Evidence of Purposeful Evolution

Darwin's evolution challenged!

A recently published article in Nautre challenged evolution theory suggesting DNA repair was the more likely candidate driving evolutionary development than the environmental conditions thought to be the driver of natural selection. In some sense the two may be linked, but this study showed how epigenome-associated mutation bias reduced the occurrence of deleterious mutations, challenging the prevailing paradigm that mutation is a directionless force in evolution.

Quantitative assessment of DNA gain and loss through DNA double-strand break (DSB) repair processes suggests deletion-biased DSB repair causes ongoing genome shrinking in A. thaliana, whereas genome size in barley remained nearly constant.

Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DSB repair. Alu elements are the most abundant transposable elements (capable of shifting their positions) containing over one million copies dispersed throughout the human genome.

The emergence of recombination-activating genes (RAGs) in jawed vertebrates endowed adaptive immune cells with the ability to assemble a diverse set of antigen receptor genes. Innate Natural Killer (NK) cells are unable to express RAGs or RAG endonuclease activity during ontogeny. They exhibit a cell-intrinsic hyperresponsiveness, but a diminished capacity to survive following virus-driven proliferation, a reduced expression of DNA damage response mediators, and defects in the repair of DNA breaks. However, RAG expression in uncommitted hematopoietic progenitors and NK cell precursors marks functionally distinct subsets of NK cells in the periphery, demonstrating a novel role for RAG in the functional specialization of the NK cell lineage. 

The most active region of Human Chromosome 19 has a long history of recombinations that define the expression patterns of telomeric and centromeric proportions of Killer-cell immunoglobulin-like receptor (KIR) gene's encoding receptors. KIR's bind cells presenting MHC class 1 HLA haplotype combinations, that vary significantly across tissues in different population groups. Further, the deletion rate in Zinc Finger clusters (ZNF) located around 19q13.42, near KIR and C19MC between 51,012,739 and 55,620,741 are about twofold higher than the background deletion rate. 

The relationship between deletions and mutation may indeed play a direct role in rapidly evolving, innate immunity. This may just begin to explain the speed at which global populations can respond and survive pandemics caused by the likes of COVID-19. And, the '19' in its nomenclature may go beyond time to the very chromosome responsible for innate immune diversity.

Immunity keeping p53 in check!

In a 2012 study on the topology of the human and mouse m6A RNA methylomes, Gene Ontology (GO) analysis of differentially expressed genes (DEG's) indicated a noteworthy enrichment of the p53 signaling pathway: 22/23 genes had differentially expressed splice variants, of which 18 were methylated. Moreover, 15 other members of the signaling pathway, which were not significant DEG's, exhibited significant differential isoform expressions. For example, isoforms of MDM4, needed for p53 inactivation were downregulated. Similar pro-apoptotic effects were observed in other pathway genes including MDM2, FAS and BAX. Higher apoptosis rate in HaCaT-T cells resulted with knockdown of m6A subunit METTL3, which also reversed a significant decrease in p53 activity. Modulation of p53 signaling through splicing may be relevant to induction of apoptosis by silencing of METTL3. 

Then, in 2019 a study of arsenite-induced human keratinocyte transformation demonstrated that knockdown of METTL3 significantly decreased m6A level, restored p53 activation and inhibited cellular transformation phenotypes in the-transformed cells. Further, m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. m6A also upregulated expression of negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, the study revealed the novel role of m6A in mediating human keratinocyte transformation by suppressing p53 activation and sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.

Finally in 2021 a discovery that YTHDF2 is upregulated in NK cells upon activation by cytokines, tumors, and cytomegalovirus infection. Ythdf2 deficiency in NK cells impaired its anti-tumor and anti-viral activity in vivo. YTHDF2 maintains NK cell homeostasis and terminal maturation, correlating with modulating NK cell trafficking and regulating Eomes, respectively. It promotes NK cell effector function and is required for IL-15-mediated NK cell survival and proliferation by forming a STAT5-YTHDF2 positive feedback loop. Analysis showed significant enrichment in cell cycle, division, and division-related processes, including mitotic cytokinesis, chromosome segregation, spindle, nucleosome, midbody, and chromosome. This data supports roles of YTHDF2 in regulating NK proliferation, survival, and effector functions. Transcriptome-wide screening identified Tardbp (TDP-43) to be involved in cell proliferation or survival as a YTHDF2-binding target in NK cells.

Downregulation of METTL3, which in spinal cord contributes with YTHDF2 to modulate inflammatory pain may upregulate differentially expressed p53 network splice variants that oppose YTHDF2 induced downregulation of p53, via PRDM2 leading to apoptotic or diseased cells. In diseased environments cytokines may upregulate YTHDF2 in NK cells leading to downregulation of p53 and cytoskeletal transformation that may be sufficient, at an immune synapse to advance cytolysis.

p53 signals may inform selections of cells and tissue that prime NK cells for advanced, personalized immune therapy. 

p53 in Transition, Covid19, Cancer and Immunity

p53's trajectory, sensitivity and function influences different outcomes in stages of transition of developing pluripotent or embryonic stem cells that can inform tumorigenesis and immune response. 

Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation, and DNA damage-induced apoptosis was p53-independent. 

Human (induced) pluripotent stem cell differentiation, from endoderm toward mesoderm was driven by a DNA damage-induced, time-sensitive, p53 transcriptional program. In cells passing through epithelial-to-mesenchymal transition DNA damage prevents the normal reduction of p53 levels, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. 

From the blastocyst, villous cytotrophoblasts undergo a partial epithelial to mesenchymal transition (EMT) when they differentiate into extravillous cytotrophoblasts and gain the capacity to migrate and invade. Extravillous cytotrophoblast invasion involves a cellular transition from an epithelial to mesenchymal phenotype. TWIST, an emerging gene of interest strongly influences p53 to complete EMT.  

p53 is necessary for cells to initiate EMT, but attenuation of its levels by MDM2 is also necessary for expression of the mesenchymal phenotype. Downregulation of p53 may be directly controlled by this transition as the EMT factor TWIST1 can bind p53 leading to its MDM2-dependent degradation. During definitive endoderm differentiation, downregulation of p53 may be necessary for the normal transcriptional program to proceed. The unscheduled stabilization of p53, caused by DNA damage may result in a transcriptional perturbation driving differentiation away from definitive endoderm.

Using KRAS-driven pancreas tumor-derived cancer cells as a model of p53 loss, p53 deletion can promote immune tolerance through the recruitment of both myeloid and Treg cells. Enrichment of these suppressive cell populations enhanced the protection of p53-null cancer cells from immune-mediated elimination. 

Tumor-derived VEGF through VEGFR2 and NRP-1 creates a perivascular niche to regulate the initiation and stemness of skin tumors and autocrine VEGF promotes survival and invasion of prostatic, pancreatic cancer and glioblastoma cells, particularly for cancer stem-like cells in a NRP-1-dpendent enhanced EMT manner. 

A recent SARS-CoV2 update may point to anti-apoptotic affects that occur through the axis inactivation of p53 and mitochondrial apoptotic pathway as mediated by NRP-1, in endothelial cells of Zebra Fish. Decreased levels of p53 might suppress caspase cleavage and therefore downregulate apoptosis (a feature of Covid19). Data showed that p53 is the downstream signaling molecule of PI3K/Akt pointing at MDM2 as a signaling component in NRP-1 survival signaling. NRP-1 was shown as a host factor for SARS-CoV-2 infection and in a successful Covid19 phase trial, for critical care patients injection of apoptotic cells induced signaling to restore immune homeostasis.  

Even brief reactivation of endogenous p53, in p53-deficient tumors can produce complete tumor regressions. Primary response to p53 reactivation was not apoptosis, but the induction of a cellular senescence program associated with differentiation and upregulation of inflammatory cytokines. 

Elimination of senescent tumors, by Natural Killer (NK) cells occurred as a result of signal cooperation associated with p53 expression or senescence, which regulate NK cell recruitment and other signals that induce NKG2D ligand expression on tumor cells. p53 expression enhances CCL2-dependent NK cell recruitment to the tumors.

A feature of several NK cell activating receptors resides in their capacity to detect self molecules induced in conditions of cellular stress. This is the case for NKG2D, which interacts with various ligands, including CCL2 that are expressed at low levels in most tissues but are overexpressed upon initiation of cellular distress, for example, after initiation of the DNA damage response.

Codondex is working to identify p53 status in cells isolated from TME tissue samples that can be cocultured to educate NK cells to stimulate a desired immune response. 

p53 vasoregulation and NK cell depletion in SARS-CoV2

p53 has earned first prize in the academic stakes. It is also the most mutated gene in cancer and elephant's have 20 copies, which probably explains their surprisingly low rate of cancer. Its associations to innate immunity, particularly Natural Killer (NK) cells through the mechanics of vasoconstriction-dilation have become a point of interest in COVID19 patients.

Remarkably COVID19 has inspired the global scientific community to focus a significant portion of its aggregate research toward the impact of  SARS-CoV2 (CoV2). For the first time in history global research is singularly focused because a large number of other protein's and gene's are affected by CoV2 binding Ace2. The Ace2 receptor is important in systems of vasoconstriction-dilation and has wide ranging impact.

CoV2 binding Ace2 reduces its availability to convert Angiotensin1 to Angiotensin 1-7 (Ang1-7) or Angiotensin 1-9 (Ang1-9), which primarily interact via MAS and Angiotensin2 Receptor (AT2R) respectively. These have been linked to signaling and stretch caused by vasoconstriction-dilation, mitochondrial dysfunction, mitochondrial fission as well as cardiac and vascular remodeling.

Ang1-7 and Ang1-9 interactions with MAS or AT2R cell surface receptors have been linked to signaling events that drive p53 binding DNA and transcription. Myocyte stretching activates p53 and p53-dependent genes, leading to the formation of Angiotensin II (Ang II) and apoptosis. AngII, stimulates phosphorylation of p53 (on serine 15) and CREB (on serine 133) and signaling converges on the p53-CRE enhancer to stimulate Bradykinin receptor 2 (BK2) gene transcription. BK2 is a key element in the p53 related kallikrein-kinin system (KKS) of vasodilation that counters the Renin-Angiotensin-Aldosterone-System (RAAS) of vasoconstriction. 

Aldosterone was shown to induce mitochondrial dysfunction and podocyte injury mediated by p53/Drp1-dependent mitochondrial fission. In neuronal cells p53 dependent declines in Drp1 and parkin contribute to altered mitochondrial morphology and cell death. Parkin, via Pink1 activity binds depolarized mitochondria to induce autophagy of mitochondria. Mutations in both Drp1 and Pink1 were fatal in Drosophila models. These events also implicate a direct functional link to chronic inflammation in ageing between p53 and expression levels of ICAM1 on endothelial and NK cells required to bind targets. The p53 mediated negative regulation of autophagy is Pink1 dependent and experiments have shown that mitochondrial antigens, recognized by NK cells presented on MHC's are Pink1 and parkin dependent. 

Severe COVID-19 patients have highly elevated Bradykinin and AngII, perhaps an indication of elevated p53 trends that have been discovered in these patients. Under normal circumstances, on endothelial cells Bradykinin would act as a potent vasodilator via its BK2 receptor. However, since Ang (1-7) potentiates Bradykinin action on BK2 receptors its near absence may reduce KKS vasodilation. On the other hand RAAS, also via p53 and elevated AngII primarily interacts with AT1R to promote vasoconstriction.  

NK cells through their Renin Angiotensin System may counter-regulate target cells in response. However, in COVID19 patients depletion of NK cells, invasion of Neutrophils and endothelial cell damage, in part through elevated p53 autophagy and apoptosis is the overwhelming nasty work of CoV2 against the backdrop of dysregulated blood pressure in tissue.

 

 

p53 in the SARS-CoV2 Storm

Coronavrius induced cytokine storm

A massive simulation and analysis using the supercomputer at Oakridge led scientists to more accurately identify the general idea that a Covid19 induced "cytokine storm" is responsible for disease progression. After detailed genetic analysis they specifically predicted that Bradykinin (BK) initiated the storm. If correct, this would help improve treatment directions for admitted patients. 

BK receptors are coded by BDKRB1 and BDKRB2 (BK2) gene's that operate in a kallikrein-kinin system (KKS), like the Renin Angiotensin System (RAS) as another potent regulator of blood pressure. BK is a part of the vasopressor system that induces hypotension and vasodilation, it is degraded by ACE and enhanced by angiotensin1-9, which is produced by ACE2 the receptor that SARS-COV2 binds. BK has been implicated as being active in the metabolic response to stress.

Similar to angiotensin peptides, BK is produced from an inactive pre-protein kininogen through activation by serine protease kallikrein (KKL). KLK1-KLK15 are mostly represented as a cluster of serine proteases on chromosome 19, with different tissue distributions. 

KLK's 1-15 further evidence a convergence on chromosome 19, associated closely with a large number of genes involved in blood pressure. KLK's are located at 19q13.41, an active transposon region with a 2x background deletion rate clustered near Zinc Fingers and KIR's (Killer cell like receptors). Chr19 is also associated with MHC precursors around which innate immunity and Natural Killer (NK) cell signaling developed. A link was confirmed in mice uterine NK cells that regulated local tissue blood pressure by at least Angiotensin Type 1 Receptor (AT1R) partly in response to mechanical stretch of vasoconstriction and vasodilation induced by uterine NK's internal RAS. 

A study of BK2 confirmed a conserved p53 binding site (rat, mouse and human) and p53-mediated activation of the BK2 promoter was augmented by transcriptional co-activators, CBP/p300. The results demonstrated BK2 promoter as a target of the p53-mediated activation and suggested a new physiological role for p53 in the regulation of G protein-coupled receptor (GPCR) gene expression. 

A follow up study, by the same group explained that Angiotensin II (AngII), the product of Angiotensin-Renin-Angiotensin1-Ace stimulates the phosphorylation of p53 (on serine 15) and CREB (on serine 133), and that AngII signaling converges on the p53-CRE enhancer to stimulate BK2 gene transcription.  

The convergence revealed that AT1R signaling activated CREB phosphorylation and in vivo assembly of p-CREB on the BK2 promoter in conjunction with histone hyperacetylation. It confirmed that AngII stimulates BK2 gene transcription in IMCD3 cells via AT1R. Thus, under conditions of augmented AngII and AT1R signaling, BK expression will be enhanced, thereby maintaining a balance of these two powerful counter-regulatory systems representing a novel form of cross-talk between GPCR's that link RAS and KKS, crucially here via p53.

This combined research indicates that activation of BK2 on endothelial cells, which is mediated by p53 dependent RAS-KKS cross-talk may also implicate AT1R on NK cells to secrete growth disrupting or growth promoting factors in response. SARS-CoV2 bound to ACE2 reduces its availability to convert angiotensin 1 to angiotensin 1-9, which normally enhances BK. The resulting imbalance and increase in circulating angII may directly implicate a NK cells' AT1R RAS response to a CoV2-ACE2 bound cell with disabled p53 promoter of GPCR expression. This cell with a crippled BK2 KKS, hypotension-vasodilation response offers no counter to the the AngII induced NK RAS vasoconstrictive function, affecting local tissue blood pressure thus failing to become a NK target. 

Blood Pressure by Natural Killer Cells and SARS-CoV2

A meta-analysis of gene expression signatures for diastolic, systolic blood pressure and hypertension found that out of 7717 unique, related genes 34 were most differentialy expressed across 7017 individuals from 6 international studies. No less than 20% of the 34 gene's, were located on Chromosome 19. Enrichment analysis for the diastolic and systolic gene group's associated to Natural Killer (NK) Cell mediated cytotoxicity and 13 other pathways including antigen processing and inflammatory response, pointing strongly to innate and adaptive immunity. 

I covered the NK origin of MHC and antigen immunity and reproduction at Chromosome 19 on a previous blog, now the meta-analysis adds infection, immunity and blood pressure to this location. Evolutionary detectives tracked events from Chromosome 19 to 1,6 and 9 via transposon re-combination events, which provided further direction for interpreting the blood pressure meta-analysis. A review of the genes and pathways involved increasingly characterized innate immunity as an integrated core component of almost every aspect of our skeletal, circulatory, tissue and neuronal systems. 

Blood pressure is enormously complex, but its governance of entropy under the mechanical laws of molecular diffusion and disassociation reign supreme. Renin-Angiotensin (RAS) genes are widely recognized to be the cornerstones needed for blood pressure. Innate immune cells including NK have been confirmed to possess and express RAS genes. Macrophages, a member of innate immune system have been linked to angiotensin signaling neuropathic pain as well as bacterial infection inducing pain suppression by angiotensin 2 receptor (AT2R).  Maternal NK cells AT1R and AT2R have been implicated in the control of localized blood pressure in placental tissues leading to preeclampsia a condition in pregnancy.

In various studies, including in disease conditions it has been shown and suggested that different male:female ratios between AT1R and AT2R in monocytes and other innate cells is an important factor in the determination of blood pressure that has been extensively studied in heart and lung conditions. AT2R plays a critical role in satellite cell differentiation and skeletal muscle regeneration via myoblasts, which may be the reason it's expressed ubiquitously in developing fetal tissue. It's likely that balance between AT1R and AT2R signaling is critical for normal muscle regeneration.  

In addition to the role of NK cells in blood pressure a study using lung-intravital microscopy linked pulmonary NK cells to the control of neutrophil intravascular motility, response to acute inflammation and diminished pathogenic accumulation. NK cell derived IFN-γ plays an important role in the activation and maturation of monocytes into macrophages and dendritic cells, an amplifying mechanism in the early innate inflammatory response. Angiotensin II can induce rapid neutrophil infiltration via AT1R that also stimulates leucocyte–endothelium interactions. Inhibited IFN-γ signaling ameliorated Angiotensin II induced cardiac damage, which led to a finding that NK-cells play an essential role in the induced vascular dysfunction.

Ace2 Expression Patterns in Covid19

Pathophysiology of Covid19 demonstrates that NK cells are depleted and neutrophils infiltrate into lung tissue leading to tissue damage and escalation of the disease. By SARS-CoV2 binding the Ace2 receptor of vascular epithelial and other cells, the conversion of Angiotensin II is blocked (image above), therefore upregulated. Increased levels of Angiotensin II were shown to induce NK cells to release IFN-γ. On recruitment to inflammatory sites, NK-cells release IFN-γ and engage with monocytes in a reciprocal program of activation in which monocytes mature into macrophages and dendritic cells. NK exhaustion results and is a known outcome that may relate to IFN-γ levels. However, in patients with high expression of Ace2, NK cell counts are lower and cytokine expressions do not show up during the initial disease state pointing to the role of accumulating Angiotensin II.  

Covid19 Meta Analysis

The image above shows distribution of expression (y axis) for ACE2, PCSKs (blood pressure mediation) and TMPRSS2 (CoV2 S1 cleavage) across lung cell types (x axis). It completes the picture that Corin-Furin mediated control over blood pressure normalization is a significant component of Covid19 disease progression and NK cells are a central player.

Molecules targeting RAS are a major focus of inhibitory or complementary therapeutic design, but a modified NK cell that is shielded from SARS-CoV2 may be the tool-in-the-shed our immune systems need. 

Is The Natural Killer our Anti-Cell?

Lymphocyte

Natural Killer (NK) cells may be our innate, anti-cell sentinels that first arise in the yolk sac between day 8 and 10. Simultaneously maternal, uterine NK cells promote fetal development by secreting growth promoting factors. Distinct sets of NK cells mature during early fetal development by associating with differentiating cells and tissue environments. Typically NK cells mature in around 25-35 days. By comparison, the developing embryonic heart begins pumping blood around day 22. The transcriptome, lineage and variety of each terminally differentiated embryonic NK cell and whether, in adults they continue to exist is unknown and genetically undefined.

The prospect of innate immune memory is a developing interest that has been shown to link innate and adaptive cell sets by epigenetic responses. Natural Killer cells have been a particular focus of this research because they were once considered innate and non-adaptive. Recently there is an emerging body of evidence that suggests otherwise. Whether NK cell memory arises and which NK cells participate in its establishment is not established science. Without more research whether and to what extent very narrow NK cell protein expressions distinguish cell and tissue lineages will continue unanswered. However, some good research supports the important idea of NK memory and conserved lineage.

A study on the heterogeneity of NK cells, by transcriptome in human bone marrow identified distinct NK populations, including one expressing higher levels of immediate, early genes indicative of a homeostatic activation.  Analysis identified a transitional population between CD56bright and CD56dim NK cells. Most interesting they reported on a donor with GATA2-T354M mutation who exhibited a reduced percentage of CD56bright NK cells with altered transcriptome and elevated cell death indicating the smaller number of CD56bright NK cells were contributing to the donors disease progression.

In a mouse model of CMV infection, a specific population of NK cells expands, contracts after control of the virus and generates long-lived “memory” NK cells that are more protective during a second encounter. Other reports indicate antigen specificity and antibody dependent NK mediated cytotoxicity in autoimmune disease by a sub-population of NK cells expressing a combination of specific receptors that was associated with apoptosis and the depletion of IgG in individuals with autoimmune thyroid disease.

Coronavirus Lung Cells

In Covid19 admitted patients NK cells and lymphocytes were depleted including by apoptosis and exhausted. This late stage condition developed for some time prior to admission. The published research identified two receptors as particularly important for CoV2 viral entry into a cell. Ace2 to which CoV2 binds and TMPRSS2 that cleaves its Serine protein enabling entry. Only one report, so far provided useful information about expression of these genes in NK cells of lungs. From the 57,020 lung cells on the UMAP plot only NK cells expressing Ace2-TMPRSS2 connected Muscle, Fibroblasts, T-Cells and Macrophages.

A four year experiment tracked bar-coded NK cells introduced to rhesus macaque's. The results indicated that during homeostasis and moderate proliferative stress, peripheral, compartmentalized, self-renewal can maintain the composition of distinct, differentiated NK cell sub-populations.

Blunted categorizations of NK receptor expressions may be a sub-set limitation, but the good news is that more recent research is defining NK cells in increasingly diverse ways. We are also learning to identify diseases that infect NK cells including influenza, that induces apoptosis, HIV, Hepatitis c and Epstein Bar. A study found peptide specific recognition of human cytomegalovirus strains control expansion of adaptive NK cells. Another Covid19 study compared the transcriptome in lung tissues of older patients, including NK cells and concluded that genes induced by SARS-CoV-2 infection tend to increase in expression with aging, and vice versa.

Although still a little abstract for science, its plausible that a cell contracting a disease could recall its NK 'memory cell', its NK anti-cell counterpart to annihilate it because it differentiated beyond its NK anti-cell phenotype range. If true, an indiscriminate disease that also targeted the NK anti-cell and eliminated it would prevent annihilation of its phenotype differentiated counterpart cells. By example, SARS-CoV2 would also bind the Ace2-TMPRSS2 expressing NK anti-cell, infect and kill it then NK immune resistance would be compromised and cell differentiation may rapidly progress to become lethal.

Does SARS-CoV2 Strangle P53 to kill Natural Killer Immunity?

Codondex iScore

It's intriguing to speculate why p53 regulates the cardiac transcriptome and the ATIP gene, at least to transcribe ATIP1 a human isoform of the Angiotensin II AT2 Receptor Interacting Protein? The gene's different names describe its various functions including; Mitochondrial tumor suppressor 1 (MTSG1) or Microtubule-Associated Scaffold Protein 1 (MTUS1). Expression of MTUS1 was reported lost in various types of human malignancies such as colon, ovarian, head-and-neck, pancreas, breast cancers, bladder, gastric, and lung cancers.

AT2 is a receptor associated with Renin-Angiotensin System (RAS) and vasodilation that confers potential benefits in pathogenic conditions. It is not highly expressed on Natural Killer (NK) cells, but has been detected and was shown in a rat model of myocardial infarction to reduce injury and have dampening effects on inflammation. The AT2 receptor, in combination with ATIP1 is also reported as a tumor suppressor. New experimental evidence showed interaction defects between ATIP1 and two mutant forms of the AT2 receptor identified in cases of mental retardation. The studies point to a functional role of the AT2-ATIP1 axis in cognition.

p53 is a major transcription factor and the TP53 gene is the most mutated in cancer. It has been the extended subject of this blog and our research into relationships between p53 and Natural Killer cells. Our interest is p53 genetic signatures used to select specific diseased cells to co-culture and educate NK cells that can be applied as custom, target therapy against various diseases.

As reported in a previous blog entry, RAS is a cornerstone of blood pressure which has been directly implicated in Coronavirus patients whose NK cells have also been severely depleted. Transcriptomic analyses identified several immune pathways and pro-inflammatory cytokines induced by SARS-CoV-2 infection evidencing a sustained inflammation and cytokine storm. Pathway analysis revealed that patient’s lymphopenia may be caused by activation of apoptosis and p53 signaling pathway in lymphocytes.

If, as reported the RAS of NK cells contributes to the control of blood pressure via the localized ratio of AT1:AT2 expression. Then, in the capillaries of tissue micro-environments the delicate balance of vasoconstriction<>vasodilation signaling may be exploited by NK cells scanning the affected cells to deliver their innate inflammatory response. Cytokine directed vascular changes that effect local blood pressure may be another varied contributor.

So, with these time-bound, localized, micro-pressure effects in progress how does an innate NK cell go about killing an infected target that may have also motivated its AT1:AT2 response? AT2 was found to be an AT1 antagonist, which adds complexity to the function and ratio effects of this diverse protein. ATIP is an agonistic factor of AT2 to exert opposing effects to AT1, such as organ-protective and anti-inflammatory effects. We know ATIP's including ATIP1 binds AT2's cytoplasmic tail and impacts signaling that results from Angiotensin II (AngII) binding AT2's extracellular domain. We also know p53 transcribes at least ATIP1 and that ATIP3 is directly implicated in microtubule organization.

ATIP1 has been previously reported to localize either at the mitochondria or the Golgi. These apparently discordant results might be reconciled if ATIP1 were a microtubule-associated protein. Indeed, mitochondrial organelles are highly enriched on microtubules that ensure their intracellular transport. On the other hand, the Golgi apparatus is located at the nuclear periphery close to the microtubule organizing center (MTOC). ATIP1 might thus associate with microtubules and provide a direct link between the AT2 receptor at the cell membrane and the cytoskeleton.

The prospect that RAS related signaling and p53 are tied in both lymphocytes and broader cell biology is tantalizing. Among the cellular signaling pathways, p53 plays a prominent role in RAS. Activation of p53 increases Angiotensin and AT1 expression. Conversely, AngII activates p53 pathway to mediate its downstream cellular effects. Mechanically, stretch-mediated release of AngII induces myocyte apoptosis by activating p53 that enhances the local renin-angiotensin system and decreases the Bcl-2-to-Bax protein ratio in the cell. 

In Cardiometabolic Disease pathogen-associated, molecular pattern–mediated metabolic reprogramming can be considered a manifestation of innate immune signal reprogramming a conserved phenomenon that changes how we think about the biology and function of the innate immune network including indispensable acetylation that destabilizes p53 and its inhibitor MDM2.

In Coronavirus patients, SARS-CoV2 binding ACE2 receptors may modify processes associated with the ACE2/Ang-(1-7)/Mas axis and acute, chronic inflammation, including reported leukocyte influx. The mechanical stretch of vasoconstriction<>vasodilation could cause AngII to activate p53. Therefore, if SARS-CoV2 impedes the AngII-ACE2-AT2-ATIP1 pathway that under normal circumstances in NK cells may direct microtubules to form an immune synapse for target killing, then the rapidly multiplying virus binding ACE2 may simultaneously render NK ineffective and by mechanical-stretch affect cardiomyocytes by p53 activation, which would lower AT2 ratios, including in vascular epithelial cells to induce apoptosis or G2/M cell death. A potent cocktail indeed!

SARS-CoV2, Blood Pressure and Natural Killer Cells

The Cardiac Cycle

A beating heart is the first sign of life in the developing fetus. Cells of the heart's ventricular and aortic cavities express various proteins with non-active natriuretic and diuretic peptides in a pattern associated with blood pressure. Activated Corin or Furin proteins cleave these peptides into shorter active forms. The cleavage site often defines how these peptides influence cells and the cellular processes while circulating in blood. But, the heart is not the only place this mix of proteins and peptides are manufactured. From early to late pregnancy Corin level changes were greater in women who developed gestational hypertension, commensurate with diastolic and mean arterial blood pressure and it got doctors and scientists thinking?

NT-proBNP is a pro-hormone, a Corin or Furin dependent peptide from the same molecule as activated BNP. Both are released when pressure changes inside the heart. Circulating levels of NT-proBNP were higher in early and late-onset preeclampsia. BNP mRNA and protein were also detected in placentas from women with preeclampsia and controls. In normal pregnancies, BNP in plasma is stable, but in severe preeclampsia it is elevated. In 181 preeclampsia patients higher levels of Corin were expressed, also secreted from synctiotrophoblasts and extravillous trophoblasts of the placenta.

The secretion of NT-proBNP, and BNP mRNA and receptors were investigated in cultured primary trophoblasts. Low levels of NT-proBNP were found in the supernatants of term, but not first-trimester trophoblasts. In preeclampsia patients Corin mRNA and protein in uterine tissue were significantly lower, but plasma Corin higher, compared to normal pregnancies. These apparently conflicting reports may relate to local blood pressure.

A paper studying Atrial Natriuretic Peptide (ANP) identified that Corin and ANP promoted trophoblast invasion and spiral artery remodeling in the developing placenta. Pregnant Corin or ANP-deficient mice developed high blood pressure and proteinuria, characteristics of preeclampsia. Further, trophoblast invasion and uterine spiral artery remodeling were markedly impaired. Consistently ANP potently stimulated human trophoblasts invading in Matrigels. That Corin is up-regulated with stromal cell decidualization and strongly localized provides compelling evidence to support localized, but not circulating Corin activating ANP within these uterine cells. This may then invite trophoblast and Natural Killer (NK) cell invasion and the ultimate handshake of fetal trophoblasts with maternal endothelial cells lining spiral arteries of the developing placenta.

In early pregnancy NK cells expand to become the largest population of immune cells in decidua lining the uterus (uNK). They are closely associated with the development of blood vessels including spiral artery remodeling and possess a functional Renin- Angiotensin system, cornerstones of blood pressure. ANP antagonizes Angiotensin II receptor type 1 (AT1) leading to vasoconstriction. The ratio of uNK cells expressing AT1 markedly changed between gestation day 6 and 10. At day 10-12 ANP strongly co-localized to uNK cells at implantation sites, immediately after spiral arterial modification. Expression of vasoregulatory molecules by uNK cells suggests they contribute to the changes in blood pressure that occur between days 5 and 12 coincidental with their expansion during normal pregnancy in mice.

Studies of patients with Pulmonary Arterial Hypertension have also implicated BNP in the decline of NK cells and CD8+ T-Cells. A similar depletion was recently reported in Coronavirus  patients and expression of NT-proBNP was significantly elevated.  It is reasonable to infer that blood pressure and NK cells are associated especially in the tight confines of pulmonary capillaries or placental tissue.  Perhaps its their capacity to express molecules that participate in Corin activated vasoregulation through natriuretic peptides and to be immunoreactive, or perhaps their reaction to kill infected cells results from it - who knows?

In addition to the well documented natriuresis, diuresis and vasodilation, BNP may also modulate immune and inflammatory reaction to cardiac injury. BNP depletes monocytes, B lymphocytes and NK cells in peripheral blood. BNP regulates the chemotaxis of monocytes and production of inflammatory molecules by macrophages. BNP may promote cardiac neutrophil infiltration and also have direct effects on matrix remodeling and wound healing. All of these characteristics have been observed in lungs of critical care and deceased Coronavirus patients.

What does COVID-19 have to do with heart attack?

Ground Glass Opacity's in Lungs

Natural Killer (NK) cells are depleted, but neutrophils are elevated in the lungs of hospitalized Coronavirus patients the world over. This is the sign of immune system chaos that typically precedes disease progression. How COVID-19 generates this condition is unknown, but surviving NK cells express NKG2A inhibitory receptors and are exhausted.

Typically patient CT scans reveal "Ground Glass Opacity's", fuzzy areas in lung scans that identify affected tissues. Without recovery at this point, the disease advances, tissues of the lungs can become infected, pneumonia may set it and soon after the patient may die.

It was recently published by doctors at Northwestern and UCLA that in around 20% of COVID-19 cases Troponin enzyme was elevated and correlated with very high mortality rates. Troponin is almost exclusively expressed in heart attack patients, so what does it have to do with COVID-19?

A different study tracked NK cells in lungs and linked pulmonary inflammation with depleted NK cells and elevated neutrophils. It found pulmonary NK cells control neutrophil intravascular motility and response to acute inflammation. Intriguingly, in a model of experimental myocardial infarction, NK cell depletion resulted in increased neutrophilic pathology in the lungs of mice, raising the question of how this influence is mediated. The study failed to identify the function of Ly6G, which by June 2019 remained unclear to the scientists what role it might play in the transfer of information between NK cells and neutrophils. A different team recently published a joint report showing lymphocyte antigen 6 family member E (Ly6E) impaired Coronavirus fusion and conferred immune control of viral disease. The link to Ly6 in these different reports may be important to front-line teams working to identify treatments.

In 2013 a joint China-Japan team had already published; "Lung Natural Killer Cells Play a Major Counter-Regulatory Role in Pulmonary Vascular Hyperpermeability After Myocardial Infarction". The report documented the counter-intuitive action of NK cells in lungs of mice induced with heart attack. Similarities to the reported behavior of NK cells and neutrophils in late stage COVID-19 patients expressing Troponin is remarkable. The team rescued the respiratory phenotype in NK cell–depleted mice by the adoptive transfer of NK cells from wild-type mice, but not from IL-10 knockout mice. All this may explain why preliminary successes have been achieved treating patients with plasma transfers or from patients who had recovered from Corona or with Mesenchymal stem cells.

Ly6 is only present in mice, but human neutrophils express the structurally related Ly6G molecule CD177 (19q13.31), a member of the Ly6/uPAR (urokinase plasminogen activator receptor) family. Interestingly, antibodies against CD177 have been shown to inhibit neutrophil transmigration across an endothelial monolayer, potentially by interfering with an interaction between Ly6G and PECAM1.

One interesting approach, at the right dosage may be to deploy a broad anti-venom aimed at 3TFx toxins because of their close resemblance to COVID-19 and Ly6 protein structures especially at the S1-CTD contact point. If anti-venom performs anywhere close to COVID-19 binding or connector domains it may impede it's entry to cells.

HCoV binding ACE2

A scientific conflict is brewing over the use ACE receptor inhibitor Captoptril against COVID-19 binding ACE. The drug is an angiotensin-converting-enzyme inhibitor derived from a peptide discovered in the venom of the lancehead viper (Bothrops jararaca). This debate over ACE2 upregulation may be preventing its broader use in patients despite its potential to reduce the capacity of COVID-19 to bind cells.

Its well known that several toxins and venoms can also lead to heart muscle injury, which COVID-19 seems to be emulating. The combination of viral immune response and false toxin signalling, that raises Troponin levels seems sufficient to trigger the immune system chaos that precedes typical disease progression and self-induced (possibly autoimmune) infection.