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26.  Highly Pathogenic Avian Influenza Virus H5N1 Infects Alveolar Macrophages without Virus Production or Excessive TNF-Alpha Induction 
PLoS Pathogens  2011;7(6):e1002099.
Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine. In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.
Author Summary
Alveolar macrophages (AM), which reside in the alveolar lumen, usually dampen down the host immune response to incoming pathogens. However, they are thought to increase inflammation during highly pathogenic avian influenza virus (HPAIV) H5N1 infections, which cause severe and often fatal disease in humans. This is based on experiments with human macrophages cultured from monocytes rather than with human AM. Here we show that human AM, collected via broncho-alveolar lavage from healthy volunteers, can become infected with HPAIV H5N1. However, this results in neither induction of the pro-inflammatory cytokine TNF-alpha nor virus production. Therefore, AM are most likely not responsible for the excessive cytokine response or high viral load during human HPAIV H5N1 infections as assumed previously. These data significantly changes our insight into the pathogenesis of HPAIV H5N1 pneumonia in humans, indicating that other cells than AM must be responsible for the excessive pro-inflammatory cytokine profile observed during HPAIV H5N1 infections.
PMCID: PMC3121882  PMID: 21731493
27.  Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis 
EMBO Molecular Medicine  2011;3(4):222-234.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c+MHC-IIintCD40int dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
PMCID: PMC3377067  PMID: 21328541
Mycobacterium; SapM; tuberculosis; vaccine; BCG
28.  High mobility group box-1 recognition: The beginning of a RAGEless era? 
EMBO Molecular Medicine  2010;2(6):193-195.
High mobility group box 1 (HMGB1) is a molecular alarm signal that triggers an immune response when released. It was assumed that the receptor for advanced glycation end-products (RAGE) would mediate the signal to the immune system. Recently pattern recognition receptors that are triggered by molecules of bacterial origin (the Toll-like receptor (TLR) family) were shown to also respond to HMGB1. Now two papers establish the TLR4–HMGB1 axis as proinflammatory, eventually leading to disparate conditions like seizures or skin cancer. These reports add a new twist to our understanding of the mode of action of the alarm signal HMGB1.
PMCID: PMC3377318  PMID: 20535746
HMGB1; inflammation; TLR4
29.  House dust mite allergen induces asthma via TLR4 triggering of airway structural cells 
Nature medicine  2009;15(4):410-416.
Barrier epithelial cells and airway dendritic cells (DC) make up the first line of defence against inhaled substances like house dust mite (HDM) allergen and endotoxin. We hypothesized that these cells need to communicate to cause allergic disease. Using irradiated chimeric mice, we demonstrate that TLR4 expression on radioresistant lung structural cells is required and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony stimulating factor, interleukin-25 and IL-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.
PMCID: PMC2789255  PMID: 19330007
30.  Both Conventional and Interferon Killer Dendritic Cells Have Antigen-Presenting Capacity during Influenza Virus Infection 
PLoS ONE  2009;4(9):e7187.
Natural killer cells are innate effector cells known for their potential to produce interferon-γ and kill tumour and virus-infected cells. Recently, B220+CD11cintNK1.1+ NK cells were found to also have antigen-presenting capacity like dendritic cells (DC), hence their name interferon-producing killer DC (IKDC). Shortly after discovery, it has already been questioned if IKDC really represent a separate subset of NK cells or merely represent a state of activation. Despite similarities with DCs, in vivo evidence that they behave as bona fide APCs is lacking. Here, using a model of influenza infection, we found recruitment of both conventional B220− NK cells and IKDCs to the lung. To study antigen-presenting capacity of NK cell subsets and compare it to cDCs, all cell subsets were sorted from lungs of infected mice and co-cultured ex vivo with antigen specific T cells. Both IKDCs and conventional NK cells as well as cDCs presented virus-encoded antigen to CD8 T cells, whereas only cDCs presented to CD4 T cells. The absence of CD4 responses was predominantly due to a deficiency in MHCII processing, as preprocessed peptide antigen was presented equally well by cDCs and IKDCs. In vivo, the depletion of NK1.1-positive NK cells and IKDCs reduced the expansion of viral nucleoprotein-specific CD8 T cells in the lung and spleen, but did finally not affect viral clearance from the lung. In conclusion, we found evidence for APC function of lung NK cells during influenza infection, but this is a feature not exclusive to the IKDC subset.
PMCID: PMC2747012  PMID: 19784375
31.  Effect of Cigarette Smoke Extract on Dendritic Cells and Their Impact on T-Cell Proliferation 
PLoS ONE  2009;4(3):e4946.
Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells including neutrophils, macrophages,T lymphocytes, and dendritic cells (DCs). The relative contributions of these various inflammatory cells to airway injury and remodeling are not well documented. In particular, the potential role of DCs as mediators of inflammation in the smoker's airways and COPD patients is poorly understood. In the current study we analyzed the effects of cigarette smoke extract on mouse bone marrow derived DC and the production of chemokines and cytokines were studied. In addition, we assessed CSE-induced changes in cDC function in the mixed lymphocyte reaction (MLR) examining CD4+ and CD8+ T cell proliferation. Cigarette smoke extract induces the release of the chemokines CCL3 and CXCL2 (but not cytokines), via the generation of reactive oxygen species (ROS). In a mixed-leukocyte reaction assay, cigarette smoke-primed DCs potentiate CD8+T cell proliferation via CCL3. In contrast, proliferation of CD4+T cells is suppressed via an unknown mechanism. The cigarette smoke-induced release of CCL3 and CXCL2 by DCs may contribute to the influx of CD8+T cells and neutrophils into the airways, respectively.
PMCID: PMC2655711  PMID: 19293939
32.  Sustained desensitization to bacterial Toll-like receptor ligands after resolutionof respiratory influenza infection 
The World Health Organization estimates that lower respiratory tract infections (excluding tuberculosis) account for ∼35% of all deaths caused by infectious diseases. In many cases, the cause of death may be caused by multiple pathogens, e.g., the life-threatening bacterial pneumonia observed in patients infected with influenza virus. The ability to evolve more efficient immunity on each successive encounter with antigen is the hallmark of the adaptive immune response. However, in the absence of cross-reactive T and B cell epitopes, one lung infection can modify immunity and pathology to the next for extended periods of time. We now report for the first time that this phenomenon is mediated by a sustained desensitization of lung sentinel cells to Toll-like receptor (TLR) ligands; this is an effect that lasts for several months after resolution of influenza or respiratory syncytial virus infection and is associated with reduced chemokine production and NF-κB activation in alveolar macrophages. Although such desensitization may be beneficial in alleviating overall immunopathology, the reduced neutrophil recruitment correlates with heightened bacterial load during secondary respiratory infection. Our data therefore suggests that post-viral desensitization to TLR signals may be one possible contributor to the common secondary bacterial pneumonia associated with pandemic and seasonal influenza infection.
PMCID: PMC2271005  PMID: 18227219
33.  Ischemia of the lung causes extensive long-term pulmonary injury: an experimental study 
Respiratory Research  2008;9(1):28.
Lung ischemia-reperfusion injury (LIRI) is suggested to be a major risk factor for development of primary acute graft failure (PAGF) following lung transplantation, although other factors have been found to interplay with LIRI. The question whether LIRI exclusively results in PAGF seems difficult to answer, which is partly due to the lack of a long-term experimental LIRI model, in which PAGF changes can be studied. In addition, the long-term effects of LIRI are unclear and a detailed description of the immunological changes over time after LIRI is missing. Therefore our purpose was to establish a long-term experimental model of LIRI, and to study the impact of LIRI on the development of PAGF, using a broad spectrum of LIRI parameters including leukocyte kinetics.
Male Sprague-Dawley rats (n = 135) were subjected to 120 minutes of left lung warm ischemia or were sham-operated. A third group served as healthy controls. Animals were sacrificed 1, 3, 7, 30 or 90 days after surgery. Blood gas values, lung compliance, surfactant conversion, capillary permeability, and the presence of MMP-2 and MMP-9 in broncho-alveolar-lavage fluid (BALf) were determined. Infiltration of granulocytes, macrophages and lymphocyte subsets (CD45RA+, CD5+CD4+, CD5+CD8+) was measured by flowcytometry in BALf, lung parenchyma, thoracic lymph nodes and spleen. Histological analysis was performed on HE sections.
LIRI resulted in hypoxemia, impaired left lung compliance, increased capillary permeability, surfactant conversion, and an increase in MMP-2 and MMP-9. In the BALf, most granulocytes were found on day 1 and CD5+CD4+ and CD5+CD8+-cells were elevated on day 3. Increased numbers of macrophages were found on days 1, 3, 7 and 90. Histology on day 1 showed diffuse alveolar damage, resulting in fibroproliferative changes up to 90 days after LIRI.
The short-, and long-term changes after LIRI in this model are similar to the changes found in both PAGF and ARDS after clinical lung transplantation. LIRI seems an independent risk factor for the development of PAGF and resulted in progressive deterioration of lung function and architecture, leading to extensive immunopathological and functional abnormalities up to 3 months after reperfusion.
PMCID: PMC2335107  PMID: 18366783
34.  The Balance between Plasmacytoid DC versus Conventional DC Determines Pulmonary Immunity to Virus Infections 
PLoS ONE  2008;3(3):e1720.
Respiratory syncytial virus (RSV) infects nearly all infants by age 2 and is a leading cause of bronchiolitis. RSV may employ several mechanisms to induce immune dysregulation, including dendritic cell (DC) modulation during the immune response to RSV.
Methods and Findings
Expansion of cDC and pDC by Flt3L treatment promoted an anti-viral response with reduced pathophysiology characterized by decreased airway hyperreactivity, reduced Th2 cytokines, increased Th1 cytokines, and a reduction in airway inflammation and mucus overexpression. These protective aspects of DC expansion could be completely reversed by depleting pDCs during the RSV infection. Expansion of DCs by Flt3L treatment enhanced in CD8+ T cell responses, which was reversed by depletion of pDC.
These results indicate that a balance between cDC and pDC in the lung and its lymph nodes is crucial for the outcome of a pulmonary infection. Increased pDC numbers induced by Flt3L treatment have a protective impact on the nature of the overall immune environment.
PMCID: PMC2249704  PMID: 18320041
35.  GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells 
PLoS Biology  2007;5(12):e329.
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
Author Summary
Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells. The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check. The current study focuses on the role of two key transcription factors—FOXP3 and GATA3—in controlling the commitment of these cells. We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells. We show that IL-4–induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter. We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.
It is shown that Th2 responses prevent the generation of inducible Tregs. This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter. This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.
PMCID: PMC2222968  PMID: 18162042
36.  Specific Migratory Dendritic Cells Rapidly Transport Antigen from the Airways to the Thoracic Lymph Nodes 
Antigen transport from the airway mucosa to the thoracic lymph nodes (TLNs) was studied in vivo by intratracheal instillation of fluorescein isothiocyanate (FITC)-conjugated macromolecules. After instillation, FITC+ cells with stellate morphology were found deep in the TLN T cell area. Using flow cytometry, an FITC signal was exclusively detected in CD11cmed-hi/major histocompatibility complex class II (MHCII)hi cells, representing migratory airway-derived lymph node dendritic cells (AW-LNDCs). No FITC signal accumulated in lymphocytes and in a CD11chiMHCIImed DC group containing a CD8αhi subset (non–airway-derived [NAW]-LNDCs). Sorted AW-LNDCs showed long MHCIIbright cytoplasmic processes and intracytoplasmatic FITC+ granules. The fraction of FITC+ AW-LNDCs peaked after 24 h and had reached baseline by day 7. AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs. Compared with intrapulmonary DCs, AW-LNDCs had a mature phenotype and upregulated levels of MHCII, B7-2, CD40, and intracellular adhesion molecule (ICAM)-1. In addition, sorted AW-LNDCs from FITC-ovalbumin (OVA)–instilled animals strongly presented OVA to OVA-TCR transgenic T cells. These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.
PMCID: PMC2195883  PMID: 11136820
antigen-presenting cells; endocytosis; fluorescein isothiocyanate; respiratory mucosa; lymph nodes
37.  Immunologists getting nervous: neuropeptides, dendritic cells and T cell activation 
Respiratory Research  2001;2(3):133-138.
It is increasingly recognised that the immune and nervous systems are closely integrated to optimise defence systems within the lung. In this commentary, the contribution of various neuropeptides such as substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and somatostatin to the regulation of T cell activation is discussed. These neuropeptides are released not only from nerve endings but also from inflammatory immune cells such as monocytes, dendritic cells, eosinophils and mast cells. On release they can exert both direct stimulatory and inhibitory effects on T cell activation and also indirect effects through their influence on the recruitment and activation of professional antigen-presenting dendritic cells. Neuropeptides should therefore be included in the conceptual framework of the immune regulation of T cell function by dendritic cells.
PMCID: PMC2002076  PMID: 11686876
calcitonin gene-related peptide; dendritic cells; substance P; T cells; vasoactive intestinal peptide

Results 26-37 (37)