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.