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.

 

 

$100,000 Biohunt

Some of the past research on neoantigen and p53 antibodies in immunity has been encouraging. The data is enormously complex, but keeps pointing to TP53's great potential. To this end, we were anxious to start our mega-experiment, but were delayed by C19, now I'm glad to report we are well underway. In co-operation with researchers at UCLA we aim to determine whether Codondex transcript analysis, of TP53 can predict the best tumor tissue selection for most effective Natural Killer (NK) cell priming, activation and cell killing, including in autologous tumor micro environments.

We're hoping to to achieve a result along the path toward our ambitious clinical goal. We aim to prove that a specifically selected section from biopsied tissue can be used to effectively prime autologus NK cells for patient reapplication and disease treatment. 

This co-culture vs. sequencing challenge uses sections (T1-T8) taken from each of two tumors. Each section is co-cultured with 2 treated NK cell and one naive NK cell line and tests the efficacy of NK cell cytotoxicity against tumor cell and tumor tissue in killing assays. Separately, by sequencing TP53 of each selection and computing Codondex iScore(TM) algorithm we hope to identify specific features of each tissue selection that point computed results to research outcomes.

Co-culture vs. Sequencing Challenge

To better understand the analysis and encourage research contributions we are inviting applicants for first grants directed toward this objective. 

Codondex tools analyse genetic sequences at an arbitrary number of nucleotides. The tool provides an easy way to observe fine repetitive details of small subsequences contained within a gene. We compute various metrics for each subsequence including 'Inclusiveness', which measures the total occurrences of every computed smaller subsequence is found within the subsequence of interest. 

Our primary interest is intronic, non-coding DNA in multi-transcript genes. In these systems we create a transcript list, which we call the Vector, that is sorted by Codondex i-Score. This metric looks at Inclusiveness scaled by the length of the subsequence, to better account for intrinsic probability of finding smaller subsequences within progressively longer ones. Using this we look at the way order of this vector changes from subsequence to subsequence. Large changes in these vectors then prompts us to tag them for further investigation as it represents large deviation from transcript similarity, with this subsequence being labelled a Key Sequence. 

Codondex is proposing 3 grants for open problems to aid in our journey towards a more biologically useful platform. These 3 problems span statistical analysis, data acquisition and biological relevance of various aspects that are integral to our platform. 

Applicants should inquire further and sign up here.

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. 

The Cancer Effect On P53 and Natural Killer Cells

Naked Mole Rat Without NK Cells

New evidence elucidated the mechanisms of growth promoting and disrupting modes of Natural Killer (NK) cells especially in Tumor Micro Environments (TME). In a previous article on cancer exploiting modes of reproduction I explained that NK cells have an innate capacity to effect reproduction during the early stages of Blastocyst implantation and placental development. These include NK cells secreting growth promoting factors and near simultaneous disruption of epithelial cells lining spiral arteries, that penetrate the decidua to expose maternal blood to extravillous cytotrophoblasts of the developing fetal placenta.

Extravillous cytotrophoblast invasion involves a cellular transition from an epithelial to mesenchymal phenotype. Villous cytotrophoblasts undergo a partial epithelial to mesenchymal transition (EMT) when differentiating into extravillous cytotrophoblasts and gain the capacity to migrate and invade. 

An antagonistic relationship between transforming growth factor (TGF)-β and tumor necrosis factor (TNF)-α provides a glimpse into cancers influence over NK cells to permit and promote survival, extravasatation and metastasis. Multiple studies report synergism between TGF and TNF in EMT to cancer. Spatiotemporal regulation of DeltaNp63 by TGF regulated miRNAs is essential for cancer metastasis and the interplay between different p63 isoforms, related mutant p53 proteins, and p53 family members can largely determine overall anti-metastatic activity.

Mutant p53 that is co-expressed with WT p53 in human bronchial epithelial cells inhibits the response of  cancer cells to TGF. However, overexpression of mutant p53 on a p53-null background is able to attenuate the response to TGF. Mutant p53 is also implicated is chronic inflammation, a well-accepted hallmark of cancer. Numerous studies showed that the TME has a significant impact on perpetuation of the chronic inflammatory state. Mutant p53 is able to enhance the activation of NF-κB by TNFα, while concomitantly suppressing the pro-apoptotic effect of TNFα, which leads to increased invasiveness of the cancer cells. 

Blockade of TNF improved NK cell activation, hypertension, and mitochondrial oxidative stress in a preclinical rat model of preeclampsia. Rats with depleted NK cells had decreased serum and placental levels of TNF support that NK cells secrete TNF, indicating an autocrine effect. Additionally findings suggest that elevated decidual TGF suppresses the activation of specific subsets of decidual NK cells which in turn contributes to pathology associated with the onset of preeclampsia. A study looked at the cytokine profile of NK cells in early human pregnancy including TGF  and TNF and found two highly specific NK cells in peripheral blood and decidua were most likely responsible for maintenance of pregnancy by regulation of maternal immune function. 

The cancer resistant Naked Mole Rat possess no NK cells, but has a stable p53 half-life more than 10X  that found in human and mouse embryonic fibroblasts. The stability of p53 was determined to result from protein extrinsic factors yet response to DNA damage was classic, via p53 sequestration from nucleus to cytoplasm. How mole rats reproduce without decidual NK cells is an open question? p53 stability may have demoted a need for the mammalian equivalent of allorecognition at the site of Blastocyst implantation. The need for the maternal-fetal decidual NK cell mediation may have been replaced by more direct mechanisms. One option is via pheromone sensing. 

p53 and two related transcription factors p63 and p73 exhibit significant sequence similarity, can bind to the same consensus sites in DNA and activate the same target genes. However, unlike p53, with a paramount role in cancer suppression, p63 and p73 play more central developmental roles. p63 and p73 nullizygosity results in lethality, but p73-nullizygosity also causes pheromone-sensing defects that impede breeding.

Increasingly evidence points to the intricate interplay of p53, its related proteins and NK cells as primary targets for reproductive success and reducing death through cancer.

Oil and Water and Cellular Function

Genetic DNA are single acid nucleotide's stringed along a sugar-phosphate spine that winds around proteins, called histones and collapses into a chromosome assembly. At specific 'gene' locations DNA are often unwound and replicated into smaller, related RNA strings that can be incorporated by clustered proteins to attract and assemble amino acid combinations that may fold into functional proteins. Aqueous proteins aggregate in complex units and interact with DNA, RNA, amino acids and other proteins to build life on planet earth. 

Entropy can disrupt the order of liquid-liquid phase separation (LLP) and other density based separations that govern events effecting DNA and are central to cellular bio-physics. Since the discovery of DNA in 1869 and its double helix structure in 1953, research has been directed to decipher the vast string assemblies of billions of these ordered acid combinations that govern cells of different species. Recently research has more beautifully described how orders of short repeating DNA sequences govern cellular mechanics and provides insight to the delicate balance in aqueous separations.

Chromosomes of cells that divide and replicate are tethered via centromere including concentrated short, ordered DNA combinations repeated at extending distances along the sugar-phosphate spine. They attract proteins and other epigenetic factors that may direct the cells centrosome - a protein tube geared to a vast cytoskeleton spindle to move chromosomes and the cells skeletal structure in response to activity on its centromere and distant regions.

Intron regions of genes are considered regulatory since exons or DNA coding regions, when replicated into RNA exclusively translate combinations of amino acids for protein. The intron regions of yeast centromeres were found to promote formation of centromeric heterochromatim - DNA wound around histones and methylated to repress regions and maintain lineage during replication. 

A study of centromere heterochromatin surprisingly showed that distant euchromatic regions enriched in repressed methylated genes also interacted with the hierarchical organization of centromeric DNA. These 3D spacial interactions are likely mediated by LLP (similar to how oil and vinegar separate in salad dressing), resulting liquid-like fusion events and can influence the fitness of individuals. Repressed gene's were identified as Transposable Elements (TE's), sequences often associated with pathogenic DNA insertions that have been persistently retained.  

A study found 96.3% of TEs enriched in 156 gene bodies overlapped introns, in line with the normally observed distribution of introns and exons in the human genome. Across cells in different tissues, genes that are consistently replicated are less likely to be associated with TE's. Multiple TE's in tissue-specific, active regulatory regions are enriched in intron enhancer sequences to attract and bind protein transcription factors as master replication regulators.

TE's have mostly been analyzed by the frequency of short identical repeating sequences, but methods have not revealed the full extent of the TE repeat hierarchy. When any part of TE's are replicated and released from their sugar-phosphate spine the hierarchy of repeats may effect dissociation. Codondex built a uniform analytic to tease out the inherent hierarchy of repeating sequences that may expose separation potential whether or not the DNA is classified as a TE.  

As outlined, repressed DNA regions with more frequent repeats are less actively replicated into RNA. Therefore, actively transcribed regions yield more RNA for coding proteins and edited intron RNA can accumulate to concentrate in the liquid nucleus, be transported to the cytoplasm or be degraded. A cell's machinery must be finely tuned to process the RNA remnant of DNA replication, but mutations and aberrant separations can disrupt the order of these finely tuned micro-organisms. 

If repeats define a universal separation hierarchy that is heavily weighted toward regulatory introns then de novo chromosome and gene repeat analysis may identify distant and centromeric influences to the centrosome. The iScore(TM)  algorithm repeatedly explodes any DNA or RNA string into its ordered, theoretical hierarchy of repeats until the smallest required string length and may provide a structural basis for liquid separations. A repeat-hierarchy, for any gene would have to also relate to its chromosome repeats for inherent, universal influence over 3D spacial interaction and potentially cell function.

The complete record of repeats for an average length gene explodes to 100,000,000+ ordered strings representing its iScore signature. If a repeat hierarchy does exit for aqueous aggregations, a gene transcripts' intron iScore should be sufficient to measure and compare its inherent repeat potential to other transcripts. Significant consecutive iScore variations with any of the 100,000,000+ strings could be used to expose systemic, structural separation differences for that transcript in context of other transcripts in their aqueous environments. 

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.