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People are constantly exposed to airborne fungal spores, including Aspergillus fumigatus conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense A. fumigatus conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the A. fumigatus conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII+ antigen-presenting cells, including IE-DCs, and CD11b+ phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of A. fumigatus conidia increased the number of CD11b+ phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b+ phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b+ phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b+ phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b+ phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.

Immunostimulatory properties of extracellular heat shock proteins 70 kDa (HSP70) became interesting for investigators a long time ago. However, in recent years a series of works showing a significant relation of the immunostimulating effects of recombinant HSP70 to contamination of the protein samples with bacterial endotoxins (lipopolysaccharide, LPS) has been published. The authors showed that intensive elimination of LPS from the protein samples resulted in inversion of immunostimulating effects of HSP70 to immunosuppressive activity of the protein. Nevertheless, at present the conception of immunostimulating, proinflammatory action of extracellular HSP70 is the most common. In this work, we studied immunomodulatory effects of exogenous HSP70 in a mouse model of allergic inflammation of airways. We also analyzed the dynamics of the level of the extracellular pool of HSP70 in the site of inflammation. The results demonstrated a considerable content of extracellular HSP70 in bronchoalveolar lavages with dynamics reflecting the stages of development of the induced inflammation. Oropharyngeal injection of exogenous HSP70 in the acute phase of allergic inflammation of airways resulted in significant suppression of the inflammatory process, which conforms to published data demonstrating an immunosuppressive activity of the extracellular pool of HSP70.

Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall afterAspergillus fumigatusconidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+or Ly6G+neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h afterA. fumigatusinfection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates uponA. fumigatusinfection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.