Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy

<div><p>Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP⁺ signals were also EEA1⁺. Peak association with late endosomes occurred at 15 min when 60% of NP⁺ signals were LAMP1⁺. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.</p></div

3D automated image processing and analysis of z stacks acquired by confocal or STED microscopy using scikit-image.

<p><b>(A)</b> Raw microscope images of IAV-infected human DCs were processed to extract features such as the cell boundary, the nucleus, the viral particles and endosomal vesicles (method I). The extracted features were compared to each other using several methods to determine subcellular localization of IAV nucleoprotein (NP) (method II), or to assess colocalization of NP with endosomal compartments (method III). <b>(B)</b> Z stacks were analyzed as a whole to preserve the three-dimensional volume, taking into account overlapping features present in subsequent slices that may be counted repeatedly if slices were assessed individually. <b>(C)</b> The total number of NP⁺ signals in each volume of a cell was quantified (n = 60 cells per condition), with median values indicated by a red line. NP⁺ signals were not significantly different from 5 to 10 min, suggesting quantification of input virus, whereas increased significantly at 15 and 30 min, suggesting newly synthesized NP. Statistical differences were assessed using an unpaired <i>t</i> test: ** <i>p</i> < 0.01.</p

Trafficking of IAV particles to LAMP1⁺ late endosomes in DCs peaked at 15 min post exposure to IAV.

<p><b>(A)</b> DCs were labeled with primary antibodies against HLA-DR (blue), IAV NP (green) and LAMP1 (red). All images were acquired by STED microscopy on a Leica SP8. Images were deconvolved using Huygens Professional. Merged images of HLA-DR, IAV NP and LAMP1 from one cell per condition and a insert at 7x magnification of NP and LAMP1 are shown (n = 30 cells per condition). Arrow heads point to LAMP1⁺ NP⁺ signals. Scale bar = 5 μm. <b>(B)</b> The percentage of NP⁺ signals in each volume of a cell also coinciding with LAMP1⁺ signals out of total NP⁺ signals was quantified using the Python script (n = 3–30 cells per condition) with median values indicated by a red line. LAMP1⁺NP⁺ signals peaked at 15 min post infection. Statistical differences were assessed using an unpaired <i>t</i> test: ** p < 0.01, *** p < 0.001, n.s., not significant.</p

Early trafficking events of IAV upon entry in human DCs.

<p>The schematic summarizes the endosomal trafficking pathway of IAV upon entry in human DCs, beginning with binding of IAV to receptors on the cell surface. Endocytosed IAV were targeted to EEA1⁺ early endosomes within 5 min, followed by LAMP1⁺ late endosomes where membrane fusion could take place. Release of viral ribonucleoproteins (vRNPs) led to nuclear translocation where viral replication could proceed.</p

Improved resolution in visualization of viral trafficking in human DCs using STED microscopy with deconvolution.

<p><b>(A)</b> Confocal (left panel) and STED (right panel) images of a DC 4 hours post infection with IAV, stained with antibodies against IAV NP (green) and EEA1 (red). Scale bar = 5 μm. <b>(B)</b> An image of a DC 0 min post infection with IAV, stained with antibodies against HLA-DR (blue), IAV NP (green) and LAMP1 (red) acquired by STED microscopy before (top panel) and after (bottom panel) deconvolution using Huygens Professional. Scale bar = 5 μm. <b>(C)</b> Full width at half maximum (FWHM) values of a representative NP signal from confocal, STED and deconvolved STED images was determined.</p

Early trafficking events of IAV upon entry in human DCs.

<p>The schematic summarizes the endosomal trafficking pathway of IAV upon entry in human DCs, beginning with binding of IAV to receptors on the cell surface. Endocytosed IAV were targeted to EEA1⁺ early endosomes within 5 min, followed by LAMP1⁺ late endosomes where membrane fusion could take place. Release of viral ribonucleoproteins (vRNPs) led to nuclear translocation where viral replication could proceed.</p

Kinetics of NP subcellular trafficking after entry in human DCs.

<p><b>(A)</b> DCs were cultured with no virus, or infected with IAV for 4 hours in the absence or presence of NH₄Cl. After 4 hours, cells were adhered on to Alcian blue-coated coverslips for 20 min and fixed with 4% PFA. DCs were blocked with 1% goat serum and permeabilized with 0.1% Triton X-100. DCs were labeled with primary antibodies against HLA-DR (blue), IAV nucleoprotein NP (green) and the nucleus was counterstained with DAPI (gray). All images were acquired by confocal microscopy on a Leica LSM700. Scale bar = 5 μm. <b>(B)</b> For earlier time points, DCs were first adhered to Alcian blue-coated coverslips for 20 min, exposed to IAV at an MOI of 25 for 60 min at 4°C to allow virus particles to attach to cell membrane, and incubated at 37°C for 0–30 min, allowing a more synchronized entry pattern. Scale bar = 5 μm. <b>(C)</b> The percentage of intracellular or nuclear NP⁺ signals relative to total NP⁺ signals in each volume of a cell was quantified using the Python script (n = 3 cells per condition). NP⁺ signals were in the nucleus as early as 10 min post exposure to IAV, with a majority of NP⁺ signals in the nucleus after 30 min.</p

Trafficking of IAV particles to EEA1⁺ early endosomes in DCs occurred at 5 min post exposure to IAV.

<p><b>(A)</b> DCs were labeled with antibodies against HLA-DR (blue), IAV nucleoprotein NP (green) and EEA1 (red). All images were acquired by STED microscopy on a Leica SP8. Images were deconvolved using Huygens Professional. Merged images of HLA-DR, IAV NP and EEA1 from one cell per condition and a insert at 7x magnification of NP and EEA1 are shown (n = 30 cells per condition). Arrow heads point to EEA1⁺ NP⁺ signals. Scale bar = 5 μm. <b>(B)</b> The percentage of NP⁺ signals in each volume of a cell also coinciding with EEA1⁺ signals out of total NP⁺ signals was quantified (n = 10–60 cells per condition) with median values indicated by a red line. EEA1⁺NP⁺ signals peaked at 5 min post infection. Statistical differences were assessed using an unpaired <i>t</i> test: ** p < 0.01, **** <i>p</i> < 0.0001.</p

mDCs but not pDCs are susceptible to IAV infection.

<p>(<b>A</b>) pDCs (red) and mDCs (blue) were continuously exposed to IAV for 1, 6 or 24 hr and the frequency of IAV+ DCs was measured by flow cytometry using a rabbit polyclonal antibody raised against IAV/X31. Graph shows mean±SD percent of IAV+ CD123+ CD14− pDCs and CD11c+ CD14− mDCs (n = 7). Differences between IAV infection of mDCs and pDCs were assessed using paired t test: n.s. no significant difference, ** p<0.01, *** p<0.001. (<b>B</b>) DCs were treated with NH₄Cl, then exposed to IAV for 24 hr at 4°C or 37°C. The frequency of IAV+ DCs and level of CD86 expression was determined by flow cytometry. One representative experiment of six is shown. Dot plots show live DCs and numbers in each quadrant depict the frequency of positive DCs. (<b>C</b>) DCs were exposed to IAV for 1 hr, washed 3 times to remove free virus and allowed to adhere to coverslips. Surface HLA-DR (red) was labeled before fixation to visualize the plasma membrane. After permeabilization, virus was stained using an anti-IAV antibody (green) and the nucleus was stained using DAPI (blue). The entire volume of each cell was analyzed using confocal microscopy (100× 1.47NA oil objective, 6× digital zoom) and one single optical slice through the center of the cell is shown with arrowheads pointing to virus structures. Scale bar 10 µm. (<b>D</b>) The frequency of IAV+ DCs after 1 hr of virus exposure was determined by analyzing entire z-stacks of DCs and counting the number of cells that had virus associated with them, either on the membrane (white) or intracellularly (black). The graph shows average frequency of IAV+ DCs±SD, with 100–300 DCs analyzed per donor and condition (n = 4). (<b>E</b>) DCs were treated with LPS, TLR7/8L, IFNα or nothing for 24 hr and the MxA expression was determined by flow cytometry using intracellular staining with a monoclonal anti-MxA antibody. Graph shows MFI±SD of MxA with isotype control subtracted (n = 3).</p

IAV infected and uninfected mDCs have similar CMV antigen load.

<p>(<b>A</b>) mDCs were exposed to infectious IAV, HI IAV or untreated for 4 hr, washed and exposed to increasing doses of whole, γ-irradiated CMV for an additional 3 hr. DCs were then washed, adhered to coverslips and fixed with PFA. After permeabilization, samples were stained for CMV pp65 (green), IAV (red) and HLA-DR (blue). The entire volume of each cell was analyzed using confocal microscopy (100× 1.47NA oil objective, 7× digital zoom) and one single optical slice is shown through the center of the cell with arrowheads pointing to CMV pp65+ structures. Scale bar 5 µm. (<b>B</b>) The frequency of CMV pp65+ uninfected mDCs (blue), IAV infected mDCs (black) or HI IAV stimulated mDCs (white) after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of cells that had virus associated with them. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3). (<b>C</b>) The average number of CMV pp65+ puncta per cell after 3 hr of CMV exposure was determined by analyzing entire z-stacks of DCs and counting the number of green (CMV) puncta per nucleated cell, assessed by DAPI staining. The graph shows average frequency of CMV positive DCs ± SD, with 100 cells analyzed per donor and condition (n = 3).</p