Virus and mouse infection
The RSV A2 (ATCC, VR-1540) strain was propagated in HEp-2 cells (ATCC), as described previously [15], and the viral titer was determined by plaque assay. Female nude mice (BALB/c background) aged at 6–8 weeks were purchased from the Chongqing Medical University Animal Laboratory (China) and housed in individually filtered cages under accredited specific pathogen-free conditions. The mice were infected intranasally with 100 μl of a stock suspension of 6 × 107 PFU/ml RSV under sodium pentobarbital anesthesia. Mock-infected mice were inoculated intranasally with 100 μl of HEp-2 cell culture supernatant. All protocols were approved by the Committee on the Ethics of Animal Experiments of Chongqing Medical University (permit number SYXK-(YU) 2012-0001), and all methods were carried out in accordance with relevant guidelines and regulations. The study is reported in accordance with ARRIVE guidelines.
Experimental design and sample collection
Intraperitoneal (i.p.) injections of anti-HMGB1 neutralizing antibodies (400 μg/kg) (R&D Systems, USA) dissolved in phosphate-buffered saline (PBS) or control antibodies were performed daily from days 20 to 27 after RSV incubation in nude mice.
AMD3100 (Sigma-Aldrich, USA), a specific blocker of CXCR4, was dissolved in PBS and administered subcutaneously at 20 mg/kg daily from days 20 to 27 after RSV infection in nude mice. The control group received a sham injection of PBS.
On day 28 after RSV incubation, the mice were sacrificed to collect bronchoalveolar lavage fluid (BALF) and lungs for analyses of protein, RNA, and histopathology.
Real-time quantitative polymerase chain reaction (PCR)
Total RNA was extracted from the lung tissues with TRIzol reagent (Invitrogen, CA), and cDNA was synthesized using a PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative PCR was performed on an ABI PRISM 7900 HT system (Applied Biosystems, Foster City, CA, USA). Mouse HMGB1 primers were: forward, 5’-CCAAGAAGTGCTCAGAGAGGTG and reverse, 5’-GTCCTTGAACTTCTTTTTGGTCTC; CXCL12 primers were: forward, 5’-TGCATCAGTGACGGTAAACCA and reverse: 5’-TTCTTCAGCCGTGCAACAATC; β-actin primers were: forward, 5’-TGGCATTGTTACCAACTGGGAC and reverse: 5’-TCACGGTTGGCCTTAGGGTTC.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of CXCL12 in BALF was measured using mouse-specific ELISA kits (Sizhengbai, Beijing, China). The concentration of HMGB1 in BALF was determined using an HMGB1 ELISA kit (Shino-Test Corporation, Kanagawa, Japan).
Western blotting
Total protein was extracted from lung tissues using a total protein extraction kit (KeyGEN, Nanjing, China). Samples containing equal quantities of protein were separated on 8% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies against CXCR4 (1:500, Santa Cruz, USA) or β-actin (1:5 000; 4abio, China). An alkaline phosphatase-conjugated goat anti-mouse antibody (1:5 000; MultiSciences, China) was used to detect the presence of the respective protein bands. Signals were quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) and normalized relative to β-actin.
Inflammatory cell counts in BALF
Briefly, mice were anesthetized with urethane (15 mg/10 g body weight, i.p.). BALF was collected following six lavage washes with 0.5 ml of ice-cold PBS for inflammatory cell counting, as described previously [15].
Airway resistance detection
AHR was assessed in conscious and unrestrained mice by whole-body plethysmography (Emca instrument, All Medicus, France). Briefly, each mouse was placed in a plastic chamber and exposed to aerosolized PBS, followed by increasing concentrations of aerosolized methacholine solution (3.125, 6.25, 12.5, 25, and 50 mg/ml; Sigma, USA) dissolved in PBS at 3-min exposures. Bronchoconstriction was recorded for an additional 5 min after each dose of methacholine. The highest enhanced pause (Penh) value obtained during each methacholine challenge was expressed as the proportion of basal Penh observed in response to PBS challenge.
Lung histopathology
The left lung lobes of mice were fixed in 10% (v/v) neutral-buffered formalin for 24 h, embedded in paraffin, cut into 5-μm-thick sections, and stained with hematoxylin and eosin (H&E) (Sigma-Aldrich, Sigma MHS-16, and Sigma HT110-1-32, respectively, USA) to evaluate RSV-associated pulmonary histopathology. The infiltration of inflammatory cells around the airway was scored, as described previously [16].
Flow cytometry
The prepared single-cell suspensions were blocked with rat serum for 30 min and then stained with antibodies against mouse CD45, CD3, CD19, CD49b or isotype control conjugated with PE-Cy7, PerCP-cy5.5, FITC, or PE for 30 min, respectively. The indicated antibodies were obtained from eBioscience (San Diego, CA, USA). Subsequently, the stained samples were measured on a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).