Ifn γ

The murine liver and ileum were fixed in 4% paraformaldehyde overnight at 4°C, dehydrated, soaked in xylene, embedded in paraffin in sequence, and then sliced into 4-μm sections. Paraffin sections were dewaxed with xylene and then dehydrated with different concentrations of ethanol. Sections were subjected to H&E staining and immunohistochemical as well as immunofluorescence staining. The following primary antibodies were used for immunohistochemical staining: IL-1β (Bioss, China), IL-6 (Bioss), IFN-γ (Bioss), IL-17A (Bioss), Caspase-1 (ABclonal, China) and NLRP3 (ABclonal). For immunofluorescence on tissues, ZO-1 (Abcam, UK), occludin (Abcam), F4/80 (Abcam), and Alexa Fluor 555 (BBI, China) were used. Furthermore, images were obtained under a microscope (AxioObser Z1, Germany) at a magnification of ×200, and positive results were quantified using ImageJ software (Free Software Foundation Inc., Boston, MA).

Whole cell extracts were prepared using RIPA buffer (Cell Signaling Technology, Beverly, MA) with protease inhibitor cocktail (Roche, Switzerland). Equal amounts of cell lysates were separated on 10% SDS–polyacrylamide gel, and were then electrotransferred to polyvinylidene difluoride membrane (GE Life sciences, Pittsburgh, PA). The membranes were blocked with 5% skim milk in 0.1% TBS-Tween20 for 1 h at 25°C prior to incubation with primary antibodies against PD-L1 (Proteintech, Rocky Hill, NJ), IFN-γ (Bioss, Woburn, MA), TNF-α (Santa Cruz Biotechnology), phospho-STAT1 (S727) (Cell Signaling Technology, Beverly, MA), STAT1 (Cell Signaling Technology), phospho-STAT3 (Y705) (Santa Cruz Biotechnology), STAT3 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology). Primary antibodies were incubated for overnight at 4°C after blocking. For antibodies detection, goat-anti-rabbit IgG-HRP antibody (Santa Cruz Biotechnology) was incubated for 1 h at 25°C. The blots were developed using chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA) and Signals were detected using ImageQuant LAS-4000 (Fujifilm, Tokyo, Japan). GAPDH was used as a loading control.

IL-33, TSLP, IL-12p70, and IL-6 levels in BMDCs supernatants; HDM-specific IgE, IFN-γ, IL-13, and IL-17A in BALF samples; IL-33, TSLP, IL-13, IL-17A, CCL11 and CXCL1 in lung homogenates; IL-33 and TSLP in HBE cell supernatants were measured using ELISA kits (HDM-specific IgE, Sigma: St. Louis, Missouri, USA; IL-33, TSLP, IFN-γ, IL-13, IL-17A, CCL11, and CXCL1, Bioss Inc., Woburn, MA, USA), as directed by the manufacturers

Purified dNK cells from all groups were incubated for 20 h before harvesting. Equal amounts of protein from total-cell lysates were separated by 10% sodium dodecyl surface-polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked at room temperature for 3 h in 7% (w/v) nonfat dry milk dissolved in tris-buffered saline/Tween^® 20 (TBS-T) buffer. Membranes were incubated with primary antibodies for 2B4 (Bioss, China), p-2B4 (Bioss, China), SHP-2 (Proteintech, China), Fyn (Proteintech, China), p-ERK (Abcam, England), p-P38 (Abcam, England), IFN-γ (Bioss, China), TNF-α (Bioss, China), horseradish peroxidase (HRP)-goat-anti-rabbit immunoglobulin G (IgG) (Proteintech, China), HRP-goat-anti-mouse IgG (Proteintech, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech, China) overnight at 4 °C with gentle rocking. Membranes were washed with TBS-T five times and then incubated with the appropriate secondary antibody for 3 h at room temperature. Immune complexes were then visualized with an enhanced chemiluminescence (ECL) detection kit (F. Hoffmann-La Roche, Ltd., Switzerland). Protein expression levels were quantified in ImageJ software (Rawak Software, Inc., Germany).

For immunoblots and immunofluorescence, the procedures described in Rao, Farooqui, Zhang, et al. (2018) were followed. For immunoblot control, rat/mouse amyloid‐β1‐42 synthetic peptide (Abcam; ab120959) was used. Since formalin‐fixed aged brain samples tend to generate high autofluorescence, sodium borohydride and CuSO₄ treatments were included as our standard procedures for immunofluorescence. For immunohistochemistry, we used the SuperPicture 3rd gen IHC kit (Thermo Fisher Scientific) following the manufacturer's protocol. We used the following primary antibodies: Actin (Santa Cruz Biotechnology; Cat. number, sc‐1616), amyloid‐β (referred as B‐4; Santa Cruz, sc‐28365), amyloid‐β (referred as NAB228; Cell Signaling Technology; #2450), amyloid‐β (referred as D54D2; Cell Signaling Technology, #8243), COX‐2 (Santa Cruz, sc‐7951), GFAP (Thermo Fischer Scientific, Rb‐087‐A), GSK3α+β (Cell Signaling Technology, # 5676T), Iba1 (Abcam, ab178846), IFN‐γ (Bioss antibodies, bs‐0480R), IL1‐β (Bioss, bs‐6319R), IL‐6 (Bioss, bs‐0379R), IL10 (Bioss, bs‐0698R), NFκB 65kd (Santa Cruz, sc‐372), phosphor‐p38MAPK [T180+Y182] (Bioss, bs‐2210R), p21^WAF1/CIP1 (Santa Cruz, sc‐817), p‐GSK3α (S21) (Cell Signaling Technology, #9316), p‐GSK3β (S9) (Biorbyt, # orb393067), p‐TAU(S262) (EnoGene; E011111), p‐TAU (S404) (Santa Cruz, sc‐12952), and TNF‐α (Bioss, bs‐2081R).