Respiratory mononuclear phagocytes during steady state and sarcoidosis

Each day, our lungs inhale thousands of liters of air containing not only essential oxygen but also smoke particles, dust, allergens, and potentially dangerous pathogens. In order to tolerate harmless antigens and initiate an immune response against pathogens, a well-organized network of immune cells is required in the respiratory tract. Mononuclear phagocytes (MNPs), comprised of macrophages, monocytes, and dendritic cells, line the respiratory mucosa and are equipped with tools to recognize and rapidly respond to foreign materials. Despite MNPs act as sentinels at the mucosal barrier, infections and inflammation can affect the lungs. Pulmonary sarcoidosis is a T cell-driven inflammatory disease characterized by granuloma formation. The causative antigen is yet to be identified but MNPs are known to be involved in the pathogenesis of sarcoidosis. A theory is that the antigen is taken up by macrophages. Macrophages also produce vast amounts of the proinflammatory cytokine tumor necrosis factor (TNF). Dendritic cells on the other hand, take up the antigen and transport it to the lymph nodes where they activate naïve T cells. Circulating monocytes have an important role in cytokine production. However, the role of respiratory monocytes during sarcoidosis is less well studied. We hypothesized that pulmonary MNPs are crucial in maintaining a steady state of antiand pro-inflammatory processes in healthy individuals. This dynamic process is disturbed during sarcoidosis, thereby contributing to pathogenesis. Hence, our aim was to study MNPs in the respiratory tract during steady state and sarcoidosis. First, we investigated MNPs in the human respiratory tract. We found profound differences in distribution of seven MNP subsets between blood and the respiratory tract both during steady state and sarcoidosis. We observed an increase in frequencies of blood and respiratory monocytes in sarcoidosis patients compared to healthy controls. Additionally, monocytes from sarcoidosis patients showed an inflammatory profile with upregulation of genes related to inflammatory pathways. Intriguingly, MNPs from the lungs of sarcoidosis patients produced TNF without external stimulation to a significantly higher degree than that of MNPs from healthy controls. In contrast to previous observations, we found that pulmonary monocytes contributed more to TNF production than macrophages. Additionally, we associated higher frequencies of monocytes in the circulation as well as high numbers of intrinsically TNF producing monocytes at time of diagnosis with progressive disease development in sarcoidosis. In conclusion, we have mapped the MNP network in several anatomical locations of the respiratory tract during steady state and sarcoidosis. We also identified pulmonary monocytes to play an important role in disease pathogenesis in sarcoidosis. That knowledge can help to design new treatment options in sarcoidosis to favor disease resolution and improve quality of life

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

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

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

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

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

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