Influenza A virus interacts with mucin on human airway tissue

IAV tropism depends on HA binding specificity and the host sialylation pattern. The distribution of terminal Sias in α2-6 and in α2-3 linkages varies along the respiratory tract, and changes with age and developmental stage [19, 23]. Human respiratory tract sialylation patterns have been extensively studied on paraffin embedded tissues, which are lacking much of the secreted mucus layer [23, 24]. Here we examine glycosylation and IAV binding to frozen human trachea/bronchus tissues that were frozen and embedded in optimal cutting temperature (OCT) compound. This treatment preserves the secreted mucus layer in a natural state, enabling both immunohistochemistry and virus binding studies [25]. Secreted mucus forms a visible lining on the epithelium of human bronchial tissues, detected by Periodate Acid Schiff staining (Figure 1, PAS, dashed line indicates secreted mucus). Potential receptors for human IAV on secreted airway mucus were detected with Sambucus nigra lectin (SNA), which binds to Siaα2-6Gal/GalNAc, or with TKH2 antibody, which bind to Siaα2-6GalNAc on O-linked glycans (Sialyl Tn) (Figure 1, SNA & TKH2, outlined dark brown staining). Sialyl Tn is a glycan epitope that is abundant on mucins but infrequent in other tissues [26]. TKH2 staining is confined to the secreted material lining the epithelium and the glands (Figure 1, TKH2), further confirming that this material represents the secreted mucus layer. In order to test the ability of IAV to bind secreted mucus, these tissues were incubated with 600 HAU of two seasonal virus strains, A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2), and a clinical isolate of the pandemic A/SD/1/2009(SOIV). All three virus strains bound to secreted mucus as well as to the underlying ciliated cells (Figure 1, lower panels, dashed lines). Removal of Sias from the tissues by enzymatic cleavage with Arthrobacter ureafaciens sialidase (Figure 2, AUS) significantly reduces virus binding to the mucus, confirming specific binding to sialylated receptors. Similarly, truncation of the Sia side chain by mild sodium periodate treatment [27] reduces virus binding to the mucus (Figure 2, NaIO4). These findings confirm that the secreted mucus layer presents sialylated decoy receptors for binding by IAV and other pathogens.

Figure 1 IAV binds to secreted mucus in human trachea tissues. Frozen human trachea tissue sections were stained with Hematoxylin and Eosin (H&E), periodic acid Schiff (PAS, mucin staining in pink), Sambucus nigra agglutinin (SNA, binds to Siaα2-6Gal/GalNAc) or TKH2 antibody (binds to Sialyl Tn: Siaα2-6GalNAc on O-linked glycans). Dashed lines specify the location of secreted mucus, which is preserved in the frozen tissues, as seen in H&E and PAS staining. Both SNA and TKH2 bound to the secreted mucus (dark brown staining), indicating abundant potential ligands for IAV binding. TKH2 binding was confined to the lining of the epithelium and the glands, further confirming that the secreted mucus layer is adequately preserved in these tissues. Binding of IAV to the tissues was tested by incubating 600 HAU of virus 1.5 h at room temperature. All three strains bound to the secreted mucus layer (dashed line), and to ciliated cells (cells stained in dark brown). Virus was detected by anti-NP antibodies. Boxed area is enlarged below each image. Scale bar indicates 500 μm, scale bar of enlarged area indicates 50 μm. Full size image

Figure 2 IAV binding to secreted mucus is Sia-dependent. Human trachea tissue sections were treated with Arthrobacter ureafaciens sialidase (AUS), which cleaves Sias, or with mild sodium periodate (NaIO4), which truncates the Sia side chain. Both treatments reduce IAV binding to the secreted mucus on human trachea tissues compared to untreated control tissues, confirming that IAV binding to the secreted mucus is Sia-dependent. Dashed lines specify location of virus binding to secreted mucus. Scale bar indicates 50 μm. Full size image

Human mucin protects cells from infection in vitro

Since IAV can both bind and cleave sialylated epitopes, we tested whether sialylated mucins can effectively protect underlying cells from IAV infection in vitro. Confluent monolayers of MDCK cells in 16-well Lab-Tek chamber slides were overlaid with human salivary mucins (HSM), porcine submaxillary mucins (PSM) or buffer. The mucin content of HSM is similar to that of human airway epithelium submucosal glands (Additional file 1A) [15]. HSM preparation is enriched by acid precipitation of mucins and filtration of saliva samples [28] (Additional file 1B-C). Thus the HSM preparation is a good representation of the mucus of human upper respiratory system. The Sia contents of HSM and PSM samples were determined by DMB-HPLC (Additional file 1D), and MDCK cells were overlaid with mucus containing a known amount of Sias. Total Sia content in the wells was 12,000 pmol/well (high), 3,200 pmol/well (medium) or 1,500 pmol/well (low). The protection efficacy of HSM and PSM against IAV infection of the underlying cells was determined by challenging the cells with 109 TCID 50 of four IAV strains: A/PR/8/34(H1N1), A/SD/1/2009(SOIV), A/SD/17/2008(H1N1), and A/Aichi/2/68(H3N2) for 1 h at 37°C. Cells were then washed to remove both virus and mucus, and fresh DMEM-TPCK media was added. The cells were incubated for additional 5.5 h at 37°C, fixed and stained for viral nuclear proteins. The number of infected cells was quantified in twenty randomly selected images from each sample, and the infection rate relative to buffer coated cells was determined (Figure 3). For all four IAV strains, coating of cells with HSM at medium or high Sia content significantly reduces the infection of underlying cells compared to buffer coated cells (Figure 3, compare PBS (0) to HSM (3200) and HSM (12000 pmol), P<0.05). Only three of the tested IAV strains were significantly inhibited by HSM at low Sia content: A/PR/8/34(H1N1), A/SD/17/2008(H1N1) and A/Aichi/2/68(H3N2) (Figure 3A and C-D, compare PBS (0) to HSM (1500 pmol), P<0.05). Notably a dose effect of Sia-content in the HSM layer was observed for three of the IAV strains (A/PR/8/34(H1N1), A/SD/2009(SOIV) and A/Aichi/2/68(H3N2)) where higher Sia-content resulted in fewer infected cells. The numbers of infected cells were 60-95% lower in monolayers coated with HSM (high), 40-65% lower in monolayers coated with HSM (medium), and 40-50% lower in monolayers coated with HSM (low), depending on the strain (Figure 3A-B and D, see Additional file 2 for complete statistical analysis). In contrast to HSM, coating cells with PSM typically did not result a significant reduction in the number of infected cells (Figure 3A-B and D). However, a mild reduction (15-25%) in number of cells infected by A/SD/17/2008(H1N1) was observed in PSM coated monolayers (Figure 3C, P<0.05).

Figure 3 HSM and oseltamivir have additive inhibitory effects. MDCK cells were layered with PSM or HSM at 1,500, 3,200, 12,000 pmol Sia/well or with PBS buffer as control. The cells were challenged for 1h at 37°C with 109 TCID 50 of (A) A/PR/8/34(H1N1), (B) A/SD/1/2009(H1N1), (C) A/SD/17/2008(H1N1), or (D) A/Aichi/2/68(H3N2) in the presence (gray bars) or absence (black bars) of 1 μM oseltamivir. Infected cells were identified by staining with anti-NP antibodies, and quantified in twenty randomly selected images from each sample. Experiments were repeated three times, for each experiment the number of infected cells in the PBS-coated sample was set to 1, and the relative number of infected cells for each treatment was calculated. Lower number of infected cells was observed in HSM coated monolayers compared to PBS-coated monolayers for all tested virus strains (A-D, P<0.05). Dose effects of Sia content in HSM-coated samples were observed for three IAV strains (A-B, D). Significant reduction in the number of infected cells in PSM-coated monolayers was observed only for one strain (C, P<0.05). With exception of the A/SD/1/2009(H1N1) strain, oseltamivir did not inhibit infection of cell coated with either PBS or PSM (A, C-D, gray bars). In contrast, addition of oseltamivir to HSM-coated samples further reduced infection of A/PR/8/34(H1N1), A/SD/1/2009(H1N1) strains (A-B). Both of the oseltamivir-insensitive strains, A/SD/17/2008(H1N1) and A/Aichi/2/68(H3N2), were not affected by addition of the drug (C-D). Data was analyzed by 3-way ANOVA, corrected for multiple comparisons using Tukey’s HSD (see Additional file 2 for complete statistical analysis data). Error bars represent standard deviation. *P<0.05, **P<0.005. Full size image

High Sia-content PSM (high) is comprised of 10,200 pmol Neu5Gc and 1,800 pmol Neu5Ac (Additional file 1D). However, the presentation of Sia differs between PSM [17] and HSM [13–15]. Despite having a similar Neu5Ac content, PSM (high) and HSM (low) did not have the same inhibitory effect on IAV infection. Two strains, A/PR/8/34(H1N1) and A/Aichi/2/68(H3N2) were not significantly inhibited by coating monolayers with PSM (high), however coating monolayers with HSM (low) significantly reduced the number of infected cells (Figure 3A and D, P<0.0005, and P<0.0462, respectively). Numbers of cells infected by A/SD/17/2008(H1N1) were reduced in monolayers coated with both PSM (high) and HSM (low), however, fewer infected cells were observed in HSM-coated monolayers compared with PSM-coated monolayers (Figure 3C, P<0.0504). In contrast coating monolayers with either PSM (high) or HSM (low) did not significantly reduce the number of cells infected with A/SD/1/2009(SOIV) (Figure 3B). Thus Sia content, type, and presentation are all important factors for inhibition of IAV infection. Furthermore, mucus inhibition of IAV infection is strain-dependent.

Inhibition of IAV neuraminidase by oseltamivir increases the protective effect of HSM but not PSM

Since the virus NA can potentially cleave sialylated receptors presented on secreted human mucus, inhibition of NA activity may enhance the protective effect of mucus. In order to test this hypothesis, 1 μM oseltamivir was added to IAV prior to challenging the mucus-coated cells. The most prominent reduction of infection rate by oseltamivir was observed for the pandemic A/SD/2009(SOIV) strain. A reduction of ~60% in the number of infected cells was observed in monolayers coated with either buffer or PSM (Figure 3B, PBS and PSM P<0.05). This could be attributed to oseltamivir inhibition of the secondary Sia-binding site found on the virus N1 neuraminidase [29], rather than to the neuraminidase enzymatic activity. However, the number of infected cells in HSM-coated monolayers was further reduced to <10% upon addition of oseltamivir, even with monolayers that were coated with HSM (low). Thus oseltamivir and HSM have an additive inhibitory effect (HSM + oseltamivir compared with HSM, or with PBS + oseltamivir, P<0.05, see Additional file 2 for complete statistical analysis). The number of infected cells in PSM (high)-coated monolayers and HSM (low)-coated monolayers were reduced to a similar extent by the addition of 1 μM oseltamivir (Figure 3B, 24±8% and 11±7%, respectively, P<0.0853).

Similar results were obtained for the seasonal H1N1 strain (A/PR/8/34(H1N1)). Although the number of infected cells in PSM- or PBS-coated monolayers was not significantly reduced by oseltamivir, 25-30% reduction in the number of infected cells was observed (Figure 3A, gray bars). However, the number of infected cells in HSM-coated monolayers was further reduced to <5% even in monolayers coated with only a low Sia-content HSM (Figure 3A, gray bars, P<0.05).

The clinical isolate A/SD/17/2008(H1N1) contains the oseltamivir-resistant mutation H275Y in the NA gene, as determined by cDNA sequencing (data not shown). An overall reduction of 12-44% in the number of cells infected by this strain was observed in oseltamivir-supplemented samples (Figure 3C, compare gray and black bars for each treatment). However, this effect was mostly not statistically significant, and all of the oseltamivir-supplemented samples had similar number of infected cells regardless of the mucus content (Figure 3C, compare all gray bars). Thus, the oseltamivir effect seen with this strain is likely attributed to inhibition of a secondary Sia binding site of N1 neuraminidase. In contrast, infection with A/Aichi/2/68(H3N2) strain was not affected by addition of 1 μM oseltamivir (Figure 3D, gray bars).

Taken together this suggests that NA sialidase activity is important to release the virus from the HSM layer.

Direct evidence for cleaving of Sias by viral Neuraminidase

In order to demonstrate that our viruses can cleave Sias from HSM, IAV was incubated with HSM and PSM conjugated magnetic beads. HSM and PSM were biotinylated and captured in streptavidin magnetic beads. As a control, biotinylated polyacrylamide-Galβ1-3GalNAc (T antigen) conjugated beads were also prepared. Beads were incubated with 50 μl A/PR/8/34(H1N1) (2048 HAU), A/Aichi/2/68(H3N2) (600 HAU), or DMEM-TPCK buffer for 1.5 h at room temperature to allow cleavage of sialylated beads. The beads were then extensively washed to remove both virus and cleaved (released) Sias molecules, and were then fixed with formalin. Sia content of the beads was analyzed by DMB-HPLC (Figure 4). Neu5Ac comprises 100% of the Sias in HSM samples, in contrast, Sias from PSM samples consist of ~30% Neu5Ac and 70% Neu5Gc (Additional file 1D). Both virus strains cleaved Neu5Ac from HSM, reducing the total Sia content by 40-60% compared to beads incubated with buffer alone (Figure 4, P<0.001). In contrast, cleavage of Sias from PSM coated beads was less effective. Both virus strains reduce Neu5Ac content by 15-23% (Figure 4, P<0.012), and only A/Aichi/2/68(H3N2) cleaved Neu5Gc (Figure 4, hatched bars, P=0.03). To our knowledge, this is the first direct demonstration that NA can cleave Sias from mucus, and we show that IAV effectively cleaves Neu5Ac from HSM but is ineffective at cleaving Sias from PSM.

Figure 4 IAV effectively cleaves sialylated HSM. Magnetic beads-conjugated to HSM or PSM were incubated with A/PR/8/34(H1N1), A/Aichi/2/68(H3N2), or buffer at room temperature to allow cleavage of sialylated beads. After 1.5 h incubation the Sia content of the beads was analyzed by DMB-HPLC, and is expressed as percent of Sia content in buffer-incubated beads. Solid bars represent Neu5Ac content, and hatched bars represent Neu5Gc content. Both viruses reduce Neu5Ac content of HSM by 40%-60%, in contrast, only mild cleavage of PSM Sias was observed. ***P<0.001, **P<0.012 *P=0.03 values indicate the significance in difference between Sia content in the virus-treated samples and the corresponding buffer control (two-tailed T-Test, n=3). Full size image

Cleaving specificity of viral neuraminidase

The cleaving preference for Sia type was tested for six IAV strains using different substrates: Neu5Ac, Neu5Gc, and 2-keto-3-deoxynononic acid (Kdn), each linked to the fluorescent reporter 4-methyl-umbelliferyl (4MU, Figure 5) [30]. Virus (32-64 HAU) was diluted 10-fold in MES buffer and incubated with 4MU-Sia substrates (0-5,000 pmol) for 1 h at 37°C. The enzymatic activity of each virus NA was determined by quantifying the release of fluorescent 4MU compound. To account for spontaneous release of 4MU due to instability of the 4MU-Sia compounds (see Additional file 3), MES buffer was added instead of virus. Fluorescence in these samples was deemed background. As expected, all of the tested virus strains cleaved Neu5Ac (Figure 5A, black diamonds), and did not cleave Kdn (Figure 5A, black circles), which typically is not found in this unmodified form on mammalian tissues [31, 32]. Interestingly, all six viral strains cleaved Neu5Gc as well, although to lesser extent than Neu5Ac (Figure 5A, black squares). This is surprising since the same IAV strains were ineffective at cleaving Neu5Gc from PSM (Figure 4). The viruses showed different susceptibility to inhibition by oseltamivir. Enzymatic activity of A/PR/8/34(H1N1), A/Denver/1/57(H1N1), and A/SD/1/2009(SOIV) was abolished by addition of 1 μM oseltamivir (Figure 5A and B, top panels). As expected, A/SD/17/2008(H1N1) and A/SD/21/2008(H1N1), both carrying the oseltamivir-resistant mutation H275Y in the NA gene, were only partially inhibited by 1 μM oseltamivir (Figure 5A and B, bottom panels). A/Aichi/2/68(H3N2) was also not sensitive to oseltamivir inhibition (Figure 5A and B, bottom right graphs). Importantly, all viruses were inhibited by high (17.5 μM) oseltamivir concentration (Figure 5B, P<0.01), thus enabling us to effectively block NA activity in order to study HA interactions with mucus (see below).

Figure 5 Sia cleavage preference and susceptibility to oseltamivir inhibition. (A) The cleaving preference of IAV NA was tested by incubation of IAV with the fluorescent reporter, 4-methyl-umbelliferyl (4MU), linked to Neu5Ac, Neu5Gc, or 2-keto-3-deoxynononic acid (Kdn) as substrate. All virus strains cleaved Neu5Ac and Neu5Gc but not Kdn. Background from spontaneous degradation of the 4MU-Sia compounds was subtracted from the results. (B) Virus susceptibility to oseltamivir inhibition was tested by incubation of IAV with 2.5 nmol 4MU-Neu5Ac in the presence of 0, 1 or 17.5 μM oseltamivir in triplicates. Certain strains were only partially inhibited by 1 μM oseltamivir, however, all of the strains were inhibited by 17.5 μM oseltamivir. Substrate cleavage was quantified by measuring fluorescence from the released 4MU reporter compound. *P<0.05, **P<0.01 (two-tailed T-Test, n=3). Full size image

HSM directly inhibits viral neuraminidase

The ability of HSM and PSM to compete 4MU-Neu5Ac for the virus NA activity was tested by incubating virus with 4MU-Neu5Ac in the presence of mucus with 10 nmole Sia content (Figure 6). HSM but not PSM competitively inhibits the cleavage of 4MU-Neu5Ac by A/Aichi/2/68(H3N2) virus (Figure 6A). Similarly, HSM (4.7 nmol Sia content) inhibited the cleavage of 4MU-Neu5Ac (0.1 nmol) by A/Denver/1/57(H1N1), A/Aichi/2/68(H3N2), and A/SD/1/2009(SOIV). For all viruses PSM did not inhibit cleavage of 4MU-Neu5Ac (Figure 6B). Since NA affinity to 4MU-Neu5Ac compound is high [30], these findings further confirm that HSM is effectively bound by the enzymatic pocket of IAV NA.

Figure 6 HSM inhibits IAV cleavage of the 4MU-Neu5Ac reporter substrate. (A) Cleavage of 4MU-Neu5Ac reporter substrate by A/Aichi/2/68(H3N2) was tested in the presence of HSM from two donors, PSM (10 nmol Sia), or PBS buffer. HSM from both donors inhibited the cleavage of 4MU-Ne5Ac. In contrast PSM did not inhibit cleavage of 4MU-Neu5Ac, similar to the buffer control. (B) Cleavage of 0.1 nmol 4MU-Neu5Ac by three IAV strains was tested in the presence of HSM, PSM (4.7 pmol Sia), or PBS buffer in triplicates. All virus strains were inhibited by HSM but not by PSM. Bars represent standard error, *P<0.05, **P<0.01 (two-tailed T-Test, n=3). Full size image

Direct binding of IAV to HSM on magnetic beads array

IAV binding to sialylated mucus was tested incubating virus with HSM and PSM conjugated to magnetic beads. As control, we used magnetic beads conjugated to a non-sialylated mucus-like polyacrylamide polymer. A/PR/8/34(H1N1), A/SD/1/2009(SOIV) and A/Aichi/2/68(H3N2) viruses (32-64 HAU) were incubated with the beads for 1 h at 37°C. In order to avoid cleavage of sialylated epitope and release of the virus, 16 μM oseltamivir was added to the virus and to the wash buffer. Following incubation, the beads were washed extensively to remove both unbound virus and oseltamivir. NA regains normal activity once oseltamivir was removed (data not shown). In order to quantify the bead-bound virus, each sample was incubated with 10 nmol 4MU-Neu5Ac compound for 30 min at 37°C, in the absence of oseltamivir. The release of fluorescent 4MU compound directly correlates with the number of virions in the sample. All three tested strains bound to HSM, but only A/PR/8/34(H1N1) bound to PSM (Figure 7). In addition virus binding to magnetic beads conjugated to an array of sialylated polyacrylamide polymer standards was tested (Additional file 4). The virus-binding pattern to the standard array was in agreement with previous reports [33–36] (Additional file 4). This confirms that the glycan array method produces reliable results. Thus the balance between HA binding- and NA cleaving- of the sialylated mucus protective layer determines the ability of mucus to protect underlying cells from infection.