The ACE2 Receptor for COVID-19 Entry Is Expressed on the Surface of Hematopoietic Stem/Progenitor Cells and Endothelial Progenitors As Well As Their Precursor Cells and Becomes Activated in Nlrp3 Inflammasome-Dependent Manner By Virus Spike Protein - a Potential Pathway Leading to a “Cytokine Storm”

Background. The SARS-CoV-2 pandemic, with its high mortality, has become an urgent clinical problem. It is well established that SARS-CoV-2 enters human cells after binding to the angiotensin-converting enzyme 2 (ACE2) receptor and utilizes a spike protein (S) for attachment and entry into the cells, leading to their lysis or damage. This infection damages several organs, including lungs, heart, blood vessels, kidneys, and intestines and may lead to a fatal complication known as a “cytokine storm”, which is the result of uncontrolled hyperactivation of the innate immunity-initiated response and the release of several pro-inflammatory cytokines. An important potential aspect of SARS-CoV-2 infection is damage to the stem cell compartment, which may lead to severe complications from the infection. The ACE2 receptor has been described as being expressed on the surfaces of certain types of stem cells, including specified hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). These cells could be potentially damaged and lysed after virus entry or could undergo pyroptosis due to hyperactivation of Nlrp3 inflammasomes. Supporting this possibility, we recently reported that all Nlrp3 inflammasome components are expressed in HSCs (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-10005-w). Hypothesis. We hypothesized that one of the triggers of a cytokine storm could be interaction of the ACE2 receptor with the SARS-CoV-2 spike protein, leading to hyperactivation of Nlrp3 inflammsomes in target cells including population of stem cells. Materials and Methods. Experiments were performed on human stem cells at different levels of specification, including very small CD34⁺CD133⁺lin^–CD45^– cells, which may become specified into HSCs and EPCs, as well as human CD34⁺Lin^–CD45⁺ HSCs and CD34⁺ CD133⁺ KDR⁺ CD31⁺ EPCs. These cells were phenotyped for expression of ACE2 and the SARS-CoV-2 entry-facilitating transmembrane protease TMPRSS2 at the mRNA level and by FACS at the protein level. Next, we exposed these cells to the NCP-CoV (2019-nCoV) spike protein (S1+S2 ECD, expressed with a His-tag; Sino Biological) at a concentration of 10 nM. After 16 h of incubation, the cells were lysed, and total RNA was isolated for qRT-PCR analysis of Nlrp3 and essential Nlrp3 inflammasome components, including ASC, caspase 1, IL1b, and IL18. In some experiments, UCB-derived HSCs were plated into 96-well plates and stimulated with NCP-CoV (2019-nCoV) spike protein, as described above, alone or together with angiotensin 1-7 or the anti-inflammatory heme oxygenase 1 (HO-1) activator CoPP. Results. First, we observed that the ACE2 receptor and SARS-CoV-2 entry-facilitating transmembrane protease TMPRSS2 are expressed by all types of stem cells evaluated in our studies. Moreover, we detected activation of Nlrp3 inflammasomes in response to viral spike protein. This activation was inhibited by exposure of the stimulated cells to angiotensin 1-7 or CoPP. Conclusions. We envision that, in addition to directly infecting target cells, virus can hyperactivate the Nlrp3 inflammasome in stem cells, which may trigger their pyroptosis. Therefore, since we still do not have an effective SARS-CoV-2 vaccine in hand, the results presented in our current work suggest that inhibition of the Nlrp3 inflammasome by the small-molecule inhibitor MCC950 or application of Nlrp3 inflammasome inhibitors, such as Ang (1-7) or heme oxygenase 1 activators, could find potential clinical application to prevent the onset of a cytokine storm and cell pyroptosis.