Material and Methods
Allergenic extracts . Lyophilized cat dander extract (CDE) (lots#253320, 351876, and 392583) was purchased from Greer Labs (Lenoir, NC). The level of endotoxin in CDE was measured using a LAL chromogenic endotoxin quantitation kit (Thermo Scientific, Hudson, NH), and was less than 0.1 pg/µg CDE protein, hence unlikely to contribute significantly to inflammation33.
Protocols used for animal studies. C57Bl/6 mice were anesthetized with an intraperitoneal injection of a low dose of xylazine/ketamine anesthetic mixture for intranasal administration of CDE and sacrificed by a lethal dose of intraperitoneal xylazine/ketamine. The protocol was approved by the IACUC of Baylor College of Medicine.
CDE Multiple Challenge Model (CDE-MCM) to induce allergic sensitization. Naïve wild type (WT) mice were sensitized by five intranasal challenges of CDE (100 µg/60µl) on days 0, 1, 2, 3, and 4. After a rest period of 7 days, these mice were challenged with an intranasal dose of CDE or phosphate-buffered saline (PBS) on day 11 and sacrificed at 2 h, 4 h,16 h, 28 h, 40 h, and 72 h post-CDE challenge. Some mice challenged with CDE on day 11 were orally treated with 15 mg/kg body weight of ladarixin on days 11, 12, and 13 (Fig 1A ).
CDE Single Challenge Model (CDE-SCM) to induce innate lung inflammation without sensitization . WT mice were intranasally challenged with a single dose of 100 µg/60µl of CDE and administered orally 15 mg/kg body weight of ladarixin simultaneously and sacrificed after 16 h or 28 h post-CDE challenge (Fig 1B ).
Ladarixin . GMP human-use grade ladarixin was used in all studies performed in this manuscript and was a gift from Dompe pharmaceutical company (Dompé farmaceutici, L’Aquila, Italy).
Processing of mouse BALF and lung tissue samples. The BALF and lung were obtained as described previously34.
Bone marrow cells isolation . Bone marrow cells were isolated from the femur and tibia bones of the mouse. After red blood cells lysis by red blood cell lysing buffer (Sigma-Aldrich, St. Louis, MO), the bone marrow cells were frozen for subsequent experiments.
qRT-PCR analysis . Total RNA from mouse lungs, bone marrow cells, or BALF cells were extracted with an RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized using a cDNA Synthesis kit (Qiagen). Amplification by real-time PCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using SYBR Green PCR Master Mix Kit (Bio-Rad Laboratories) to examine lung mRNA expression of Cxcl1, Cxcl 2, Cxcl 3, Cxcl 4, Cxcl 5 , Cxcl7, Cxcr1 , Cxcr2 . Gata3, Il1b, Il13, Il17, Il23, Il4, Il5, Il6, Rorc , and Tgfb1. These primers were purchased from Integrated DNA Technologies (Coralville, IA).
Measurement of IL23 protein in the lung tissue . Mouse lungs were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA) and sonicated, then the content of IL23 in the lung lysate was measured using a DuoSet ELISA development kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol.
Lung single cells isolation . Lung tissues from mice were cut into small pieces and incubated with liberase-thermolysin medium (Sigma-Aldrich) in 42 µg/mL and 20% heat-inactivated fetal bovine serum in Hanks balanced salt solution at 37°C for 20 mins. After passing the digested lung through a 70 µm cell strainer, ice-cold PBS was added to neutralize the enzyme activity. After several wash steps with PBS, the lung single cells were used for flow cytometry analysis or culture system.
Flow cytometry analysis with lung single cells. Lung single cells were pre-incubated with TruStain FcX solution (BioLegend, San Diego, CA) for 10 mins at 4°C, and stained with fluorophore-conjugated antibodies Fixable Viability Dye eFluor 780 (eBioscience, San Diego, CA), CD45 monoclonal antibody-pacific orange (Thermo Fisher Scientific, MA, USA, #MCD4530, clone 30-F11), anti-mouse CD3-Alexa Fluor 700 (Biolegend, #100216, clone 17A2), rat anti-mouse CD4-Brilliant Violet 520 (BD Biosciences, #563106, clone RM4.5), PE rat anti-mouse CD181 (CXCR1) (BD Biosciences, San Jose, CA, #566383, clone U45-632), BV711 Rat Anti-Mouse CD182 (CXCR2), (BD Biosciences, #747812, clone V48-2310), BV786 rat anti-mouse Siglec-F (BD Biosciences, #740956, clone E50-2440), Spark YG 593 anti-mouse Ly-6G Antibody (Biolegend, #127668, clone 1A8), Spark NIR™ 685 anti-mouse/human CD11b Antibody (Biolegend, #101278, clone M1/17) at 1:100 dilution in flow stain buffer containing FBS for 30 mins at 4°C, then cells were permeabilized with Fixation/Permeabilization kit (BD Biosciences) according to the protocol from vendor and stained with intracellular cytokine specific antibodies Rat anti-mouse Rorγt- Brilliant Violet-650 (BD Biosciences, #564722, clone Q31-378), mouse anti-GATA3 Alexa Fluor F488 (BD Bioscience #560163, clone L50-823,) Rat anti-mouse IL4-PE/Cyanine7 (BD Biosciences, #560699, clone 11B11), anti-mouse/anti-human IL5-Allophycocyanin (APC) (BD Biosciences, #554396, clone TRFK5), IL13 monoclonal antibody ((eBio13A), Brilliant Ultra Violet 805, eBioscience), Rat Anti-Mouse IL17A-PE (BD Biosciences, # 559502, clone TC11-18H10), and rat anti-mouse IL23 p19 Alexa Fluor 647 (BD Biosciences, # 565317, clone N71-1183) for 45 mins at 4°C. After washing, a flow cytometer was performed using a high-parameter Cytek®Aurora Flow Cytometer. Staining specificity was determined by fluorescence minus one (FMO) control to enhance the reliability of the gating analysis. Absolute cell numbers were quantified using Precision true count beads (BioLegend). The flow cytometry data was analyzed using FlowJo 10.8.1 Software. FC plots showing gating strategies and FMO are shown in sup Fig 1 . Neutrophils were identified as live CD45+ CD11b+ Ly6G+ Siglec F- cells. Eosinophils were identified as live CD45+ CD11b+ Siglec F+ Ly6G- cells. CD4+ T-cells were identified as live CD45+ CD3+ CD4+ cells.
Measurement of serum total IgE and CDE-specific IgE. The methods have been described previously34. Briefly, the plates were coated with CDE overnight or rat anti-mouse IgE (BD Biosciences) for 2 h. After blocking with sea block buffer for 2 h, the serum from the mice were added onto the plate. After washing, biotin-conjugated rat anti-mouse IgE (BD Biosciences) were plated onto the plate and incubated with avidin-conjugated alkaline phosphatase for 45 mins at 4°C (Sigma-Aldrich). Fluorescence intensities were measured with AttoPhos Substrate Solution (Promega, Madison, WI) using the Varioskan LUX reader (Thermo Fisher Scientific).
Long amplicon quantitative-PCR (LA-qPCR). DNA damage in the lung tissue was quantified by LA-qPCR as previously described35,36. Genomic DNA extraction from the mouse lung was performed using the genomic-tip 20/G kit (Qiagen) per the manufacturer’s protocol. After precise quantitation of the DNA by Pico Green (Invitrogen, Carlsbad, CA), the genomic DNA was digested with the E. coli enzymes Fpg and Nei to induce strand breaks at the sites of the unrepaired oxidized base lesion. Gene-specific LA-qPCR analyses for measuring DNA damage were performed using Long Amp Taq DNA polymerase (New England Biolabs) for long amplicon (LA) and Green mix (New England Biolabs) for short amplicon (SA). The PCR condition for LA was optimized at 94 °C for 30 s (94 °C for 30 s, 60 °C for 30 s depending on the oligo annealing temperature, 65 °C for 10 min) for 25 cycles and 65 °C for 10 min. The PCR conditions for SA were 94 °C for 30 s (94 °C for 30 s, 58°C for 20 s, and 68 °C for 30 s) for 25 cycles and 68 °C for 5 min. The amplified products were then visualized on agarose gels and quantitated with an ImageJ automated digitizing system (National Institutes of Health). In this assay, we amplified a long amplicon (6.5 kb of DNA polymerase β (pol β) and 8.7 kb of β-globin ) vs. the short amplicons (250 and 200 bp, respectively).
The sequence of the oligos:
LA-qPCR forward primer for mouse pol β ; TATCTCTCTTCCTCTTCACTTCTCCCCTGG
LA-qPCR reverse primer for mouse pol β ; CGTGATGCCGCCGTTGAGGGTCTCCTG
LA-qPCR forward primer for mouse β-globin ; TTGAGACTGTGATTGGCAATGCCT
LA-qPCR reverse primer for mouse β-globin ; CCTTTAATGCCCATCCCGGACT
SA-qPCR forward primer for mouse pol β ; TATGGACCCCCATGAGGAACA
SA-qPCR reverse primer for mouse pol β ; AACCGTCGGCTAAAGACGTG
SA-qPCR forward primer for mouse β-globin ; ACACTACTCAGAGTGAGACCCA
SA-qPCR reverse primer for mouse β-globin ; ATACCCAATGCTGGCTCCT
Statistical Analysis. The statistical analysis was performed by unpaired t-test for comparison of two groups or ANOVA for three or more groups using the software package GraphPad Prism 6 (GraphPad Software, San Diego, CA). The results are shown as mean ± SEM. All statistical analyses indicated data as significant at p < 0.05. *=P< .05, **=P< .01, ***=P< .001, ****=P< .0001.