The Effects of H2S and Recombinant Human Hsp70 on Inflammation Induced by SARS and Other Agents In Vitro and In Vivo.

The ongoing epidemic caused by SARS-CoV-2 infection led to the search for fundamentally new ways and means to combat inflammation and other pathologies caused by this virus. Using a cellular model of lipopolysaccharide (LPS)-induced sepsis (human promonocytes), we showed that both a hydrogen sulfide donor (sodium thiosulfate, STS) and a recombinant Heat shock protein 70 (rHsp70) effectively block all major inflammatory mediators when administrated before and after LPS challenge. The protective anti-inflammatory effect of rHsp70 and H2S was also confirmed in vivo using various animal models of pneumonia. Specifically, it was found that rHsp70 injections prevented the development of the acute respiratory distress syndrome in highly pathogenic pneumonia in mice, increased animal survival, and reduced the number of Programmed death-1 (PD-1)-positive T-lymphocytes in peripheral blood. Based on our model experiments we developed a combined two-phase therapeutic approach for the treatment of COVID-19 patients. This procedure includes the inhalation of hot helium-oxygen mixtures for induction of endogenous Hsp70 in the first phase and STS inhalation in the second phase. The use of this approach has yielded positive results in COVID-19 patients, reducing the area of lung lesions, restoring parameters of innate immunity and T-cell immune response against coronavirus infection, and preventing the development of pulmonary fibrosis and immune exhaustion syndrome.

Neurotoxic Effects of Aß6-42 Peptides Mimicking Putative Products Formed by the Angiotensin Converting Enzyme.

Angiotensin converting enzyme (ACE) is involved in proteolytic processing of the amyloid-ß(Aß) peptide implicated in the development of Alzheimer's disease (AD) and known products of ACE-based processing of Aß42 are characterized by reduced aggregability and cytotoxicity. Recently it has been demonstrated that ACE can act as an arginine specific endopeptidase cleaving the N-terminal pentapeptide (Aß1-5) from synthetic Aß peptide analogues. In the context of proteolytic processing of full length Aß42, this suggests possible formation of Aß6-42 species. The aim of this study was to test a hypothesis that some N-terminally truncated Aß peptide(s) could retain aggregability and neurotoxic properties typical for Aß42. We have investigated aggregability of two amyloid-ß peptides, Aß6-42 and isoD7-Aß6-42, mimicking potential proteolytic products of Aß42 and isoD7-Aß42, and evaluated their effects on the repertoire of brain Aß binding proteins, and cytotoxicity towards neuroblastoma SH-SY5Y cells. Aggregability of isoD7-Aß6-42 and Aß6-42 was higher than that of full-length peptides Aß42 and isoD7-Aß42, while the repertoire of mouse brain Aß binding proteins dramatically decreased. Aß6-42 and isoD7-Aß6-42 exhibited higher neurotoxicity towards SH-SY5Y cells than Aß42 and isoD7-Aß42, respectively. They effectively stimulated production of ROS and NO, and also TNFα secretion by cells. Thus, our results suggest that ACE-dependent processing of full-length Aßs could result in formation of more pathogenic peptides.

The Fate of Exogenous Human HSP70 Introduced into Animal Cells by Different Means.

Over the last decade, it has become evident that in mammals, including humans, heat shock protein 70 (HSP70), apart from its intracellular localization, is found in extracellular space, where it may execute various protective functions. Furthermore, the upregulation of HSP70 family members can be beneficial in the prevention and treatment of various human neurodegenerative diseases and cancer. Here, we demonstrate that recombinant human HSP70 after intranasal administration can penetrate various brain regions of mice in its native form and subsequently undergo rapid degradation. It was also shown that labeled HSP70 added to culture medium of different human and mouse cell lines enters the cells with strikingly different kinetics, which positively correlates with the basic levels of membrane bound Toll-like receptors (TLR) that are characteristic of these cell lines. HSP70 administration does not significantly modulate the level of TLR expression at the protein or RNA level. The degradation of the introduced recombinant HSP70 after entering the cells is likely proteasome-dependent and varies significantly depending on the cells type and origin. These results should be considered when developing HSP70-based therapies.

Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels.

It has been previously reported that pretreatment with exogenous heat shock protein 70 (Hsp70) is able to protect cells and animals from the deleterious effects of bacterial lipopolysaccharide (LPS) produced by Gram-negative bacteria. However, the effects of Hsp70 pretreatment on lipoteichoic acid (LTA) challenge resulted from Gram-positive bacteria infection have not been fully elucidated. In this study, we demonstrated that preconditioning with human recombinant Hsp70 ameliorates various manifestations of systematic inflammation, including reactive oxygen species, TNFα, and CD11b/CD18 adhesion receptor expression induction observed in different myeloid cells after LTA addition. Therefore, exogenous Hsp70 may provide a mechanism for controlling excessive inflammatory responses after macrophage activation. Furthermore, in a rat model of LTA-induced sepsis, we demonstrated that prophylactic administration of exogenous human Hsp70 significantly exacerbated numerous homeostatic and hemodynamic disturbances induced by LTA challenge and partially normalized the coagulation system and multiple biochemical blood parameters, including albumin and bilirubin concentrations, which were severely disturbed after LTA injections. Importantly, prophylactic intravenous injection of Hsp70 before LTA challenge significantly reduced mortality rates. Thus, exogenous mammalian Hsp70 may serve as a powerful cellular defense agent against the deleterious effects of bacterial pathogens, such as LTA and LPS. Taken together, our findings reveal novel functions of this protein and establish exogenous Hsp70 as a promising pharmacological agent for the prophylactic treatment of various types of sepsis.

Exogenous mammalian extracellular HSP70 reduces endotoxin manifestations at the cellular and organism levels.

In this study, we checked whether HSP70 preparations of different origins are able to protect model animals (rats) from endotoxic shock and modify the response of myeloid cells to lipopolysaccharide (LPS) challenge. It was shown that HSP70 preparations can effectively protect organisms from endotoxic shock by strongly decreasing mortality and restoring both homeostasis and various hemodynamic characteristics. At the cellular level, HSP70 preparations significantly inhibit LPS-induced reactive oxygen species production in various myeloid cells and decrease NO expression in macrophages, which is enhanced after LPS priming. In parallel, HSP70 preconditioning partially normalizes neutrophil apoptosis, which is disturbed as a result of LPS stimulation. These results suggest that the antiseptic actions of HSP70 preparations are probably realized at the level of receptor membrane complexes of myeloid cells, which represent the major target of LPS action. Taken together, our findings show that extracellular mammalian HSP70 may play an important role in innate immunity modulation and stimulation of endogenous protective mechanisms, both at the cellular and organism levels, which make this protein a promising base for the development of efficient antiseptic drugs.