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. 2004 Aug;78(16):8565-72.
doi: 10.1128/JVI.78.16.8565-8572.2004.

Pulmonary collectins modulate strain-specific influenza a virus infection and host responses

Samuel Hawgood  1 , Cynthia Brown, Jess Edmondson, Amber Stumbaugh, Lennell Allen, Jon Goerke, Howard Clark, Francis Poulain
Affiliations

Pulmonary collectins modulate strain-specific influenza a virus infection and host responses

Samuel Hawgood et al. J Virol. 2004 Aug.

Abstract

Collectins are secreted collagen-like lectins that bind, agglutinate, and neutralize influenza A virus (IAV) in vitro. Surfactant proteins A and D (SP-A and SP-D) are collectins expressed in the airway and alveolar epithelium and could have a role in the regulation of IAV infection in vivo. Previous studies have shown that binding of SP-D to IAV is dependent on the glycosylation of specific sites on the HA1 domain of hemagglutinin on the surface of IAV, while the binding of SP-A to the HA1 domain is dependent on the glycosylation of the carbohydrate recognition domain of SP-A. Here, using SP-A and SP-D gene-targeted mice on a common C57BL6 background, we report that viral replication and the host response as measured by weight loss, neutrophil influx into the lung, and local cytokine release are regulated by SP-D but not SP-A when the IAV is glycosylated at a specific site (N165) on the HA1 domain. SP-D does not protect against IAV infection with a strain lacking glycosylation at N165. With the exception of a small difference on day 2 after infection with X-79, we did not find any significant difference in viral load in SP-A(-/-) mice with either IAV strain, although small differences in the cytokine responses to IAV were detected in SP-A(-/-) mice. Mice deficient in both SP-A and SP-D responded to IAV similarly to mice deficient in SP-D alone. Since most strains of IAV currently circulating are glycosylated at N165, SP-D may play a role in protection from IAV infection.

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Figures

FIG. 1.
FIG. 1.
SP-D neutralizes IAV infection of MDCK cells. MDCK cells were grown to confluence in 96-well plates and incubated with a standard dose of IAV in the presence of increasing concentrations of collectins. After 16 h, the cells were stained for IAV and IAV fluorescent foci were counted and expressed as a percentage of total cells assessed by DAPI staining. The data shown are averaged from two separate experiments. Filled squares, recombinant mouse SP-D and X-79; empty circles, recombinant human SP-A and X-79; filled triangles, recombinant mouse SP-D and X-79Δ167; filled circles, recombinant human SP-A and X-79Δ167.
FIG. 2.
FIG. 2.
Weight change in mice after inoculation with X-79 virus. Mice were weighed daily after intranasal inoculation with X-79 virus or uninfected allantoic fluid. WT mice given X-79 (open circles) and SP-D−/− mice given sterile allantoic fluid (shaded circles) or anesthetic only (shaded squares) gained weight at the same rate as uninfected controls or infected SP-A−/− mice (data not shown). Infected SP-D−/− mice (open squares) failed to gain weight for the first 7 days after inoculation and then recovered. Data are means ± standard errors of the means (n = 10 mice/group). The weights of infected SP-D−/− mice were significantly different from those of infected WT and SP-A−/− mice and uninfected SP-D−/− mice from days 2 to 14 (P < 0.05).
FIG. 3.
FIG. 3.
BAL neutrophil counts 2 days after X-79 inoculation. BAL neutrophil counts were significantly elevated after inoculation of X-79 compared to results with allantoic fluid only for all three genotypes (P < 0.05; n = 5 mice/group). The neutrophil count was significantly greater for infected SP-D−/− mice than for infected WT and infected SP-A−/− mice (*, P < 0.002; n = 5 mice/group). Cotreatment of SP-D−/− mice with X-79 and recombinant mouse SP-D significantly reduced the neutrophil response to IAV (**, P < 0.05; n = 5 mice/group). (A) WT with allantoic fluid; (B) WT with X-79; (C) SP-A−/− with allantoic fluid; (D) SP-A−/− with X-79; (E) SP-D−/− with allantoic fluid; (F) SP-D−/− with X-79; (G) SP-D−/− with X-79 and recombinant mouse SP-D. Data are means ± standard errors of the means; n = 5 mice/group).
FIG. 4.
FIG. 4.
Viral load assessed by HA mRNA is increased in SP-D−/− mice during infection with X-79. Three-week-old mice were inoculated intranasally with X-79 virus and sacrificed 2, 4, 6, and 11 days later. HA mRNA levels in lungs were determined by quantitative real-time RT-PCR. Open bars, SP-A−/− mice; shaded bars, SP-D−/− mice; filled bars, WT mice. Data are means ± standard errors of the means (n = 10 mice/group). *, P < 0.005 compared with WT mouse results. #, P < 0.05 compared to WT mouse results.
FIG. 5.
FIG. 5.
Viral load assessed by HA mRNA is similar in SP-D−/− and SP-AD−/− mice during X-79 infection. Three-week old mice were inoculated intranasally with X-79 virus and sacrificed 2, 4, or 6 days later. Lung HA mRNA levels were determined by quantitative real-time RT-PCR. Open bars, SP-AD−/− mice; shaded bars, SP-D−/− mice; filled bars, WT mice. Data are means ± standard errors of the means (n = 5 mice/group). The viral load was significantly greater in SP-D−/− and SP-AD−/− mice than in WT mice at all time points. The viral load was significantly greater in SP-D−/− mice than in SP-AD−/− mice on day 2, but there were no differences on days 4 and 6.
FIG. 6.
FIG. 6.
Lung MIP-2 levels are significantly higher in SP-D−/− mice after X-79 inoculation. MIP-2 levels in lung homogenates were measured by ELISA 2, 4, and 6 days after intranasal inoculation with X-79 virus. MIP-2 levels in uninfected mice or after inoculation with sterile allantoic fluid ranged from undetectable to 200 pg/g. The control values did not differ significantly between genotypes (data not shown). After X-79 inoculation, MIP-2 levels were significantly higher in SP-D−/− mice (filled bars) than in WT (open bars) and SP-A−/− (shaded bars) mice. There were no significant differences between WT and SP-A−/− mice. Data are means ± standard errors of the means (n = 5 mice/group). *, P < 0.0001 compared with WT results.
FIG. 7.
FIG. 7.
Lung IL-6 levels are significantly higher in SP-D−/− mice after inoculation with X-79. IL-6 levels in lung homogenates were measured by ELISA 2, 4, and 6 days after intranasal inoculation with X-79 virus. IL-6 was detectable in uninfected mice but did not differ between genotypes and did not change after inoculation with sterile allantoic fluid (not shown). After X-79 inoculation, IL-6 levels were significantly higher (*, P < 0.001) in SP-D−/− mice (filled bars) than in WT (open bars) and SP-A−/− (shaded bars) mice on day 2. There were no significant differences between WT and SP-A−/− mice on day 2. On day 4, IL-6 levels were higher in both SP-A−/− (P < 0.002) and SP-D−/− (*, P < 0.001) mice compared to WT results. The levels in SP-D−/− mice were significantly higher than those in SP-A−/− mice on day 4, but on day 6, IL-6 levels were higher in SP-A−/− mice than in both WT and SP-D−/− mice (#, P < 0.01). Data are means ± standard errors of the means (n = 4 to 7 mice/group).
FIG. 8.
FIG. 8.
SP-D cotreatment reduces the cytokine response to X-79 virus in SP-D−/− mice. MIP-2 (open bars) and IL-6 (black bars) were measured by ELISA 2 days after inoculation of SP-D−/− mice with X-79 ± 10 μg of recombinant mouse SP-D or human albumin. (A) WT infected mice; (B) SP-D−/− infected mice; (C) SP-D−/− infected mice with 10 μg of recombinant mouse SP-D; (D) SP-D−/− infected mice plus 10 μg of human albumin. Cotreatment with recombinant mouse SP-D (C) significantly reduced the MIP-2 and IL-6 to levels similar to those for infected WT mice. Human albumin had a small but significant effect on MIP-2 levels but no effect on IL-6 levels. Data are means ± standard errors of the means (n = 5 mice/group). *, P < 0.05 compared to results for SP-D−/− mice with X-79 without cotreatment.
FIG. 9.
FIG. 9.
Histology of the lung 6 days after inoculation with X-79 virus. Mid-sagittal sections of the right lung were prepared 6 days after inoculation with X-79 virus. (a) Twenty-seven-day-old control SP-D−/− mouse lung; (b) WT mouse lung with X-79; (c) SP-A−/− mouse lung with X-79; (d) SP-D−/− mouse lung after X-79 inoculation; (e) SP-D−/− after X-79 inoculation and cotreatment with recombinant mouse SP-D.
FIG. 10.
FIG. 10.
Weight change in mice after inoculation with X-79Δ167 virus. Mice were weighed daily after intranasal inoculation with X-79Δ167 virus (10−1 LD50). WT mice (squares), SP-D−/− mice (filled circles), and SP-A−/− mice (open circles) all had similar patterns of initial weight gain similar to the control pattern (see controls in Fig. 3) followed by weight loss from days 5 to 10 and then recovery. No differences between genotypes were observed (n = 10 per genotype).
FIG. 11.
FIG. 11.
Viral load assessed by HA mRNA is similar for all genotypes during infection with X-79Δ167. Three-week-old mice were inoculated intranasally with X-79Δ167 virus and sacrificed 2, 4, 6, and 11 days later. Lung HA mRNA levels were determined by quantitative real-time RT-PCR. Open bars, WT mice; grey bars, SP-D−/− mice; black bars, SP-A−/− mice. Data are means ± standard errors of the means (n = 10 mice/group). *, P < 0.05 compared with WT results.
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