Human Blood and Tonsil Plasmacytoid Dendritic Cells Display Similar Gene Expression Profiles but Exhibit Differential Type I IFN Responses to Influenza A Virus Infection.

Influenza A virus (IAV) infection constitutes an annual health burden across the globe. Plasmacytoid dendritic cells (PDCs) are central in antiviral defense because of their superior capacity to produce type I IFNs in response to viruses. Dendritic cells (DCs) differ depending on their anatomical location. However, only limited host-pathogen data are available from the initial site of infection in humans. In this study, we investigated how human tonsil PDCs, likely exposed to virus because of their location, responded to IAV infection compared with peripheral blood PDCs. In tonsils, unlike in blood, PDCs are the most frequent DC subset. Both tonsil and blood PDCs expressed several genes necessary for pathogen recognition and immune response, generally in a similar pattern. MxA, a protein that renders cells resistant to IAV infection, was detected in both tonsil and blood PDCs. However, despite steady-state MxA expression and contrary to previous reports, at high IAV concentrations (typically cytopathic to other immune cells), both tonsil and blood PDCs supported IAV infection. IAV exposure resulted in PDC maturation by upregulation of CD86 expression and IFN-α secretion. Interestingly, blood PDCs secreted 10-fold more IFN-α in response to IAV compared with tonsil PDCs. Tonsil PDCs also had a dampened cytokine response to purified TLR ligands compared with blood PDCs. Our findings suggest that tonsil PDCs may be less responsive to IAV than blood PDCs, highlighting the importance of studying immune cells at their proposed site of function.

Human hantavirus infection elicits pronounced redistribution of mononuclear phagocytes in peripheral blood and airways.

Hantaviruses infect humans via inhalation of virus-contaminated rodent excreta. Infection can cause severe disease with up to 40% mortality depending on the viral strain. The virus primarily targets the vascular endothelium without direct cytopathic effects. Instead, exaggerated immune responses may inadvertently contribute to disease development. Mononuclear phagocytes (MNPs), including monocytes and dendritic cells (DCs), orchestrate the adaptive immune responses. Since hantaviruses are transmitted via inhalation, studying immunological events in the airways is of importance to understand the processes leading to immunopathogenesis. Here, we studied 17 patients infected with Puumala virus that causes a mild form of hemorrhagic fever with renal syndrome (HFRS). Bronchial biopsies as well as longitudinal blood draws were obtained from the patients. During the acute stage of disease, a significant influx of MNPs expressing HLA-DR, CD11c or CD123 was detected in the patients' bronchial tissue. In parallel, absolute numbers of MNPs were dramatically reduced in peripheral blood, coinciding with viremia. Expression of CCR7 on the remaining MNPs in blood suggested migration to peripheral and/or lymphoid tissues. Numbers of MNPs in blood subsequently normalized during the convalescent phase of the disease when viral RNA was no longer detectable in plasma. Finally, we exposed blood MNPs in vitro to Puumala virus, and demonstrated an induction of CCR7 expression on MNPs. In conclusion, the present study shows a marked redistribution of blood MNPs to the airways during acute hantavirus disease, a process that may underlie the local immune activation and contribute to immunopathogenesis in hantavirus-infected patients.

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry.

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.