Virus and mouse infection
The RSV A2 (ATCC, VR-1540) strain was propagated in HEp-2 cells (ATCC) as previously described , and the viral titer was determined by plaque assay. Female nude mice (on a BALB/c background) aged at 6-8 weeks were purchased from the Chongqing Medical University Animal Laboratory 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 100μl of culture supernatant of HEp-2 cell at the same time. The protocol was approved by the Committee on the Ethics of Animal Experiments of Chongqing Medical University (permit number SYXK-(YU) 2012-0001).
Experimental design and sample collection
Intraperitoneal injections of an anti-HMGB1 neutralizing antibody at 400 μg/kg (R&D Systems, USA) dissolved in phosphate-buffered saline (PBS) or control antibodies were performed daily from day 20 to 27 after RSV incubation in nude mice.
AMD3100 (Sigma-Aldrich, USA), specific blocker of CXCR4, was dissolved in PBS and administered subcutaneously at 20 mg/kg daily from day 20 to 27 after RSV infection in nude mice. The control group received a sham injection of PBS at the same time.
On day 28 after RSV incubation, the mice were sacrificed to collect bronchoalveolar lavage fluid (BALF) and lungs for analyses of the protein, RNA and histopathology.
Real-time quantitative PCR analysis
Total RNA was extracted from the lung tissues with the TRIZOL reagent (Invitrogen, CA), and cDNA was synthesized by a PrimeScript RT reagent kit (TaKaRa, Japan). Quantitative PCR was performed on ABI PRISM 7900 HT system (Applied Biosystems, Foster City, CA). The primers for mouse HMGB1 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.
The concentration of CXCL12 in the BALF were measured using mouse-specific ELISA kits (Sizhengbai, Beijing, China). The concentration of HMGB1 in the BALF were determined using an HMGB1 ELISA kit (Shino-test Corporation, Kanagawa, Japan).
The total protein was extracted from the lung tissues using a total protein extraction kit (KeyGEN, Nanjing, China). Samples containing equal quantities of protein were separated on 8% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and then transferred onto PVDF membranes (Millipore, Billerica, MA). 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 (1:5000; MultiSciences, China) was used to detect the presence of the respective protein bands. Signals were quantified using Quantity One software (Bio-Rad, Hercules, CA) and normalized relative to β-actin.
Inflammatory cell counts in BALF
Briefly, mice were anesthetized with urethane at 15 mg/10 g body weight intraperitoneally. BALF was collected following six times lavage washes with 0.5 ml ice-cold PBS for inflammatory cell counting as previously described .
Airway resistance detection
AHR was assessed in conscious and unrestrained mice by means of whole-body plethysmography (Emca instrument, All Medicus, France). Briefly, each mouse was placed in a plastic chamber and exposed to an aerosolized PBS, following by increasing concentrations of aerosolized methacholine solution (3.125, 6.25, 12.5, 25, 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 Penh (enhanced pauses) value obtained during each methacholine challenge was expressed as a proportion of the basal Penh value observed in response to the PBS challenge.
The left lung lobes from mice were fixed in 10% (v/v) neutral-buffered formalin for 24 hours, embedded in paraffin, cut into sections of 5μm thickness, and stained with hematoxylin and eosin (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 previously described .
The prepared single cell suspensions were blocked with rat serum for half an hour and then stained with antibody to 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, stained samples were measured on a flow cytometer, FACSCalibur (BD Biosciences). The data were analyzed by CellQuest software (BD Biosciences).