Fig 2: 3-IAld promotes resistance to VVC by inducing the IL-22/IL-18 axis via AhR. (A) IL-22, IL-1β production (pg/mg, cytokine/total proteins for each sample), and (B) fungal growth (mean log CFUs ± s.e.m.) at 7 dpi in vaginal fluids of C57BL/6 mice (n = 6 mice/group) inoculated with 5 × 106 C. albicans blastoconidia and treated subcutaneously (sc), intraperitoneally (ip), or intragastrically (ig) with 3-IAld every day starting the day of infection. (C) C57BL/6 and Ahr−/− mice (n = 6 mice/group) with VVC were ip treated with 3-IAld every day starting the day of infection and assessed for vaginal histology (Periodic Acid Shiff–staining of vaginal sections (scale bars, 100 μm) and PMN recruitment in the VF (mean % ± s.e.m., in the insets) at different dpi. Images were acquired using a microscope with a 40× objective and the analiSIS image processing software. Red arrows indicate fungi. AhR protein (D) and gene (E) expression by immunofluorescence staining of vaginal sections (Scale bars, 100 μm) and RT-PCR of vaginal tissues, respectively, in infected mice treated with 3-IAld. Images were acquired with a 40× objective and the analySIS image processing software. (F) Gene expression by RT-PCR at 3 (AhR and Cyp1a1) and 7 (Il18) dpi. *P < 0.05, **P < 0.01 and ***P < 0.001, ****P < 0.0001 treated vs. untreated (None) mice.

Fig 3: Anakinra colocalizes with AhR in Il1r1–/– cells.(A) Immunofluorescence imaging of cellular localization of FITC-anakinra in alveolar macrophages, isolated from naive C57BL/6 or Il1r1–/– mice, primed with 100 ng/mL LPS for 2 hours at 37°C, and exposed to 10 µg/mL FITC-anakinra for 60 minutes. (B) Immunofluorescence analysis of FITC-anakinra in Il1r1–/– cells exposed to FITC-anakinra at 30 and 60 minutes in the presence of 5 µM cytochalasin D. Insets, percentage positive cells. (C) Il1r1–/– MEF cells were treated with 10 µg/mL anakinra for 0, 2, and 4 hours and assessed for AhR and IL1Ra expression by immunoblotting with specific antibodies. (D) Immunofluorescence imaging of alveolar macrophages from naive C57BL/6 and Il1r1–/– mice primed with 100 ng/mL LPS for 2 hours at 37°C, exposed to 10 µg/mL FITC-anakinra for 60 minutes, and stained with anti-AhR antibody. (E) Immunofluorescence imaging of MEF cells stained with anti-AhR antibody and DAPI and exposed to 10 µg/mL FITC-anakinra for 60 minutes. (F) Il1r1–/– MEF cells were treated with 10 µg/mL anakinra, lysed, immunoprecipitated with anti-AhR antibody, and assessed for IL1Ra and AhR expression by immunoblotting with specific antibodies. None, untreated cells. Representative images, acquired with a fluorescent microscope (BX51), and immunoblots from 2 independent experiments are shown. DAPI was used to detect nuclei. Sections were examined using a Zeiss Axio Observer Z1.

Fig 4: Anakinra activates a xenobiotic sensing pathway via IDO1.(A) H1L1 cells were treated with different doses of kynurenine and anakinra for 6 hours and assessed for luciferase assay. (B) Representative immunoblots of IL1RA, HSP90, AIP, and AhR and (C) ARNT and AhR in cell lysates in which AhR was immunoprecipitated from Il1r1–/– MEF cells treated with 10 µg/mL anakinra or 10 µM FICZ. In B and C, data are representative of 1 out of 2 independent experiments and the relative densitometric analysis are reported. (D and E) Il1r1–/– MEF cells were treated with 10 µg/mL anakinra for 2 and 8 hours and analyzed for gene expression by a custom QuantiGene plex gene expression assay. Fold changes are reported as heatmap for 2 and 8 hours (D) and histograms for 8 hours (E) (data are representative of 1 out of 2 independent experiments). (F) Il1r1–/– MEF cells were treated with 10 µg/mL anakinra and analyzed for mRNA expression of selected genes by RT-PCR (n = 3 independent samples) and protein expression of IDO1 and SOCS3 by immunoblotting (representative experiment). The relative densitometric analysis is reported. (G) Il1r1–/– MEF cells were treated with either 10 µg/mL anakinra for different times or 10 ng/mL IFN-? as positive control for 48 hours and assessed for kynurenine production by ELISA. (H) Il1r1–/– MEF cells were treated with 10 µg/mL anakinra in the presence or absence of 10 µM epacadostat and assessed for kynurenine production by ELISA and Cyp1a1 gene expression by RT-PCR (n = 3 independent samples). *P < 0.05, **P < 0.01, ***P < 0.001, treated versus untreated (None or 0 h) cells. One-way ANOVA, Bonferroni post hoc test.

Fig 5: Anakinra promotes AhR transcriptional activity.(A–C) AhR–/– mice were infected and treated with anakinra as in legend to Figure 1 and assessed for immunoblotting of LC3b and p62 (A), cytokine production (ELISA) in lung homogenates (B), and lung histology (PAS staining) at 7 days after infection (C). Scale bar: 200 µm. Data represent 3 independent experiments. Each in vivo experiment includes 6 to 8 mice per group, pooled before analysis. NS, not statistically significant, treated versus untreated (None) cells. One-way ANOVA, Bonferroni post hoc test. (D and E) Ex vivo purified alveolar macrophages and total lung cells from Ahr–/– mice infected and treated with anakinra were assessed for Nox4 and p22phox expression (RT-PCR) (n = 3 independent samples) (D) and H2O2 production (DHR staining) (n = 2 independent samples) (E). (F) Illustration of predicted binding sites from the ALGGEN-PROMO database and the Eukaryotic Promoter Database. (G) Il1r1–/– and Il1r1+/+ MEF cells were treated with 10 µg/mL anakinra. ChIP assay was performed with AhR antibody. IgG was used as negative control. qPCR was conducted at the promoter regions of Nox4. Data are technical replicates of 1 representative out of 2 independent experiments.