HMGB1, microRNA, Inflammation, and Coagulation Charles Lewis, MD MPH

NOTICE: This is a largely theoretical discussion of agents that may be useful in the treatment of COVID-19,

sepsis and cytokine storm. It is being posted to suggest potential lines of inquiry for researchers interested

in conducting laboratory research or use in well-controlled, sanctioned clinical trials. It should not be

assumed to be correct. It should not be construed as medical advice.

In COVID-19, there is both inhibition of immune processes and hyperactivity of immune systems. In the

simplest terms, the adaptive immune system is down-regulated making the production of protective

antibodies inefficient, while parts of the innate immune system are quiescent and other parts go into hyper-

drive, causing tissue injury while not effectively stemming viral replication.

One part of innate immunity that is upregulated is illustrated below.

ZBP1 is an intracellular protein that is part of the innate immune system. It is activated by the presence of

viral RNA or DNA. Both ZBP1 and TRIF (not shown here) activate RIPK3. RIPK3 activates the “necrosome”

which activates numerous functions that result in the death of cells infected by a virus. As part of this

process, HMGB1 is released from the cell. HMGB1 act locally as a Damage-Associated Molecular Pattern

(DAMP) signal, promoting a higher level of inflammation. ZBP1 also activates Interferon Related Factors,

IRF3 and IRF7, which are nuclear transcription factors for the expression of interferon α and β (IFNα and

IFNβ). ZBP1 also activates JNK. SARS-CoV-2 is a single stranded RNA virus. Thus, presence of SARS-CoV-2

RNA in the cells activates ZBP1.

The SARS-CoV viral protein Papain-Like Protease (PLPro) inhibits the production of IFNα and IFNβ. IFNα

and IFNβ are essential to both innate and adaptive immunity. In adaptive immunity, they stimulate T-

helper cells which help coordinate B-cell production of antibodies, among other functions. These cytikines

also stimulate Natural Killer cell function.

Another SARS-CoV viral protein, ORF3a also activates the NRLP3 inflammasome via TRAF3,1 and ACS.

This stimulates the assembly of the NRLP3 inflammasome that promotes the pyroptosis form of

programmed cell death. NLRP3 is a cellular stress sensor. Activation of the NLRP3 inflammasome leads to

Casp1 activation and IL-1β and IL-18 release and active secretion of HMGB1. In this process HMGB1 is

released along with inflammatory cytokines. HMGB1 binds with TLR4 and TRL2, stimulating additional

inflammatory activity.

This virus may also causes inflammation by depleting ACE2, an anti-inflammatory protein. ACE2 is the

protein the SARS-CoV-2 binds to on the surface of the alveolar cells and certain other cells of the body.

Without an adequate anti-viral and innate immune signalling response from the target cells of SARS-CoV-2,

i.e.; the alveolar endothelial cells, there is delayed protective immune response and poorly control over virus

proliferation. There is likely little cytokine signaling to myelogenous immune cells, (NK cells, Monocytes, T

helper cells) until the infected cells start necroptosis or form inflammasomes that induce pyroptosis. Under

these cascades, there is the late release of IL-1, IL-33 and DAMPSs including HMGB1, ATP and

mitochondrial DNA.

Once DAMPS are released, they activate macrophages and monocytes. HMGB1 and IL-1 and other DAMPs

thus can act as delayed stimuli for immune response. HMGB1 is ligand for the pattern recognition receptors

(PRRs) TLR4 and TLR2 receptors that promote the inflammatory response in white blood cells. TLR4

activation by DAMPS activates NK-κB as well as other transcription factors for the inflammatory response

that produce TNFα and other cytokines.

DAMPs are largely released as a part of pyroptosis and necroptosis; it can be understood that these

cytokines signal the need for “a cleanup crew” to breakdown and clear up dead and injured cells after injury.

This mechanism did not evolve to act as a primary immune mechanism to get rid of a virus, but rather as

demolition after injury, prior to repair. When this process becomes the primary immune response, it

functions through tissue destruction. Thus, DAMPs act as cytokines for macrophages to move into the

alveoli, where they clear up dead cells, but also cause injury. Additionally, filling the alveoli with white blood

cells, the leaking of serous fluid, and the injury they provoke can cause acute respiratory distress syndrome

(ARDS). It is neutrophils and macrophages that have migrated to the lungs which are the major source of

cytokines fueling the inflammatory reaction.2

Over-production of TNFα or vigorous releases of HMGB1 fuel the acceleration of the inflammatory

response. It can become like a fire that will burn until it runs out of fuel. While TNFα and INF appear in

hours, and can remit as quickly, HMGB1 release from the cell nucleus into the cytoplasm and subsequent

release from the cell can take one to two days. After that, HMGB1 remains in the blood stream for several

days, and can affect most of the organs as well as white blood cells. In the lungs it causes neutrophil

infiltration, edema and alveolar injury.

In SARS and severe influenza, the development of a sepsis-like lung disease can develop. There is

recruitment of macrophages to the lung and destruction of the alveolar endothelium. In severe COVID-19

there are cytokine storm-like processes, however we now understand that the primary disease is vascular,

involving the endothelium, and the lung disease is at least usually a secondary effect.

There is emerging evidence that there are two different presentations of severe COVID-19; a wet and a dry

form.3 Recognizing these two presentations of severe COVID-19 not just important in the respiratory

management of the disease, but also because it suggests that the two different presentations may involve

different immune pathways.

In the dry form, which is more common and less severe, the amount of fluid in the lung is not greatly

elevated and the lungs have near normal compliance. The patient is able to breathe, but does not get

sufficient oxygen. It is thought that there may be either vasoconstriction of the pulmonary venules so that

the blood is not getting sufficiently oxygenated, and/or there may be micro-thrombi, blocking blood flow to

the pulmonary capillaries. Another additional theory is that the red blood cells are not carrying oxygen

normally. In the dry form of COVID-19, the lung maintains good compliance (elasticity) and the patient can

move air in and out of the lungs without excessive difficulty, but is not having good gas exchange in the

lungs. Intubation with the use of a ventilator with PEEP (positive end expiratory pressure) may not be

helpful and may be dangerous to the patient; however oxygen therapy is typically required. The dry form

appears to be the more common, or perhaps may just appear earlier in the disease.

In the wet form, the lung has a high level of fluid in the alveoli. This presentation is like the typical ARDS

(acute respiratory distress syndrome) with macrophage and neutrophil infiltration, edema and alveolar

injury. Here therapy with a ventilator and PEEP to expand the alveoli may be helpful.

In COVID and influenza, OxPL (oxidized phospholipids) are released from virally infected cells as a result of

oxidative stress.4 This response is protective, as it allows the mounting of the TLR4 (Toll-like receptor-4)

triggered immune defense.5 TLR4 is a cell surface protein that is activated by DAMPS and PAMPs

(pathogen-associated molecular patterns). Several Toll-like Receptors activate MyD88. MyD88 mediates

several downstream events culminating in the release of NF-κB, a transcription factor for TNFα, IL-1β, IL6,

and IL-12. MyD88 also promotes the transcription for INFα and INFβ.

As a secondary trigger, intracellular TLR3 (Toll-like receptor-3) response is activated by the presence of

single-stranded RNA (ssRNA) in the cell. TLR3 triggers TRIF, which is a MyD88-independent pathway that

also promotes INF-α and INF-β.

Viral ssRNA is sensed in the cell by RIG-I-like receptors (RLR), which causes ubiquitination of STING,

which promotes the phosphorylation and activation of TRAF3.6 TRAF3 promotes activation of TBK1/IKKε

which promotes NF-κB and activates IRF-3 dimerization, which that allows IRF-3 to enter the nucleus,

where it is a transcription factor for interferon that is essential to mounting an adequate antiviral defense.7 8

Both SARS-CoV and SARS-CoV-2 viruses code for a viral enzyme, Papain-like Protease (PLPro - EC

3.4.22.B50). This protein inhibits the ubiquitination of STING, and the phosphorylation of TRAF3. Thus,

the virus can defeat much of the TLR3 – TRIF – TRAF3 pathway for expression of interferon, and thereby

inhibit the activation of NK cells and T cells and their production of antibodies to the virus. PLPro also

interferes with the TLR7 signalling pathway.9 Viral protein inactivation of IFNα and IFNβ depress innate

immunity and limit T-cell activation towards the formation of antibodies by B cells.

Another SARS-CoV viral protein, ORF3a also known as Viroporin 3a, activates the NOD-like receptor P3

(NRLP3) inflammasome. Viporin acts as an ion channel allowing K+ efflux and release of ROS from the

mitochondria.10 The inflammasome activates Caspase 1, which cleaves pro-IL-1β into the active

proinflammatory cytokine IL-1β.11 This action may be further upregulated by HMGB1 TLR2 activation.12

ORF3a activates ACS and thus pyroptosis – thus can induce programmed cell death. Why would it be to

virus’s evolutionary advantage to kill its host cell on which it is dependent for reproduction?

It appears that SARS-CoV-2 can also infect lymphocytes. Although lymphocytes bear very low levels of

ACE2, the virus appear to be able to enter these cells via CD147 (aka basigin).13 CD147 is essential for T-cell

and NK cell development and differentiation and mediates the specific type of immune target these cells

recognize.14 Thus, the coronavirus may cause lymphopenia by inducing pyroptosis in lymphocytes or

common lymphoid precursor cells. Viral ORF3a may thus help eliminate Natural Killer cells and T cells

essential for anti-viral defense.

Red blood cells bear very high levels of CD147, however, these cells lack the cellular machinery to reproduce

viruses.

(Adapted from KEGG Influenza pathway, modified to reflect COVID and viral proteins PLPro and ORF3a)

In a study of mice infected with SARS virus, mice with MyD88 knockout had a mortality rate over 90%

within 6 days, while all the wild-type test mice survived the infection.15 Clearly the MyD88 pathway is

critical to survival of this infection.

In a subsequent study, mice with TLR4, TLR3, or TRAM knockout mice, were infected with the SARS virus.

They were found to lose more weight as a result of the infection than wild-type mice, but there were no

deaths. When TRIF (toll-like receptor adapter molecule 1) knock-out mice were infected with the SARS virus

they had reduced lung function, more lung injury, higher viral loads and higher mortality than wild-type

mice. Thus the TRIF pathway is an important for survival from SARS COVID infections.16

If both TRIF and MyD88 pathways are impaired, there is little way to fight these infections. SARS-CoV viral

papain-like protease (PLPro) is a protein that impairs the adaptive immune system, by inhibiting the

formation of interferon alpha and beta. If the virus can defeat the TRIF pathway at the level of TRAF3 via its

enzyme PLPro,17 that leaves the NFκB pathways. As a result of an incompetent response to the virus and

inability to adequately mount T cell activation and antibody response, there is then a cycling upregulation of

IL-1, IL-6, TNFα, and other cytokines. The recruitment of monocytes and macrophages causes tissue injury

and activation of the NF-κB activates JAK/STAT1 and activation of the inflammasome, which cause the

release of HMGB1. HMGB1 activates and reinforces TLR4, creating a positive feedback loop of cytokine

release, inflammations, macrophage recruitment, and injury but without activation of NK and T-cells.

It can thus be understood that inhibiting the immune response early in the TLR3 and TLR4 pathways in

viral target cells, wherein activation of MyD88 or TRIF are inhibited, it impedes the ability to signal for the

development of an appropriate immune response. Production of interferon α and β, the activation of NK

cells, and development of T1 helper cells and of antiviral antibodies are essential to mount a defense and

recovery from SARS, influenza and COVID.

TLR4 also mediates the MAPK signalling pathway and AP-1 which promote the chemotaxis and survival of

white blood cells. AP-1 signally may worsen tissue damage in the lungs and endothelial cells. While some

aspects of the pathway are essential to forming immunity, INF-β production from non-infected cells

stimulated by DAMPs can stimulate the JAK/STAT1 signalling pathway, which further promotes HMGB1

migration from the nucleus to the cytoplasm. Cell death also releases HMGB1, which like OxPL, activates

TLR4. Thus HMGB1 can create a positive feedback that ramps up tissue injury. Thus, incomplete TLR4

response in infected cells mutes the immune response and allows viral proliferation; excessive immune

response promotes severe life-threatening damage by monocytes and macrophages in response to PAMPS

such as HMGB1 later in the disease process.18 HMGB1 is a potent chemokine and activator of immune

mediated cell destruction that plays an important role in the lung damage occurring in SARS, influenza and

presumably COVID-19.

Medication:

The beta blocker labetalol has been identified as potentially having SARS-CoV viral papain-like protease

(PLPro) enzyme inhibitory effects. 19 Thus, it may help prevent SARS-CoV-2 immune evasion. Additionally,

labetalol lowers blood pressure and should protect the heart from arrhythmias caused by long QTc,20 (both

common issues in patients with severe COVID-19) and thus may reduce risk of sudden cardiac death

associated with this disease.

I suggest a trial of labetalol in appropriate COVID-19 patients. I suggest a starting treatment dose at about

150 mg/ day in divided doses for a 70 kg adult.

Cytokines in COVID-19

In a Chinese study comparing severe and critical ICU COVID-19 patients to those with less serious illness,

those with severed disease had higher blood levels of interleukin-2 (IL-2), IL-7, granulocyte-colony

stimulating factor (GSCF), interferon-γ inducible protein 10 (IP10), monocyte chemoattractant protein 1

(MCP1) (aka; CCL2)), macrophage inflammatory protein 1-α (MIP1α), and tumor necrosis factor-α

(TNFα).21

In a separate study of bronchoalveolar lavage fluid and monocytes of COVID-19 patients, CCL2 (MCP-1),

CXCL10 (IP-10) CCL3 (MIP-1A) and CCL4 (MIP1B) were highly expressed.22

In SARS-COV, the spike protein triggers ACE2 signalling, which activates the Ras →ERK→AP-1 pathway.

TNFα transcription is induced by AP-1 as well as by other inflammatory transcription factors.

TNFα strongly induces numerous inflammatory molecules. In breast cancer cells, exposure to TNFα was

found to increase the transcription of CCL2 by over 60 times, CXCL10 by 29 times, and complement C3 by

six times.23 TNFα strongly induces the expression of fibrosis-associated CCL2. CCL2 is a chemokine for

monocytes and macrophages. Additionally, CCL2 limits the survival of antibody forming cells in the lymph

nodes.24 CCL2 was associated with severe lung injury and fibrosis in SARS.25

Treatment:

Apigenin, a phenolic compound present in certain edible plants, inhibits the canonical and non-canonical

NLRP3 inflammasome pathways by decreasing caspase-1 and caspase-11 enzyme expression and activity.

Apigenin strongly down-regulates CCL2.26 In tumor cells, a non-toxic dose of apigenin was found to reduce

TNFα induced transcription of IKBKε by 108%, MLKL (a mediator of the necrosome/inflammasome

pathway) by 106%, CCL2 by 97%, complement C3 by 91%, IL-6 by 88%, CXCL10 by 83%, TLR2 by 79%,

PAI-1 by 77%, the IL7 receptor by 76%, and Complement Factor B (CFB) by 68%.27 Apigenin upregulates

miR-155.28 miR-155 inhibits TNFα and the NF-κB signaling pathway in cytokine storm in animal models

and human cell macrophages.29 The down regulation of C3 and CFB by apigenin are of significant interest in

this disease, as complement activation is associated with worse outcome in mice with SARS, as discussed

below.

Cathespin S (CAT S) expression was found to be increased six times by TNFα in cancer cells in the study

above, and was down regulated by apigenin by 66%.27 CAT S is a lysosomal cysteine protease. This enzyme

has an important role in antigen presentation; it cleaves proteins into little chucks so that they can be

presented on MHC II (major histocompatibility complex II) molecules for display on the cell surface. This

allows immune cells to tell if the cell is adorned with “self” proteins or foreign proteins – in which case the

T-cells will attack the cell and promote the development of antibodies to the foreign antigen. If there is

insufficient CAT S, then large pieces of the proteins are displayed on the MHC II; this decreases immune

recognition of foreign proteins and immune response to them. If there is over-abundant CAT S, it can cause

the proteins to be cut into small fragments and be presented by the MHC II; this can trigger autoimmunity.

Thus, while up-regulation of TNFα assists in mounting an immune response, excessive levels can promote

autoimmune injury.30 Perhaps during cytokine storm, non-infected cells, over stimulated by TNFα and over-

producing CAT S, may become targets of attack by the immune system. Furthermore, CAT S from alveolar

macrophages, a cysteine protease, can also act as elastase. Thus it may promote alveolar injury similar to

that caused by neutrophil elastase, which causes emphysema. This may be especially damaging during

mechanical ventilation.31

I recommend the use of apigenin for the prevention and treatment of COVID-19, cytokine storm, sepsis, and

viral pneumonia, and herein, specifically for the prevention and treatment of severe COVID-19. The dose

estimate for apigenin for a severely ill COVID-19 patient is about 250 to 400 mg per day, while 100 mg per

day in divided doses maybe helpful for persons at high risk of severe COVID-19 associated with chronic

endothelial disease (HTN, type 2 diabetes, CAD, etc.). Preferably apigenin should be used in combination

with other agents, such as pomegranate juice, as discussed below.

Coagulation/Complement Cascade

In another mouse study of SARS-CoV, mice with complement C3 knock-out mice (C3-/-) were infected with

the SARS virus. Wild-type (C3+/+) mice when infected with the virus generally have viral infection in the

lungs, lose weight and have an increase in inflammatory cytokines, but do not die. With SARS infection C3-/-

mice has less weight loss, reduced lung pathology and lower cytokine levels in both the lung and blood. The

viral load in the lungs of the C3-/- mice was no different than in the C3+/+ mice. This suggests that the

complement cascade while exacerbating the pathology of SARS provides little if any benefit in clearing the

disease. Thus therapy that inhibits the complement cascade may be helpful in treating the disease.32

There are multiple pathways for activation of the complement system; some of the pathways are closely

aligned with coagulation. Microbes can activate C3, but it is also activated by plasmin and thrombin. When

C3 becomes activated, it activates C5. Thrombin may activate C5 directly.

Complement factor B mediates the alternative pathway activation of both C3 and C5. The alternative

pathway is activated by infection. Apigenin down-regulates TNFα induced upregulation of C3 and CFB.

In severe SARS-CoV infections there is diffuse alveolar damage, vascular leakage into the alveolar spaces,

premature breakdown of fibrin clots and possible micro-hemorrhage in the lungs. Similar pathology is seen

with severe strains of influenza, including the 1918 and 2009 H1N1 influenza viruses. In the 1918 H1N1

pandemic, many young patients died a hemorrhagic death. With the outbreak of SARS in 2002, we were

fortunate that the outbreak was small, and there were a limited number of cases. With COVID-19, were have

found, that while presenting as a pulmonary disease with hypoxia, it likely primarily affects the endothelial

cells of the vasculature, and microvasculature, causing vasoconstriction and micro emboli to the lungs.

COVID-19 patients with elevated levels of D-dimer, a fibrin degradation product, have a higher mortality.33

D-dimer is an indicator that there has been clot formation and breakdown. Heparin and warfarin inhibit

coagulation Factor Xa, an enzyme that promotes cleavage of prothrombin to thrombin, which activates C3.

Heparin therapy in COVID-19 patients appears to lower mortality.

A series of autopsy of four COVID-19 cases was performed at Tulane University Medical Center. All the

decedents had had elevated ferritin, fibrinogen, PT (prothrombin time), and very elevated D-dimer,

indicating disseminated intravascular coagulation. Nevertheless, on autopsy, thrombotic microangiopathy

was restricted to the lungs. The lungs were heavy (wet) lungs and the decedents had dilated right heart

ventricle, suggestive of right heart failure following pulmonary hypertension and pulmonary edema. There

was an abundance of megakaryocytes in the lungs, as had been found with SARS infections. These cells

produce platelets, and likely play a role in micro-thrombosis and blockage of the pulmonary capillary bed. 34

In early disease, such pulmonary capillary blockage would cause the shunting of blood through un-aerated

parts of the lung, causing hypoxia. This would be exacerbated by vasoconstriction. As the disease

progressed, the elevated capillary pressure would cause leakage of fluid into the intestinal fluid and into the

alveoli causing pulmonary edema and worsening hypoxia. Eventually, the right ventricle of the heart may

become unable to pump sufficient blood through the lungs and suffer exhaustion and fail.

There is renal injury in severe COVID-19. More than half of hospitalized COVID-19 patients with severe not

prior history of kidney disease were found to have proteinuria, hematuria and leukocyturia.35 Many COVID-

19 survivors require dialysis.

The coagulation/complement cascades is likely important in the causation of the “dry” lung manifestation of COVID-19. Virally induced endothelial injury in blood vessels causes oxidative injury, los of nitric oxide production and thus vasoconstriction, platelet activation, activation of prothrombin to thrombin as a result of activation of Factor X to Factor Xa. This causes the formation of microemboli that are filtered in the capillaries of the lung. This would explain the hypoxia with normal lung compliance seen in the dry manifestation of the disease. Heparin inhibits the enzymatic activity of Factor Xa.

As illustrated above, the coagulation cascade promotes activation of the complement system via both

thrombin (pro-fibrin production) and plasmin (pro-fibrinolysis). The complement system is part of the

innate immune system. It promotes chemotaxis, drawing more white blood cells to the area and promotes

degranulation and phagocytosis. C5 activation (by C3) promotes assembly of the Membrane Attack

Complex, which targets and kills virally infected cells; in COVID-19, the alveolar cells are the target.

Activation of the complement cascade occurs as early as the first day following inoculation of animals with

the SARS-CoV virus. Complement activation is known to increased vascular permeability, a feature of severe

SARS-CoV infection that is associated with poor outcome. Baseline complement activity increases with age,

and is consistent with the increase mortality with age in SARS. Furthermore, complement activation is

predictive of the development of ARDS. Complement activation increases inflammation and promotes lysis

of cells, causing the release of damage associated molecular patterns, (DAMPs) such as HMGB1. DAMPS

can then further activate the inflammatory cascade and the complement system.36

IL-1β and TNFα → uPA → Plasmin → C3 → C3a, C3b and C5 activation

DAMPs → C1, C4 → C3 → C3a, C3b and C5 activation

Viral endothelial injury → X → Xa → Thombin → C3

Tissue plasminogen activator (tPA) (EC 3.4.21.68) also activates plasminogen as does uPA. Kallikrein also

promotes plasminogen.37 Later in the disease course of severe SARS, there is fibrin degradation and

bleeding into the alveoli. This may be followed by lung fibrosis in survivors.

Medications:

Anti-coagulation therapy likely needs to be part of the treatment of regimen of hospitalized patients with

COVID-19. Acetylsalicylic acid, P2Y12 inhibitors, and glycoprotein IIb/IIIa antagonists, may reduce lung

injury and mortality in COVID without increased bleeding risk.38 Nevertheless, many studies have failed to

find a survival benefit using anticoagulant therapy in sepsis.39

As shown in the figure above, heparin (LMWH) is a logical choice in the treatment of severe COVID-19 for

the prevention of coagulopathy and complement activation.

Statin drugs also impact the PAI-1 and tPA. Statins induce tPA and inhibits plasminogen activator inhibitor-

1. Thus, statins have an anti-fibrosis effect on wound healing by promoting the degradation of fibrin

products.40 41 Atorvastatin has been found to increase the expression of ACE2 in the heart of animals.42 43

This may or may not be a class effect. Statins also downregulate ICAM-1, a protein whose expression is

required for Killer T-cells to adhere and eliminate viral-infected cells.44 I suggest that simvastatin and other

statin medications be avoided during the course of COVID. If they are used, I suggest they be used at very

low dose, i.e.; 5 mg of simvastatin per day.

Urokinase plasminogen activator (uPA) has been found to play a central activating role in causing fibrosis

and lung injury in SARS.45 uPA activates the conversion of plasminogen to plasmin which also activates C3

via activation of plasminogen to plasmin. uPA also activates uPAR (plasminogen activator urokinase

receptor) which induces cell adhesion, migration, and proliferation.46 The inflammatory cytokines IL-1β 47

and TNF-α 48 induce the activation/expression of uPA.

A literature search for readily available medications that inhibit uPA revealed amiloride as a potential

candidate. 49 This is an anti-hypertensive drug, and this seems to be a unique feature of this medication. This

medicine may also inhibit tissue kallikrein,50 an enzyme that upregulates plasminogen.51 This is not the

drug’s mechanism of action as an anti-hypertensive medication, but rather a side effect that may be useful in

treating ARDS in SARS and severe influenza. It may reduce injury and help prevent late effects of the

disease.

In COVID-19, the development of hypertension is part of the disease pathology in severe disease. Thus

treatment is often needed for hypertension and congestive heart failure in severely and critically ill COVID

patients. Amiloride is a potassium-sparing diuretic. Hypokalemia has been found in 93 percent of patients

with severe or critical COVID-19, as a result of urinary loss of potassium resulting from degradation of

ACE2.52 Amiloride may decrease potassium loss; however, potassium levels will still need to be monitored. I

suggest a trial using low-dose amiloride (2.5 mg/day) for its anti-uPA effect. Higher doses may have adverse

effects, especially if the patient is not anticoagulated.

HMGB1

HMGB1 is a moderator of inflammation and survival of phagocytic white blood cells, and causes prolonged,

vigorous tissue destruction in influenza and SARS, and likely COVID-19. HMGB1 is a key pathway in sepsis

and septic shock and mediates organ damage. HMGB1 is a nuclear protein that leaks into the cytoplasm in

inflammation and the out of the cell in cell injury of cell death. In cancer, HMGB1 promotes cancer cell

proliferation, migration and tissue invasion. It is a DAMP (damage-associated molecular pattern) that

activates TLR4 (and TLR2) and induces inflammatory cytokines in cytokine storm. DAMPs create an

inflammatory cascade (HMGB1/TLR4/ MyD88/NF-κB) that can result in tissue destruction.

Hypoxia/reperfusion, oxygen glucose deprivation, oxidative stress, and other stressors can cause the

acetylation of the nuclear HMGB1 promoting its translocation into the cytosol and then into the

extracellular space. Pathogen-associated molecular pattern (PAMP) molecules, such as LPS and Ox-PL,

which is released in viral infections such as influenza A and SARS, can activate the TLR4 pathway. The

TLR4 pathway can activate the JAK/STAT1 pathway that also promotes the acetylation and translocation of

HMGB1 from the cell’s nucleus to the cytoplasm.

Pyroptosis, a form of induced programmed cell death that is part of the innate immune response to protect

against intracellular microbial infection. In pyroptosis there is inhibition of internal pathogen reproduction

and promotion of phagocytosis. Pyroptosis involves TLRs, Casp1 activation the inflammasome and NOD-

like receptors. Extracellular ATP, oxidized mitochondrial DNA, mitochondrial ROS, and leakage of

cardiolipin from the mitochondria together promote the activation of the NRLP3 inflammasome, which

activates pro-caspase-1 to Casp-1. Casp-1 activates pro-IL1β and pro-IL-18 to IL1β and IL-18. TLR4 can also

induce IL-1β processing via Casp-8. Activation of the TNF receptor promotes pro-IL-1β and NLRP3

inflammasome transcription via nuclear factor-κB (NF-κB) activation.

MicroRNAs (miR) are non-coding small hairpin-shaped sections of RNA that bind to specific mRNAs, and

prevent the mRNA from moving through the ribosome. They thus inhibit the production of the protein for

that the mRNA translates. This is an important way the cell stops the production of various proteins when

there is a sufficient supply or when there is no further need for that protein. Many medications act by

inhibiting enzymes or inhibiting or activation cell receptors. Other proteins are considered non-drugable.

Promoting or inhibiting the production of miR may allow pharmacologic control of some traditionally “non-

drugable” proteins and pathways. Thus, I explored miRNA induction as a potential treatment of virally

induced cytokine storm.

Several miR have been identified that, some experimentally in cell culture and others by chemical modeling,

that down-regulate HMGB1 protein translation. Additionally, several compounds have been identified that

either up or down-regulate the production of miR that reduce HMGB1 activity. HMGB1 promotes an

increase in the expression of miR-21 and miR-129-2, and some of its effects are mediated by these

microRNA. It may be possible to intervene by inhibiting these. Certain miR have also been identified to be

in higher concentration in patients with cytokine storm or sepsis, and these may provide clues to treatment,

however, with this type of data, it is essential to understand whether the rise is part of the cause of injury,

part of the response to injury.

Human pulmonary/cardiac microvascular endothelial cells (HMVECs) are available that can be grown in

cell culture. Cultured HMVECs grown in the presence of thrombin have increased permeability. Over-

expression of miR-126 decreased endothelial space, thus preventing he increase in permeability.53 In mice,

over-expression of miR-126 decreased serum levels of IL-6 and TNF-α in a model of sepsis, and lowered the

mortality rate. In humans, miR-126 has been found to be down-regulated in patients with hospitalized with

sepsis, and the degree of down-regulation correlated to the severity of sepsis early in the disease

progression.54 In a separate study, miR-126 was found to protect HCMECs against hypoxia/reoxygenation

injury and to decrease inflammatory Reactive oxygen species ROS, and reduce the expression of IL6, IL10

and TNFα, while increasing the vasodilator NO, and the antioxidant SOD.55

The table below summarizes the effect of several microRNA on HMGB1 and cytokine storm relevant to

COVID-19, and list some agents that impact the expression of these miR. Those estimated to be most

important in COVID-19 and are highlighted in bold. Note that that the medications metformin and the PPI,

esomeprazole as well as carnosic acid inhibit the expression of miR that reduce HMGB1 translation and thus may worsen COVID-19 disease. All PPIs may have this effect. Olive oil and vitamin D deficiency may

increase HMGB1 activity, and thus perhaps should also be avoided.

Pomegranate Juice: In a gene assay, pomegranate juice up-regulated genes involved in cell adhesion

such as E-cadherin, intercellular adhesion molecule 1 (ICAM-1) and down-regulates genes involved in cell

migration such as hyaluranan-mediated motility receptor (HMMR) and type I collagen. It up-regulated

microRNAs including miR-335, miR-205, miR-200, and miR-126, and down-regulates miR-21 and miR-

373. Pomegranate juice reduces the level of pro-inflammatory cytokines/chemokines such as IL-6, IL-

12p40, IL-1β and RANTES. 56 In an in vivo study, rats receiving pomegranate juice had significantly down-

regulated proinflammatory enzymes nitric oxide synthase and cyclooxygenase-2 messenger RNA (mRNA)

and protein expression. NF-κB and VCAM-1 mRNA and proteins expression were suppressed. There was

also inhibition of phosphorylation of PI3K/AKT and mTOR expression and increased the expression of miR-

126.57 Pomegranate juice also is expected to down-regulates HMGB1 via miR-200a and likely by miR-20558

and also reduces HMGB1 downstream activity as a result of reduced expression of miR-21 and let-7c. 59

I recommend the use of pomegranate juice (but not other pomegranate extracts) for the prevention and

treatment of cytokine storm, sepsis, and viral pneumonia, and herein, specifically for the prevention and

treatment of severe COVID19. I suggest trials beginning with 4 to 8 ounces of pomegranate juice or about 4

to 10 grams of freeze dried pomegranate juice powder per day; higher end doses may be required for more

severe disease and larger patients.

miRNA and HMGB1 60 61 62 63 64 65 66 67 68 69

miRNA MicroRNA Effect Upregulates (Desirable)

Downregulates (Undesirable)

miR-126 Decreases serum IL-6 and TNF-α in sepsis, decreased intracellular permeability in cell culture. Protects from hypoxia/re-oxygenation/ reperfusion injury

Pomegranate juice70 Mango polyphenols71 72

miR-129-5p Suppresses HMGB1, RAGE Metformin Downregulated by 40%73

miR-193a-3p74 Targets HMGB1, TGF-β, and HYOU1 Resveratrol

Metformin lowers by 55%75

mir-200a Lowers HMGB1 expression Pomegranate Juice

miR-34a Suppresses HMGB1 (Downregulated in septic shock)

Honokiol, resveratrol and n3-PUFA Pomegranate rind76 EGCG

Carnosic acid77 (Present in rosemary and sage) Quercetin Curcumin

miR-376a Lowers HMGB1 expression Esomeprazole, (proton pump inhibitors)78

miR-15579 Inhibits the NF-κB signaling pathway and TNFα.

Apigenin80 Resveratrol81

Quercitin82 Pomegranate

extract

miR-320a, miR-325 and miR-505

Lower HMGB1 expression

miRNA MicroRNA Effect Upregulates (Undesirable)

Downregulates (Desirable)

miR-21 83 HMGB1 promotes miR-21 and NF-κB pathway activity

Oleic acid (olive oil) DIM, Choline, and Folic acid up-regulate. Curcumin Vit. D3 deficiency n-3 fatty acid deficiency

Resveratrol Avoid vitamin D3 and n-3 fatty acid deficiencies

miR-129-2 HMGB1 increases

let-7c HMGB1 increases EGCG Quercetin curcumin

Pomegranate Juice, Resveratrol

Note: Not intended to be a comprehensive listing of miRNA related activity, but rather list those

encountered that are pertinent to HMGB1 and cytokine storm in viral pneumonia and COVID-19.

Summary

Medications that may be helpful for COVID-19 and similar cytokine storm, DIC, sepsis conditions, dose

estimate per day for a 70 kg adult:

1. Low molecular weight heparin (LMWH) All treatment patients should be heparinized and monitored

with aPTT as per usual hospital protocol. This may already be part of the hospital’s usual care for

COVID patients. I recommend that all patients (test and control patients) without contraindication

be placed on LMWH.

2. Labetalol 150 mg (75 mg/BID)

3. Amiloride 2.5 mg QD

4. Vitamin D3 500 IU QD

Natural Agents that may be helpful in the prevention or treatment of severe COVID with daily dose. In

addition to those agents already discussed, I also recommend ginger juice as an antiviral agent for use in

COVID infection.

Divided into 4 or five doses per day:

1. Pomegranate Juice: 3 ml/kg day of juice or 100 mg of powder per Kg Juice Source Powder Source

2. Apigenin 250 mg Source

Notes:

Freeze dried pomegranate juice may be used as a substituted for pomegranate juice, using about 1.5 grams

per ounce of juice.

A small dose of vitamin D3 is recommended daily as 25 hydroxyvitamin D3 is required for new WBC

function as new cells are created daily, but the body stores vitamin D as 1-25 hydroxyvitamin D3. Studies of

vitamin D3 use in influenza support the use of low doses, but not higher doses.

I am unaware of any other contraindication to using these medications together or together with the natural

agents listed above.

I also recommend avoidance of statin medications, proton pump inhibitors, and metformin during the

course of this disease, as well as avoidance of olive oil, sage, rosemary and curcumin/turmeric as a result of

their miRNA activity on HMGB1. Since oleic acid may remain in the body for an extended time, it may be

prudent to avoid it if there is high risk of COVID infection morbidity.

These agents are not intended to displace of other anti-SARS-CoV-2 medications. Antiviral agents such as

Camostat mesilate84 or remdesivir may be helpful, especially early in the disease. The focus here in is to

improve the immune response to the disease.

https://www.amazon.com/POM-Wonderful-Pomegranate-Juice-Count/dp/B004O4BB4Whttps://www.amazon.com/Navitas-Organics-Pomegranate-Powder-oz/dp/B001TNW23Uhttps://www.amazon.com/Swanson-Apigenin-Prostate-Supplements-Capsules/dp/B001TEIJIQ/

Additional notes:

TMPRSS2, the host cell protein on cells that that mediates uptake of the SARS-CoV-2 virus after binding to

ACD2, is inhibited by plasminogen activator inhibitor-1 (PAI-1).85 PAI-1 in inhibited by statin drugs.

TMPRSS2 is even more strongly inhibited by antithrombin, which is activated by heparin86 in physiologic

levels of calcium. PAI-1 expression greatly augmented by TNF-α, and this greatly down-regulated by

apigenin.

Apigenin is a Src-tyrosine kinase inhibitor that inhibits activation of SHP1 and SHP2, thus inhibiting the

IDO mediated downregulation of Th1 and NK cell immune activity. 87

Apigenin reduces NLRP3 inflammasome activation.88

Please let me know if tested in patients. Thanks dr.charles.lewisgmail.com

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