Fig 1: Transcription Factor IRF-4 Links IL-1R and IL-23R Pathways for Enhanced IL-17 Production in Dermal γδT Cells(A) Differential gene expression in immature fetal Vγ4T17 cells and Vγ6T17 cells (1.5-fold cutoff).(B) Immature fetal Vγ6T17 cells express higher mRNA levels of IL-1r, IL-23r, and IL-7r than Vγ4T17 cells.(C) Skin single-cell suspensions were stained for IL-1R and IL-23R on dermal Vγ4 and Vγ6 T cells. Flow plots gated on CD3+γδTCRintVγ4 or CD3+γδTCRintVγ6 cells are representative of at least two independent experiments with similar results.(D) Sorted Vγ4 and Vγ6 T cells from cultured CD2-cre;Stat3f/f or control Stat3f/f skin γδ T cell lines were stimulated with IL-23 or IL-23 plus IL-1β for 3h. The IL-1R mRNA expression levels were determined by real-time PCR analysis. Summarized data are representative of at least two independent experiments with similar results. Each experiment includes at least two mice from each strain. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001 (unpaired Student’s t test).(E) IL-1R expression levels on dermal Vγ4 and Vγ6 T cells from CD2-cre;Stat3f/f or control Stat3f/f mice were assessed by flow cytometry. Flow histograms gated on CD3+γδTCRint Vγ4 or CD3+γδTCRintVγ6 cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Stat3f/f or control Stat3f/f strains.(F) Sorted Vγ4 and Vγ6T cells from cultured CD2-cre;Stat3f/f or control Stat3f/f skin γδT cell lines were stimulated with IL-23 or IL-23 plus IL-1β for 3 h. The IRF-4 mRNA expression levels were determined by real-time PCR analysis. Summarized data are representative of at least three independent experiments with similar results. Each experiment includes at least three mice from each strain. Data are shown as mean ± SEM. *p < 0.05 (unpaired Student’s t test).(G) Sorted γδT cells from cultured CD2-cre;Rictorf/f or control Rictorf/f mice and CD2-cre;Raptorf/f or control Raptorf/f γδ T cells were stimulated with IL-1β for 3 h, and the IRF-4 mRNA expression levels were determined by real-time PCR analysis. Summarized data are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from each strain. Data are shown as mean ± SEM. *p < 0.05 (unpaired Student’s t test).(H) Cultured dermal γδT cells from WT mice were transfected with IRF-4 siRNA and/or fluorescein isothiocyanate (FITC)-labeled control siRNA. Cells were then stimulated with IL-1β plus IL-23 for intracellular IL-17 production. Flow plots gated on CD3+γδTCR+ cells are representative of at least three independent experiments with similar results. Percentages of IL-17-producing γδT cells are shown as mean ± SEM. **p < 0.01 (unpaired Student’s t test).(I) Sorted Vγ6T cells from cultured CD2-cre;Stat3f/f or control Stat3f/f skin γδT cell lines were stimulated with IL-23 plus IL-1β for 3 h. The RelA mRNA expression levels were determined by real-time PCR analysis. Summarized data are representative of at least two independent experiments with similar results. Data are shown as mean ± SEM. **p < 0.01, ***p < 0.001 (unpaired Student’s t test).
Fig 2: JNK1 is necessary for inflammasome activation by Aldara® cream but not upon TLR7 or TLR7/8 stimulation. (A) JNK1 expression in Mapk8fl/fl, CD11cCre+ Mapk8fl/fl BMDCs and LysMcre+ Mapk8fl/f l BMMs. (B) Mapk8fl/fl, Mapk8∂M and Mapk8∂DC mice were shaved and then treated daily with a topical dose of Aldara® cream (62.5 mg/d) for eight consecutive days. Mice were sacrificed 4h after the last application. (C, D) Epidermis thickness (μm) was measured on MGG-stained sections. Results were pooled and collected from two independent experiments (treated mice n = 7 or 8 or 10 or 11 according to mice groups and untreated n = 5). (E) Representative MGG-stained slides from each experimental group. (scale bar = 50 μm, 200X magnification). (F, G) Expression of inflammatory markers in whole skin samples was quantified by RTqPCR. Results were pooled from two independent experiments (treated mice n = 5 or 6 or 9 according to mice groups and untreated n = 6). Results are expressed as median and interquartile range and each dot represents an individual mouse. Statistical analysis was performed using Mann-Whitney test, ns, non significant, * p < 0.05, **p < 0.01, ***p < 0.001. (H) TNF, (I) CXCL1and (J) IL12p40 production by BMMs and (K) IL-23 production by BMDCs stimulated overnight by Aldara cream (1/2500 dilution in DMSO), R837 (8μg/ml) or R848 (5μg/ml) were quantified by ELISA. (L) IL-1β production by BMMs primed for 3h with LPS (100 ng/ml) and then stimulated by Aldara cream (1/100 dilution in DMSO), R837 (8μg/ml) or R848 (10μg/ml) were quantified by ELISA. Results are from one experiment representative of two independent experiments. Results are expressed as median and interquartile range and each dot represents an experimental replicate. Statistical analysis was performed using Mann-Whitney test, **p < 0.01.
Fig 3: STAT3 Signaling Differentially Regulates Effector Function of Different Subsets of Dermal γδT Cells(A) Whole skin cell suspensions from CD2-cre;Stat3f/f or control Stat3f/f mice were stimulated with IL-1β, IL-23, or IL-23 plus IL-1β for 48 h. Intracellular IL-17 production by dermal γδT cells was determined by flow cytometry. Flow plots gated on CD3+γδTCRint cells, Vγ4, or Vγ6 T cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Stat3f/f or control Stat3f/f strains. Percentages of total γδT17, Vγ4T17, and γδ6T17 cells are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired Student’s t test).(B) Cultured skin γδT cell lines from CD2-cre;Stat3f/f or control Stat3f/f mice were stimulated with IL-23 or IL-1β for 30 min. p-Stat3, p-mTOR, p-AKT, and p-S6 were examined by flow cytometry. Flow histograms gated on CD3+γδTCR+Vγ4 or CD3+γδTCR+Vγ6 cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Stat3f/f or control Stat3f/f strains.
Fig 4: mTORC2 Deficiency in Dermal γδT Cells Leads to Accumulation of Dysfunctional Mitochondria and Production of Mitochondria ROS(A) γδT cell lines from C57BL/6 WT mice were stimulated with phorbol 12-myristate 13-acetate (PMA)+ionomycin in the presence of varying concentrations of 2-DG, OXA, or AGI for 24 h. Intracellular IL-17 and IFN-γ were examined by flow cytometry. Flow plots gated on CD3+γδTCR+ cells are representative of at least three independent experiments with similar results. Each experiment includes at least three WT mice.(B) Sorted skin Vγ4 or Vγ6 (Vγ4−) T cells from cultured γδT cell lines established from CD2-cre;Rictorf/f or control Rictorf/f mice were blotted with indicated molecules including HIF-1α, PFKFB3, IDH2, and HK1.(C) Whole skin cell suspensions from CD2-cre;Rlctorf/f or control Rictorf/f mice and CD2-cre;Raptorf/f or control Raptorf/f were stained with 2-NBDG. Expression of 2-NBDG was analyzed by flow cytometry. Flow histograms gated on CD3+γδTCRint cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from each strain.(D) Whole skin cell suspensions from CD2-cre;Rictorf/f or control Rictorf/f mice and CD2-cre;Raptorf/f or control Raptorf/f were stained with MitoTracker Green and MitoTracker Red. Flow plots gated on CD3+γδTCRint cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from each strain. Percentages of MitoTracker Green+/hi and MitoTracker Red+ dermal γδT cells are shown as mean ± SEM. **p < 0.01 (unpaired Student’s t test).(E and F) Whole skin cell suspensions from CD2-cre;Rictorf/f or control Rictorf/f mice were stained with MitoSOX for mitochondria ROS production (E) and DCFDA for total ROS production (F). Flow plots (E) or histograms (F) gated on CD3+γδTCRint cells are representative of at least two independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Rictorf/f or control Rictorf/f strains. Percentage of MitoSOX+(E) and mean fluorescence intensity (MFI) of DCFDA+ (F) dermal γδ T cells are shown as mean ± SEM. *p < 0.05 (unpaired Student’s t test).(G) Skin γδT cell lines from Rictor cKO mice were stimulated with IL-1β plus IL-23 in the presence of varying concentrations of NAC for 24 h. Intracellular IL-17 was examined by flow cytometry. Flow plots gated on CD3+γδTCR+ cells are combined from two independent experiments with similar results. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 (one-way ANOVA).
Fig 5: mTORC2-Mediated Signaling Pathway Is Critical in IL-1β-Induced Dermal γδT Cell Activation(A and B) Sorted Vγ6 (Vγ4−) or Vγ4T cells from cultured skin γδT cell lines were stimulated with IL-1β for 30 min. p-AKT and p-S6 were examined by western blot (A) or flow cytometry (B). Western blot analysis is representative of two independent experiments with similar results. Flow histograms gated on CD3+γδTCR+ cells are representative of at least three independent experiments with similar results. Each experiment includes at least two WT mice.(C) Schematic of mTORC2 with representative histogram showing abolished mTORC2 activity (p-AKT) in Rictor-deficient dermal γδT cells upon IL-1β stimulation. Flow histogram was gated on CD3+γδTCR+ cells.(D) Whole skin cell suspensions from CD2-cre;Rictorf/f or control Rictorf/f mice were labeled with CFSE and then stimulated with IL-1β or IL-23 plus IL-1β for 3 days. CFSE dilution by dermal γδT cells was determined by flow cytometry. Flow plots gated on CD3+γδTCRint cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Rictorf/f or control Rictorf/f strains. Proliferated dermal γδ T cells are shown as mean ± SEM. **p < 0.01, ***p < 0.001 (unpaired Student’s t test).(E) Whole skin cell suspensions from CD2-cre;Rictorf/f or control Rictorf/f mice stimulated with IL-23 plus IL-1β at Indicated time points and Intracellular IL-17 production by dermal γδT cells were assessed by flow cytometry. Flow plots gated on CD3+γδTCRint cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Rictorf/f or control Rictorf/f strains. Percentages of IL-17-producing γδT cells are shown as mean ± SEM. **p < 0.01, ***p < 0.001 (unpaired Student’s t test).(F) Cultured skin γδT cell lines from CD2-cre;Rictorf/f or control Rictorf/f mice and CD2-cre;Raptorf/f or control Raptorf/f were stimulated with IL-23 for 30 min. p-STAT3 was examined by flow cytometry. Flow histograms gated on CD3+γδTCR+ cells are representative of at least three independent experiments with similar results. Each experiment includes at least two mice from CD2-cre;Rictorf/f or control Rictorf/f and CD2-cre;Raptor’7’ or control Raptorf/f strains.
Supplier Page from BioLegend for Recombinant Mouse IL-23 (carrier-free)