Journal: Frontiers in immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: FIGURE 1 SP-A inhibits IAV infection independent of its glycosylation. RAW264.7 cells were cultured overnight at a density of 2 x 105 cells/well in 24-well plates and infected with either IAV PR8 (A-F) or IAV Phil82 (F). Cells were incubated with indicated concentration of virus in 1:1 (v/v) PBS/DMEM and then allowed to proceed in DMEM/10% FBS. At the endpoint of the following experiments cells were harvested, stained with NP antibodies, and analyzed by flow cytometry. (A) SP-A inhibits IAV infection in a concentration-dependent manner. RAW264.7 were pretreated with increasing concentration of 5, 10, 25 and 50 mg/mL SP-A for 5 hours and then infected with IAV PR8 at MOI=2 in the presence of respective concentration of SP-A. The percentage of infected cells was determined by flow cytometry 10-hous after infection. N=4, t-test, **p < 0.05. (B) IAV overcomes SP-A inhibition at high MOI. RAW264.7 cells were incubated in the presence or absence of 50 mg/mL of SP-A for 5 hours, then infected with IAV PR8 at MOI 1, 2, and 5 in 1:1 PBS/DMEM in the presence or absence of 50 mg/mL SP-A and infection was assessed by flow cytometry. N=6, t-test, **p < 0.05 (C) Either pretreatment of macrophages or IAV with SP-A inhibits infection. RAW264.7 cells were incubated with 50 mg/mL SP-A in indicated combinations before or during infection. N=2 independent experiments per condition. (D) De-glycosylation of SP-A was accomplished using EndoF and confirmed by visualization of molecular weight reduction of monomeric and oligomeric forms of SP-A on Western blots using a polyclonal SP-A antibody. (E) The inhibitory effect of SP-A does depend on its glycoconjugate moiety. De-glycosylation enhances the inhibitory effect SP-A on IAV infection. RAW264.7 cells were treated with either native of de-glycosylated (DCHO) SP-A and infected with IAV at MOI=2 and infection assessed at 6 and 12 hrs. after infection. N=4, t-test, *p < 0.05; **p < 0.01, NS, not significant. (F) SP-A delays temporal infection with both H1N1 and H3N2 strains of IAV. RAW264.7 were infected with either IAV H1N1 strain PR8 or H3N2 strain Phil82 following a 5-hour treatment with no or either 10 or 50 mg/mL SP-A. Infection was then assessed by flow cytometry at 3, 12, or 24 hours after infection. Representative histograms of n=2 independent experiments per treatment are shown.

Article Snippet: After washing, plates were incubated with 1:5000 dilutions of rabbit anti-SP-A polyclonal antibodies followed by washing and incubation with 1:10,000 dilution of an HRP-conjugated anti-rabbit antibody (BioRad Cat No 170-6515).

Techniques: Infection, Glycoproteomics, Cell Culture, Incubation, Concentration Assay, Virus, Staining, Cytometry, Inhibition, Molecular Weight, Western Blot

Journal: Frontiers in immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: FIGURE 2 SP-A binds HA in a calcium and glycosylation independent manner. SP-A binding to HA was carried out in static solid phase assays (A-C) and under flow using surface plasmon resonance (SPR) (D-F). For static assays, recombinant PR8 HA (1 mg/well) was adsorbed onto 96-well Immobilon-2 plates in 0.05 M bicarbonate buffer (pH 9.6) overnight at 4°C. Coated plates were incubated with albumin control or SP-A at room temperature for 1 hour. Plates were washed in binding buffer and incubated with a polyclonal SP-A antibody. Bound SP-A was visualized colorimetrically using HRP- conjugated anti-rabbit antibody and tetramethylbenzidine. Albumin was used as non-specific control. (A) SP-A binding to HA does not depend on the presence of calcium. HA and albumin control coated plates were washed and incubated with increasing concentration of 10, 25, 50, and 100 mg/mL of SP-A in the presence or absence of 5 mM EDTA. N=4 of 2 independent experiments. (B) De-glycosylation (DCHO) of HA does not prevent SP-A binding. Plates coated with DCHO recombinant HA, or albumin were washed, and then incubated with increasing concentration of 10, 50, and 100 mg/mL of SP-A. N=2. (C) De-glycosylation (DCHO) of SP-A does not prevent binding to HA. Recombinant HA or albumin control coated plates were incubated with increasing concentration of 10, 50, 75, and 100 mg/mL of SP-A or DCHO SP-A. N=4 of 2 independent experiments. (D-F) Calcium does not impact kinetic and binding parameters of SP-A binding to HA. For SPR assays, recombinant HA with a carboxy-terminal hexa- histidine tag was immobilized onto Nicoya nitrilotriacetic acid (NTA) sensor chips. Increasing concentration of 10, 20, 40, or 80 mg/mL of SP-A analyte in the presence (D) or absence (E) of 0.5 mM CaCl2 was injected at 20 mL/min for 300 sec to obtain on-rates and switched to buffer without SP-A for an additional 600 sec to obtain off-rates. Binding sensograms were acquired using a 2-channel Nicoya OpenSPR instrument. Curves were fitted according to 1:1 interaction model to obtain kinetic and binding parameters (F). Data shown are representative of N=3 independent experiments.

Article Snippet: After washing, plates were incubated with 1:5000 dilutions of rabbit anti-SP-A polyclonal antibodies followed by washing and incubation with 1:10,000 dilution of an HRP-conjugated anti-rabbit antibody (BioRad Cat No 170-6515).

Techniques: Glycoproteomics, Binding Assay, SPR Assay, Recombinant, Incubation, Control, Concentration Assay, Injection

Journal: Frontiers in immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: FIGURE 3 SP-A delays IAV infection at endosomal level. (A) SP-A delays synthesis of HA. RAW264.7 cells were treated with SP-A for five hours and then infected with IAV at MOI=15. Cells were harvested 1, 2, 4, 6, 12, and 24-hours after infection to obtain cell extracts for Western blot analysis using a polyclonal rabbit anti-HA antibody and a fluorescent IRDye800CW goat anti-rabbit secondary antibody. Blots were probed with an anti-mouse actin antibody and IRDye680RD goat anti-mouse antibody as loading control. Blots were quantitated by fluorescence densitometry using a LICOR instrument to obtain band intensity graphs and densitometry data for HA band intensity were normalized to actin. N=3, *p < 0.05 and ***p < 0.001 assessed by 2-way ANOVA. (B) To monitor trafficking to acidified endosomes, RAW264.7 cells were treated with SP-A for 5 hours and infected with IAV MOI=15 for 15, 35, 60, 90 and 240 minutes. Cells were harvested, fixed, permeabilized, and stained with an antibody recognizing a pH- dependent epitope that is exposed upon conformational transition of HA to the fusion competent form (pHHA). SP-A suppressed detection of the low pH HA conformation over the first 30 min of infection and delayed synthesis of new HA. N=4, *****p < 0.00005 assessed by tailed t-test. (C, D) SP-A treatment results in reduced infection and retention of NP in a punctate perinuclear compartment. Immunofluorescence microscopy was used to visualize the effect of SP-A on localization of viral NP. Cells were treated with 50 mg/mL SP-A for 5 hours and then infected with IAV for 4 hours. Cells were then fixed and stained with FITC-Cholera Toxin B subunit to stain the cell membrane, antibodies to NP to visualize IAV, and DAPI to stain the nucleus (C). Stained cells were counted across 10 microscopic fields at 63x magnification using a Nikon Eclipse confocal microscope to obtain the distribution of NP between nuclear and cytosolic compartments (D).

Article Snippet: After washing, plates were incubated with 1:5000 dilutions of rabbit anti-SP-A polyclonal antibodies followed by washing and incubation with 1:10,000 dilution of an HRP-conjugated anti-rabbit antibody (BioRad Cat No 170-6515).

Techniques: Infection, Western Blot, Control, Staining, Microscopy, Membrane

Journal: Frontiers in immunology

Article Title: Surfactant protein A alters endosomal trafficking of influenza A virus in macrophages.

doi: 10.3389/fimmu.2023.919800

Figure Lengend Snippet: FIGURE 4 Differential reduction in uptake of endocytic cargo and binding of IAV PR8 to cell membrane in the presence of SP-A. Endocytic and binding experiments were performed using RAW264.7 cells (A-E) or isolated RAW264.7 membrane (F). Raw264.7 cells were incubated with Dextran 10,000 or transferrin conjugated with either the pH sensitive dye pHRodo (A, B) or FITC (C, D) in 1:1 DMEM in the presence or absence of SP-A, for 15 minutes at room temperature. The media were replaced with dye-free 1:1 DMEM and cells incubated with PHRodo- and FITC-labeled molecules were chased at 37°C for 10, 20, 30, and 45-minutes and 5, 10, 15, 30, and 45-minutes at 37°C to monitor rates of endosomal acidification and uptake, respectively. (A, B) SP-A does not alter acidification rate for either Dextran (A) or transferrin (B) tracked endosomes, N=6. (C) SP-A reduced level of FITC-Dextran over the first 5-minutes, whereas SP-A appeared to delay uptake of FITC-transferrin between 5-20-minutes reaching a similar plateau after 30-minutes. Differences were significant for FITC-Dextran only. N=6, *p<0.05. (D) and (E) SP-A suppresses uptake rate of Alexa Fluor488-labeled PR8. RAW264.7 cells were incubated with IAV with SP-A throughout infection or pre-treatment only. Cells were infected with Alexa Fluor488-labeled PR8 at MOI=15. The uptake of the labeled virus was monitored for 5, 15, 30, and 45-minutes at 37°C by flow cytometry. N=6 per condition per time point, **p < 0.005 and ****p < 0.001 for vehicle vs SP-A throughout; ++, p < 0.005 and +++, p<0.0005 for vehicle vs SP-A pretreatment; ##, p < 0.05 for SP-A throughout vs SP-A. (F) To assess differences in membrane binding, immulon-2 flat bottom plates were coated overnight at 4°C with 12.5 mg/ml of Raw264.7 cell membranes and then washed and incubated with PBS, 5 x 106 ffc of PR8, or 50 mg/ml SP-A for 2- hours at room temperature. The membranes were then washed, and incubated for an additional 2-hours with PR8, pre-formed PR8+SP-A complex, or SP-A to evaluate PR8 binding in a combination of conditions shown on the x-axis on panel F graph. Plates were washed and bound PR8 visualized spectrophotometrically using a polyclonal anti-HA antibody and HRP-conjugated anti-rabbit antibody as described in Materials and Methods. N=8, ****p < 0.00001, ***p < 0.001. Statistical differences were assessed by 2-way ANOVA.

Article Snippet: After washing, plates were incubated with 1:5000 dilutions of rabbit anti-SP-A polyclonal antibodies followed by washing and incubation with 1:10,000 dilution of an HRP-conjugated anti-rabbit antibody (BioRad Cat No 170-6515).

Techniques: Binding Assay, Membrane, Isolation, Incubation, Labeling, Infection, Virus, Cytometry