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 after Aspergillus fumigatus conidium 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 after A. fumigatus infection 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 upon A. fumigatus infection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

Reactive oxygen species (ROS) produced by phagocytic cells of the innate immune system play an important role in the first line of defense protecting the host from pathogens. The NADPH oxidase multi-subunit complex is the main source of ROS in all types of the phagocytes. Formation of the membrane-associated enzyme complex and its activity are dependent on many different factors controlling both intensification and suppression of the ROS production rate. However, the evidences are emerging in recent years indicating existence of poorly studied mechanisms of restriction of ROS generation level in phagocytes directed at protection of host tissues in the sites of inflammation from destruction caused by the oxygen free radicals. Our previous data and results of other authors demonstrate that a mechanism of the limitation of ROS production by phagocytes may by connected with immunomodulating activity of extracellular pool. of HSP70. In the present work, we used inhibitors of NADPH oxidase and in vitro cultures of different phagocytes to study a possible relationship between down-regulating effect of exogenous HSP70 on ROS generation and the interaction of the protein with the enzyme subunits. Our results confirmed the literature data concerning the ability of extracellular HSP70 to modulate NADPH oxidase activity and demonstrated for the first time an inhibitory effect of the protein on intracellular ROS generation in phagocytes.

Bogorodskiy et al. Intraepithelial Dendritic Cells Contact Phagocytes 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.

Predictive classification of metabolites from natural products holds immense promise for developing new therapeutics to treat diseases that involve cell-surface proteins. Cardiac glycosides and monoterpene indole alkaloids are metabolite compounds that can be extracted from plants and have therapeutic applicability in the treatment of irregular heartbeats and pain sensitivity through their interactions with ion pumps and neurotransmitter receptors. We seek to combine the results of analytical experiments involving ion mobility mass spectrometry (IM-MS) with computational studies involving open-source cheminformatics software. Our goal for this project is to predict the collision cross section (CCS) values of modeled structures and match with our IM-MS experimental results. Using a standard off-the-shelf cheminformatics software (RDKit), we can rapidly generate molecular ensembles based on simple SMILES strings and calculate different geometric properties that allow us to cluster these ensembles and obtain the most representative conformers to match with experiment. Using an open-source quantum chemistry software (Psi4), we can generate Mulliken charges of the representative conformers to conduct CCS calculations and compare with IM-MS experiments. We are currently applying this method to a suite of cardiac glycosides and monoterpene indole alkaloids to create a library of ion mobility data for rapid classification and identification of mixtures of plant-based toxin molecules. This combined experimental and computational approach will allow scientists to rapidly analyze and identify toxins in unknown mixtures based on their mobilities and CCS values.