Protocols for animal use were approved by the Institutional Animal Care and Use Committee at Southern Illinois University School of Medicine and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as established by the National Institutes of Health. Experiments employed 9–10 weeks old male C57BL/6J, global AhR null (AhRKO) Bradfield strain (Schmidt et al., 1996) and 20 months old male and female APP/PS1 mice procured from the Jackson Laboratory (Bar Harbor, ME) and bred at Southern Illinois University School of Medicine animal facilities. All animals were group housed and entrained to a control 12:12 h light: dark schedule with food and water provided ad libitum.

All chemicals were prepared and stored according to manufacturer recommendations unless otherwise noted. Sodium chloride (Cat: 7647-14-5), Sodium phosphate, Dibasic, Anhydrous (Cat: 7558-79-4), Potassium chloride (Cat: 7447-40-7), Potassium phosphate (Cat: 7778-77-01) were obtained from Sigma-Aldrich Co. (St. Louis, MO), Triton X-100 (Cat: 807426) were obtained from MP Biomedicals (Santa Ana, CA).

Primary hippocampal cell cultures were prepared from postnatal day 0–1 C57BL6/J and AhRKO pups by adopting the method previous described (Gutiérrez-Vázquez and Quintana, 2022). Hippocampi were dissected and dissociated by papain (EC 3.4.22.2; Brain Bits) treatment, followed by trituration with sterile glass pipettes. Hippocampal cells were grown on poly-D-lysine (0.05 mg/ml) coated T-75 flasks that contains NbAstro medium (Brain Bits) (Neurobasal, 10% Horse serum, Glutamax and 1% Penicillin/Streptomycin) at 37°C in humidified 5% CO₂ for (DIV 10–14). The cell culture medium was changed on the day after seeding and then every 3 days. Upon achieving approximately 90% confluence, adherent astrocytes were detached using Trypsin (0.25%) (Cat: 15050057; Gibco, United States) and subsequently related, maintaining them in serum-free media for an additional 1–2 days prior to exposure to 5 μM oligomeric Aβ (1-42) (AnaSpec; Cat: 20276). The purity of cultured astrocyte cells was determined by staining with polyclonal glial fibrillary acidic protein (GFAP).

The lyophilized peptide powder was reconstituted by the addition of 1.0% NH₄OH, followed by dilution with sterile PBS to achieve an approximate concentration of 100 μM. This mixture was incubated for 24 h at 4°C. Subsequently, it was centrifuged at 14,000 rpm for 10 min at 4°C, and the supernatant was collected as the oligomeric Aβ peptide, as previously described by Sondag et al. (2009), Dahlgren et al. (2002).

Male mice aged 9–10 weeks were anesthetized with isoflurane and placed on a stereotaxic frame (David Kopf Instruments). A bilateral craniotomy was performed, and 2 μl of 0.5 μg/μl Aβ (1-42) or PBS was administered into the CA1 region of the hippocampus (AP = −1.70 mm, ML = ± 1.2 mm, DV = 1.53 mm) using a 5–10 μL gas-tight Hamilton syringe operating at a flow rate of 0.4 μL/min. Subsequent to the injection, the needle was left for a further 5 min before being gradually retracted. The skin of the mice was subsequently sealed with surgical staples and mice were observed for full recovery. Histological analyses were performed on treated animals 2 weeks after injection.

The expression 2–2.5 × 10⁶ astrocyte cells per T-25 flask were lysed in Trizol (Fisher Scientific, Hampton, NH, United States) and the aqueous layer was collected following chloroform extraction with RNA isolation protocol. RNA concentrations were measured using the Nanodrop spectrophotometer and cDNA was synthesized. SYBR green-based real-time reverse transcriptase PCR was carried out on Quant-Studio real-time PCR system. Values for genes of interest were normalized using GAPDH as the housekeeping gene and the relative levels of mRNA were determined using the ΔΔCt method. Primers sequence for real time PCR is listed in Supplementary Table 1.

The expression 2–2.5 × 10⁶ astrocyte cells per T-25 flask were seeded. Cell culture media from primary hippocampal astrocytes were collected following exposure to Aβ (1-42) ELISA was used to quantify levels of IL-1β (Cat BMS6002; Invitrogen), IL-10 (Cat BMS614; Invitrogen) and TNF-alpha (Cat BMS607-3; Invitrogen) secretion following manufacturer directions.

A total of 50,000 hippocampal astrocyte cells per 4-well glass slide (1.7 cm² per well) were washed with PBS and subsequently fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. Cells were permeabilized with PBST (0.1M PBS with 0.25% TritonX-100) three times for 7 min, followed by a 1 h incubation in a solution of 10% normal goat serum and 1% bovine serum albumin (BSA). After incubation, primary antibodies were applied to the cells at 4°C overnight. Cells were rinsed with PBST (0.1M PBS with 0.25% TritonX-100) before incubation with secondary antibodies in the dark at room temperature for 2 h. The cells were subsequently subjected to a final wash three times for 7 min in PBST and were cover slipped with ProLong™ Gold antifade reagent containing DAPI. Staining was examined using confocal microscopy, and quantified with National Institute of Health Image J Software 1.48 (RRID:SCR_003070). For immunoreactivity quantification, a threshold value was set and maintained for all images. The percentage area covered and GFAP mean intensity were then calculated. For astrocyte total area size (μm²) quantification, Z-stack images containing five optical slices at an interval of 1 μm using a 40x oil immersion objective (numerical aperture 1.3) with adjusted pinhole size of 0.81 AU on a confocal microscopy system (Zeiss LSM800) at a resolution of 1,024 × 1,024 pixels were taken and a surface was built around each astrocyte stained with GFAP, and astrocyte size was determined. Primary antibodies used included: Chicken polyclonal glial fibrillary acidic protein antibody (1:1000 biosensis Catlog:C-1373-50), Rabbit monoclonal IgG2 anti-C3 (1:100 Invitrogen Catlog:PA5-21349) while secondary antibodies used were Goat anti-chicken IgY H&L (Alexa Fluor^® 594) 1:1000, Goat anti-rabbit IgG (H + L) (Alexa Fluor^® 488) 1:1000.

For tissue staining, 20 μm hippocampal sections were taken using a cryostat (Model HM525 NX, ThermoFisher Scientific). Serial sections were extracted from every sixth section of the hippocampus. Hippocampal slices were subjected to immunofluorescence using a chicken polyclonal glial fibrillary acidic protein (GFAP) antibody at a dilution of 1:500. Sections were permeabilized in PBST (0.1M PBS with 0.25% Triton X-100) and subsequently washed three times for 10 min each in sodium borohydride in PBS (1 mg/ml) for antigen retrieval. To mitigate non-specific binding, slices were subjected to three washes of 10 min each with PBST and subsequently incubated in a solution of 10% normal goat serum and 1% BSA for 1 h. The slices were subsequently treated overnight in primary antibody under a humidity chamber at 4°C. On the subsequent day, slices were washed four times for 10 min in PBST and incubated with the secondary antibody. Incubate Goat anti-chicken IgY H&L (Alexa Fluor ^® 594) at a dilution of 1:1000 for a duration of 2 h. The sections were subsequently subjected to a final wash of four cycles, each lasting 10 min, in PBST and were then coverslipped with ProLong™ Gold antifade reagent containing DAPI. Z-stack images containing five optical slices at an interval of 1 μm using a 40x oil immersion objective (numerical aperture 1.3) with adjusted pinhole size of 0.81 AU on a confocal microscopy system (Zeiss LSM800) at a resolution of 1,024 × 1,024 pixels were obtained, and the morphology of astrocytes was assessed with Imaris software. For astrocyte soma size, 40X z stacks Confocal images were imported into the imaris software and a 3D surface were built around the immunofluorescence z-stacks based on GFAP staining of the cells, the total soma size were then determined and was exported.

Hippocampal sections were obtained via a cryostat (Model HM525 NX, ThermoFisher Scientific). Slides were rinsed with RNase-free PBS for 5 min prior to adhering to the manufacturer’s stated methods (Cat QVT4646B), incubated in Protease QF at 40°C for 10 min, followed by three washes in PBS, each lasting 1 min. Post-washing, mRNA in the tissue was hybridized by incubating with a probe targeting AhR at 40°C for 2 h. Sections were subsequently rinsed three times with the manufacturer’s wash buffer for 2 min, with frequent agitation. Subsequent to washing, the slides were incubated for 30 min at 40°C with the pre-amplifier mix solution, followed by a further 30 min incubation at 40°C with the amplifier mix solution. The slides were subsequently rinsed three times with fresh wash buffer, each for a duration of 2 min. The tissue was then incubated with the labeled probe mix solution for 30 min at 40°C, followed by a final wash with wash buffer for 10 min. Subsequent to RNA labeling, tissue slices were treated overnight at 4°C with a chicken polyclonal primary antibody against glial fibrillary acidic protein (GFAP). On the next day, slices were rinsed four times for 10 min in PBST and incubated with the secondary antibody for 2 h. The slides were subsequently cleaned with PBS and coverslipped using ProLong™ Gold antifade reagent containing DAPI. Z-stack images containing five optical slices at an interval of 1 μm were acquired using a 40x oil immersion objective (numerical aperture 1.3) with adjusted pinhole size of 0.81 AU for the red and green channel, while the blue channel had 0.95 AU on a confocal microscopy system (Zeiss LSM800) at a resolution of 1,024 × 1,024 pixels and imported into Zeiss ZEN 3.8 software to manually count the number of AhR puncta co-labeled with GFAP stain.

Prism (GraphPad Software, Inc., La Jolla, CA; RRID:SCR_002798) software was used for statistical analyses. Data are presented as mean ± SEM. Unless otherwise stated, all cell culture experiments were repeated at least three times, from separate dissections. One-way or two-way ANOVA with Tukey’s post-hoc tests were utilized to identify significant differences between groups, where appropriate. Statistical significance was defined as p < 0.05.

To assess the inflammatory effects of Aβ (1-42), primary hippocampal astrocyte cultures were established. Purity of cultures was verified using the astrocyte marker, GFAP. Roughly 96% of the cells were positive for GFAP, and the morphology of the cultured astrocyte cells resembled the established in vitro cellular structure of astrocytes (Figures 1A, B). Furthermore, AhR was constitutively expressed in astrocyte cultures taken from C57BL6/J mice, but absent in cultures established from global AhRKO mice (Figure 1C). To examine the inflammatory effects of Aβ (1-42) on astrocyte cells, a time course induction of proinflammatory cytokine transcripts for TNF-α and IL-1β was determined. Inflammatory cytokine transcripts peaked 4–8 h after treatment with Aβ (1-42), suggesting these timepoints are optimal for experiments exploring inflammatory cytokines at the mRNA level (Figure 1D). Taken together, this result indicates that Aβ (1-42) induces inflammation in astrocytes in a time-dependent manner in vitro.

Astrogliosis is characterized by structural and molecular changes in astrocytes. The impacts of AhR activation on Aβ (1-42) -induced morphological changes, as well as induction of reactive astrocyte phenotype, were examined. To establish that AhR is activated effectively in astrocytes, the canonical downstream target Cyp1a1 was assessed. AhR agonists, FICZ (250 nM) and TCDD (10 nM) increased CYP1a1 transcript levels, in an AhR-dependent manner (Figure 2A). Subsequently, pretreatment of astrocytes with FICZ attenuated the reactive astrocyte marker induced by Aβ (1-42), as C3 gene expression and immunoreactivity were significantly reduced when compared to Aβ (1-42) only treatment group (Figures 2B, E, F); meanwhile, transcript levels of S100A10 remained unaffected by FICZ treatment (Figure 2C). C3 transcript levels induced by Aβ (1-42) were similar in Wild Type and AhRKO-derived astrocytes; however, the ratio of C3 to S100A10 transcripts was higher in AhRKO astrocytes after Aβ (1-42) treatment (Figure 2D). The deletion of AhR in astrocytes increased GFAP immunoreactivity induced by Aβ (1-42), which is another indicator for astrocyte reactivity (Figures 3A, B). Surprisingly, astrocyte size was unaffected by Aβ (1-42) treatment in both Wild Type and AhRKO-derived astrocytes (Figure 3C). Thus, our results suggest that activation of AhR by FICZ helps suppress Aβ (1-42) -induction of a reactive astrocyte phenotype.

Cytokine profiling was conducted to better elucidate the effects of AhR signaling on Aβ-induced reactive astrocytes. Aβ markedly elevated transcripts levels and secretion of TNF-α and IL-1β; however, prior activation of AhR in astrocyte cell culture with FICZ [2 h before Aβ (1-42)] substantially reduced both transcript levels and secretion of TNF-α and IL-1β. Alternatively, the anti-inflammatory cytokines IL-10 and IFN-β were not significantly changed (Figures 4A, B). To assess whether the protective effects of FICZ on astrocytes are dependent on AhR activation, an analysis of proinflammatory cytokines (TNF-α and IL-1β) was conducted in astrocytes derived from AhRKO mice. In the absence of AhR, the protective effects of FICZ were diminished, as secreted levels of TNF-α and IL-1β were markedly increased in astrocytes lacking AhR, even in the presence of FICZ, suggesting that the effects of FICZ are dependent upon the presence of AhR (Figure 4C). Furthermore, AhR-deficient astrocytes exposed to Aβ (1-42) exhibited significant increases in the transcript levels of TNF-α and IL-1β when compared to Aβ (1-42) treated WT controls (Figure 4D). In a similar manner, the levels of secreted TNF-α were significantly elevated in astrocytes derived from AhRKO mice compared to Aβ (1-42) treated WT controls, whereas no differences were observed in IL-1β secretion levels (Figure 4E). The anti-inflammatory cytokine, IL-10 showed no significant changes; however, the increased transcript expression of IFN-β induced by Aβ (1-42) treatment was diminished in AhR-deficient astrocytes exposed to Aβ (1-42) (Figures 4D, E). These results imply that AhR signaling could be controlling astrocyte responses to inflammatory stimuli by inhibiting neurotoxic cytokines.

To assess the effect of AhR deficiency on the inflammatory response of astrocytes in their native environment, structural alterations in male mice astrocytes were evaluated following CNS inflammation induced by Aβ in vivo. In this neuroinflammatory model, a substantial population of hippocampal astrocytes exhibits inflammatory-induced morphological alterations 14 days after intrahippocampal Aβ oligomer injection. Aβ generated an increase in astrocyte soma size compared to the control-treated group; furthermore, these structural alterations were exacerbated in astrocytes from male AhRKO mice, which had more hypertrophic characteristics than Aβ-treated wild-type controls (Figures 5A, B). To assess the significance of astrocyte AhR function in an AD mouse model that approximates AD progression, AhR levels were assessed in astrocytes of both males and females aged APP/PS1 mice. Twenty-months-old male APP/PS1 mice exhibited a significant increase in AhR transcript levels in astrocytes compared to age-matched WT controls (Figures 5C, D). Also, aged female APP/PS1 mice demonstrated reduced AhR transcript levels when compared to male APP/PS1 mice, suggesting a sex-specific difference in AhR expression within the astrocytes of aged APP/PS1 mice. Thus, these findings indicate that AhR deletion in mice increased astrocyte cell immunological response to inflammatory stimuli in an in vivo system, similar to results of the in vitro model.