Journal: bioRxiv

Article Title: A novel interoceptive subfornical organ to infralimbic cortex circuit relays airway inflammation effects on fear extinction

doi: 10.1101/2025.07.16.664367

Figure Lengend Snippet: (A) Single-cell RNA-sequencing analysis of cellular diversity in the SFO reveals 13 transcriptomic cell classes with two microglial populations. Data shown in a tSNE embedding of 7,950 cells with color-coded cell identity. (B) Log-normalized expression of Il17ra in SFO cell classes. (C) Violin plot of log-normalized expression of Il17ra in SFO cell classes. Data in A-C reanalyzed and plotted from a previous study (Pool et al, 2020) [56]. (D) Representative image from RNAscope in-situ hybridization on SFO slices using probes for Il17ra and ionized calcium binding protein IBA1 showing co-localization of IL17ra +ve puncta (magenta) with IBA1 +ve puncta (yellow) and DAPI stained cells (blue). Inset shows low magnification 20X image of the whole SFO with the dotted line indicating area shown in the magnified 40X image. D3V= dorsal 3 rd ventricle (E) Bar graph shows quantified % colocalization of Il17ra +ve puncta with IBA1 +ve (73%), GFAP +ve (25.3%) puncta and HuC/D +ve soma (1.1%). shows Images for Il17ra -GFAP and Il17ra -HuC/D in-situ hybridization. N= 2-5 SFO slices (F) Schematic showing the experimental layout for FACS measurements on SFO and PFC enriched tissue. Mice were intratracheally administered HDM ± IgG or αC5aR1 to generate mild versus severe AI. SFO and PFC tissue were collected 3h following the last HDM administration. Bottom panel shows representative image for gating of CD11b +ve /P2RY12 +ve cells. Mean fluorescence intensity (MFI) of samples from mild and severe AI mice was not significantly different. (G) Quantitative PCR (qPCR) assessment of Il17ra mRNA expression in FACS sorted P2RY12 +ve SFO microglia from severe AI and mild AI mice. Significantly higher Il17ra mRNA expression was observed in P2RY12 +ve SFO microglia from severe compared to mild AI mice (t test: t 14 = 2.445, p= 0.028, N= 8 mice per group). Il17ra expression was not significantly different in P2RY12 +ve microglia from the prefrontal cortex (H) or the P2RY −ve cells from the SFO (I) (J) Strategy for the generation of Cx3cr1 cre/ERT2 : IL17ra fl/fl mice and the experimental timeline showing administration of tamoxifen, followed by a 2-week recovery period to enable establishment of peripheral Cx3cr1 +ve macrophages. Subsequently, mice underwent intratracheal HDM ± IgG or αC5aR1 administration followed by behavioral testing for FC-EXT. Control mice were Il17ra fl/fl but lacked Cre/ERT2 (Cre −ve ). (K) FACS sorted P2RY12 +ve SFO microglia from Cre +ve and Cre −ve Cx3cr1 cre/ERT2 Il17ra fl/fl mice were analyzed for Il17ra mRNA expression using qPCR. Bar graph shows negligible Il17ra transcript levels in Cre +ve vs Cre −ve mice N= 11 (Cre −ve ), 9 (Cre +ve ). (L) Severe-AI phenotype was generated in Cre +ve and Cre −ve Cx3cr1 cre/ERT2 : Il17ra fl/fl mice followed by FC-EXT. For fear acquisition, no significant group difference was observed in freezing between the severe-Cre +ve and severe-Cre −ve mice (RM-ANOVA, time (F 1,27 = 345.7, p<0.0001) but no genotype (F 1,27 = 0.264; p = 0.611) or interaction (F 1,27 = 0.154; p =0.69), Conditioned freezing (CF) 24 h later was not significantly different (t 27 = 1.98; p = 0.06). For extinction, severe-Cre +ve mice showed reduced freezing. Fear extinction over 5 days revealed the effects of time (F 2.242, 60.53 = 2.614, p=0.07) and treatment (F 1, 27 =6.798, p=0.015) but there was no interaction (F 4,108 = 0.9308, p=0.449) N= 13 (Cre +ve ), 16 (Cre −ve ). (M) Freezing during early extinction learning (E1) was not significantly different between severe-Cre +ve and severe-Cre −ve groups (t 27 = 1.508; p = 0.143). (N) Severe AI-Cre +ve mice showed significantly lower freezing during late extinction retrieval (E5) (t 27 = 3.28; p = 0.002) as compared to the severe AI-Cre −ve group. (O) Pie charts show the percent distribution of mice within each group that achieved extinction (defined as <30% freezing on E5). Note: In this cohort freezing was higher in FC-EXT, potentially contributed by genotype and strain differences (C57/Bl6). Data are represented as mean ± sem. N= 16 (severe-Cre −ve ), 13 (severe-Cre +ve ) mice (*p<0.5)

Article Snippet: The Cx3cr1 CreER (JAX: 020940) transgenic mouse line in which the expression of Cre recombinase is under the control of the Cx3cr1 promoter was crossed with the floxed Il17ra mouse line B6.Cg- Il17ra tm2.1Koll /J (JAX: 031000).

Techniques: RNA Sequencing, Expressing, RNAscope, In Situ Hybridization, Binding Assay, Staining, Fluorescence, Real-time Polymerase Chain Reaction, Control, Generated

Journal: bioRxiv

Article Title: A novel interoceptive subfornical organ to infralimbic cortex circuit relays airway inflammation effects on fear extinction

doi: 10.1101/2025.07.16.664367

Figure Lengend Snippet: Representative 20x and 40x images from RNAscope in-situ hybridization on SFO slices using probes for Il17ra and GFAP (A) and immunohistochemistry with neuronal marker HuC/D (B). Arrows in (A) show scattered co-localization of Il17ra +ve puncta (magenta) with GFAP +ve puncta (yellow), DAPI stained cells shown in blue. No co-localization of Il17ra +ve puncta (magenta) was evident with HuC/D +ve neurons (green) (B). Graph (C) and Images (D-F) are from Il17ra in situ-hybridization performed in PFC slices. Representative 20x images using Il17ra probe with neuronal marker HuC/D (D) and IBA1 (E) co-localized with Il17ra puncta. Arrows in (D, E) show scattered co-localization of Il17ra +ve puncta (magenta) with HuC/D +ve cells (green) and IBA1 +ve puncta (yellow) (E). No co-localization was observed with GFAP +ve puncta (yellow) (F). Graph (C) shows percent expression of Il17ra with IBA1 (43.8%), HuC/D (20.3%) and GFAP (not detected) within the prefrontal cortex.

Article Snippet: The Cx3cr1 CreER (JAX: 020940) transgenic mouse line in which the expression of Cre recombinase is under the control of the Cx3cr1 promoter was crossed with the floxed Il17ra mouse line B6.Cg- Il17ra tm2.1Koll /J (JAX: 031000).

Techniques: RNAscope, In Situ Hybridization, Immunohistochemistry, Marker, Staining, Expressing

Journal: Journal of Ayurveda and integrative medicine

Article Title: Withaferin A reduces pulmonary eosinophilia and IL-25 production in a mouse model of allergic airways disease.

doi: 10.1016/j.jaim.2025.101144

Figure Lengend Snippet: Fig. 2. In lung tissue of HDM-challenged mice, WFA reduces IL-25 and increases lung Il17ra mRNA expression. (A) IL-33 was measured in whole lung homogenates using ELISA. Data are presented as mean pg/mL IL-33 ± SEM. (n = 3 mice per group). *p < 0.050 in HDM-challenged mice compared to saline-challenged mice for both VC and WFA treatment groups. (B) IL-25 was measured in whole lung homogenates using ELISA. Data are presented as mean pg/mL IL-25 ± SEM. *p < 0.050 in VC-treated vs WFA-treated HDM-challenged mice (n = 3 mice per group). *p < 0.050 in WFA-treated mice comparing saline-challenge to HDM-challenge. (C and D) Il17ra and Il17rb mRNA expression was measured in whole lung RNA using RT-PCR. Data are presented as fold change in Il17ra (C) or Il17rb (D) mRNA expression ± SEM relative to Gapdh mRNA expression and to the mean of the saline-challenged VC-treated mice. (n = 2–3 mice per group). *p < 0.050 Il17ra mRNA expression in WFA-treated vs VC-treated HDM-challenged mice.

Article Snippet: 50 ng cDNA/ sample was assayed using a QuantStudio6 Flex (Applied Biosystems) using TaqMan primers specific for murine Il17ra (Mm00434214_m1) and Gapdh (Mm99999915_g1) (ThermoFisher Scientific).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Saline, Reverse Transcription Polymerase Chain Reaction