Determinants of host susceptibility to murine respiratory syncytial virus (RSV) disease identify a role for the innate immunity scavenger receptor MARCO gene in human infants

Abstract

Background: Respiratory syncytial virus (RSV) is the global leading cause of lower respiratory tract infection in infants. Nearly 30% of all infected infants develop severe disease including bronchiolitis, but susceptibility mechanisms remain unclear.

Methods: We infected a panel of 30 inbred strains of mice with RSV and measured changes in lung disease parameters 1 and 5days post-infection and they were used in genome-wide association (GWA) studies to identify quantitative trait loci (QTL) and susceptibility gene candidates.

Findings: GWA identified QTLs for RSV disease phenotypes, and the innate immunity scavenger receptor Marco was a candidate susceptibility gene; targeted deletion of Marco worsened murine RSV disease. We characterized a human MARCO promoter SNP that caused loss of gene expression, increased in vitro cellular response to RSV infection, and associated with increased risk of disease severity in two independent populations of children infected with RSV.

Interpretation: Translational integration of a genetic animal model and in vitro human studies identified a role for MARCO in human RSV disease severity. Because no RSV vaccines are approved for clinical use, genetic studies have implications for diagnosing individuals who are at risk for severe RSV disease, and disease prevention strategies (e.g. RSV antibodies).

Keywords: Haplotype; Infectious disease; Innate immunity; Lung; Promoter; SNP; Single nucleotide polymorphism.

Fig. 1

Lung phenotypes in representative mouse strains with mild and severe disease after infection with RSV. Numbers of PMNs (a) and monocytes (b) found in BALF from C3H/HeJ (mild disease) and BALB/cJ (severe disease) mice following vehicle or RSV infection (1, 5 days PI). Mean ± SEM (n = 6–12 mice/group). Groups were analyzed by 3-way ANOVA and Student-Newman-Keuls a posteriori tests. *P < 0.05 versus vehicle. ⁺ P < 0.05 versus C3H/HeJ. (c) Representative H&E stained lung sections from C3H/HeJ (mild disease) and A/J (severe disease) mice following vehicle and RSV infection (1, 5 days PI). (d) Representative AB/PAS double stained lung sections from BTBR_T + _tf/J (mild disease) and SJL/J (severe disease) mice following vehicle and RSV infection (1, 5 days PI). Arrows = areas of increased airway inflammation and bronchial epithelial proliferation (hyperplasia). AV = Alveoli, BR = bronchus or bronchiole, BV = blood vessel. Bar = 100 μm. (e) Circular node and edge representation of relatedness between RSV response phenotypes among all mouse strains. Pearson correlation coefficients (R) noted on edges (solid lines) indicate associations between phenotypes. R values ≥ 0.48, P < 0.05; R values ≥ 0.73, P < 0.01.

Fig. 2

Lung disease phenotypes in mice infected with RSV. Maximum numbers of BALF monocytes (a), lymphocytes (b), and PMNs (c), and RSV-N mRNA expression (d) in lung homogenates. Bars, means ± SEM (n = 6–12/group). Horizontal brackets (monocytes) indicate means are not significantly (P < 0.05) different from each other (1-way ANOVA and Student-Newman-Keuls comparisons of means).

Fig. 3

Genome-wide haplotype association map of BALF monocytes in 30 mouse strains infected with RSV and Marco haplotype clustering and monocytic inflammatory response to RSV infection. (a) Manhattan plot for maximum mean BALF monocytes in 30 strains of mice. X axis, chromosome number and cumulative genomic position; Y axis, − Log₁₀ P values. The rectangle on chromosome 1 identifies a locus significantly associated with RSV-induced monocyte infiltration. (b) Detailed view of the chromosome 1 QTL for monocyte response to infection. The gene Marco is indicated within the locus. (c) Hierarchical clustering of 29 mouse strains based on 87 Marco SNPs (http://phenome.jax.org/SNP). Numbers represent uncertainty in genotype changes after clustering (Φ), where Φ = 1 − cluster entropy / crude entropy. If, after clustering, all strains in the cluster have the same genotype then Φ = 1; if Φ = 0, the genotype distribution within the cluster is as variable as the genotype distribution across all strains. (d) Box plots of numbers of BALF monocytes following RSV infection in strains with Marco haplotypes 1, 2, and 3. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Error bars above and below the box indicate the 90th and 10th percentiles. Outlying points are shown for haplotype 1. *P < 0.05 versus Marco haplotype 1 (1-way ANOVA and Student-Newman-Keuls comparisons of means). (e) Differential pulmonary disease phenotypes after RSV infection in Marco^+/+ and Marco^−/− mice. Means (± SEM) of monocytes (left) and PMNs (right) recovered in BALF after vehicle or RSV infection. *P < 0.05 versus vehicle; ⁺ P < 0.05 versus Marco^+/+ mice (3-way ANOVA with Student-Newman-Keuls comparisons of means). (f) Pulmonary pathology 1 and 5 days following vehicle control or RSV infection in Marco^+/+ and Marco^−/− mice. Arrows indicate areas of increased airway inflammation and bronchial epithelial proliferation (hyperplasia). AV = Alveoli, BR = bronchus or bronchiole, BV = blood vessel. Bar = 100 μm.

Fig. 4

Effect of a genetic polymorphism on in vitro MARCO promoter activity. (a) Deletion analysis of the MARCO promoter compared to pGL3 basic vector. *P < 0.05 versus − 300-Luc; group sizes = 4. (b) Mutagenesis analysis of the MARCO rs1318645 variant (− 156G) compared to pGL3 basic vector. Bars (means ± SEM) represent luciferase activity in basic vector, control − 156 C allele, and mutant − 156 G constructs. Flanking sequence for MARCO rs1318645 is shown at top with the NRF2 response element boxed. *P < 0.05 versus − 156 C (P = 0.001); group sizes = 4. Statistical analyses, one-way ANOVA and Student-Newman-Keuls comparisons of means. (c) Luciferase activity of RSV infected MARCO reporter constructs. MARCO promoter constructs were transiently transfected and cells infected with RSV (MOI of 4). Bars (means ± SEM) represent luciferase responses after no RSV (control) or 3–72 h post-infection (PI). Cells were harvested 3–72 h PI and relative luciferase activity assessed. *P < 0.05 versus RSV-infected − 300_C (WT); ⁺ P < 0.05 versus No RSV control; group sizes = 6. Statistical analyses, 2-way ANOVA and Student-Newman-Keuls comparisons of means. (d) Gel shift analysis of MARCO promoter variants. Gel shift analysis of MARCO − 156 CC (wild type), GC and GG (variant) oligos show increased overall binding in the presence of the G allele. Incubation with NRF2 antibody shows reduced MARCO:NRF2 specific binding with the variant G allele. This suggests the MARCO G allele affects efficient binding to the NRF2 ARE site, and may alter transcriptional activity. A representative gel shift is shown; group sizes n = 3. Single arrowhead, overall binding; double arrowhead, MARCO:NRF2 specific binding. FP is free radiolabeled probe, B is bound, NRF2 is NRF2 antibody, IgG is control serum, and Cold is competition without antibody (40 ×).

Fig. 5

Effect of the MARCO rs1318645 C/G polymorphism on human RSV disease severity. (a) Representative electropherograms of the MARCO promoter and putative NRF2 binding site with the rs1318645 C/G polymorphism. (b). Role of MARCO rs1318645 C/G in disease severity among two populations of infants infected with RSV. Infants with mild or severe RSV disease were genotyped for the MARCO SNP and determined to be homozygous for the major (C) or minor (G) allele, or heterozygous. χ² analysis of disease severity among the three genotypes was done independently for both populations and the two combined. *P < 0.05 versus the CC and CG genotypes.