Mouse
Mouse studies were conducted at the University of California, San Francisco (UCSF). Mice were housed in specific pathogen-free facilities in temperature- and humidity-controlled animal rooms, with ambient temperature maintained between 19 and 23°C and relative humidity ranging from 40% to 60%. Mice were maintained under a 12:00–12:00 light–dark cycle, with lights on from 7:00 a.m. to 7:00 p.m., and were group housed with ad libitum access to food and water. When necessary, mice were purchased from the Jackson Laboratory. By default, mice fed at UCSF received the PicoLab Rodent Diet 20 5053 irradiated diet. For experiments requiring the use of special diets, diets were irradiated and purchased from Research Diets. The HFD was 60 kcal% fat (D12492) and the control LFD was 10 kcal% fat (D12450J). The MFD was 45 kcal% fat (D08091803). The 30 kcal% fat diet was D20072301. The Western diet was D18053102 and the matched control diet was D18053101. Mice designated as obese in this study were fed the HFD for at least 10 weeks, starting at 6 to 7 weeks of age, and weighed at least 38 g (on a cage average). For short-term HFD feeding, 6-week-old mice received HFD for 7 days before IMQ challenge, unless otherwise noted. Unless otherwise noted, mice remained on their assigned diet throughout IMQ treatment. For HFDIn fed, lean mice were placed on HFD starting 3 days before IMQ challenge and remained on this diet until 1 day after initiation. For LFDIn fed, obese mice were placed on LFD starting 3 days before IMQ challenge and remained on LFD until 1 day after initiation. For HFDout fed, lean mice were maintained on LFD, except for two separate periods when they were switched to HFD: first, 7 to 3 days before the onset of IMQ and, later, 3 to 6 days after the onset, during disease progression. For LFDout fed, obese mice were maintained on HFD except for two separate periods when they were switched to LFD: first 7–3 days before IMQ initiation and later 1–6 days after initiation during disease progression. For HFDDODO fed, mice were placed on HFD for the entire disease onset phase (extending from 7 days before to 6 days after initiation of IMQ) and then returned to LFD for the recovery and rechallenge phases. C57BL/6J (000664), B6.SJL-Ptprca Pepcb/BoyJ (002014)56, Cloth1−/− (002216)57, Tcra−/− (002116)58B6.129S2-H2dlAb1-Ea/J (MHCIIΔ/Δ) (003584)59, B2m−/− (002087)60, Tcrd−/− (002120)61, Cd4create (022071)62 And Nlrp3−/− (021302)63 mice were purchased from the Jackson Laboratory. Il1r1fl/fl (ref. 64) And Cd4creERT2Ai14 mouse65.66 were provided by the Scharschmidt laboratory at UCSF. All mice used for the studies were male. All procedures involving animals were performed in accordance with protocols approved by the UCSF Institutional Animal Care and Use Committee (IACUC).
Type 17 acute skin inflammation model induced by IMQ
IMQ-induced type 17 acute skin inflammation was generated in mice of different ages fed with LFD or HFD. Briefly, mice were anesthetized using isoflurane and 15 mg of cream containing 5% IMQ (TARO) was applied daily to the dorsal and ventral sides of the mouse ear for 6 days (ref. 22). Ear thickness was measured daily with a digital micrometer (Mitutoyo). Then, 24 hours after the last application, the mice were euthanized and the ears were collected for flow cytometry, RNA extraction, histological analysis, and other downstream applications. For the rechallenge experiments, after the last day of IMQ treatment during the initial challenge, mice were allowed to recover. Ear thickness was measured weekly to assess recovery. Unless otherwise noted, after 25–30 days, mice were subjected to 15 mg of cream containing 5% IMQ for 4–6 consecutive days. To inhibit NLRP3 activation, MCC950 sodium (S7809, Selleckchem) was prepared in 50 mg ml−1 in water and 75 μg was administered daily to each mouse topically, in synchronization with the application of IMQ. For experiments involving the back skin, mice were anesthetized with isoflurane 1 day before IMQ treatment and an area of approximately 2.5 cm2 The area was shaved and treated with Nair Hair Removal Cream, which was quickly rinsed off with water. Under isoflurane anesthesia, 50 mg of cream containing 5% IMQ (TARO) was applied daily to the prepared back skin for 6 consecutive days. For re-challenge experiments, shaving and hair removal were performed only before the initial challenge and were not repeated until the second IMQ challenge.
Histology of lesional skin of the ear
Ear skin was collected and immersed in 4% paraformaldehyde (PFA) overnight. The tissues were then embedded, sectioned and stained with H&E by the Histology and Light Microscopy Core at the Gladstone Institutes. Images were taken using a Zeiss microscope. The scale bar was added by converting pixels to µm using ImageJ.
Assays of serum cytokines, hormones and IL-1β
Serum was collected and submitted to Eve Technologies for quantification using Luminex-based assays. For tissue IL-1β measurements, individual whole ears were collected and processed separately. Each spike was minced in 400 μl of lysis buffer at 4°C and homogenized using a Precellys Cryolys tissue homogenizer. Homogenates were clarified by centrifugation, and 100 μl of clarified lysate from each sample was submitted to Eve Technologies for Luminex-based cytokine quantification. IL-1β concentrations were calculated from standard curves and are reported in pg ml−1 of clarified lysate generated from a homogenized whole ear in a fixed volume of lysis buffer.
Glucose Tolerance Test
Fasting was induced for 2 hours. Glucose (2.0 g kg−1intraperitoneally) was injected and serum was collected for glucose measurements. Blood glucose was monitored using a Nova Max Plus glucometer.
Semaglutide injections
Mice received semaglutide (Selleck Chemicals, S9697) by daily intraperitoneal injection at a dose of 120 µg per kg. Treatment was initiated at the start of HFD or LFD exposure and continued daily until the end of IMQ treatment. Semaglutide was dissolved in a vehicle consisting of PBS:kolliphor:DMSO in a ratio of 18:1:1. Control mice received daily intraperitoneal injections of vehicle alone. The injection volume did not exceed 150 μl per mouse.
Ear immune cell suspensions
Dissected mouse ear skin was separated into dorsal and ventral halves and incubated in 1 ml of digestion buffer. Mouse skins were incubated in 1 ml of digestion buffer (RPMI-1640 medium with 2% FCS and 1 × penicillin–streptomycin–glutamine solution (10378016, Roche) containing 0.2 mg ml−1 liberase (05401020001, Roche), 0.2 mg ml−1 DNase I (DN25-100MG, Sigma-Aldrich) and 0.5 mg ml−1 hyaluronidase (LS002592, Worthington). After 30 minutes of incubation, the skin samples were chopped into fine pieces (1–2 mm).3) and incubated for another 30 min. The suspension was then passed through a 40 µm sieve to remove clumps and undigested debris. The flow was centrifuged at 500 rcf for 5 min. The pellet containing the stromal vascular fraction was washed once in 10 ml of RPMI and the resulting isolated cells were prepared for fluorescence-activated cell sorting (FACS) analysis directly or first stimulated with PMA and ionomycin in the presence of Brefeldin A (Cell Activation Cocktail; BioLegend) for 1.5 h at 37°C for subsequent intracellular cytokine staining and then prepared for FACS analysis.
In vivo antibody treatments
The following antibodies were used: anti-mouse CD4 (BioXCell, GK1.5), isotype control for anti-mouse CD4 (rat IgG2b, BioXCell, LTF-2), anti-mouse Ly6G (BioXCell, 1A8) and isotype control for anti-mouse Ly6G (rat IgG2a, BioXCell, 2A3). For the anti-CD4 experiments: the antibodies were injected intraperitoneally 2 days before, the same day and 2 days after the start of the IMQ challenge at a dose of 0.1 mg per mouse. For the rechallenge experiment, anti-CD4 or isotype control antibodies were injected intraperitoneally on days 29, 32, and 35 after the initial IMQ challenge using the same dose. For the anti-Ly6G experiments: the antibodies were injected intraperitoneally 3 days before, 1 day before, 1 day after, 3 days after and 5 days after the start of the IMQ challenge at a dose of 0.1 mg per mouse. Injection volumes never exceeded 150 µl. For the LPS-induced lung inflammation model, antibodies were injected intraperitoneally 3 days before, 1 day before, 1 day after, and 3 days after LPS challenge at a dose of 0.1 mg per mouse. Injection volumes never exceeded 150 µl.
Adoptive transfer of CD4+ T cells
CD4+ T cells were isolated from the spleens of WT mice using Mouse CD4+ T cell isolation kit (StemCell) and 100,000 cells were transferred intravenously into Cloth1−/− mouse. After an 8-week reconstitution period, mice were fed LFD or HFD for 1 week. Mice were then subjected to IMQ treatment for 6 days to induce acute skin inflammation type 17. Disease severity was assessed by ear thickness measurements and immune responses were analyzed by flow cytometry.
Flow cytometry
Fc block (2.4G2, 553141, BD) was diluted in FACS buffer and added to each sample 5 min before performing surface staining. The eBioscience eFluor780 fixable viability dye was used to distinguish living cells from dying or dead cells. Cell surfaces were stained for 30 min at 4°C. For intracellular staining, cells were treated with fixation and permeabilization reagents from eBioscience and labeled with the appropriate antibodies before being analyzed. Data were analyzed using Aurora Cytek (v.4.0.1) and FlowJo (v.10 and v.11, FlowJo) software. Cells were sorted on the Cytek Aurora CS system. The following antibodies were used: CD45.2 (104, 109839, BioLegend), CD197 (4B12, 120120, BioLegend), CD11c (N418, 117322, BioLegend), CD11b (M1/70, 101224, BioLegend), CD44 (IM7,…
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