SARS-CoV-2 Entry Receptor ACE2 Is Expressed on Very Small CD45- Precursors of Hematopoietic and Endothelial Cells and in Response to Virus Spike Protein Activates the Nlrp3 Inflammasome

Abstract

Angiotensin-converting enzyme 2 (ACE2) plays an important role as a member of the renin-angiotensin-aldosterone system (RAAS) in regulating the conversion of angiotensin II (Ang II) into angiotensin (1-7) (Ang [1-7]). But at the same time, while expressed on the surface of human cells, ACE2 is the entry receptor for SARS-CoV-2. Expression of this receptor has been described in several types of cells, including hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), which raises a concern that the virus may infect and damage the stem cell compartment. We demonstrate for the first time that ACE2 and the entry-facilitating transmembrane protease TMPRSS2 are expressed on very small CD133⁺CD34⁺Lin^-CD45^- cells in human umbilical cord blood (UCB), which can be specified into functional HSCs and EPCs. The existence of these cells known as very small embryonic-like stem cells (VSELs) has been confirmed by several laboratories, and some of them may correspond to putative postnatal hemangioblasts. Moreover, we demonstrate for the first time that, in human VSELs and HSCs, the interaction of the ACE2 receptor with the SARS-CoV-2 spike protein activates the Nlrp3 inflammasome, which if hyperactivated may lead to cell death by pyroptosis. Based on this finding, there is a possibility that human VSELs residing in adult tissues could be damaged by SARS-CoV-2, with remote effects on tissue/organ regeneration. We also report that ACE2 is expressed on the surface of murine bone marrow-derived VSELs and HSCs, although it is known that murine cells are not infected by SARS-CoV-2. Finally, human and murine VSELs express several RAAS genes, which sheds new light on the role of these genes in the specification of early-development stem cells. Graphical Abstract •Human VSELs and HSCs express ACE2 receptor for SARS-CoV2 entry. •Interaction of viral spike protein with ACE2 receptor may hyperactivate Nlrp3 inflammasome which induces cell death by pyroptosis. •SARS-CoV2 may also enter cells and eliminate them by cell lysis. •What is not shown since these cells express also Ang II receptor they may hyperactivate Nlrp3 inflammasome in response to Ang II which may induce pyroptosis. Our data indicates that Ang 1-7 may have a protective effect.

Keywords: ACE2; COVID19; Cytokine storm; Hematopoietic stem cells; Nlrp3 inflammasome; Pyroptosis; SARS-CoV-2; Spike protein; VSELs.

Graphical Abstract

•Human VSELs and HSCs express ACE2 receptor for SARS-CoV2 entry. •Interaction of viral spike protein with ACE2 receptor may hyperactivate Nlrp3 inflammasome which induces cell death by pyroptosis. •SARS-CoV2 may also enter cells and eliminate them by cell lysis. •What is not shown since these cells express also Ang II receptor they may hyperactivate Nlrp3 inflammasome in response to Ang II which may induce pyroptosis. Our data indicates that Ang 1–7 may have a protective effect.

Fig. 1

Gating strategy for sorting human CD34⁺ and CD133⁺ VSELs and HSCs by FACS. UCB-derived VSELs and HSCs were isolated from human UCB mononuclear cells (MNCs) following magnetic isolation of CD34 or CD133-positive cells and immunostaining. UCB derived populations of MNCs was visualized by dot plot showing forward scatter (FSC) vs. side scatter (SSC) signals, which are related to the size and granularity/complexity of the cells. Cells from region P1 were further analyzed for CD45 and Lin expression. The population of CD45⁺/Lin⁻ objects was included in region P2. Cells from region P2 were analyzed for CD45 and CD34 expression and subsequently sorted into Lin⁻/CD34⁺/CD45⁻ cell (VSEL, region P3) and Lin⁻/CD34⁺/CD45⁺ cell (HSC, region P4) subpopulations (Panel a) or CD45 and CD133 expression and sorted into Lin⁻/CD133⁺/CD45⁻ cell (VSEL, region P3) and Lin⁻/CD133⁺/CD45⁺ cell (HSC, region P4) subpopulations (Panel b)

Fig. 2

Expression of SARS-CoV-2 entry receptors and selected RAAS genes in purified human VSELs and HSCs. Expression of ACE2, AGTR1, AGTR2, MAS1, CMA1, RENIN and TMPRSS2 mRNAs in UCB MNC and UCB purified VSELs and HSCs as measured by RT-PCR. To evaluate relative expression, comparative ΔCT method was employed. Results are combined from three independent purification of UCB VSELs and HSCs. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Expression of ACE2 on human CD34⁺ and CD133⁺ VSELs and CD34⁺ and CD133⁺ HSCs. Expression of ACE2 was evaluated on human UCB-derived VSELs and HSC by FACS and RT-PCR. Panel a. Detection of ACE2 on Lin⁻/CD34⁺/CD45⁻ VSELs (second raw) and Lin⁻/CD34⁺/CD45⁺ HSCs (first raw). Panel b. Detection of ACE2 on Lin⁻/CD133⁺/CD45⁻ VSELs (second raw) and Lin⁻/CD133⁺/CD45⁺ HSCs (first raw). Results from flow cytometry are presented as a percentage of ACE2⁺ cells. The data represent the mean value ± SEM for two independent experiments (upper part of Panel a and b). The expression of ACE2 gene was detected in purified mRNA from sorted human VSELs and HSCs by reverse transcription polymerase chain reaction (RT-PCR). Samples containing only water instead of cDNA and samples without reverse transcriptase were used in each run as negative controls. Representative agarose gels of the RT-PCR amplicon are shown (lower part of Panel a and b)

Fig. 4

Gating strategy for sorting murine VSELs and HSCs by FACS. BM-derived VSELs and HSCs were isolated from murine BM total nucleated cells (TNCs) following immunostaining for Sca-1, CD45, and the hematopoietic lineage marker (Lin). BM-derived TNCs were visualized by dot plot showing forward scatter (FSC) vs. side scatter (SSC) signals, which are related to the size and granularity/complexity of cells. Cells from region P1 are further analyzed for Sca-1 and Lin expression. The population of Sca-1⁺/Lin⁻ objects was included in region P2 and subsequently sorted into CD45⁻ and CD45⁺ subpopulations based on CD45 marker expression. Region P3 shows Sca-1⁺/Lin⁻/CD45⁻ cells (VSELs). Region P4 shows Sca-1⁺/Lin⁻/CD45⁺ cells (VSELs)

Fig. 5

The relative expression of mRNAs for ACE2, RAAS peptides and receptors, and components of the Nlrp3 inflammasome in highly purified murine bone marrow-derived VSELs and HSCs. Real-time PCR quantitation of FACS-sorted murine VSELs and HSCs in comparison with mononuclear cells; *P < 0.05, **P < 0.01, ***P < 0.001. In order to evaluate relative expression, the comparative ΔCT method was employed. Results are presented as mean ± SEM. Panel a. Differences in the expression of mRNAs for angiotensin-converting enzyme 2 (ACE2), the type 1 angiotensin II receptor (AT1), the type 2 angiotensin II receptor (AT2), the proto-oncogene Mas (MAS), the type 3 angiotensin II receptor (AT3), and transmembrane protease 2 (TMPRSS2). Panel b. Differences in the expression of mRNAs for renin (REN), angiotensinogen (AGT), and angiotensin-converting enzyme (ACE)

Fig. 6

Human CD34⁺ HSC activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) and Angiotensin 1–7 on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived HSCs in comparison to mononuclear cells; *P < 0.05, **P < 0.01, ***P < 0.001. In order to evaluate relative expression, comparative ΔCT method was employed. Results are presented as mean ± SEM. Differences in the expression of mRNAs for NLRP3, AIM2, ASC, IL-1beta, IL-19 and NLRP1 after 16 h exposure to SARS-CoV-2 Spike alone and in the presence of Angiotensin 1–7

Fig. 7

Human CD34⁺ VSELs activate Nlrp3 inflammasome in response to SARS-Cov-2 spike protein. Effect of NCP-CoV (2019-nCoV) Spike protein (S1 + S2 ECD, His tag) on the expression of inflammasome related genes. Real-time PCR quantitation of FACS sorted human UCB derived VSELs in comparison to mononuclear cells; *P < 0.05, **P < 0.01, ***P < 0.001. In order to evaluate relative expression, comparative ΔCT method was employed. Results are presented as mean ± SEM. Differences in the expression of mRNAs for NLRP3 after 16 h exposure to SARS-CoV-2 Spike protein

Fig. 8

Murine VSELs express mRNAs for elements of the Nlrp3 inflammasome. Real-time PCR quantitation of FACS-sorted murine VSELs and HSCs in comparison with mononuclear cells; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. In order to evaluate relative expression, the comparative ΔCT method was employed. The relative quantity of target gene was normalized to the endogenous β2 microglobulin gene. Results are presented as mean ± SEM. Graphs represent differences in the expression of mRNAs for NACHT, LRR, and PYD domain-containing protein 3 (NLRP3); caspase 1 (CASP1); interleukin 1β (IL-1β); interleukin 18 (IL-18); gasdermin (GSDM); and high-mobility group protein B1 (HMGB1)