n = 3 per group). (B–C) Representative flow cytometry plots and quantification of GFP + B-ALL and GFP − B cells. (D) GSVA analysis of transcriptional changes in BM B-ALL cells isolated from mice in (A). (E) Gene Ontology (GO) enrichment analysis of DEGs between Ctrl and IL-27 groups in (A) (adjusted P < 0.05, |log 2 fold change| > 1). The outer ring indicates enriched GO terms, the middle ring represents relative gene expression changes in the IL-27 group, and the inner ring shows background gene counts, with colour intensity reflecting enrichment significance. Enriched pathways include interferon-γ-mediated signalling, interferon-β responses, adhesion-related processes, protozoan defence responses, calmodulin-dependent kinase signalling, and negative regulation of STAT tyrosine phosphorylation. (F–G) Expression of apoptosis- and cell cycle-related genes in BM B-ALL cells from (A). (H) Experimental design. WT and IL-27R −/− mice transplanted with GFP + B-ALL cells were treated with AAV-Ctrl or AAV-IL-27 (n = 4 for WT Ctrl group; n = 4 for WT IL-27 group; n = 5 for IL-27R −/− Ctrl group; n = 5 for IL-27R −/− IL-27 group). (I–J) Representative flow plots and quantification of BM B-ALL cells. (K–L) Flow cytometry plots and quantification of BM B-ALL cells in CD4 Cre ;IL-27R fl/fl , CD19 Cre ;IL-27R fl/fl , and Lyz2 Cre ;IL-27R fl/fl mice (n = 2 for CD4 Cre Ctrl group; n = 2 for CD4 Cre IL-27 group; n = 3 for CD19 Cre Ctrl group; n = 3 for CD19 Cre IL-27 group; n = 3 for Lyz2 Cre Ctrl group; n = 3 for Lyz2 Cre IL-27 group). (M) Flow cytometry plots and quantification of BM B-ALL cells in Rag1 −/− mice (n = 3 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Flow Cytometry, Isolation, Gene Expression, Phospho-proteomics, Expressing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 reshapes the BM niche, reducing B-cell support and leukaemogenesis. (A) Pseudotime analysis of BM B-cell subsets (C0–C10). (B) Expression of representative marker genes across B-cell clusters in (A). (C–G) BM cells were harvested 8 days after AAV-Ctrl or AAV-IL-27 administration for RNA-seq analysis (n = 3 per group). (C) CIBERSORTx analysis showing increased pre-pro-B and cycling pro-B subsets after IL-27 treatment. (D) KEGG pathway enrichment analysis highlighting alterations in adhesion-related pathways. (E–F) Heatmaps of transcription factors (E) and adhesion molecules (F). (G) RNA-seq analysis of genes shown in the figure in BM cells. (H) RNA-seq analysis of genes shown in the figure in human MSCs with or without hIL-27 treatment (n = 2 per group). (I) qPCR analysis of genes shown in the figure in murine MSCs with or without IL-27 (n = 3 per group). (J) RNA-seq analysis of genes shown in the figure in BM Gr-1 + cells 3 weeks after AAV-Ctrl or AAV-IL-27 administration (n = 2 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; unpaired Student's t-test.

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Expressing, Marker, RNA Sequencing

Journal: eBioMedicine

Article Title: Interleukin-27 remodels the bone marrow niche to suppress B-cell development and leukaemia progression in mouse models

doi: 10.1016/j.ebiom.2026.106239

Figure Lengend Snippet: IL-27 exhibits therapeutic potential while modulating B-cell compartments. (A–B) Experimental design of combination therapy in WT (A) and Rag1 −/− (B) mice, with corresponding Kaplan–Meier survival analysis. (C–D) Experimental design of IL-27R −/− CAR-T cell therapy combined with AAV-Ctrl or AAV-IL-27 in B-ALL-bearing mice, with Kaplan–Meier survival curves. B-ALL cells were transplanted either prior to chemotherapy (C) or after chemotherapy (D). (E–F) NSG mice transplanted with human G-PBSCs were injected intramuscularly with AAV-Ctrl or hIL-27. 10 days later, frequencies and absolute numbers of B cells in the BM and spleen were analysed (n = 5 per group). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; ns, not significant; log-rank test (A–D) and unpaired Student's t-test (E–F).

Article Snippet: Cells were treated with recombinant murine IL-27 (51107-M08H, Sino Biological) or human IL-27 (50 ng/mL) for 24 h prior to RNA extraction.

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

doi: 10.3389/fimmu.2026.1802599

Figure Lengend Snippet: Effect of the administration of Lactiplantibacillus plantarum ATCC 10241 cell-free supernatant (LpCFS) on broncho-alveolar fluid (BAL) macrophage-enriched adherent cells. BALB/c mice (6-week-old) received aerosol administration of LpCFS or vehicle control for three consecutive days. On day four, BAL macrophage-enriched adherent cells were isolated, cultured, and ex vivo challenged with antibiotic-sensitive (PaS) or multidrug-resistant (PaR) Pseudomonas aeruginosa strains or lipopolysaccharide (LPS). One day after the challenges, the levels of IFN-γ, IL-10, and IL-27 were measured using ELISA. Data are presented as mean ± SEM. Statistical analysis was performed using Student’s t-test. Differences were considered statistically significant at p < 0.05 (*). The same control cohort was used for the experiments in Figures 3 , .

Article Snippet: Interferon (IFN)-β (Mouse IFN-beta ELISA Kit), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit), TNF-α (Mouse TNF alpha ELISA Kit, High Sensitivity, BMS607-2HS), IL-6 (Mouse IL-6 ELISA Kit, BMS603-2), IL-1β (Mouse IL-1 beta ELISA Kit, BMS6002), IL-27 (Mouse IL-27 p28/IL-30 ELISA Kit Quantikine, M2728), CCL-2(Mouse CCL2/JE/MCP-1 ELISA Kit Quantikine, MJE00B), and IL-10 (Mouse IL-10 Quantikine ELISA Kit) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).

Techniques: Aerosol, Control, Isolation, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

doi: 10.3389/fimmu.2026.1802599

Figure Lengend Snippet: Effect of the therapeutic administration of Lactiplantibacillus plantarum ATCC 10241 cell-free supernatant (LpCFS) on the resistance to Pseudomonas aeruginosa respiratory infection. BALB/c mice (6-week-old) were challenged with antibiotic-sensitive (PaS) or multidrug-resistant (PaR) P. aeruginosa strains and then received aerosol administration of LpCFS or vehicle control for three consecutive days. Three days after the last LpCFS administration the levels of TNF-α, IL-6, IFN-γ, IL-10 and IL-27 in broncho-alveolar fluid (BAL) samples were determined. Basal levels of cytokines in BAL from uninfected mice receiving vehicle alone are included for comparison (gray lines). Data are presented as mean ± SEM. Statistical analysis was performed using Student’s t-test. Differences were considered statistically significant at p < 0.05 (*).

Article Snippet: Interferon (IFN)-β (Mouse IFN-beta ELISA Kit), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit), TNF-α (Mouse TNF alpha ELISA Kit, High Sensitivity, BMS607-2HS), IL-6 (Mouse IL-6 ELISA Kit, BMS603-2), IL-1β (Mouse IL-1 beta ELISA Kit, BMS6002), IL-27 (Mouse IL-27 p28/IL-30 ELISA Kit Quantikine, M2728), CCL-2(Mouse CCL2/JE/MCP-1 ELISA Kit Quantikine, MJE00B), and IL-10 (Mouse IL-10 Quantikine ELISA Kit) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).

Techniques: Infection, Aerosol, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

doi: 10.3389/fimmu.2026.1802599

Figure Lengend Snippet: Effect of the administration of live (LV1505) and heat-killed (HK1505) Lacticaseibacillus rhamnosus CRL1505 on broncho-alveolar fluid (BAL) macrophage-enriched adherent cells. BALB/c mice (6-week-old) were nasally treated with LV1505, HK1505, or vehicle control for three consecutive days. On day four, BAL macrophage-enriched adherent cells were isolated, cultured, and ex vivo challenged with antibiotic-sensitive (PaS) or multidrug-resistant (PaR) Pseudomonas aeruginosa strains or lipopolysaccharide (LPS). One day after the challenges, the levels IFN-γ, IL-10, and IL-27 were measured using ELISA. Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test. Differences were considered statistically significant at p < 0.05 (*), p < 0.01 (**). The same control cohort was used for the experiments in Figures 3 , .

Article Snippet: Interferon (IFN)-β (Mouse IFN-beta ELISA Kit), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit), TNF-α (Mouse TNF alpha ELISA Kit, High Sensitivity, BMS607-2HS), IL-6 (Mouse IL-6 ELISA Kit, BMS603-2), IL-1β (Mouse IL-1 beta ELISA Kit, BMS6002), IL-27 (Mouse IL-27 p28/IL-30 ELISA Kit Quantikine, M2728), CCL-2(Mouse CCL2/JE/MCP-1 ELISA Kit Quantikine, MJE00B), and IL-10 (Mouse IL-10 Quantikine ELISA Kit) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).

Techniques: Control, Isolation, Cell Culture, Ex Vivo, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

doi: 10.3389/fimmu.2026.1802599

Figure Lengend Snippet: Effect of the prophylactic administration live (LV1505) and heat-killed (HK1505) Lacticaseibacillus rhamnosus CRL1505 on the resistance to Pseudomonas aeruginosa respiratory infection. BALB/c mice (6-week-old) received prophylactic intranasal administration of LV1505 or HK1505 for three days and then were challenged with antibiotic-sensitive (PaS) or multidrug-resistant (PaR) P. aeruginosa strains. Three days after the infection, the levels of TNF-α, IL-6, IFN-γ, IL-10 and IL-27 in broncho-alveolar fluid (BAL) samples were determined. Basal levels of cytokines in BAL from uninfected mice receiving vehicle alone are included for comparison (gray lines). Data are presented as mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test. Differences were considered statistically significant at p < 0.05 (*), p < 0.05 (**).

Article Snippet: Interferon (IFN)-β (Mouse IFN-beta ELISA Kit), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit), TNF-α (Mouse TNF alpha ELISA Kit, High Sensitivity, BMS607-2HS), IL-6 (Mouse IL-6 ELISA Kit, BMS603-2), IL-1β (Mouse IL-1 beta ELISA Kit, BMS6002), IL-27 (Mouse IL-27 p28/IL-30 ELISA Kit Quantikine, M2728), CCL-2(Mouse CCL2/JE/MCP-1 ELISA Kit Quantikine, MJE00B), and IL-10 (Mouse IL-10 Quantikine ELISA Kit) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).

Techniques: Infection, Comparison

Journal: Frontiers in Immunology

Article Title: Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

doi: 10.3389/fimmu.2026.1802599

Figure Lengend Snippet: Effect of the combined prophylactic administration of heat-killed (HK1505) Lacticaseibacillus rhamnosus CRL1505 and the therapeutic administration of Lactiplantibacillus plantarum ATCC 10241 cell-free supernatant (LpCFS) on the resistance to Pseudomonas aeruginosa respiratory infection. BALB/c mice (6-week-old) received prophylactic intranasal administration of HK1505 for three days, then were challenged with antibiotic-sensitive (PaS) or multidrug-resistant (PaR) P. aeruginosa strains and received aerosol administration of LpCFS or vehicle control for three consecutive days after the infection. Three days after the last LpCFS administration, the levels of TNF-α, IL-6, IFN-γ, IL-10 and IL-27 in broncho-alveolar fluid (BAL) samples were determined. Data are presented as mean ± SEM. Statistical analysis was performed using Student’s t-test. Differences were considered statistically significant at p < 0.05 (*), p < 0.05 (**) compared with vehicle control. † significant at p < 0.05 (*) between the indicated groups.

Article Snippet: Interferon (IFN)-β (Mouse IFN-beta ELISA Kit), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit), TNF-α (Mouse TNF alpha ELISA Kit, High Sensitivity, BMS607-2HS), IL-6 (Mouse IL-6 ELISA Kit, BMS603-2), IL-1β (Mouse IL-1 beta ELISA Kit, BMS6002), IL-27 (Mouse IL-27 p28/IL-30 ELISA Kit Quantikine, M2728), CCL-2(Mouse CCL2/JE/MCP-1 ELISA Kit Quantikine, MJE00B), and IL-10 (Mouse IL-10 Quantikine ELISA Kit) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (ELISA) technique kits following the manufacturer’s recommendations (R&D Systems, MN, USA).

Techniques: Infection, Aerosol, Control

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