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BACKGROUND: Airborne pathogens such as virus particles undergo elimination from the respiratory tract by mucociliary clearance and phagocytosis by immune cells. The data about phagocytic cell type infiltration and stimuli that attract phagocytic cells to conducting airway are required for the anti-virus immune response mechanism understanding and the treatment strategy development. AIM: To detect the role of the receptor-binding domain of SARS-CoV-2 in neutrophil immune response activation in conducting airway mucosa after 100 nm particles application. MATERIALS AND METHODS: C57BL/6 mice received an oropharyngeal application of fluorescent 100 nm particles suspended in the receptor-binding domain of SARS-CoV-2 solution. 24 hours after, conducting airways of mice were dissected and subjected for immunohistochemistry as whole-mounts. Three-dimensional images of conducting airway regions were obtained using confocal microscopy. Quantitative image analysis was performed to estimate the ingestion activity of neutrophils in conducting airway mucosa. RESULTS: Neutrophil migration to conducting airway mucosa was detected in case of the application of particles in receptor-binding domain solution, but not in phosphate buffer or bovine serum albumin solution. Receptor-binding domain solution alone also induced neutrophil migration to conducting airway mucosa. Infiltrating conducting airway wall mucosa neutrophils contributed to particles internalization. CONCLUSIONS: The receptor-binding domain of SARS-CoV-2 can activate the neutrophil-mediated response in conducting airway mucosa.

BACKGROUND: Daily, people inhale airborne viral particles, some of which have a size of about 100 nm, such as particles of SARS-CoV-2. Kinetics of such 100 nm particle distribution in the respiratory tract is important, however, not a properly investigated question. AIM: To estimate the dissemination of inert viral particles based on the analysis of the spatial distribution of fluorescent 100 nm particles in the mouse lungs at different time points after the application. MATHERIALS AND METHODS: Fluorescent particles of 100 nm size were applied to C57BL/6 mice. 6, 24, 48 and 72 hours after, lungs were excised and fixed. Lung lobes were stained with immunohistochemistry as whole-mounts and then underwent optical clearance. Three-dimensional images of whole-mount mouse lung lobes were acquired using confocal laser scanning microscopy. RESULTS: 6 hours after the particle application particles were detected in lungs both as single particles and as particle agglomerates. Particles were both free and internalized by phagocytic cells. 24 hours after the application particles were detected both in bronchial lumen and in the alveolar space. Particles were detected in the mouse lungs up to 72 hours after the application. CONCLUSIONS: Reaching the respiratory tract of mammalian, inert particles which size equal to SARS-CoV-2 particle size distribute both in bronchi and in alveoli and undergoes internalization of phagocytic cells.

SARS-CoV-2-induced infection is still dangerous. Mouse models are convenient to the investigation of virus-activated immune response mechanisms. However, mice are not proper model organisms to study COVID-19 due to decreased interaction affinity between the SARS-CoV-2 receptor-binding domain (RBD) and mouse angiotensin-converting enzyme 2 (ACE2) compared with human ACE2. In the present study, we propose a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system. To mimic the effects of RBD– ACE2 high-affinity interaction, mice received the ACE2 inhibitor MLN-4760. To simulate virus loading, we applied 100 nm particles suspended in the solution of RBD via the oropharyngeal route to mice. In this model, MLN-4760 application enhanced neutrophil egress from the bone marrow to the bloodstream and RBD attracted neutrophils to the luminal side of the conducting airway epithelium. By contrast, inert 100 nm particles were not potent to stimulate neutrophil recruitment to the conducting airway mucosa. Using this model, and by altering the dosage of the ACE2 inhibitor, nanoparticles, and RBD, one can adapt it to investigate different COVID-19 states characterized with mild or severe airway inflammation.StatementThis study presents a mouse model that allows estimating the influence of SARS-CoV-2 on the immune system and investigates immune cell-model virus particle interactions in the conducting airway mucosa.

Vaccination protects against COVID-19 via the spike protein receptor-binding domain (RBD)-specific antibody formation, but it also affects the innate immunity. The effects of specific antibody induction on neutrophils that can cause severe respiratory inflammation are important, though not completely investigated. In the present study, using a mouse model mimicking SARS-CoV-2 virus particle inhalation, we investigated neutrophil phenotype and activity alterations in the presence of RBD-specific antibodies. Mice were immunized with RBD and a week after a strong antibody response establishment received 100 nm particles in the RBD solution. Control mice received injections of a phosphate buffer instead of RBD. We show that the application of 100 nm particles in the RBD solution elevates neutrophil recruitment to the blood and the airways of RBD-immunized mice rather than in control mice. Analysis of bone marrow cells of mice with induced RBD-specific antibodies revealed the increased population of CXCR2+CD101+ neutrophils. These neutrophils did not demonstrate an enhanced ability of neutrophil extracellular traps (NETs) formation compared to the neutrophils from control mice. Thus, the induction of RBD-specific antibodies stimulates the activation of mature neutrophils that react to RBD-coated particles without triggering excessive inflammation.

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.

Background Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores. Methods Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G+ neutrophils and MHC II+ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways. Results Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application. Conclusions Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.