Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jul 30;9:1586.
doi: 10.3389/fimmu.2018.01586. eCollection 2018.

Entry Inhibition and Modulation of Pro-Inflammatory Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant Protein D

Affiliations
Free PMC article

Entry Inhibition and Modulation of Pro-Inflammatory Immune Response Against Influenza A Virus by a Recombinant Truncated Surfactant Protein D

Mohammed N Al-Ahdal et al. Front Immunol. .
Free PMC article

Abstract

Surfactant protein D (SP-D) is expressed in the mucosal secretion of the lung and contributes to the innate host defense against a variety of pathogens, including influenza A virus (IAV). SP-D can inhibit hemagglutination and infectivity of IAV, in addition to reducing neuraminidase (NA) activity via its carbohydrate recognition domain (CRD) binding to carbohydrate patterns (N-linked mannosylated) on NA and hemagglutinin (HA) of IAV. Here, we demonstrate that a recombinant fragment of human SP-D (rfhSP-D), containing homotrimeric neck and CRD regions, acts as an entry inhibitor of IAV and downregulates M1 expression considerably in A549 cells challenged with IAV of H1N1 and H3N2 subtypes at 2 h treatment. In addition, rfhSP-D downregulated mRNA levels of TNF-α, IFN-α, IFN-β, IL-6, and RANTES, particularly during the initial stage of IAV infection of A549 cell line. rfhSP-D also interfered with IAV infection of Madin Darby canine kidney (MDCK) cells through HA binding. Furthermore, rfhSP-D was found to reduce luciferase reporter activity in MDCK cells transduced with H1+N1 pseudotyped lentiviral particles, where 50% of reduction was observed with 10 µg/ml rfhSP-D, suggestive of a critical role of rfhSP-D as an entry inhibitor against IAV infectivity. Multiplex cytokine array revealed that rfhSP-D treatment of IAV challenged A549 cells led to a dramatic suppression of key pro-inflammatory cytokines and chemokines. In the case of pH1N1, TNF-α, IFN-α, IL-10, IL-12 (p40), VEGF, GM-CSF, and eotaxin were considerably suppressed by rfhSP-D treatment at 24 h. However, these suppressive effects on IL-10, VEGF, eotaxin and IL-12 (p40) were not so evident in the case of H3N2 subtype, with the exception of TNF-α, IFN-α, and GM-CSF. These data seem to suggest that the extent of immunomodulatory effect of SP-D on host cells can vary considerably in a IAV subtype-specific manner. Thus, rfhSP-D treatment can downregulate pro-inflammatory milieu encouraged by IAV that otherwise causes aberrant inflammatory cell recruitment leading to cell death and lung damage.

Keywords: inflammation; influenza A virus; innate immunity; pseudotyped lentiviral particles; surfactant protein D.

Figures

Figure 1
Figure 1
SDS-PAGE (12% v/v) under reducing conditions showing expression and purification of a recombinant surfactant protein D (rfhSP-D). The neck and carbohydrate recognition domain regions were expressed in Escherichia coli BL21 (λDE3) pLysS. (A) Following induction with 0.5 mM IPTG, a ~20 kDa band appeared being overexpressed compared to uninduced sample. Following denaturation–renaturation cycle, the rfhSP-D was purified on an affinity column to homogeneity after elution with EDTA as fractions F1, F2 and F3 (B). A rabbit polyclonal antibody raised against full-length SP-D purified from human bronchoalveolar lavage (C) recognized the purified rfhSP-D, but not BSA that was used as a negative control protein.
Figure 2
Figure 2
ELISA to show binding of rfhSP-D to (A) pH1N1 and (B) H3N2: microtiter wells were coated with different concentrations of rfhSP-D (5, 2.5, 1.25, and 0.625 µg/ml). 20 µl of concentrated pH1N1 or H3N2 virus (1.36 × 106 pfu/ml) was diluted in 200 µl of PBS + 5 mM CaCl2 and 10 µl of diluted virus was added to all the wells, and probed with either monoclonal anti-influenza virus H1 or polyclonal anti-influenza virus H3 antibody. VSV-G pseudotyped lentivirus was used as a negative RNA virus control. The data were expressed as mean of three independent experiments done in triplicates ± SEM.
Figure 3
Figure 3
Cell-binding assay to show binding of (A) pH1N1 and (B) H3N2 pre-incubated with rfhSP-D to A549 cells. Microtiter wells were coated with A549 cells (1 × 105 cells/ml) and incubated overnight at 37°C. Varied concentrations of pre-incubated rfhSP-D (10, 5, 2.5, and 1.25 µg/ml) with pH1N1 and H3N2 virus were added to the corresponding wells, followed by incubation at room temperature for 1–2 h. After fixing the cells with 4% paraformaldehyde solution, monoclonal anti-influenza virus H1, or polyclonal anti-influenza virus H3 were added to corresponding well. Maltose-binding protein (MBP) was used as a negative control protein. The data were expressed as mean of three independent experiments done in triplicates ± SEM.
Figure 4
Figure 4
Far western blot analysis to show rfhSP-D binding to purified (A) pH1N1 and (B) H3N2: 10 μl of concentrated virus (1.36 × 106 pfu/ml) was first run on the SDS-PAGE under reducing conditions, and then transferred onto a nitrocellulose membrane and incubated with 5 µg of rfhSP-D. The membrane was probed with anti-rabbit SP-D polyclonal antibodies. rfhSP-D bound to HA (70 kDa) and M1 (27 kDa) in the case of both pH1N1 and H3N2 subtypes. (C) ELISA to show the binding of rfhSP-D to purified recombinant hemagglutinin (HA) (μg/ml). VSV-G was used as a negative control. The data were expressed as mean of three independent experiments carried out in triplicates ± SEM. Significance was determined using the unpaired one-way ANOVA test (***p < 0.0001) (n = 3).
Figure 5
Figure 5
rfhSP-D restricts replication of (A) pH1N1 and (B) H3N2 in target human A549 cells. M1 expression of both pH1N1 and H3N2 influenza A virus (IAV) (MOI 1) after infection of A549 cells at differential time points at 2 and 6 h. A549 cells were incubated either with pre-incubated pH1N1 and H3N2 with (10 µg) or without purified rfhSP-D. Cell pellets were harvested at 2 and 6 h to analyze the M1 expression of IAV. Cells were lysed, and purified RNA extracted was converted into cDNA. Infection was measured via qRT-PCR using M1 primers and 18S was used as an endogenous control. Results shown are normalized to M1 levels at 2 h untreated. Significance was determined using the unpaired one-way ANOVA test (**p < 0.01, ***p < 0.001, and ****p < 0.0001) (n = 3). (C) Western blotting to shown M1 expression in both untreated (cells + virus) and treated (cells + virus + 10 µg/ml rfhSP-D) following 6 h incubation. Titration assay to show the anti-IAV activity of rfhSP-D (10 µg/ml), using both pH1N1 (D) and H3N2 (E) subtypes. A549 cells were infected with pH1N1/H3N2 (MOI 1) for 24 h. Then, the supernatants were collected and virus titers measured using a TCID50 assay. Treatment with rfhSP-D reduced viral titers by approximately 40%, suggesting that rfhSP-D acts as an entry inhibitor.
Figure 6
Figure 6
Differential mRNA expression profile of A549 cells challenged with pre-incubated (A) pH1N1, (B) H3N2 with rfhSP-D, and (C) expression levels of type I interferon (IFN) subtypes in both untreated and treated samples. The expression levels of cytokines and chemokine were measured using qRT-PCR and the data were normalized via 18S rRNA expression as a control. The relative expression (RQ) was calculated by using cells only time point as the calibrator. The RQ value was calculated using the formula: RQ = 2−ΔΔCt. Assays were conducted in triplicates and error bars represents ± SEM. Significance was determined using the unpaired one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) (n = 3).
Figure 6
Figure 6
Differential mRNA expression profile of A549 cells challenged with pre-incubated (A) pH1N1, (B) H3N2 with rfhSP-D, and (C) expression levels of type I interferon (IFN) subtypes in both untreated and treated samples. The expression levels of cytokines and chemokine were measured using qRT-PCR and the data were normalized via 18S rRNA expression as a control. The relative expression (RQ) was calculated by using cells only time point as the calibrator. The RQ value was calculated using the formula: RQ = 2−ΔΔCt. Assays were conducted in triplicates and error bars represents ± SEM. Significance was determined using the unpaired one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001) (n = 3).
Figure 7
Figure 7
Multiplex cytokine array analysis of supernatants that were collected at 24 h time point. A549 cells were infected with pH1N1 (A) and H3N2 (B), treated with 10 µg/ml of rfhSP-D. Cytokines (TNF-α, IL-6, IL-10, IL-1α, IFN-α, and IL-12p40), chemokine (eotaxin), and growth factors (GM-CSF and VEGF) were measured using a commercially available MagPix Milliplex kit (EMD Millipore). Assays were conducted in triplicates and error bars represent ± SEM (n = 3); significance was determined using unpaired one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).
Figure 8
Figure 8
(A) Western blotting to show the expression of influenza A virus-hemagglutinin (HA) protein in purified H1+N1 pseudotyped lentiviral particles and cell lysate at 24 and 48 h. The presence of HA was identified at 70 kDa. (B) Far western blotting to show rfhSP-D binding in both purified H1+N1 pseudotyped lentiviral particles and cell lysate at 24 and 48 h. HA was evident at 70 kDa when probed with rfhSP-D. (C) Luciferase reporter activity of purified H1+N1 pseudotyped lentiviral particles at 24 and 48 h, and (D) Luciferase reporter activity of rfhSP-D treated MDCK cells transduced with these lentiviral particles. Significance was determined using the unpaired one-way ANOVA test (*p < 0.05 and ****p < 0.0001) (n = 3).
Figure 8
Figure 8
(A) Western blotting to show the expression of influenza A virus-hemagglutinin (HA) protein in purified H1+N1 pseudotyped lentiviral particles and cell lysate at 24 and 48 h. The presence of HA was identified at 70 kDa. (B) Far western blotting to show rfhSP-D binding in both purified H1+N1 pseudotyped lentiviral particles and cell lysate at 24 and 48 h. HA was evident at 70 kDa when probed with rfhSP-D. (C) Luciferase reporter activity of purified H1+N1 pseudotyped lentiviral particles at 24 and 48 h, and (D) Luciferase reporter activity of rfhSP-D treated MDCK cells transduced with these lentiviral particles. Significance was determined using the unpaired one-way ANOVA test (*p < 0.05 and ****p < 0.0001) (n = 3).

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Nayak A, Dodagatta-Marri E, Tsolaki AG, Kishore U. An insight into the diverse roles of surfactant proteins, SP-A and SP-D in innate and adaptive immunity. Front Immunol (2012) 3:131.10.3389/fimmu.2012.00131 - DOI - PMC - PubMed
    1. Kishore U, Greenhough TJ, Waters P, Shrive AK, Ghai R, Kamran MF, et al. Surfactant proteins SP-A and SP-D: structure, function and receptors. Mol Immunol (2006) 43(9):1293–315. - PubMed
    1. Crouch E, Hartshorn K, Horlacher T, McDonald B, Smith K, Cafarella T, et al. Recognition of mannosylated ligands and influenza A virus by human surfactant protein D: contributions of an extended site and residue 343. Biochemistry (2009) 48:3335–45.10.1021/bi8022703 - DOI - PMC - PubMed
    1. Hartshorn KL, Webby R, White MR, Tecle T, Pan C, Boucher S, et al. Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D. Respir Res (2008) 9:65–9921–9–65.10.1186/1465-9921-9-65 - DOI - PMC - PubMed
    1. Hartshorn KL, Crouch EC, White MR, Eggleton P, Tauber AI, Chang D, et al. Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses. J Clin Invest (1994) 94:311–9.10.1172/JCI117323 - DOI - PMC - PubMed

Publication types

Feedback