The Role of NLRP3 Inflammasome Activation in The Pathogenesis of Post-vaccine Long Haul Symptoms

Below is a peer reviewed study, which shows the plausible key role of NLRP3 inflammasome activation by spike protein ACE2 receptor interaction in the pathogenesis of post-vaccine long haul symptoms:

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.

Discussion
The most important message of this report is that human early-development stem cells deposited in adult tissues, known as very small embryonic-like stem cells (VSELs), like specified HSCs or EPCs, express on their surface the ACE2 receptor for entry of SARS-CoV-2 as well as the entry-facilitating protease TMPRSS2. In addition, these cells express AT1R, which indicates that they can be challenged by Ang II stimulation. We also report for the first time that the virus-derived recombinant spike protein (S) activates the Nlrp3 inflammasome in human VSELs and HSCs.

Our results are highly relevant to the potential long-term effects of SARS-CoV-2 infection, as the virus may damage VSELs by i) direct entry and cell lysis or ii) induction of cell death by pyroptosis due to hyperactivation of the Nlrp3 inflammasome in response to S protein or to excessive Ang II stimulation by AT1R. Nlrp3 inflammasome-induced cell death by pyroptosis is a result of activation of caspase 1, the release of IL-1β and IL-18, the perturbation of mitochondrial function, the creation of pores in the cell membrane by gasdermin D, and the release of several alarmines or danger-associated molecular pattern molecules (DAMPs) that subsequently amplify an uncontrolled immune response [16, 42–44]. This response involves secretion from other cells of several pro-inflammatory cytokines such as. IL-6 or TNF-α and mediators as well as activation of the complement and coagulation cascades [45–47]. If uncontrolled, this process may end in a cytokine storm and fatal organ damage.

It is well known that the innate immune response and activation of the Nlrp3 inflammasome are important defense mechanisms during the first days of infection, until acquired immunity responds with the production of antibodies. However, on the other hand hyperactivation of this intracellular protein complex may induce a cytokine storm, with detrimental effects, leading to multi-organ failure. We have proposed three scenarios for how this could happen. First, the SARS-CoV-2 spike protein (S), after binding to cell surface-expressed ACE2, directly triggers Nlrp3 inflammasome activation. In fact, we show for the first time that S protein binding to ACE2 on human cells contributes to activation of the Nlrp3 inflammasome. Importantly, we found that activation of the Nlrp3 inflammasome in human cells after the interaction of ACE2 with the SARS-CoV-2 S protein is ameliorated in the presence of Ang (1–7). Second, as previously reported, after engaging the AT1 receptor, Ang II activates the Nlrp3 inflammasome in lung, kidney cells, and cardiomyocytes, and excessive activation of the Ang II–AT1R axis in these cells may lead to pyroptosis [12, 48, 49]. Since, as shown in our studies, both VSELs and HSCs highly express AT1R, there is i) an increase in Ang II activity in patients infected with SARS-CoV-2 and ii) a lack of protective Ang (1–7)–Mas signaling due to blockade and downregulation of ACE2 by viral proteins, which may hyperactivate the Ang II–AT1R axis. Third, recognition and interaction of the complement cascade with SARS-CoV-2 releases several potent cleavage fragments, including C3a and C5a anaphylatoxins as well as the C5bC9 membrane attack complex (MAC), which may also directly trigger activation of the Nlrp3 inflammasome in target cells including population of stem cells.

Therefore, the potential damage of VSELs after virus entry or exposure to Nlrp3 inflammasome hyperactivating mediators may have a negative effect on the regenerative potential of SARS-CoV-2-infected individuals and create potential long-term health problems in survivors. This, however, will require further study. In support of this possibility, ACE2 function and Ang (1–7)–MasR signaling, which are perturbed by SARS-CoV-2 infection, play a role in the proliferation of several stem cell types, including HSCs, EPCs, and skeletal muscle cells [6, 15, 50]. Moreover, since several complications from endothelium have been already reported, it is important to assess the effects of infection on hematopoiesis and lymphopoiesis and also take into account damage of more primitive precursors of these cells that are VSELs.

Another important observation is that RAAS mediators may play a role in the development of VSELs. As mentioned in the introduction, the role of RAAS in the development of putative hemangioblasts has been reported in several old publications [39, 41, 51, 52]. These cells were purified for example from human para-aortic splanchnopleura as CD143+, CD34−, and CD45− population [53]. They expressed the ACE receptor (CD143), however they were not evaluated for the expression of SARS-Cov-2 entry receptor ACE2. In our studies human VSELs, which may correspond to hemangioblasts in in vitro assays, are present among the population of human ACE2+, very small CD133+CD34+Lin−CD45− cells. These cells, which have been isolated from adult bone marrow, mobilized peripheral blood, and umbilical cord blood, according to several independent reports, have been proposed to serve in postnatal tissues as a backup stem cell population involved in tissue or organ rejuvenation [26, 33–35, 54]. Importantly in appropriate experimental models they can become specified into functional HSCs and EPCs.

In conclusion, 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 applications to prevent onset of a cytokine storm and cell pyroptosis [23]. In further support of this possibility, encouraging results have already been obtained in employing antibodies against a Nlrp3 inflammasome-activation product, IL-1β. Finally, based on our results demonstrating ACE2 expression on the surface of VSELs, further studies of infection with live virus will be needed to address whether the virus may enter these cells. Finally, the expression of several genes involved in RAAS in VSELs raises the possibility that this system is involved in the development and specification of early-development stem cells.

Since key findings from a REACT19 survey of 967 vaccine injured folks included autoimmunity as being a possible risk factor for COVID vaccine injury and that new onset autoimmunity seems to be diagnosed in the vaccine injured at very high rates, below are studies showing the role of NLRP3 in autoimmune diseases:

Role of the NLRP3 inflammasome in autoimmune diseases

Highlights

  • NLRP3 inflammasome is composed of NLRP3, ASC and caspase-1.
  • NLRP3 inflammasome activation leads to the IL-1β and IL-18 release and pyroptosis.
  • NLRP3 inflammasome plays a key role in immunity and autoimmune diseases.
  • NLRP3 inflammasome acts as a promising therapeutic target for autoimmune diseases.

Therapeutic potential of the target on NLRP3 inflammasome in multiple sclerosis

Highlights

  • NLRP3 inflammasome activation contributes to the pathogenesis of MS.
  • NLRP3 inflammasome is a key mediator in the progression of MS and EAE.
  • NLRP3 inflammasome inhibitors reduce the secretion of IL-1β and alleviate MS and EAE.
  • NLRP3 inflammasome could be a potential therapeutic target for MS treatment.

Below are links to webpages showing peer reviewed studies that show that all ingredients of the natural supplement Quadramune (available online on Amazon, Walmart, BuyQuadramune.com and youcanordernow.com) inhibit NLRP3, thereby showing why Quadramune will likely alleviate post-vaccine long haul symptoms, as supported by this clinical study results and as also specified in Amazon review comments:

https://areyoucovidmune.com/covidmune/nlrp3-and-pterostilbene/

https://areyoucovidmune.com/covidmune/sulforaphane-and-nlrp3/

https://areyoucovidmune.com/covidmune/egcg-and-nlrp3/

https://areyoucovidmune.com/covidmune/thymoquionone-and-nlrp3/