Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Identification of Distinct Human TH17 Cell Subsets and Generation of Stable TH17 Clones from PBMC for Functional Characterization. A Schematic representation of the workflow to generate T H 17-IL22 + /IFNg + and T H 17-IL-10 + clones used to perform bulk ATAC-seq and RNA-seq data sets. In brief, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using density gradient centrifugation. The samples were enriched for CD4 + CCR6 + CXCR3- TH17 cells, referred to as “bulk TH17 cells.” Viable IL-17-producing cells were isolated by flow cytometry following a 3-hour stimulation with PMA and ionomycin using a IL-17 capture assay. The single TH17 cell clones were sorted into 384-well plates and expanded with allogeneic γ-irradiated feeder cells and phytohemagglutinin in complete medium containing IL-2. After approximately ten days, clones were transferred to 96-well plates for expansion, and following 2–3 weeks, their cytokine profiles were analyzed. T cell clones were then evaluated at two stages: day 0 (resting state) and day 5 (activated state). On day 5, they were stimulated for 48 hours with anti-CD3 and CD28, followed by an additional 3 days in uncoated plates. On both evaluation days, cells underwent further stimulation — 5 hours for protein analysis and 2 hours for RNA and chromatin-accessibility (ATAC-seq) analysis. Only TH17 clones exhibiting a stable cytokine profile after two rounds of resting and reactivation were selected for RNA-seq and ATAC-seq analysis. B Intracellular staining of IL-17 and IFNγ (top) and IL-22 and IL-10 (bottom) in a T H 17-IL10 + clone (right) and a T H 17-IL22 + /IFNg + clone (left) in the resting state (Day 0) and 5 days post-activation (Day 5). Numbers in quadrants indicate percent cells. C Frequency of IL-17+, IL-10+, IFNγ+, and IL-22+ cells among 6 independent TH17-IL-22 + /IFNγ + (left) and TH17-IL-10 + (right) clones at Day 0 and Day 5. Each symbol represents an individual T cell clone ( n = 6); data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01 (one-way ANOVA). TH17 clones were selected for RNA and ATAC-seq analysis based on the following criteria: ≥50% IL-17A+ cells at Day 0, ≥15% IL-22+ cells at Day 0 and Day 5, ≥15% IFNγ+ cells at Day 0 and Day 5 for TH17-IL-22 + /IFNγ + clones; ≥50% IL-17A+ cells at Day 0, ≥15% IL-10+ cells at Day 5 for TH17-IL-10 + clones

Article Snippet: Viable IL-17-producing bulk TH17 cells were sorted by flow cytometry using the IL-17 cytokine secretion assay (Miltenyi Biotec#130–094–536) following 3 h of stimulation with phorbol 12-myristate 13-acetate (PMA) (0.2 μM) and ionomycin (1 μg/ml) (both from Sigma-Aldrich), according to the manufacturer’s instructions.

Techniques: Clone Assay, Functional Assay, RNA Sequencing, Isolation, Gradient Centrifugation, Flow Cytometry, Irradiation, Staining, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Aiolos and Eos drive distinct human TH17 functional states

doi: 10.1007/s00018-026-06089-1

Figure Lengend Snippet: Identification of Distinct Human TH17 Cell Subsets and Generation of Stable TH17 Clones from PBMC for Functional Characterization. A Schematic representation of the workflow to generate T H 17-IL22 + /IFNg + and T H 17-IL-10 + clones used to perform bulk ATAC-seq and RNA-seq data sets. In brief, peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood using density gradient centrifugation. The samples were enriched for CD4 + CCR6 + CXCR3- TH17 cells, referred to as “bulk TH17 cells.” Viable IL-17-producing cells were isolated by flow cytometry following a 3-hour stimulation with PMA and ionomycin using a IL-17 capture assay. The single TH17 cell clones were sorted into 384-well plates and expanded with allogeneic γ-irradiated feeder cells and phytohemagglutinin in complete medium containing IL-2. After approximately ten days, clones were transferred to 96-well plates for expansion, and following 2–3 weeks, their cytokine profiles were analyzed. T cell clones were then evaluated at two stages: day 0 (resting state) and day 5 (activated state). On day 5, they were stimulated for 48 hours with anti-CD3 and CD28, followed by an additional 3 days in uncoated plates. On both evaluation days, cells underwent further stimulation — 5 hours for protein analysis and 2 hours for RNA and chromatin-accessibility (ATAC-seq) analysis. Only TH17 clones exhibiting a stable cytokine profile after two rounds of resting and reactivation were selected for RNA-seq and ATAC-seq analysis. B Intracellular staining of IL-17 and IFNγ (top) and IL-22 and IL-10 (bottom) in a T H 17-IL10 + clone (right) and a T H 17-IL22 + /IFNg + clone (left) in the resting state (Day 0) and 5 days post-activation (Day 5). Numbers in quadrants indicate percent cells. C Frequency of IL-17+, IL-10+, IFNγ+, and IL-22+ cells among 6 independent TH17-IL-22 + /IFNγ + (left) and TH17-IL-10 + (right) clones at Day 0 and Day 5. Each symbol represents an individual T cell clone ( n = 6); data are shown as mean ± s.e.m. * P < 0.05, ** P < 0.01 (one-way ANOVA). TH17 clones were selected for RNA and ATAC-seq analysis based on the following criteria: ≥50% IL-17A+ cells at Day 0, ≥15% IL-22+ cells at Day 0 and Day 5, ≥15% IFNγ+ cells at Day 0 and Day 5 for TH17-IL-22 + /IFNγ + clones; ≥50% IL-17A+ cells at Day 0, ≥15% IL-10+ cells at Day 5 for TH17-IL-10 + clones

Article Snippet: PE IL-17 Secretion Assay- detection kit , Miltenyi , 130-094-536.

Techniques: Clone Assay, Functional Assay, RNA Sequencing, Isolation, Gradient Centrifugation, Flow Cytometry, Irradiation, Staining, Activation Assay

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: FACS-sorted Tregs from patients with asthma and healthy controls (n = 25 per group for all panels) were evaluated for suppressive capacity, intracellular cytokine expression, and secreted cytokine production. (A, B) Suppressive capacity. Representative flow cytometry plots of responder T-cell proliferation and quantitative analysis of Treg-mediated suppression. Tregs from asthma patients exhibited reduced suppressive efficiency compared with controls. (C) Intracellular cytokine expression. Frequencies of Tregs expressing anti-inflammatory cytokines (IL-10, TGF-β) and pro-inflammatory cytokines (IFN-γ, IL-17). Asthma-derived Tregs showed decreased IL-10 + and TGF-β + populations and increased IFN-γ + and IL-17 + populations relative to controls. (D) Secreted cytokine production. Concentrations of IL-10 and active TGF-β1 in 72-h culture supernatants from highly purified Tregs. Asthma Tregs secreted lower levels of both cytokines compared with healthy controls. Data are presented as the mean ± SD, with each dot representing an individual sample. Statistical significance was determined using Welch’s unpaired t test or one-way ANOVA followed by Tukey’s post hoc test, as appropriate. For panels (B, C), P -values were Bonferroni-corrected for multiple comparisons. Significant differences are indicated by asterisks (**** P < 0.001).

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Purification

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A, B) Purity and batch consistency analysis of KQS-1. HPLC analysis of KQS-1 production batches. (A) Quantitative analysis of purity across three independent production batches (n = 5 replicates per batch). Individual data points (black dots) represent single measurements, and the vertical distribution shows batch uniformity. The red dashed line denotes the minimum purity requirement of 95%. Statistical analysis by one-way ANOVA indicates no significant variation between batches (ns, P > 0.05), confirming high-level production consistency. (B) Representative HPLC chromatogram overlay of three batches showing consistent peak profiles and identical retention times, confirming the chemical stability and absence of batch-to-batch variation in the manufacturing process. (C, D, E) Impact of PBS vehicle on the function and viability of asthma patient–derived Tregs. Assessment of PBS treatment on FACS-sorted CD4 + CD25 + CD127 − Tregs isolated from asthma patients (n = 25 patients per group). (C) Treg suppressive efficiency (%) measured by the inhibition of responder T-cell proliferation. No significant difference was observed between the untreated control and the PBS vehicle group. (D) Frequencies (%) of intracellular anti-inflammatory (IL-10, TGF-β) and pro-inflammatory (IFN-γ, IL-17) cytokines determined by flow cytometry after 48 h. Comparisons were adjusted using the Bonferroni correction; no significant alterations were detected across all markers. (E) Treg survival rate (%) after 48 h of culture in the presence or absence of PBS. In all panels, box-and-whisker plots represent the median, interquartile range (IQR), and range, whereas individual black dots denote data from independent patient samples. Statistical significance was evaluated using Welch’s unpaired t test. ns indicates P > 0.05, confirming that PBS serves as a neutral vehicle for KQS-1.

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Derivative Assay, Isolation, Inhibition, Control, Flow Cytometry, Whisker Assay

Journal: Life Science Alliance

Article Title: Dectin-1 epigenetic reprogramming rescues senescent-like Treg function in allergic asthma

doi: 10.26508/lsa.202503552

Figure Lengend Snippet: (A) Suppression assays demonstrating that KQS-1–treated Tregs exhibit enhanced inhibition of responder T-cell proliferation compared with vehicle-treated controls. (B, C) Cytokine profiling after 48-h KQS-1 exposure. ELISA analysis of culture supernatants shows a shift toward an anti-inflammatory phenotype, characterized by increased IL-10 and TGF-β1 production and reduced IFN-γ and IL-17 levels. (D) Treg survival analysis in patient-derived cells. Representative and quantitative assessment of Annexin V + /PI − apoptotic Tregs after 48-h treatment with KQS-1 or vehicle control. Paired samples are connected by lines. Data are presented as the mean ± SD. (A, B, C, D) Sample sizes: n = 8 (A, B, C) and n = 12 (D). Statistical significance was determined using unpaired or paired two-tailed t tests, as appropriate, with Bonferroni correction for multiple comparisons where applicable. ** P < 0.01, *** P < 0.001.

Article Snippet: Human IL-10, TGF-β1, IFN-γ, and IL-17 ELISA kits (R&D Systems) were used for the detection of cytokines.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Licoisoflavone B alleviates psoriasis via SCD1-targeted lipid metabolism reprogramming and suppression of Th17/IL-17–mediated inflammation

doi: 10.3389/fphar.2026.1754729

Figure Lengend Snippet: Integrated workflow for target prediction and experimental validation of Lico B in psoriasis. Bioinformatic analyses, including WGCNA module construction, gene–trait correlation analysis, multi-database target intersection (GEO, GeneCards, TTD), molecular docking, and SPR analysis were used to identify candidate targets of Lico B. Subsequent in vitro and in vivo experiments—comprising Western blotting, qPCR, immunofluorescence staining, and flow cytometry in IL-17–stimulated keratinocytes and IMQ-induced psoriatic mice—were conducted to validate the therapeutic mechanisms of Lico B.

Article Snippet: The IL-17 group was treated with 100 ng/mL IL-17 (Proteintech; HZ-1113); the Lico B group received 9 μM Lico B (MCE; HY-N3388); the IL-17 + Lico B group was co-treated with IL-17 and Lico B; and the control group was treated with PBS.

Techniques: Biomarker Discovery, In Vitro, In Vivo, Western Blot, Immunofluorescence, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Licoisoflavone B alleviates psoriasis via SCD1-targeted lipid metabolism reprogramming and suppression of Th17/IL-17–mediated inflammation

doi: 10.3389/fphar.2026.1754729

Figure Lengend Snippet: Schematic illustration of lico B treatment in psoriasis. Lico B modulates lipid metabolic pathways by suppressing SCD1-dependent metabolic reprogramming and reducing lipid droplet accumulation in keratinocyte, while simultaneously attenuating the TH17/IL-17 axis. Through concurrent regulation of keratinocyte metabolism and inflammatory cytokine production, Lico B ameliorate psoriatic skin pathology.

Article Snippet: The IL-17 group was treated with 100 ng/mL IL-17 (Proteintech; HZ-1113); the Lico B group received 9 μM Lico B (MCE; HY-N3388); the IL-17 + Lico B group was co-treated with IL-17 and Lico B; and the control group was treated with PBS.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Licoisoflavone B alleviates psoriasis via SCD1-targeted lipid metabolism reprogramming and suppression of Th17/IL-17–mediated inflammation

doi: 10.3389/fphar.2026.1754729

Figure Lengend Snippet: Integrated workflow for target prediction and experimental validation of Lico B in psoriasis. Bioinformatic analyses, including WGCNA module construction, gene–trait correlation analysis, multi-database target intersection (GEO, GeneCards, TTD), molecular docking, and SPR analysis were used to identify candidate targets of Lico B. Subsequent in vitro and in vivo experiments—comprising Western blotting, qPCR, immunofluorescence staining, and flow cytometry in IL-17–stimulated keratinocytes and IMQ-induced psoriatic mice—were conducted to validate the therapeutic mechanisms of Lico B.

Article Snippet: The IL-17 group was treated with 100 ng/mL IL-17 (Proteintech; HZ-1113); the Lico B group received 9 μM Lico B (MCE; HY-N3388); the IL-17 + Lico B group was co-treated with IL-17 and Lico B; and the control group was treated with PBS.

Techniques: Biomarker Discovery, In Vitro, In Vivo, Western Blot, Immunofluorescence, Staining, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Licoisoflavone B alleviates psoriasis via SCD1-targeted lipid metabolism reprogramming and suppression of Th17/IL-17–mediated inflammation

doi: 10.3389/fphar.2026.1754729

Figure Lengend Snippet: Schematic illustration of lico B treatment in psoriasis. Lico B modulates lipid metabolic pathways by suppressing SCD1-dependent metabolic reprogramming and reducing lipid droplet accumulation in keratinocyte, while simultaneously attenuating the TH17/IL-17 axis. Through concurrent regulation of keratinocyte metabolism and inflammatory cytokine production, Lico B ameliorate psoriatic skin pathology.

Article Snippet: The IL-17 group was treated with 100 ng/mL IL-17 (Proteintech; HZ-1113); the Lico B group received 9 μM Lico B (MCE; HY-N3388); the IL-17 + Lico B group was co-treated with IL-17 and Lico B; and the control group was treated with PBS.

Techniques: