healthmedicinet

Mice

BALB/c breeders were purchased from Harlan Laboratories (Indianapolis, IN, USA) and
bred in specific-pathogen-free facilities at University of Tennessee Health Science
Center (UTHSC, Memphis, TN, USA). Pups born on the same date were used for experiments.

All animal experiments were performed according to the Guide for the Care and Use
of Laboratory Animals and approved by the Institutional Animal Care and Use Committee
at UTHSC.

Viruses and the infection

Human RSV A2 strain was purchased from Advanced Biotechnologies Inc (Columbia, MD,
USA) and passaged in Vero cells (ATCC; Manassas, VA, USA) cultured in serum-free-media
(SFM4MegaVir; Hyclone, Logan, UT, USA). The chimeric strain rA2-19F was a gift from
Dr. Martin Moore (University of Emory, Atlanta, GA, USA) 17] and passaged in our laboratory as RSV A2 above.

Mice were infected intranasally with RSV at a dose of 10
^4.68
TCID
₅₀
(tissue culture infectious dose) per gram of body weight. Sham mice were inoculated
with culture media collected in the same way as virus stocks.

Pulmonary viral load

At various time points after primary RSV infection, lungs were isolated and stored
at ?80 °C until viral load analysis by either RT-qPCR 19] or TCID
₅₀20], 21] methods. For real time qPCR, total RNA was isolated from lungs using Qiagen RNeasy
plus mini kit (Valencia, CA, USA); and one step RT-qPCR was performed using SuperScript®
III Platinum®One-Step qRT-PCR Kit from Life Technologies (Carlsbad, CA, USA). Relative
expression of the virus NS1 gene in lungs was normalized to Hprt using ??Ct method
and the primers used were:

NS1, forward: 5?-CACAACAATGCCAGTGCTACAA-3?

NS1, reverse: 5?-TTAGACCATTAGGTTGAGAGCAATGT-3?

Hprt, forward: 5?-GGCTCCGTTATGGCGACCCG-3?

Hprt, reverse: 5?-CGAGCAAGACGTTCAGTCCTGTCC-3?

For TCID
₅₀
assay, lungs were homogenized and supernatants were used to infect Vero cells on a
96-well plate. After incubation in 37 °C at 5%CO
₂
for 7 days, the wells showing cytopathic effect were counted and TCID
₅₀
calculated as per the Spearman-Kärber method 20], 21].

Bronchoalveolar lavage fluid cellularity

Bronchoalveolar lavage fluid (BALF) was isolated in 0.25 ml (neonates) or 1 ml (adults)
of PBS containing 0.5 % BSA. BALF cells were enumerated, spun onto slides, and stained
with Hema-3 staining kit (Thermo Fisher Scientific, Waltham, MA, USA). Two unbiased
observers differentiated the cellularity using standard morphological criteria. Supernatants
were collected and stored at ?80 °C till cytokine analysis.

Pulmonary T cell profile

Lung single cells were prepared using gentleMACSâ„¢ Octo Dissociator (Miltenyi Biotec,
San Diego, CA, USA). After red blood cell lysis, the cells were stimulated for 5 h
with 5 ng/ml phorbol-12-myristate-13-acetate (PMA) and 500 ng/ml ionomycin (Sigma-Aldrich,
St. Louis, MO) in the presence of GolgiPlug (1 ?l/10
⁶
cells; BD Biosciences, Franklin Lakes, NJ). After stimulation, the cells were stained
with fixable viability dye and antibodies to CD3 (17A2), CD4 (RM4-4; Biolegend, San
Diego, CA, USA), CD8 (53–6.7), IFN? (XMG1.2), and IL4 (BVD6-24G2). Stained cells were
analyzed on a Canto II flow cytometer (BD Biosciences) and plotted with FlowJo software
(v10 for Windows; Tree Star; Ashland, OR, USA). All antibodies and viability dye were
from eBioscience (San Diego, CA, USA) unless otherwise stated.

Lung histopathology

Lungs were inflated at a constant fluid pressure of 25 cm and fixed with zinc formalin
(Thermo Fisher Scientific). After fixation, lungs were embedded and sections cut and
stained by the pathology core at UTHSC. Hematoxylin and eosin (HE) were used to identify
cellular infiltrates and periodic acid-Schiff (PAS) for mucus in the airway epithelial
cells.

Pulmonary function

Airway resistance to methacholine (MeCh; Sigma-Aldrich) was measured using the FlexiVent
FX system (Scireq, Montreal, QC, Canada). Raw data were collected by fitting into
a single –compartment model and normalized by subtracting individual baseline values
at 0 mg/ml MeCh.

Cytokine levels in the airways or the lung

The kinetics of IL13 expression in the lung after primary infection was measured using
real-time qPCR. Total lung RNA was isolated using Qiagen RNeasy plus mini kit and
reverse-transcribed to cDNA using SuperScript III first-strand synthesis system. Real-time
PCR was then performed using Power Sybr green PCR master mix (Life Technologies) and
the relative expression of IL13 were normalized to Hprt using ??Ct method. The primers
for IL13 were as follows and the primers for Hprt were as above.

Forward primer: 5?-TGATGAGCTACTACTGGTCAGC-3?

Reverse primer: 5?-GATCTCTTAGCACAAGGATGGC-3?

The level of other cytokines in the BALF including IFN?, IL12 (p40), and IL4 were
measured using Milliplex mouse cytokine/chemokine assay kit (Millipore Corporation;
Billerica, MA, USA) on Luminex 200 system (Luminex, Austin, TX, USA). IL13 level in
the BALF were measured by ELISA kit from eBioscience following manufacturer’s instructions.
Data below detection limit of each cytokine were excluded.

Statistical analysis

Data were plotted as means?±?standard errors (SEM) by Prism 6 (GraphPad Software;
La Jolla, CA, USA). Pulmonary function test results were analyzed using two-way analysis
of variance and Bonferroni post-hoc tests. All other data were analyzed using Student’s
t-test (2 groups) or multiple t test with Holm-Sidak correction (3 groups). Differences were considered significant
if p??0.05. Every experiment was repeated at least twice.