Human dendritic cells in blood and airways during respiratory viral infection

The air we inhale contains oxygen necessary for life, but also potentially harmful microorganisms, toxins and allergens. This presents an important immunological dilemma: how can our lungs quickly and selectively eliminate harmful agents without inflicting damage on the delicate tissues of the lungs? We have thus evolved a network of cells involved in immune surveillance, made up of dendritic cells (DCs), monocytes and macrophages. Together, these mononuclear phagocytes sample the lungs and airways for presence of foreign pathogens such as viruses or bacteria. Recognition of pathogenic patterns – for instance the genetic material of viruses or the lipid membrane of bacteria – triggers a cascade of events in these immune cells. They produce inflammatory mediators to signal that a source of danger has been detected, and to contain the infection while awaiting the arrival of other immune cells. DCs migrate to lymphoid organs where they present antigens to naïve T cells, thus shaping the generation of protective and adaptive immunity. Much of what we know of how our immune system functions come from studies in murine models. In this thesis, we focus our attention on human DCs. Using super resolution microscopy, we assessed the early trafficking events that take place upon internalisation of influenza A virus (IAV) by human DCs. We report that IAV trafficked via early and late endosomes in DCs, similar to epithelial cells, but with more delayed kinetics. Next, we investigated whether maturation of monocyte-derived versus bona fide DCs affects their susceptibility to IAV infection. Indeed, the two subsets of DCs are inherently different in their ability to respond to pathogenic signals by producing antiviral mediators, which protect them from IAV infection. The accessibility of human blood has improved our understanding of human DCs. However, immune cells residing at mucosal barriers are our first line of defence against respiratory viruses. Increasing data suggest that there is tissue-specific regulation of immune cells due to factors present in the local microenvironment. Hence, we performed bronchoscopies on healthy subjects and hantavirus-infected patients to characterise DCs residing in the airways and bronchial mucosal tissue. We identified several subsets of respiratory DCs at steady state, alongside alveolar macrophages and monocyte-derived cells. During acute hantavirus disease, DCs and monocytes were depleted from circulation, whereas the lungs were infiltrated with monocytes and DCs. Collectively, our findings reveal the heterogeneity of human DCs in their response to respiratory viruses, depending on their origin and anatomical location. A deeper understanding of the complex interplay between respiratory viruses and human DCs reveals how DCs contribute to immunity or pathogenesis. This knowledge may help us develop better preventive and therapeutic strategies by targeting or modulating DCs to achieve favourable immune responses

Study of influenza A virus infection in human dendritic cells and epithelial cells.

Influenza A virus (IAV) is an important pathogen which primarily targets epithelial cells of the respiratory tract, but dendritic cells (DCs) residing in the epithelium are also susceptible. DCs play a crucial role in initiating specific adaptive immune response against IAV. In order to determine if IAV replication proceeds differently in DCs and epithelial cells, the susceptibility of these two cell types to IAV, using monocyte-derived DCs (MDDCs) and A549 cell line respectively, was studied. As the maturation status of a DC is critical to its functional performance, the susceptibility of differentially stimulated MDDCs to IAV through activation of different toll-like receptors (TLRs) was also explored. MDDCs supported IAV infection at the expense of cell viability. MDDCs also responded to IAV infection by undergoing maturation. When analysed by flow cytometry, A549 cells exposed to the same dose of IAV were not well infected. Visualization of infection through immunofluorescence staining enabled detection of more infected A549 cells. Maturation of MDDCs via TLR3 and TLR4 partially protected the MDDCs from IAV infection, but not MDDCs matured via TLR7/8. The antiviral activity appears to be dependent on type I interferons (IFNs), as observed through upregulation of IFN-susceptible genes (ISG).Bachelor of Science in Biological Science

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

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

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

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

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

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

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