key: cord-1022193-f65cwcji
authors: Hasan, Djo; Shono, Atsuko; van Kalken, Coenraad K.; van der Spek, Peter J.; Krenning, Eric P.; Kotani, Toru
title: A novel definition and treatment of hyperinflammation in COVID-19 based on purinergic signalling
date: 2021-11-10
journal: Purinergic Signal
DOI: 10.1007/s11302-021-09814-6
sha: 2ade90896f12455bcc5d282292e5dfda29a9755c
doc_id: 1022193
cord_uid: f65cwcji

Hyperinflammation plays an important role in severe and critical COVID-19. Using inconsistent criteria, many researchers define hyperinflammation as a form of very severe inflammation with cytokine storm. Therefore, COVID-19 patients are treated with anti-inflammatory drugs. These drugs appear to be less efficacious than expected and are sometimes accompanied by serious adverse effects. SARS-CoV-2 promotes cellular ATP release. Increased levels of extracellular ATP activate the purinergic receptors of the immune cells initiating the physiologic pro-inflammatory immune response. Persisting viral infection drives the ATP release even further leading to the activation of the P2X7 purinergic receptors (P2X7Rs) and a severe yet physiologic inflammation. Disease progression promotes prolonged vigorous activation of the P2X7R causing cell death and uncontrolled ATP release leading to cytokine storm and desensitisation of all other purinergic receptors of the immune cells. This results in immune paralysis with co-infections or secondary infections. We refer to this pathologic condition as hyperinflammation. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function. The issue is that the half-maximal effective concentration for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. To overcome this, we selectively inhibit the P2X7Rs of the immune cells of the lymphatic system inducing clonal expansion of Tregs in local lymph nodes. Subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities suppressing systemic and (distant) local hyperinflammation. We illustrate this with six critically ill COVID-19 patients treated with lidocaine.

Hyperinflammation and acute respiratory distress syndrome (ARDS) caused by coronavirus disease 2019 (COVID- 19) have become the world's number 1 challenge. The exponential pattern in the number of severe cases in the second and third waves of the SARS-CoV-2 pandemic has shown to reach nations' maximum ICU capacities in weeks rather than months after outbreak of the disease irrespective of rigorous population-based preventive measures. In a recently published systematic review, the case fatality rates in patients in the ICU across 7 countries vary between 14.9 and 66.7%, while the case fatality rates among those who required mechanical ventilation vary between 16.7 and 97.0% [1] . In addition, the case fatality rate in a cohort of 1035 critically ill COVID-19 patients requiring extracorporeal membrane oxygenation (ECMO, artificial lungs) is alarmingly high (37.4%) [2] .

The clinical manifestations of severe COVID-19 consist of pneumonia with dyspnoea and hyperinflammation. Hyperinflammation is thought to be the basis of the development of severe and critical COVID-19 [3] [4] [5] . Currently, a clear-cut definition of hyperinflammation is lacking. Some authors describe the condition of hyperinflammation as a form of very severe inflammation with cytokine storm [6] . The criteria of hyperinflammation are not consistent and include clinical data and/or different combinations of the parameters of the activation of the pro-inflammatory response of the immune system (i.e. fever, rapid respiratory deterioration, cytokine, ferritin and/or CRP concentrations, changes in blood levels of several types of immune cells, etc., examples are presented in Table 1 ) [3, 4, 6-8, 9, 10-14] . In addition, the current definitions of hyperinflammatory syndrome do not provide an Table 1 Examples of the criteria of hyperinflammation. These criteria are not consistent and include different combinations of symptoms and laboratory parameters of the activation of the pro-inflammatory response of the immune system Author

Year of publication Macrophage activation (ferritin concentration of 700 μg/l or more) Haematological dysfunction (neutrophil to lymphocyte ratio of 10 or more or both haemoglobin concentration of 9.2 g/dl or less and platelet count of 110 × 10 9 cells/L or less) Haematological dysfunction (neutrophil to lymphocyte ratio of 10 or more or both haemoglobin concentration of 9.2 g/dl or less and platelet count of 110 × 10 9 cells/L or less) Coagulopathy (D-dimer concentration of 1.5 μg/ml or more) Hepatic injury (lactate dehydrogenase concentration of 400 U/L or more, or an aspartate aminotransferase concentration of 100 U/L or more) Cytokinaemia (defined as an IL-6 concentration of 15 pg/ml or more, or a triglyceride concentration of 150 mg/dl or more, or a CRP concentration of 15 mg/dl or more) [7] Fajgenbaum DC and June CH 2020 Very severe inflammation with cytokine storm [6] Manson JJ et al. Cytokine storm, dysregulated macrophage activation, impaired natural killer cell response, lymphopenia, elevated absolute neutrophil count and neutrophil/lymphocyte ratio and increased levels of neutrophil extracellular traps (NETs) [4] Anka AU et al. 2021 Excessive secretion of pro-inflammatory cytokines and the recruitment of pro-inflammatory cells such as granulocytes and macrophages caused by tissue injury result in a snowballing of cytokine secretion leading to a systemic inflammatory response such as macrophage activation syndrome (MAS), secondary haemophagocytic lymphohistiocytosis (sHLH-cytokine storm) [11] Cardone MC et al.

Increased plasma levels of pro-and anti-inflammatory cytokines (IL-1β, IL-6, IL-7, IL-8, IL-9, IL-10, IFN-γ, TNF), chemokines (MCP1, MIP1A, MIP1B) and growth factors (G-CSF, GM-CSF) [8] Mehta P et al. 2020

Trends in laboratory results such as increasing ferritin, decreasing platelet counts or high erythrocyte sedimentation rate 12 Freeman TL et al. (2020) 2020

Vigorous stimulation of the innate immune response activating the Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome pathway. This causes the release of the pro-inflammatory cytokines IL-6 and IL-1β [13] De Luca G et al.

explanation for the frequently observed co-infections or secondary infections in COVID-19 [15, 16] . The results of non-randomised cohort studies with controls and of retrospective observational studies suggest that IL-1 receptor blockade (anakinra) [9, 17] , monoclonal antibodies against IL-6 receptors [18] [19] [20] [21] and the combination of both drugs [22, 23] may improve survival rate in at least a subgroup of patients with COVID-19. However, in prospective randomised controlled trials with the exception of one trial with tocilizumab and sarilumab in critically patients [24] , anti-inflammatory therapy with anakinra [25] or tocilizumab [26] [27] [28] [29] [30] [31] [32] did not improve the outcome in moderate, severe and critically ill COVID-19. On December 10, 2020, an editorial commented that it is disappointing that nearly 10 months into the COVID-19 pandemic, a breakthrough treatment has not been identified [33] . Researchers of the US National Institute of Allergy and Infectious Diseases stated that although Remdesivir is effective to reduce time to recovery in hospitalised COVID-19 patients [34] and dexamethasone reduces mortality in critically ill COVID-19 patients [35] , there is no treatment for early or mild infection [36] . Moreover, dexamethasone raises concerns because it increased the 28-day mortality in patients who did not receive respiratory support [35] and it dampens the "alarm phase" of the inflammation process including the capacity of detecting pathogens in mammals by the immune system [37] . In addition, administration of methylprednisolone (1 mg/kg/day intravenously) in COVID-19 reduced the blood levels of NK cells, CD4 + and CD8 + T-cells and increases the duration of throat viral RNA detectability indicating immune cell dysfunction [38] . Furthermore, targeted anti-viral medication has failed to treat COVID-19 effectively [39] . According to the World Health Organisation, after a record-breaking development, vaccine deployment is slow and has many challenges to overcome [40] . Vaccine hesitancy is relatively high [41] even among health care workers [42, 43] . It could take more than a year to vaccinate enough people required to make an impact on SARS-CoV-2 spreading, while therapeutic measures that can immediately attenuate the course of SARS-CoV-2-related lung damage are promptly needed on a global scale. To make the matters worse, many scientists expect that SARS-CoV-2 may become endemic and is here to stay [44] .

In this report, we developed a novel definition of hyperinflammation based on purinergic signalling. Subsequently, we describe our discovery of an old drug capable of attenuating hyperinflammation and illustrate this with six critically ill patients suffering from COVID-19. Finally, we present the future development of a new and more accessible administration route for this drug as shown in Fig. 1 .

In 1929 adenylic acid (identical to adenosine) was identified [45] , and in the same year, the adenosine triphosphate (ATP) molecule was discovered and isolated [46] . Ten years later (1939) , researchers contributed to the understanding of intracellular ATP as an intracellular energy transport molecule [47] [48] [49] [50] . In 1948 and in 1959, it was reported that extracellular ATP has a different function than ATP within the cytoplasm [51, 52] . The authors showed that extracellular ATP molecules have an intercellular signalling function. The intercellular signalling by nucleotides (ATP, ADP, UTP and UDP) and nucleoside (adenosine) is referred to as purinergic signalling. The purinergic co-transmission in neurons was discovered by Geoffrey Burnstock in 1972 [53] . It took over 20 years for the importance of purinergic signalling to be accepted [54, 55] . Finally, researchers of the University of Ferrara first reported that the P2Z receptor (the former name of the P2X7R) plays an intriguing role in immunity, inflammation and cell death [56] .

The intracellular levels of ATP are high at millimolar concentrations (2-8 mM) [57], and the ATP concentrations in synaptic vesicles are even higher in the range of 5 to 100 mM [58] . In contrast, under normal resting conditions, the extracellular levels of ATP are quite low at nanomolar concentrations (<3 nM) [57, 59] . Under specific conditions, ATP release can rise by more than 1000-fold [53, 57, 60, 61] and leads to a significant increase in the extracellular levels of ATP. The resulting significant increase in extracellular nucleotides and adenosine concentrations activates their purinergic receptors inducing certain cellular functions. Examples of such conditions are membrane depolarisation (i.e. sympathetic neuron endings) [53] , mechanical stress (i.e. high mechanical power ventilation) [59] [60] [61] [62] [63] , hypoxia [64] , hyperosmosis, hypotonic and isotonic stress of endothelial cells [65] [66] [67] [68] , inflammation [69, 70] , surfactant release by alveolar epithelial type II cells [59] [60] [61] , mucine release by airway smooth muscle cells [71] , insulin release by pancreatic islet beta-cells [72, 73] , etc. There is an exception to this concept: Although a spontaneous ATP-induced inward Ca 2+ current through the P2X7R could not be detected below extracellular ATP levels of 200 μmol/ml [74] , low tonic basal activation of P2X7R at nanomolar extracellular ATP concentrations promotes serum independent cellular proliferation [75] , promotes closure of the wound area in scratch wound assay [76] , protects from apoptosis [77] , initiates anaerobic glycolysis independent of the oxygen contents [78] , etc. (Table 2 , rows 80-85). However, low tonic basal activation of the P2X7Rs by extracellular ATP does not cause a pro-inflammatory response of the immune system. Therefore, this topic is beyond the scope of this paper and will not be discussed here.

Clearance of the ATP molecule in order to avoid accumulation in the extracellular space is performed by enzymes attached to the outside of the cell membranes (ecto-enzymes) and by soluble enzymes excreted to the extracellular space ( Fig. 2) [57, [272] [273] [274] [275] . A proportion of the enzymaticbreakdown product of ATP adenosine enters the cells via the equilibrative nucleoside transporters (ENT1 and ENT2) and concentrative nucleoside transporters (CNT1 and CNT2) (Fig.  2) [57, 60, 61]. The release and subsequently clearance of the extracellular nucleotides and adenosine cause fluctuation in the extracellular levels of ATP, other nucleotides and adenosine. These fluctuations in extracellular concentrations are indispensable for the receptor resensitisation after desensitisation following receptor activation as discussed below.

The purinergic control of cellular processes including the proinflammatory and anti-inflammatory responses of the immune system is depending on the activation and the desensitisation phenomenon of the nucleotides and adenosine receptors of the immune cells [74, [276] [277] [278] [279] [280] [281] [282] . Except for the P2X7R, all other purinergic receptors, i.e. P2XRs, P2YRs and P1 receptors (adenosine receptors-AdoRs), are subject to desensitisation [279] [280] [281] [282] [283] . In addition, a certain extent of desensitisation occurs after every activation, and this desensitisation requires time to return to the state of complete resensitisation [279, 280] . The higher and the longer the stimulus of the activation, the higher the extent of desensitisation and the longer the recovery time to the state of complete resensitisation [278] . One of the P2 receptors, the P2X7 receptor, is not prone to desensitisation, and apart from the low tonic basal activation of this receptor at low nanomolar concentrations as mentioned above, the extracellular concentration of ATP required to activate this receptor is much higher. Activation of the P2X7R starts at 100 μM with an EC 50 of >1 mM [74, 279, 284] .

Summary of the effects of extracellular nucleotides and nucleoside on the innate and adaptive immune system through different purinergic receptors is presented in Table 2 Table 2 Summary of the effects of extracellular nucleotides and nucleoside on the innate and adaptive immune system through different purinergic receptors. AdoR adenosine receptor; TNF-α tumour necrosis factor alpha; FcγR receptors belonging to the immunoglobulin superfamily; IFN-γ interferon gamma; IFN-β interferon beta; MAC-1 macrophage-1 antigen comprised CD11b (integrin αM) and CD18 (integrin β2); CpG-A oligodeoxynucleotides; PARP Poly ADP ribose polymerase; FMLP N-Formylmethionyl-leucyl-phenylalanine, a chemotactic factor; COX-2 cytochrome C oxidase polypeptide II; PGE2 prostaglandin E2; MIP-1α macrophage inflammatory protein 1 alpha (MIP-1α = CCL3 chemokine ligand 3 ), MIP-1β (CCL4), MIP-2α ( C X C L 2 c h e m o k i n e C X C m o t i f l i g a n d 2 ) a n d M I P -3α(CCL20);RANTES (Regulated on Activation, Normal T cell Expressed and Secreted, CCL5); LTB4 Leukotriene B4; LTA4 Leukotriene A4; VCAM-1 vascular cell adhesion molecule 1 (CD106);ICAM-1 intercellular adhesion molecule 1 (CD54);HMGB-1 high-mobility group box 1 (belongs to danger-associated molecular patterns); MCP-1 monocyte chemoattractant protein 1 (CCL2);FoxP3 Forkhead box P3; CTL cytotoxic T lymphocyte; Th T helper cell; CTLA-4 cytotoxic T-lymphocyte-associated protein 4 (CD152); CD39 nucleoside triphosphate diphosphohydrolase 1 (NTPD1);CD735'nucleotidase (5'-NT); VEGF vascular endothelial growth factor; IDOIndoleamine-pyrrole 2,3-dioxygenase; α-SMA alpha smooth muscle actin; CTGF connective tissue growth factor (CCN2);bFGF basic fibroblast growth factor; TCRT-cell receptor; NFAT nuclear factor of activated T cells; NLRP3Nod-like receptor family pyrin domain containing 3 gene; ART2-P2X7 pathway extracellular NAD+-induced ATP-independent p2X7R activation involving ADP-ribosyltransferase 2; MMP-9 matrix metalloproteinase-9;TIMP-1 tissue inhibitor of metalloproteinase 1; LC-MS/MS liquid chromatography and tandem mass spectrometry; STAT-1 signal transducer and activator of transcription 1. Updated Endothelial cells [111] Purinergic Signalling Chemokine stromal-derived factor-1α (SDF-1α) triggered mitochondrial ATP [165] Purinergic Signalling P2X5R-deficient BMMs exhibit defective cytosolic killing of L. monocytogenes P2X5R is required for L. monocytogenes-induced inflammasome activation and IL-1β production and that defective L. monocytogenes killing in P2X5R-deficient BMMs is substantially rescued by exogenous IL-1β or IL-18. The P2X5-dependent anti-L.

[177]

Purinergic Signalling [190] Purinergic Signalling Macrophages and P2X7R-transfected HEK-293 cells

Mediates rapid uptake of beads and bacteria in the absence of serum after ATP activation 99 Mast cells Induces degranulation [200] Purinergic Signalling 114

Human embryonic kidney cells (HEK293T) Promotes paxillin and NLRP3 migration from the cytosol to the plasma membrane and facilitates P2X7R-paxillin interaction and [216] Purinergic Signalling [269, 270] 163. Coronaviruses can induce inflammation by the activation of the intracellular sensing molecules IRIG1/MDA5 [285, 286] . Reportedly, acute inflammation [69, 70] and infection with SARS-CoV-2 virus induce ATP release [287] . The vesicular exocytosis-mediated release of ATP, connexin-43 (Cx43)-mediated ATP release and pannexin-1 (Panx-1)-mediated ATP release can be triggered by the activation of Toll-like receptor 4 (TLR4) and TLR2 by pathogen-associated molecular patterns (PAMPs) and by the activation of P2X7Rs [180] [181] [182] 187] . In turn, activation of the P2X7Rs upregulates the protein expression of TLR 2, TLR3, TLR4 and TLR 5 [288] . Additionally, increased levels of TNF-α during inflammation induce ATP release via Panx-1 [289] . Proinflammatory immune response is initiated by the increase in the extracellular ATP, ADP and adenosine levels in the microenvironment of immune cells activating the P2XRs, P2YRs and AdoRs (Fig. 3) [57, 60, 169, 290] . In this case, ATP acts as a danger-associated molecular pattern (DAMP) [291, 292] . Increased ADP levels promote platelet activation and intravascular thrombosis ( Fig. 3 ) [297] including COVID-19 [298] . If these levels of extracellular ATP are accompanied by the absence of the required fluctuations f or o t h e r pu r i n e r g i c r e ce p t o r t o r ec o v er f r o m desensitisation, all P1 and P2 (other than P2X7) purinergic receptors will become fully desensitised demarcating the initiation of hyperinflammation ( Fig.3 and Table 2 , rows 120-123) [279] [280] [281] [282] [283] .

Hyperinflammation is characterised by the activation of P2X7Rs and desensitisation of other P2 receptors and AdoRs

As mentioned above, hyperinflammation starts when fluctuation of the extracellular nucleotides and adenosine no longer occurs and leads to prolonged activation of the P2X7Rs of the immune cells. Prolonged vigorous activation of the P2X7Rs leads to macropore formation and cytolysis with uncontrolled ATP release [222, 223, 227, 299] (Table 2 , rows 120-123) causing hyperinflammation with massive pro-inflammatory immune response, massive pro-inflammatory and antiinflammatory cytokine release: the cytokine storm (Fig. 3) .

In the early phase of COVID-19, hyperinflammation may be confined to the site of viral entry (i.e. airway mucosa and conjunctivae) but as viral replication and viral spreading progress, systemic hyperinflammation devel ops. The upregulation of the expression of ectonucleotidases also leads to an increase in the concentrations of other nucleotides (i.e. ADP) and adenosine. These high extracellular concentrations of nucleotides and adenosine do not show concentration fluctuations required for the recovery (resensitisation) time from desensitisation causing a state of persistent desensitisation of all P2XRs, P2YRs [279, 280, 283, 300, 301] and AdoRs [282] with the exception of P2X7Rs. Consequently, the physiological function in the affected organs and inflamma tory response of the immune system are deactivated. This leads to the failure of organ function (i.e. ARDS in the lungs as we reported earlier [61]) and the immune system (immune paralysis) rendering the host susceptible to secondary co-infections (Fig.3) . Sepsis-induced immunosuppression [302, 303] or compensatory antiinflammatory response syndrome (CARS) in critically ill patients [304] was already raised by researchers in 1996 [305] and is a well-known phenomenon in critically ill patients [302] . Secondary bacterial infections occurred in 34.4% of 274 surviving elderly patients (age over 60 years) with COVID-19 and in 81.7% of 65 deceased patients [15] . In addition, it was found that 76 co-infections with other respiratory pathogens occurred in another cohort of 354 COVID-19 patients (16 of 115 mild cases (13.9%), 33 of 155 severe cases (21.3%) and 27 in 84 critical cases (32%)) [16] . In a meta-analysis involving 118 scientific reports on patients with COVID-19, co-infection with other pathogens at admission was observed in 19% and superinfection with other pathogens during admission in the hospital in 24% [306] .

Tregs are key elements in the control of hyperinflammation [307] . Activation of AdoRA2As promotes the differentiation of naïve T-cells towards regulatory T-cells(Tregs) [112] , increases the frequency of Tregs and the expression of CTLA-4 receptor and upregulates ecto-enzymes CD39 and CD73 expression accelerating adenosine generation from extracellular ATP [118] (Table 2 , rows 25, 29, 30 and 33). This process is upset in case of desensitisation of AdoRs. In addition, activation of P2X7Rs inhibits the suppressive potential and stability of Tregs, inhibits the clonal expansion of Tregs, promotes Treg death, induces Treg depletion and reduces Treg IL-10 production ( Table 2 , rows 86-88, 106 and 107). In COVID-19 patients, significant lower Treg frequencies [308] [309] [310] , lower expression of forkhead box protein P3 (FoxP3), lower expression of transforming growth factor-β(TGF-β) and lower cytokine TGF-β secretion [309] are observed compared to healthy control. Additionally, a reduced proportion of specific SARS-CoV-2-reactive Tregs was reported [311] . The desensitisation of AdoRs and the activation of P2X7Rs may well be the underlying mechanism of the low Tregs frequency in severe and critically ill COVID-19.

As stated above, infected cells release ATP into the extracellular space. Obviously, the P2X7R antagonist blocks the activation of the P2X7Rs. Because a significant proportion of the ATP release to the extracellular space is mediated by the P2X7R (Table 2 , rows 77-79), P2X7R a ntagonism combined with the upregulated ATP hydrolysing activity of the ectoenzymes results in the decrease of the extracellular ATP concentrations. This can potentially abrogate hyperinflammation Fig. 2 Clearance of extracellular ATP and adenosine by ectonucleotidases and soluble extracellular nucleotidases . This process is indispensable to enable receptors to recover from desensitisation following receptor activation (resensitisation, see text under the heading "Purinergic signalling in inflammation and hyperinflammation" for explanation). CD39,Ecto-nucleoside triphosphate diphosphohydrolase 1-3 (ENTPD 1-3); CD73, Ecto-5′-nucleotidase (5'-NT); NPP, nucleotide pyrophosphatase/phosphodiesterase; TNAP, tissue nonspecific alkaline phosphatase; ADA, adenosine deaminase; ADK, adenosine kinase; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ADO, adenosine; ENTs, equilibrative nucleoside transporters; CNTs, concentrative nucleoside transporters Fig. 3 A schematic presentation of the activation of the purinergic receptors of the immune cells causing a pro-inflammatory response leading to hyperinflammation. Viral infection drives the controlled cellular release of ATP molecules. Increased extracellular nucleotides levels activate P2XRs and P2YRs. Upregulation of the extracellular ATP hydrolysing enzymes as depicted in Fig. 2 results in the increase of extracellular adenosine levels followed by the activation of the adenosine receptors (AdoRs). These processes initiate the physiologic pro-inflammatory response of the immune system. The green line at the bottom of the graph represents the extracellular ATP levels. The ascending part is caused by the ATP release, and the descending part results from the clearance of ATP by the extracellular or membrane-bound ATP hydrolysing enzymes. As the disease progresses and extracellular ATP levels increase above 1 mM, the P2X7R is additionally and effectively activated leading to a severe immune response. Except for P2X7Rs, all these receptors are known to be subject to desensitisation. Desensitisation of a receptor is defined as being unresponsive to activation by the ligand, resulting in (near) zero transmembrane signal transduction. A certain extent of desensitisation occurs after every activation, and this desensitisation requires time to return to the state of complete resensitisation. Increasing intensity and duration of the activation stimuli leads to increasing extent of desensitisation and duration of the recovery time to the state of complete resensitisation (represented by brown boxes with increasing size at the bottom of the graph). Severe viral infection can increase the controlled ATP release beyond the capacity of the extracellular enzymes to clear ATP and adenosine molecules. This causes a sustained high extracellular ATP and adenosine levels preventing the purinergic receptors from recovering from the state of desensitisation. The capacity to clear invading microorganisms diminishes leading to immune paralysis. In addition, prolonged high extracellular levels of ATP and activation of the P2X7R lead to macropore formation and cell death with uncontrolled release of ATP. In turn, this leads to vigorous activation of the P2X7R of the immune cells promoting massive production of cytokines ending in a cytokine storm and hyperinflammation and the concomitant immune paralysis. Moreover, P2X7R inhibition promotes the cell-autonomous conversion of CD4+ T cells into Tregs after stimulation of their T-cell receptors (TCRs) [190] . In addition, P2X7R knock-out mice, mimicking the state of complete P2X7R inhibition, show an increase in tissue Tregs, prevent Tregs death and the Tregs produce more IL-10 and TGF-β [191] . Experimental inhibition of P2X7Rs restores the Tregs levels and function ( Table 2 , rows 86-88, 106 and 107) [190] [191] [192] . Inhibition of the P2X7R or P2X7R knock-out can attenuate severe inflammation in abdominal sepsis [312] and in acute lung injury [313, 314] . Apparently, amelioration of hyperinflammation by P2X7R inhibition is based on the increased activation and clonal expansion of the anti-inflammatory Tregs population ( Table 2 , rows 86-88, 106 and 107).

Some authors proposed that the P2X7R is an ideal candida te to target in COVID-19-associated severe pneumonia [298, 315] , and others suggested that hyperactivation of the P2X7R plays a key role in the neuropathology of COVID-19 and that P2X7R antagonism may prevent or treat neurological manifestations of COVID-19 [316] .

In 2015 it was discovered that lidocaine is a P2X7R antagonist [74] , and therefore, lidocaine can potentially reduce the clinical symptoms of hyperinflammation significantly. In experimental sepsis, lidocaine improves organ failure [317] [318] [319] and survival [317] . In septic patients, lidocaine reduces neutrophil recruitment by the mitigation of chemokine-induced arrest and transepithelial neutrophil migration [320] . Neutrophil recruitment is an important facilitating process in the pathogenesis of multiple organ failure [320] and hyperinflammation in COVID-19 [321] [322] [323] [324] . In patients with skin lesions from atopic dermatitis, lidocaine increases the proportion of Tregs and upregulates the FoxP3 expression [325] . In addition, lidocaine increases the IL-10 levels in mechanically ventilated mice [326] and decreases the TNF-α in BAL, plasma and lung samples in pigs undergoing surgery for lung resection [327] .

The P2X7R antagonist dose-response relationship of lidocaine is presented in Fig.4 . The IC 50 for the inhibition of the P2X7R by lidocaine is about 66.07 μg/ml (0.28 mM) [74] where IC 50 is defined as the required extracellular concentrations of the receptor antagonist to reach an inhibitory effect of halfway between maximal activation and maximal inhibition (half-maximal inhibitory concentration). The main issue is that the IC 50 for P2X7R inhibition is much higher than the maximal tolerable plasma concentration for mammals. The maximal tolerable plasma concentration in humans is about 4.7 μg/ml (0.02 mM); this corresponds with an IC 10 or lower (<10% inhibitory concentration, Fig.4 ). Above this lidocaine plasma concentration, adverse effects in increasing severity occur as presented in Table 3 [328, 329] . Thus, systemic lidocaine plasma concentrations of >4.7 μg/ml must be avoided [328, 329] . Caveat: The inhibitory concentrations of lidocaine for P2X7R as presented in Fig.4 are not corrected for the series resistance (in the range of 1-3 MΩ) of the used whole-cell voltage clamp method with two puller microelectrodes [74] . One should bear in mind that after correction for series resistance, the reported inhibitory concentration values including IC 50 are expected to be higher [330] .

In addition to the P2X7R antagonist properties, lidocaine is also known to have several other inhibitory pharmacological targets: the voltage-gated sodium channels (VGSC: Nav1.2 [331] , Nav1.3 [332] , Nav1.4 [333] , Nav1.5 [334] , Nav1.7 [335] , 1.8 [336] and Nav 1.9 [337] ), the Toll-like receptor 2 (TLR 2) [338] , TLR4 [318] and the N-methyl-D-aspartate receptor (NMDAR) [339] .

VGSCs conduct sodium ions inward and are essential for the transduction of sensory stimuli, the generation of the action potential and the release of neurotransmitters from sensory neuron terminals. Lidocaine inhibition of VGSCs can effectively reduce pain signalling [340] . In addition, VGSCs are present on dendritic cells (maintain chemokine-induced migration) [341] , macrophages (regulate phagocytosis and endosomal pH during LPS-mediated endosomal acidification) [342] , microglia (regulate phagocytosis cytokine release ad migration) [343], neutrophils (regulate attachment, transmigration and chemotaxis) [344] and T-cells (regulate positive selection of CD4 + T cells) [345] . However, until date no relevant data have been published suggesting that other VGSC antagonists (such as HYP-17 [346] , A-803467 [347, 348] , PF-05089771 [349] , phenytoin [350] or tetrodotoxin [351, 352] ) may substitute non-steroidal anti-inflammatory drugs let alone may suppress COVID-19-related hyperinflammation [353] . A plausible reason is that during hyperinflammation-including hyperinflammation in COVID-19-the cytokine levels (i.e. IL-1β [354] , IL-6, IL-10 [355, 356] and IL-12 [357] ) are high. Reportedly, IL-1β [358] and IL-6 [359] inhibit sodium currents of VGSCs, and IL-10 downregulates the expression of VCSCs [360] . Moreover, activation of the P2X7R reduced the density and currents of VGSCs [361] . Therefore, we do not consider the inhibitory properties of lidocaine on VGSCs to be relevant for the treatment of hyperinflammation in COVID-19. At first glance, the downregulation of the expression of TLR 2 [338] and TLR 4 [318] is an important antiinflammatory mechanism directly induced by lidocaine. But at a closer look, it appeared that activation of P2X7R by the agonist cathelicidin (LL-37) leads to the upregulation of the protein expression of TLR2, TLR3, TLR4 and TLR 5 [288] . This is in line with the MyD88 (myeloid differentiation primary-response protein 88)-dependent activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) following the activation of the P2X7R by BzATP [362, 363] . The MyD88-dependent activation of NF-κB is part of the TLR4/NF-κB pathway. Therefore, it is unsurprising that the inhibition of P2X7R by its antagonists (Brilliant Blue G, A-438079 and A-740003) neutralises the abovementioned P2X7R-induced upregulation of TLRs [362] . Consequently, we argue that lidocaine inhibits inflammation directly by blocking P2X7Rs independent from the neutralisation of the P2X7R-induced upregulated TLR2 and TLR4.

The subpopulation of NMDA receptors present on the peripheral neurons are involved in nociception, and their number increases during inflammation contributing to the sensitisation of peripheral nerves to nociceptive stimuli. NMDA receptor antagonists have anaesthetic-like effects [364] . In addition, NMDA receptor antagonist can prevent hypoxic neuronal death, IL-1β and TNFα release [365] , reduce the activation of inflammatory experimental colitis [366] and suppress glial pro-inflammatory cytokine expression [367] . Moreover, the NMDA receptor antagonist memantine can increase IL-10 inhibition: 3.00 mM (702.90 μg/ml); and 98% inhibition: 10.00 mM (2343.00 μg/ml), respectively. The usual plasma concentrations in clinical settings are indicated by the green box, and the targeted concentrations in the lymph nodes are indicated by the magenta box. Note that the maximal tolerable plasma levels for human (about 4.7 μg/ml-0.02 mmol/L) are much lower than the required extracellular concentrations of lidocaine to effectively inhibit the P2X7R. Source: Okura D, et al. [74] production in BCR/CD40-activatedB-cells [368] . Lidocaine inhibits NMDA receptors [339, 369, 370] , and thus the antiinflammatory properties of lidocaine could be attributed to the inhibition of NMDA receptors. However, it has been reported that the anti-inflammatory effect in T-cell functions (inhibition of antigen-specific T-cell proliferation, T-cell cytotoxicity, Tcell migration towards chemokines and decrease in IL-2 and IFN-γ production by Th1 effector cells in favour of IL-10 and IL-13 production by Th2 cells) of the NMDA receptor antagonist ifenprodil is effective both in wild-type and in NMDA receptor (GluN1) knockout mice [371] . Moreover, it was found that KN-62, an inhibitor of Ca2+/calmodulin-dependent kinase type II and a potent P2X7R antagonist, provides neuroprotection against NMDA-induced cell death [372] . Therefore, we argue that the anti-inflammatory properties of NMDA receptor antagonists (including lidocaine) should be attributed to the inhibition of P2X7Rs rather than to the inhibition of NMDA receptors.

As mentioned above, the main issue is that the IC 50 for P2X7R inhibition is much higher than the maximal tolerable plasma concentration for mammals because P2X7Rs are indispensable for normal physiological functions (i.e. in the central nervous system [373] , the peripheral nervous system [374] and in the lungs [60, 61]). Therefore, intravenous or oral administration aimed at achieving an effective concentration of lidocaine to inhibit P2X7Rs in serum and in target organs will hamper organ functions and is potentially dangerous.

The lymphatic system is populated exclusively by trafficking immune cells, i.e. naïve T cells, activated T cells, B cells [375] , dendritic cells [376] , monocytes [377] , macrophages [378] , neutrophils [379] , mast cells [380] , eosinophils [381] and basophils [382] . We postulate that selective inhibition of the P2X7Rs of the immune cells of the lymphatic system by lidocaine suppresses hyperinflammation in two stages: stage 1, the selective inhibition of the P2X7Rs of the immune cells residing in the lymph nodes induces clonal expansion of Tregs in these lymph nodes; stage 2, subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities reducing systemic and (distant) local hyperinflammation (Fig. 1) .

The endothelium of the dermal capillaries of the skin belongs to the structural type "continuous endothelium" [383] . Although capillary walls can transport substances from blood to tissue, the absorption of substances from tissue to blood is, if any, extremely low [384] . Apparently, specialised initial lymphatics harbouring one-way valve leaflets capable of absorbing fluid and molecules from the interstitium are localised in the dermis. The absorbed lymph fluid is then propelled forward in the lymphatic network by collecting lymphatic vessels harbouring a rhythmic contracting muscle layer [385] . This system brings fluids and particles into the lymph nodes where numerous immune processes take place. The administration route to target the lymphatic system in a domestic swine model is illustrated by the subcutaneous or intradermal injection of compounds (isosulfan blue, fluorescein and radioactive technetium-99 isotope-Tc 99 ) and by tracing the extent and the transit time of the distribution of these compounds using whole body scintigraphy in pigs [386] . The absorption of intradermal application of radioactive Tc 99 into the lymph nodes is 10 times faster than after deep subcutaneous application and leads to higher concentrations in the lymph nodes related to these lymphatic vessels [386] . Radionuclide lymphoscintigraphy with molecules of different sizes after intradermal and subcutaneous injections showed that smaller particles (i.e. 99mTc-dextran and 99mTc-human serum albumin) migrate more rapidly towards the lymphatic vessels and lymphatic nodes than larger particles (i.e. radiocolloids of larger molecular size) [387] . The rate of clearance of 99m Tc-pertechnetate and 99m TcDTPA after subcutaneous and intradermal administration in the back of the hand in humans is 1 %/min and 8 to 10 %/min, respectively [387] .

The additional advantage is that the plasma concentrations of subcutaneously administered lidocaine are much lower than intravenously administered lidocaine. Intravenous administration of 2 mg/kg lidocaine in cats is almost immediately followed by a peak plasma concentration of 3.6 μg/mL [388] . In contrast, the achieved mean peak plasma concentrations after the subcutaneous administration of 30 mg/kg, 20 mg/kg and 10 mg/kg lidocaine are much lower: 1.69, 1.07 and 0.77 μg/mL, respectively [389] . Note that the applied subcutaneous dose [389] is 15, 10 and 5 times higher than the intravenous dose, respectively [388, 389] . Reportedly, the difference in the plasma concentrations after intravenous and subcutaneous administration of lidocaine is caused by the fact that, in contrast to the intravenous administration, a large proportion of the subcutaneously administered lidocaine is drained into the lymphatic system [390] [391] [392] . Obviously, this slows down the release of lidocaine to the venous blood. This is confirmed for bevacizumab in mice [390] , for trastuzumab in rats [391] and for docetaxel in rats by [392] .

As stated above, lymphatic absorption after intradermal administration is much higher than after deep subcutaneous administration [386, 387] . Practically, the intradermal infusion with lidocaine is not an accepted administration route for lidocaine. Therefore, we argue that a subdermal administration of lidocaine using a catheter inserted just beneath the dermis (subdermal infusion, Fig. 5 ) will result in higher concentrations of lidocaine in the draining local lymph nodes than a deep subcutaneous or intravenous infusion as depicted in the schematic presentation of the putative distribution of lidocaine in Fig. 6 .

In summary, by means of the subdermal administration of lidocaine, we can ensure high concentrations of lidocaine in the local lymph nodes enabling an effective inhibition of the P2X7R of the immune cells while keeping the lidocaine plasma concentrations <4.7 μg/ml(stage 1a and 1b in Fig. 1) . The induced Tregs clonal expansion in these local lymph nodes produces Tregs which migrate t h r o u g h o u t t h e b o d y c o n t r o l l i n g t h e o n g o i n g hyperinflammation (stage 2 in Fig. 1) . Obviously, the subdermal administration route may also apply to other P2X7R antagonists.

Three other P2X7R antagonists have been tested in human: CE-224,535 500 (Pfizer), AZD-9056(Astra-Zeneca) and JNJ-54175446 (Johnson and Johnson). A phase IIa study with CE-224,535 in patients with rheumatoid arthritis not responding adequately to methotrexate was recently reported [393] . Patients in the treatment arm received oral CE-224,535 500 mg twice/day for 12 weeks. Although the safety and tolerability for the compound were acceptable, CE-224,535 was not effective in this group of patients. The results of a phase II study with AZD-9056 in patients with active rheumatoid arthritis despite treatment with methotrexate or sulphasalazine was published. The treatment arm consists of oral AZD-9056 100 or 400 mg/day for 6 months [394] . The AZD-9056 used in this trial is non-lipophilic as indicated by the fact that this compound cannot penetrate the blood-brain barrier [395] . The authors conceded that "AZD-9056 does not have significant efficacy in the treatment of RA, and the P2X7 receptor does not appear to be a therapeutically useful target in RA" [394] . Recently, a randomised, placebo controlled, sequential-group, single-centre ascending dose phase I study was reported. The patients in the 5 treatment arms received 0.5, 2.5, 10, 50, 150 and 300 mg JNJ-54175446, respectively. The authors reported dose-dependent plasma levels, no serious adverse events, ex vivo attenuation of lipopolysaccharide-induced IL-1β release in peripheral blood and confirmation of passive brain penetration of JNJ-54175446 [396] . The approach of the P2X7R antagonist therapy of the above-mentioned authors is quite different from ours: While these authors directly targeted the diseased organs via the gut absorption of the drug, we target the immune cells in local lymph nodes inducing an anti-inflammatory immune response which in turn targets the diseased organs (Fig. 1 ). This is illustrated by the following study concerning a placebo-controlled, multicentre, doubleblind phase IIa study in patients with moderately to severely active Crohn's disease. The patients in the treatment arm received oral AZD-9056 200 mg/day for 28 days. The authors found a significant improvement in the Crohn's Disease Activity Index (CDAI) at day 28 [397] . In contrast to the skin, the endothelium of the mucosal capillaries of the mouth and the gastrointestinal tract are fenestrated allowing molecules to pass from the submucosal tissue into the capillaries [383] . Unlike the failure of the treatment of rheumatoid arthritis described above, the successful treatment of gut inflammation here can be attributed to the absorption of non-lipophilic oral AZD-9056 by the mucosa-associated lymphoid tissue (MALT). This is the inductive site of the mucosal immune system consisting of mesenteric lymph nodes, Peyer's patches and isolated lymph follicles [398, 399] . Although lymphatic transport to the lymph nodes of the non-lipophilic oral AZD-9056 is limited [400, 401] , AZD-9056 inhibits P2X7Rs of the local T-cells via absorption by the inductive sites of MALT. This induces a local anti-inflammatory immune response executed by the effector sites of MALT consisting of lamina propria lymphocytes and intraepithelial lymphocytes [398, 399] . 

From April 2020 until end of July 2020, two of the authors of this report (AS and TK) have successfully treated six critically ill patients with COVID-19 admitted to the ICU of the Showa University in Tokyo, Japan, with lidocaine. The lidocaine treatment was based on off-label use. The Medical Ethical Committee of the Showa University, School of Medicine, Tokyo, approved the collection, analysis and publication of patients on mechanical ventilation admitted to the ICU (protocol number 3313). The administration was initially intravenously in the two first patients, followed by subdermally (a superficially inserted subcutaneous catheter as illustrated in Fig. 5 ). In the other four patients, only the subdermal administration was further applied. The concentration of the intravenous lidocaine infusion solution is 20 mg/ml (2%), the route for continuous administration of lidocaine commonly used in daily practice. The dose for intravenous administration is 0.6 mg/kg/h as recommended earlier [402] . Due to the limited efficacy of intravenous lidocaine and based on the hypothesis of selectively targeting the inhibition of the P2X7Rs of the immune cells, the infusion in both patients was converted to subdermal infusion of 1.0 mg/kg/h (dosage as reported by Japanese researchers [403] ) after 7 and 6 days, respectively. The time course of clinical parameters of these six patients 6 Schematic presentation of the putative distribution of intravenous, oral, transmucosal (i.e. in the oral cavity) and subdermal administered lidocaine. Administration of hydrophilic lidocaine (lidocaine HCL) through a (central) venous catheter or by oral intake results in concentration gradients with the highest value in the venous blood and the lowest value in the lymph nodes. The reason is that by the time lidocaine reaches the lymph nodes, the drug is massively diluted and may never reach the effective concentration required to adequately inhibit the P2X7Rs of the immune system. In contrast, after subdermal injection of hydrophilic lidocaine, apart from a minimal absorption by the dermal capillaries, almost all the lidocaine is absorbed by the lymphatic system via the initial lymphatics. Because the fluid in the afferent collecting lymphatics originates from the interstitial fluid of the tissues, dilution of the concentration of lidocaine occurs. This fluid is then drained into the local lymph nodes. The extent of the dilution of lidocaine in the targeted lymph nodes is far less drastic compared to the (central) venous administration of the drug. We postulate that with continuous subdermal infusion, we can achieve concentrations of lidocaine in the lymph nodes sufficient to effectively inhibit the P2X7Rs of the immune cells. Theoretically, similar results may be expected from transmucosal and transdermal administration of lipophilic lidocaine base with a high concentration. Obviously, the subdermal, transmucosal and transdermal administration routes may also apply to other P2X7R antagonists is presented in Figures 7, 8, 9, 10 . In about 20% of the inserted subdermal cannulae, local subdermal indurations were observed. Whenever this occurred, the infusion cannula was removed and replaced with a new cannula at a different location. The first patient (Fig.7) , a 63-year-old male (75 kg, 168 cm), developed fever and nausea on March 27, 2020, and 3 days later, he started to cough and became dyspnoeic. After 5 days, the PCR SARS-Cov-2 test was positive, and he was admitted to the hospital with SARS-Cov-2-induced ARDS. Co-morbidities include COPD and smoking 60 cigarettes per day for more than 40 years. About 40 years earlier, the patient suffered from pneumothorax. On day 3, the patient deteriorated and was intubated and mechanically ventilated due to poor blood gases. No haemodynamic instability was observed. The CT scan showed bilateral ground glass opacities compatible with ARDS. On day 5, the patient was transferred to the ICU of the university hospital because of further respiratory deterioration. The patient received favipiravir for 14 days after admission; the patient did not receive dexamethasone. Prone position mechanical ventilation was initiated due to the progression of the respiratory disease with an extremely low PaO 2 /FiO 2 ratio of 63.3 mm Hg (severe ARDS according to the Berlin definition. The Berlin definition of ARDS includes severe PaO 2 /FiO 2 ratio ≤100 mm Hg, moderate PaO 2 /FiO 2 >100 to 200 mm Hg, mild PaO2/FiO2 >200 to 300 mm Hg, no ARDS PaO2/FiO2 >300 mm Hg [404] ). The initial ventilator settings include APRV, P high 27 cm H 2 O, T high 7.0 s, P low 0 cm H 2 O and T low 0.32 s. The Fig. 7 Patient 1, the first of the six cases with severe COVID-19 treated with subdermal lidocaine in the ICU of the Showa University, Tokyo, Japan. A 63-year-old male with COVID-19-induced ARDS, was admitted to the hospital. The CT scan showed bilateral ground glass opacities. Co-morbidities: COPD, smoking 60 cigarettes per day for more than 40 years. About 40 years before admission, the patient suffered from pneumothorax. After admission the clinical condition deteriorated requiring an ICU admission and mechanical ventilation on day 4. On day 11, continuous intravenous lidocaine of 0.6 mg/kg/h was initiated, but the patient's condition kept worsening with high pulmonary artery pressures and reduced aeration of the lung. On day 19, the continuous intravenous lidocaine of 0.6 mg/kg/h was changed to continuous subdermal lidocaine of 1 mg/kg/h. This was followed by improvement of the clinical condition, and on day 20, the aeration of the lung was improved, but the pulmonary artery pressures remained high. Despite this the P/F ratio was gradually improving, and ECMO weaning was done on day 50. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.3-0.8%). On day 99, he was weaned from the mechanical ventilator and was discharged from the ICU on day 121. CT scan on day 146 showed reduced ground glass opacities in both lungs and some interstitial change in upper and middle fields of the lung and improvement of the pneumothorax. The patient was discharged from the hospital on day 187, he went home, and he could walk but needed extra oxygen supply of 2L/min. Nine months after admission, the patient is doing well and has returned to work. The patient visited the hospital 3 months after discharge: He only uses oxygen 1 L/min to go shopping and during physical training (out-patient rehabilitation). He talked to the treating intensivist without requiring oxygen and had no shortness of breath or tachypnoea. The red-coloured labels of the legends refer to graph plots using the (left) primary Y-axis, and the black-coloured labels of the legends refer to graph plots using the (right) secondary Y-axis PaCO 2 was normal. The echocardiographic estimated pulmonary arterial systolic pressure (PASP) was 80 mm Hg. The Krebs von Lungen 6 (KL-6, a marker for lung fibrosis [405] ) plasma level was highly elevated (1299 U/mL; normal value <425 U/mL), CRP was also high (40.4 mg/L; normal value <10 mg/L), and albumin was 2.2 g/dl. The white blood cell count, platelet count and urine production were normal. On day 4, the chest X-ray was not improved. On day 6, the PaO 2 /FiO 2 ratio was slightly increased but remained low at 103 mm Hg, and the chest X-ray showed progression of the ARDS. ECMO was initiated due to exhausted ventilatory strategy. On day 9, the PaO 2 /FiO 2 ratio improved but remained low at around 153 mm Hg, but the CRP declined to around 21.8 mg/L. The patient was put on muscle relaxants. The patient's ARDS status had improved from severe to moderate ARDS. From day 10 until day 30, the ferritin levels were well >1000 ng/ml (>100 μg/dl, normal values <300 ng/ml). From day 11 until day 62, D-Dimer was very high reaching 121.9 nM/L day 14. On day 11, no improvement of the blood gases was observed, and it was decided to treat the patient with continuous intravenous lidocaine 0.6 mg/kg/h. The CRP showed a progressive decline from 19 (on day 12) to 12.8 (on day 16) and 7.4 (on day 19), but the PaO 2 /FiO 2 ratio remained poor at around 90 mm Hg (severe ARDS according to the Berlin criteria) and the chest X-ray image on day 15, 3 days after the initiation of the intravenous lidocaine infusion, deteriorated dramatically. The lidocaine plasma concentrations were 3.4 μg/ml on day 13 and 5.4 μg/ml on day 14. On day 19, the continuous intravenous lidocaine infusion was replaced by continuous subdermal lidocaine infusion of 1 mg/kg/h. Although the PaO 2 /FiO 2 ratio remained unchanged on day 20 (1 day after the switch to the continuous subdermal lidocaine), the chest X-ray improved clearly. On day 21, the lidocaine plasma concentration was 2.6 μg/ml, and albumin was 2.5 g/dl. From day 22, the PaO 2 /FiO 2 ratio was gradually improving reaching 151 mm Hg on day 34 The CT scan showed bilateral ground glass opacities. Co-morbidity: Asthma. After admission the patient's condition was deteriorating. On day 5, continuous intravenous lidocaine of 0.6 mg/kg/h was initiated, but the clinical condition and the P/F ratio kept worsening. On day 11, the intravenous lidocaine of 0.6 mg/kg/h was changed to continuous subdermal lidocaine of 1 mg/kg/h. A few days later, this was followed by improvement of the clinical condition and the P/F ratio. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.6%). The patient was discharged from the ICU on day 30 home on day 37. At 3 months after admission, the patient is doing well. The red coloured labels of the legends refer to graph plots using the (left) primary Y-axis, and the black-coloured labels of the legends refer to graph plots using the (right) secondary Y-axis (moderate ARDS). The KL-6 on day 22 dropped to 458 U/L (this is only slightly above the normal value of <450 U/l). On day 31, the CRP was low at 1 mg/L, and the lidocaine plasma concentration was 1.2 μg/ml. The muscle relaxants were discontinued. Albumin was 2.3 g/dl. On day 33, the chest Xray was further improved, and the CRP remained low at 5.5 mg/L. The patient was awake and could communicate with the nurses. On day 38, the lidocaine plasma level was 2.3. On day 43, the PaO 2 /FiO 2 ratio was increased to 214 mm Hg. According to the Berlin definition of ARDS [404] , the patient's ARDS status had changed from moderate to mild. Albumin was 2.8 g/dl. On day 50, the patient was weaned from ECMO. On day 51, the patient underwent tracheotomy. Because the clinical condition of the patient was stabilised with a low CRP of 6.3 mg/L on day 55, the continuous subdermal lidocaine was discontinued on day 57. On day 69, he developed pneumothorax requiring pleural drainage. On day 99, he was weaned from the mechanical ventilator and was discharged from the ICU on day 121. No new ECG changes were observed during treatment with lidocaine. Blood methaemoglobin (metHb) were within the normal range (0.3-0.8%). CT scan on day 146 showed reduced ground glass opacities in both lungs, some interstitial change in upper and middle fields of the lung and improvement of the pneumothorax. The patient left the hospital on day 187, he went home, and he could walk but needed extra oxygen supply of 2L/min. Nine months after admission, the patient is doing well and has returned to work. The patient visited the hospital 3 months after discharge: He only uses oxygen 1 L/ min to go shopping and during physical training (out-patient Fig. 9 Left graph: Patient 3. A 59-year-old male with respiratory distress and bilateral ground glass opacities on the CT scan. Co-morbidity: Obesity, diabetes mellitus and gout. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.4%). The patient was discharged from the ICU on day 8 and was discharged home on day 20. After 3 months, he is doing well. Right graph: Patient 4. A 51-year-old male with fever, dyspnoea and cough due to COVID-19. The CT scan showed bilateral ground glass opacities. Co-morbidity: none. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.3%). The patient was discharged from the ICU on day 8 and was disc harged home on day 28. At 3 months, he is doing well and has returned to work. The red-coloured labels of the legends refer to graph plots using the (left) primary Y-axis, and the black-coloured labels of the legends refer to graph plots using the (right) secondary Y-axis rehabilitation). He talked to the treating intensivist without requiring oxygen and had no shortness of breath or tachypnoea.

The second patient (Fig.8) is a 68-year-old male (75 kg, 164 cm) with SARS-Cov-2-induced ARDS and positive SARS-Cov-2 PCR test admitted to the university hospital. Co-morbidity is asthma. The CT scan showed bilateral ground glass opacities. Haemodynamically the patient was stable. The patient received tocilizumab on day 8 and favipiravir for 14 days; he did not receive dexamethasone. On day 2, the respiratory conditions deteriorated, and the PaO 2 /FiO 2 ratio is 118 mm Hg (moderate ARDS according to the Berlin ARDS definition [404] ). The patient was intubated and required mechanical ventilation. The initial ventilator settings include pressure control, peak inspiratory pressure 28 cm H 2 O, PEEP 13 cm H 2 O and respiratory rate 30/min. CRP was 10.6 mg/L, and KL-6 was 486 U/ml. White blood cell count, platelet count and urine production were normal. The ferritin levels remained >1000 ng/ml (100 μg/dl) during the entire ICU stay. Albumin was 2.9 g/dl. In the following 3 days, the PaO 2 /FiO 2 ratio improved to around 150 mm Hg. The PaO 2 /FiO 2 ratio dropped from 152 on day 5 to 84 mm Hg on day 6. CRP was increased to 22.9, and the KL-6 was increased to 762 U/ml. The patient was put in prone position and given muscle relaxants. Continuous intravenous lidocaine of 0.6 ml/kg/h was started. Albumin was 1.8 g/dl. On day 7, the PaO 2 /FiO 2 ratio increased to 128 mm Hg, CRP dropped to 10.3 mg/mL and the lidocaine plasma concentration was 2.2 μg/ml. From day 3 until discharge from the ICU, D-dimer values were elevated reaching 75 nM/L on day 14. On day The CT scan showed bilateral ground glass opacities. Co-morbidity: Fatty liver. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.3%). On day 14, the patient was discharged from the ICU. On day 20, the patient was discharged home and is doing well at 3 months after admission. Right graph: Patien ts 6. A 59-year-old male with fever, dyspnoea and cough due to COVID-19. CT scan showed bilateral ground glass opacities. Co-morbidity: Hypertension on medication. No new ECG changes were observed during treatment with lidocaine. MetHb were within the normal range (0.1-0.3%). On day 13, the patient was discharged from the ICU. He was discharged from the hospital on day 20, and a t 3 months after admission, he is doing well, played golf regularly and has returned to work. The red-coloured labels of the legends refer to graph plots using the (left) primary Y-axis, and the black-coloured labels of the legends refer to graph plots using the (right) secondary Y-axis 8, although the PaO2/FiO2 ratio improved from 84 to 125 mm Hg, the mechanical ventilatory strategies were exhausted, and the patient was put on ECMO. The KL-6 was increased to 845 U/L, and lidocaine plasma level was 2.9 μg/ml. The PaO 2 / FiO 2 ratio improved to 238 mm Hg on day 9, but on day 10, a sharp drop of the PaO 2 /FiO 2 ratio to 60 mm Hg was observed, and CRP was 2.0 mg/ml. The patient's ARDS status had changed from moderate to severe according to the Berlin ARDS criteria [404] . Lidocaine treatment was switched from continuous intravenous to continuous subdermal (dosage: 1 mg/kg/h). On day 14, the lidocaine plasma level was 2.7 μg/ml. KL-6 dropped to 549 U/l. On day 17, the clinical condition of the patient was improving, and the PaO 2 /FiO 2 ratio reached 158 mm Hg. The patient was weaned from ECMO. The PaO 2 /FiO 2 ratio improved further reaching 291 mm Hg on day 21, and the patient's ARDS status has changed from moderate to mild ARDS [404] . On day 22, mechanical ventilation was discontinued, and the patient was extubated. The patient was orientated, and no signs of confusion were detected. CT scan on day 25 showed persistent ground glass opacities in both lungs, some pulmonary effusion (right >left), and no signs of vascular thrombosis. In addition, no signs of deep venous thrombosis were found in the lower extremities. Lidocaine treatment was continued until discharge from the ICU on day 30. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.6%). The patient was discharged home on day 37. At 3 months after admission, the patient is doing well.

The third patient (Fig. 9left) , a 59-year-old male (109 kg, 170 cm), was admitted to the university hospital with respiratory distress and bilateral ground glass opacities on the CT scan with a positive SARS-CoV-2 test. Comorbidities include obesity (BMI 37.7 kg/m 2 ), diabetes mellitus and gout. The patient required immediate intubation and mechanical ventilation. The patient received tocilizumab on day 3 and favipiravir for 15 days and did not receive dexamethasone. The initial ventilator settings are pressure control, peak inspiratory pressure 30 cm H 2 O, PEEP 15 cm H 2 O and respiratory rate 25/min. The PaO 2 /FiO 2 ratio on admission was 160 mm Hg (moderate ARDS according to the Berlin definition [404] ), CRP was 39.3 mg/L and KL-6 was 294 U/ml. White blood cell count was increased (13.10 −9 /L) and platelet count and urine production were normal. Albumin was 2.1 g/dl. Haemodynamic parameters were stable. On the admission day, continuous subdermal lidocaine was started at 1 mg/kg/h. On day 2, the PaO 2 /FiO 2 ratio improved to 283 mm Hg, and the patient's ARDS status had changed from moderate to mild ARDS. CRP was 41 mg/L, KL-6 was 268 U/L and the lidocaine plasma level was 3.7 μg/ml. Albumin was 1.7 g/dl. On day 4, the PaO 2 /FiO 2 ratio was 302 mm Hg, and the patient's ARDS status had changed from mild ARDS to no ARDS according to the Berlin ARDS criteria. On day 5, the PaO 2 /FiO 2 ratio was improved further to 328 mm Hg, and CRP dropped to 16.4 , and the patient was extubated. The patient was orientated, no signs of confusion were detected. The patient was discharged from the ICU on day 8; CRP was 2.3 mg/ml. Albumin was 2.5 g/dl. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.4%). The patient was discharged home on day 20. After 3 months, he is doing well.

The fourth patient (Fig. 9right) is a 51-year-old male (68 kg, 175 cm). Ten days before admission, he developed fever and 2 days before admission dyspnoea and coughing. On the day of admission, the PCR SARS-CoV-2 test was positive. The CT scan showed bilateral ground glass opacities. Comorbidity is none. The patient was intubated and put on mechanical ventilation on admission. The patient received favipiravir for 14 days; he did not receive dexamethasone. On day 3, he was transferred to the university hospital because of deterioration of pulmonary condition. The initial ventilator settings include pressure control, peak inspiratory pressure 24 cm H 2 O, PEEP 12 cm H 2 O and respiratory rate 15/min. The haemodynamic conditions were stable. White blood cell count and platelet count were normal. Albumin was 2.6 g/dl. Continuous subdermal lidocaine was started immediately. On day 3, the PaO 2 /FiO 2 ratio was 214 (moderate ARDS according to the Berlin definition [404] ). KL-6 was 177 U/L, and CRP was 17.4 mg/L. On day 5, the PaO 2 /FiO 2 ratio was increased to 382 (the patient's ARDS status had changed from mild ARDS to no ARDS), and lidocaine plasma concentration was 5.2 μg/ml. CRP was 27.3mg/L. Lidocaine plasma levels on day 3 and 4 were 3.4 and 4.2 μg/ml, respectively. KL-6 was 163 U/L. The patient was extubated. The patient was orientated, and no signs of confusion were detected. The patient was discharged from the ICU on day 8, and the CRP was 9.3 mg/L. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.3%). He was discharged home on day 28. At 3 months, he is doing well and has returned to work.

The fifth patient (Fig. 10left) is a 58-year-old male (80 kg, 175 cm). Nine days before admission, he developed a sore throat. A day later, he developed fever. Two days before admission, he started coughing and was dyspnoeic. On the day of admission, the PCR SARS-Cov-2 test was positive. Comorbidity includes fatty liver. The CT scan showed bilateral ground glass opacities. The patient was initially admitted to the hospital ward. The patient received tocilizumab on day 7 and favipiravir for 10 days; dexamethasone was not prescribed. On day 3, the patient deteriorated and had to be intubated and put on mechanical ventilation. On day 4, the patient was transferred to the university hospital due to deterioration of the pulmonary condition. The initial ventilator settings include pressure control, peak inspiratory pressure 27 cm H2O, PEEP 12 cm H2O and respiratory rate 25/min. PaO 2 /FiO 2 ratio was 188 (moderate ARDS according to the Berlin definition). Haemodynamic parameters were stable, and CRP was 12.9 mg/ml. White blood cell count was increased (14.4.10 9 /L), but platelet count was normal. KL-6 was 330 U/L. Continuous subdermal lidocaine was started at 1 mg/kg/h at arrival at the ICU of the university hospital. Albumin was 2.8 g/dl. On day 5, the PaO 2 /FiO 2 ratio was unchanged, CRP was 10.4 mg/L and the lidocaine plasma level was 4 μg/ml. On day 6, the lidocaine plasma level was 3.2 μg/ml. KL-6 remained stable at 400 U/L. Albumin was 2.3 g/dl. On day 10, the respiratory insufficiency had cleared; although the PaO2/FiO2 ratio remained 184, the CRP dropped to 2.4 mg/L, and KL-6 was 322 U/L. The patient was extubated, and he was orientated; no signs of confusion were detected. On day 14, the patient was discharged from the ICU. No new ECG changes were observed during treatment with lidocaine. Blood metHb were within the normal range (0.1-0.3%). On day 20, the patient was discharged home and is doing well at 3 months after admission.

The sixth patient (Fig. 10right) is a 59-year-old male (65 kg, 175 cm) with fever, dyspnoea and cough due to COVID-19. CT scan showed bilateral ground glass opacities. Co-morbidity includes hypertension on medication. The patient was admitted to the general ward. KL-6 233 U/L, white blood cell count and platelet count were normal. Albumin was 3.6 g/dl. On day 3, there is a deterioration of the respiratory function necessitating a transfer to the ICU and mechanical ventilation. Tocilizumab was given on day 4. The patient received favipiravir for 11 days, and the patient did not receive dexamethasone. The initial ventilator settings include pressure control, peak inspiratory pressure 22 cm H 2 O, PEEP 10 cm H 2 O and respiratory rate 20/min. Continuous subdermal lidocaine of 1 mg/kg/h was initiated after admission to the ICU. Haemodynamic parameters were stable. CRP was 6.3 mg/L, and KL-6 was 263 U/L. On day 4, a progressive respiratory failure occurred requiring intubation and mechanical ventilation. PaO 2 /FiO 2 ratio was 218 mm Hg; the haemodynamic parameters remained stable. CRP was 6.3 mg/L, and the white blood count and platelet count were normal. Lidocaine plasma level was 4.6 μg/ml. On day 5, the PaO 2 /FiO 2 ratio dropped further to 164 mm Hg. Lidocaine plasma level was 3.4 μg/ml. Albumin was 3.2 g/dl. On day 9, the clinical condition of the patient improved. The ventilator settings could be decreased, the PaO 2 /FiO 2 ratio remained 207 mm Hg during the weaning period, and CRP was 0.7 mg/L. On day 10, the patient was extubated, he was orientated, and no signs of confusion were detected. On day 13, the patient was discharged from the ICU. No new ECG changes were observed during treatment with lidocaine.

Blood metHb were within the normal range (0.1-0.3%). He was discharged from the hospital on day 20, and at 3 months after admission, he is doing well, played golf regularly and has returned to work.

From July 2020 until beginning of December 2020, 14 additional critically ill patients with COVID-19-induced ARDS requiring mechanical ventilation were treated in the ICU of the Showa University with continuous subdermal lidocaine infusion (1 mg/kg/h) plus intravenous or oral dexamethasone (6 mg/day) as reported earlier [35] . Of these 20 patients, 19 survived, but an 87-year-old female patient died of invasive aspergillosis. No other patient developed secondary coinfections (unpublished data, personal communication by AS and TK).

After completing the novel definition of hyperinflammation, we developed a new approach to target the lymphatic system with continuous subdermal administration of lidocaine. This is meant to increase the anti-hyperinflammatory effect of lidocaine while avoiding toxic plasma levels. We described the treatment of six critically ill patients with COVID-19 with lidocaine. Two patients required mechanical ventilation and ECMO, and four patients were treated with mechanical ventilation. As mentioned under the heading "Introduction", the case fatality rates of patients requiring mechanical ventilation and/or ECMO are alarmingly high [1, 2] . Patient 1 and patient 2 were older than 60 years. Additionally, patient 1 had COPD and had smoked 60 cigarettes per day for more than 40 years. Patient 3 suffered from obesity and diabetes mellitus. These are serious prognostic factors for bad outcome COVID-19 [406, 407] . Patient 1 and patient 2 were initially treated with continuous intravenous lidocaine through a central venous line. In both patients, the pulmonary conditions deteriorated after the initiation of intravenous lidocaine: Patient 1 who was already on ECMO showed progressive pulmonary deterioration on the chest X-rays, and patient 2 deteriorated further necessitating the initiation of ECMO therapy. Remarkably, the pulmonary conditions of both patients improved within 48 h after the switch from intravenous to subdermal continuous lidocaine. The lidocaine plasma levels remained around 5 μg/ml. To our knowledge, these six cases represent the first observations of the promising treatment of critically ill COVID-19 patients with lidocaine targeting P2X7Rs of the immune cells in the lymphatics. All patients recovered completely from their illness. None of the patients showed the feared side effect of cardiac arrhythmia and methaemoglobinaemia during lidocaine therapy. Our findings suggest that continuous subdermal lidocaine infusion at the rate of 1 mg/kg/h has the potential to mitigate hyperinflammation and ARDS in critically ill COVID-19-patients. Obviously, although all six patients appeared to respond positively to the treatment and no severe adverse effects were observed, no final conclusions can be made on the efficacy of lidocaine in critically ill COVID-19 patients.

Researchers from Lima, Peru, reported the treatment of 28 (three mild, 21 moderate and four severe) COVID-19 patients with 0.5% lidocaine HCL solution with an intravenous dose of 1 mg/kg once a day for 2 days and 2% lidocaine HCL solution with a subcutaneous dose of 1 mg/kg once a day for 2 days [408] . The authors aimed at the improvement of pain, cough, respiratory rate and oxygen saturation. They found improvement in most patients. In severe cases, this treatment did not improve the oxygen saturation. As expected, treatment with a low daily dose of lidocaine once per day for a total treatment duration of 2 days could not adequately inhibit the P2X7Rinduced hyperinflammation in COVID-19.

Recently, a group of researchers from Strasbourg, France, announced a study entitled: "Impact of intravenous lidocaine on clinical outcomes of patients with ARDS during COVID-19 pandemia (LidoCovid): A structured summary of a study protocol for a randomised controlled trial" (ClinicalTrials.gov Identifier: NCT04609865) [409] .

Lately, an extraordinary treatment of COVID-19 ARDS was reported [410] . The authors performed lung transplantations in three critically ill COVID-19 ARDS patients: a 28-year-old female, a 62-year-old male and a 43-year-old male. The first patient underwent lung transplantation after weeks on veno-venous ECMO support with elevated pulmonary arterial pressures and severe secondary Serratia marcescens pneumonia. The second patient underwent lung transplantation after 100 days on v e n o -v e n o u s E C M O s u p p o r t c o m p l i c a t e d b y Pseudomonas aeruginosa pneumonia, haemothorax and empyema, while the third patient after 90 days on the mechanical ventilator. This patient suffered from many complications: asystolic cardiac arrest, heparin-induced thrombocytopenia, a left frontal lobe infarct of the cerebral cortex, Serratia marcescens-mediated pneumonia with bacteraemia, acute kidney injury, a left haemothorax requiring thoracotomy and lung decortication, a right pneumothorax requiring tube thoracostomy, hypernatremia associated with seizures and malnutrition. Before lung transplantation, the patient developed increasing clinical signs of pulmonary fibrosis and severe pulmonary hypertension. The first two patients are reported to have achieved independence in daily life activities several months after lung transplantation. Three months after lung transplantation, t h e t h i r d p a t i e n t m a d e i m p r o v e m e n t s i n t h e neurocognitive status and muscular strength at an inpatient rehabilitation centre.

Far less drastic is our proposed treatment of hyperinflammation in COVID-19-induced ARDS with lidocaine, an old drug that is readily available to hospitals all over the world at a low cost. In November 1948, Xylocaine was approved by the Food and Drug Administration (FDA) in the USA [411] . Lidocaine is used as a local anaesthetic [411] , treatment of chronic neuropathic pain [412] but also for the prophylaxis or treatment of ventricular arrhythmia [328, 329] . Recently, intravenous lidocaine has been administered as general anaesthetic replacing opioids in the perioperative settings [413] . Potentially, lidocaine, as a P2X7R antagonist, can abrogate hyperinflammation, can restore the capacity of the immune system to combat secondary co-infections and can improve the clinical condition in critically ill COVID-19 patients. Despite several in vitro [326, 327, 414, 415] , animal studies [319, [416] [417] [418] [419] [420] and patient cohorts [408, 421] on the anti-inflammatory properties of lidocaine, completed clinical trials which deliver a proof of concept (i.e. a randomised controlled trial) have not yet been performed. We postulate that because the maximal tolerable plasma concentration of lidocaine is much lower than the required extracellular concentration to effectively inhibit P2X7Rs, intravenous systemic administration of lidocaine simply cannot not be used to effectively treat hyperinflammation. This is a plausible reason why 5 years after the discovery of lidocaine as a P2X7R inhibitor (published in 2015) [74] the drug is still not used as an antihyperinflammatory treatment in clinical practice.

As stated in the introduction, therapeutic measures that can immediately attenuate the course of SARS-CoV-2-related lung damage are promptly needed on a global scale. In contrast to the investigational P2X7R antagonists described above, continuous subdermal infusion of 2% lidocaine solution to primarily deposit lidocaine into the lymphatics is readily available and can be used in the daily practice immediately and, in principle, even outside the ICU and is very well affordable. Therefore, this therapy deserves to be investigated in larger placebo controlled randomised clinical studies with COVID-19 patients.

However, our experience with subdermal administration of lidocaine in the ICU made clear that this method may not be routinely suitable outside hospital settings. Needless to say that high complexity and high-cost treatments (requiring highly skilled nurses and infusion pump equipment) are inaccessible to low-income COVID-19 patients in developing countries. Also, as the severity and case fatality rate of COVID-19 increase with age [406] , the case fatality rate in elderly patients in nursing homes is strikingly high, and many residents have poor access to medical care [422] . This encouraged us to explore alternative uncomplicated methods of lidocaine administration accessible to everyone, particularly elderly COVID-19 patients and COVID-19 patients in developing countries.

Recently, researchers stated in their article on targeting the P2X7R in COVID-19 that the P2X7R antagonists for human use are available only in oral form and that this might be an inefficient route of drug delivery [298] . We found a solution to this problem. Permeability of the skin and mucous membrane to water, drugs, etc. is said to be dependent on the site of the administration [423, 424] . For example, the permeability constant of the floor of the mouth (sublingual mucosa), lateral border of the tongue and buccal mucosa for tritium-labelled water is 22, 17 and 13 times as high as human skin, respectively [423] . We argue that this also applies to lidocaine. As mentioned above, the endothelium of the mucosal capillaries of the mouth and the gastrointestinal tract belong to the structural type "fenestrated endothelium" allowing molecules to pass from the submucosal tissue into the capillaries [383] . Lidocaine hydrochloride is highly soluble in water (solubility of 680 mg/ml in water) [425] and therefore will mainly be absorbed by the submucosal capillary [426] and the inductive sites of MALT [398, 399] . In contrast, the highly lipophilic lidocaine base (solubility of 4 mg/ml in water, 760 mg/ml in 95% ethanol and 790 mg/ml in chloroform) [425] is preferably absorbed by the local initial lymphatics in the submucosal tissue [426, 427] . In addition, the lymphatic drainage of the floor of the mouth is extensive, involving many lymph nodes [428] [429] [430] [431] .

We estimate that with a sublingual administration of lipophilic lidocaine base (Fig. 1) , we may reach the IC 50 of the P2X7Rs in the draining lymph nodes to control systemic hyperinflammation and avoid toxic lidocaine plasma levels ( Figs. 4 and 6) . Obviously, such solution may also apply to other P2X7R antagonists. We stress that sublingual and buccal administration of lipophilic lidocaine is different from oral administration of lidocaine. Oral administration of lidocaine is aimed at the resorption of the drug in the gastrointestinal tract (Fig.6 ).

There are other methods of targeting the immune cells in the lymphatics, i.e. transdermal administration of lipophilic P2X7R antagonist with skin penetration enhancers (i.e. alpha-terpineol [432] , ethanol [433] and lipid based nanoformulations [434] ), intravenous administration of a P2X7R antagonist using nano-sized drug delivery systems [435] , liposomes or polymer micelles [436] and oral administration of a P2X7R antagonist using delivery systems for intestinal lymphatic drug transport such as chylomicrons [437] .

Acknowledgements We dedicate this report to the memory of Professor Geoffrey Burnstock, the discoverer of purinergic signalling. This report makes clear that purinergic signalling is indispensable to see through the complicated pathophysiology of hyperinflammation in COVID-19.

We would like to thank Coosje van der Pol, PhD, for her valuable and constructive editing suggestions during the writing of this research work.

Author contribution Conception, design and data handling: DH, AS, CvK, EPK and TK Substantial contributions to the conception and design of the manuscript: DH, AS, CvK and EPK Acquisition, analysis or interpretation of data for the manuscript: DH, AS, EPK and TK Drafting and revising the manuscript: DH, AS, CvK, PJvdS, EPK and TK Drafting the manuscript: DH, AS, CvK, PJvdS, EPK and TK Revising the manuscript critically for intellectual content: DH, AS, CvK, PJvdS, EPK and TK Final approval of the version to be published: DH, AS, CvK, PJvdS, EPK and TK Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: DH, AS, CvK, PJvdS, EPK and TK Data Availability The datasets generated during and/or analysed during the current study are not publicly available due to privacy reasons but are available from the corresponding author on reasonable request.

Code availability Not applicable.

Ethics approval, consent to participate and consent for publication The Medical Ethical Committee of the Showa University, School of Medicine, Tokyo, approved the collection, analysis and publication of patients on mechanical ventilation admitted to the ICU (protocol number 3313). 

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A2A receptor signaling promotes peripheral tolerance by inducing T-cell anergy and the generation of adaptive regulatory T cells

Adenosine A2A receptor activation inhibits T helper 1 and T helper 2 cell development and effector function

A2A adenosine receptor (AR) activation inhibits proinflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence

Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptordependent and independent mechanisms

Activation of Th1 and Tc1 cell adenosine A2A receptors directly inhibits IL-2 secretion in vitro and IL-2-driven expansion in vivo

A2A adenosine receptor induction inhibits IFN-gamma production in murine CD4+ T cells

The development and immunosuppressive functions of CD4(+) CD25(+) FoxP3(+) regulatory T cells are under influence of the adenosine-A2A adenosine receptor pathway

Baicalin attenuates bleomycininduced pulmonary fibrosis via adenosine A2a receptor related TGF-beta1-inducedERK1/2 signaling pathway

From inflammation to the onset of fibrosis through A(2A) receptors in kidneys from deceased donors

Adenosine generated by regulatory T cells induces CD8(+) T cell exhaustion in gastric cancer through A2aR pathway

Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages

Adenosine promotes alternative macrophage activation via A2A and A2B receptors

The adenosine-dependent angiogenic switch of macrophages to an M2-like phenotype is independent of interleukin-4 receptor alpha (IL-4Ralpha) signaling

Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages

Regulation of macrophage function by adenosine

Adenosine A2A receptor signaling attenuates LPS-induced pro-inflammatory cytokine formation of mouse macrophages by inducing the expression of DUSP1

Adenosine suppresses activation of nuclear factor-kappaB selectively induced by tumor necrosis factor in different cell types

Netrin-1 dampens pulmonary inflammation during acute lung injury

Neuronal guidance molecule netrin-1 attenuates inflammatory cell trafficking during acute experimental colitis

A role for the low-affinity A2B adenosine receptor in regulating superoxide generation by murine neutrophils

Adenosine augments IL-10 production by macrophages through an A2B receptor-mediated posttranscriptional mechanism

Adenosine receptors in regulation of dendritic cell differentiation and function

The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6

A2B adenosine receptor activation switches differentiation of bone marrow cells to a CD11c(+)Gr-1(+) dendritic cell subset that promotes the Th17 response

Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension

A2B adenosine receptor signaling attenuates acute lung injury by enhancing alveolar fluid clearance in mice

Adenosine receptor activation induces vascular endothelial growth factor in human retinal endothelial cells

A2B adenosine receptors induce IL-19 from bronchial epithelial cells, resulting in TNF-alpha increase

Adenosine signaling increases proinflammatory and profibrotic mediators through activation of a functional adenosine 2B receptor in renal fibroblasts

Alterations in adenosine metabolism and signaling in patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis

Inhibition of A(2B) Adenosine receptor attenuates intestinal injury in a rat model of necrotizing enterocolitis

Adenosine receptor Adora2b antagonism attenuates Brucella abortus 544 infection in professional phagocyte RAW 264.7 cells and BALB/c mice

Mast cell-mediated stimulation of angiogenesis: cooperative interaction between A2B and A3 adenosine receptors

Involvement of adenosine A3 receptors in the chemotactic navigation of macrophages towards apoptotic cells

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors

A3 and P2Y2 receptors control the recruitment of neutrophils to the lungs in a mouse model of sepsis

Selfgenerated chemoattractant gradients: attractant depletion extends the range and robustness of chemotaxis

Self-generated chemotactic gradients-cells steering themselves

Directional tissue migration through a selfgenerated chemokine gradient

Where to go: breaking the symmetry in cell motility

Cell migration: sinking in a gradient

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of PI 3-kinase/Akt and NF-kappaB activation in murine BV2 microglial cells

Activation of adenosine A3 receptor alleviates TNF-alpha-induced inflammation through inhibition of the NF-kappaB signaling pathway in human colonic epithelial cells

Adenosine acts through an A3 receptor to prevent the induction of murine anti-CD3-activated killer T cells

Activation of adenosine A(3) receptor inhibits microglia reactivity elicited by elevated pressure

A3 adenosine receptor signaling influences pulmonary inflammation and fibrosis

Activation of adenosine A3 receptor inhibits inflammatory cytokine production in colonic mucosa of patients with ulcerative colitis by down-regulating the nuclear factor-kappa B signaling

Purinergic control of inflammation and thrombosis: role of P2X1 receptors

Oleic and linoleic acids induce the release of neutrophil extracellular traps via pannexin 1-dependent ATP release and P2X1 receptor activation

Pannexin-1 hemichannel-mediated ATP release together with P2X1 and P2X4 receptors regulate T-cell activation at the immune synapse

ATP induces P2X7 receptor-independent cytokine and chemokine expression through P2X1 and P2X3 receptors in murine mast cells (Article retracted in 2011 due to figure irregularities)

ATP release and autocrine signaling through P2X4 receptors regulate gammadelta T cell activation

P2X4 receptors control the fate and survival of activated microglia

Purinergic P2X4 receptors and mitochondrial ATP production regulate T cell migration

Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4-mediated calcium entry in microglia

Frontline Science: P2Y11 receptors support T cell activation by directing mitochondrial trafficking to the immune synapse

The purinergic receptor P2Y11 choreographs the polarization, mitochondrial metabolism, and migration of T lymphocytes

Purinergic regulation of the immune system

Activation of P2X(7) receptor by ATP plays an important role in regulating inflammatory responses during acute viral infection

Molecular mechanisms regulating NLRP3 inflammasome activation

Activation and regulation of the inflammasomes

P2X4 receptor regulates P2X7 receptordependent IL-1beta and IL-18 release in mouse bone marrowderived dendritic cells

Silicainduced inflammasome activation in macrophages: role of ATP and P2X7 receptor

Neutrophil P2X7 receptors mediate NLRP3 inflammasomedependent IL-1beta secretion in response to ATP

Uric acid induces caspase-1 activation, IL-1beta secretion and P2X7 receptor dependent proliferation in primary human lymphocytes

Mice lacking the purinergic receptor P2X5 exhibit defective inflammasome activation and early susceptibility to listeria monocytogenes

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

P2X(7) is a scavenger receptor for apoptotic cells in the absence of its ligand, extracellular ATP

The P2X7 receptor is an important regulator of extracellular ATP levels

P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells

P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling

ATP release due to Thy-1-integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

Purinergic P2X7 receptor regulates lung surfactant secretion in a paracrine manner

gamma-Irradiation induces P2X(7) receptor-dependent ATP release from B16 melanoma cells

P2X7-mediated ATP secretion is accompanied by depletion of cytosolic ATP

ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X(7) receptors

ATP in the tumour microenvironment drives expression of nfP2X7, a key mediator of cancer cell survival

P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration

ATP inhibits the generation and function of regulatory T cells through the activation of purinergic P2X receptors

P2X7 receptor promotes intestinal inflammation in chemically induced colitis and triggers death of mucosal regulatory T cells

The P2X7 receptor antagonist, oxidized adenosine triphosphate, ameliorates renal ischemia-reperfusion injury by expansion of regulatory T cells

Purinergic P2X7 receptor drives T cell lineage choice and shapes peripheral γδ cells

Receptor activity limits accumulation of T cells within tumors

P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 beta release

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho

Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor

Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing

P2X7 receptors induce degranulation in human mast cells

P2X7 receptors regulate NKT cells in autoimmune hepatitis

Human P2X7 receptor activation induces the rapid shedding of CXCL16

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells

P2X7 receptor-dependent tuning of gut epithelial responses to infection

Autocrine regulation of T-cell activation by ATP release and P2X7 receptors

ATP-gated ionotropic P2X7 receptor controls follicular T helper cell numbers in Peyer's patches to promote host-microbiota mutualism

Extracellular NAD+ shapes the Foxp3+ regulatory T cell compartment through the ART2-P2X7 pathway

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R

A critical role for P2X7 receptor-induced VCAM-1 shedding and neutrophil infiltration during acute lung injury

Activation of P2X7 receptors decreases the proliferation of murine luteal cells

Activation of P2X7 purinoceptor-stimulated TGF-beta 1 mRNA expression involves PKC/MAPK signalling pathway in a rat brain-derived type-2 astrocyte cell line, RBA-2

Adenosine 5'-triphosphate stimulates the increase of TGF-beta1 in rat mesangial cells under high-glucose conditions via reactive oxygen species and ERK1/2

P2X7 receptor (P2X7R) of microglia mediates neuroinflammation by regulating (NOD)-like receptor protein 3 (NLRP3)inflammasome-dependent inflammation after spinal cord injury

2020) lncRNA uc.48+ regulates immune and inflammatory reactions mediated by the P2X(7) receptor in type 2 diabetic mice

Paxillin mediates ATP-induced activation of P2X7 receptor and NLRP3 inflammasome

ADP/P2Y(1) aggravates inflammatory bowel disease through ERK5-mediated NLRP3 inflammasome activation

CD14 release induced by P2X7 receptor restricts inflammation and increases survival during sepsis

P2X7R orchestrates the progression of murine hepatic fibrosis by making a feedback loop from macrophage to hepatic stellate cells

The P2X7 receptor mediates NLRP3-dependent IL-1β secretion and promotes phagocytosis in the macrophage response to Treponema pallidum

Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages

Pannexin-1 mediates large pore formation and interleukin-1beta release by the ATP-gated P2X7 receptor

P2X7 nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes

NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells

The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7)

P2Y(13) and P2X(7) receptors modulate mechanically induced adenosine triphosphate release from mast cells

Synergistic action between inhibition of P2Y12/P2Y1 and P2Y12/thrombin in ADP-and thrombin-induced human platelet activation

ADP-mediated upregulation of expression of CD62P on human platelets is critically dependent on co-activation of P2Y1 and P2Y12 receptors

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation

receptor regulates VCAM-1 membrane and soluble forms and eosinophil accumulation during lung inflammation

Signaling through purinergic receptor P2Y(2) enhances macrophage IL-1β production

) Receptor induces L. amazonensis infection control in a mechanism dependent on caspase-1 activation and IL-1β secretion

Airway brush cells generate cysteinyl leukotrienes through the ATP sensor P2Y2

Involvement of P2X4 and P2Y12 receptors in ATPinduced microglial chemotaxis

Akt activation is involved in P2Y12 receptor-mediated chemotaxis of microglia

P2Y6 receptor antagonist MRS2578 inhibits neutrophil activation and aggregated neutrophil extracellular trap formation induced by gout-associated monosodium urate crystals

P2Y6 receptor signaling pathway mediates inflammatory responses induced by monosodium urate crystals

Responses of macrophages to the danger signals released from necrotic cells

UDP facilitates microglial phagocytosis through P2Y6 receptors

The activation of P2Y6 receptor in cultured spinal microglia induces the production of CCL2

P2Y6 receptor-mediated microglial phagocytosis in radiationinduced brain injury

Protein interacting C-kinase 1 modulates surface expression of P2Y6 purinoreceptor, actin polymerization and phagocytosis in microglia

UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

P2Y receptor signaling regulates phenotype and IFNalpha secretion of human plasmacytoid dendritic cells

Human neutrophil peptides induce interleukin-8 production through the P2Y6 signaling pathway

P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP-1-dependent manner in epithelial cells

Extracellular UDP and P2Y6 function as a danger signal to protect mice from vesicular stomatitis virus infection through an increase in IFN-beta production

Deficiency enhances dendritic cell-mediatedTh1/Th17 differentiation and aggravates experimental autoimmune encephalomyelitis

P2Y6 receptor inhibition aggravates ischemic brain injury by reducing microglial phagocytosis

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor

P2Y11 purinoceptor mediates the ATPenhanced chemotactic response of rat neutrophils

Signal transduction and white cell maturation via extracellular ATP and the P2Y11 receptor

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells

ATP gradients inhibit the migratory capacity of specific human dendritic cell types: implications for P2Y11 receptor signaling

3,1-phenylene-carbonylimino-3,1-(4-methylphenylene)-car bonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] is a non-nucleotide P2Y11 agonist and stimulates release of interleukin-8 from human monocytederived dendritic cells

Autocrine regulation of macrophage activation via exocytosis of ATP and activation of P2Y11 receptor

Involvement of P2Y12 receptor in vascular smooth muscle inflammatory changes via MCP-1 upregulation and monocyte adhesion

Role of the P2Y12 receptor in the modulation of murine dendritic cell function by ADP

Purinergic receptor P2RY12-dependent microglial closure of the injured blood-brain barrier

The P2Y12 receptor regulates microglial activation by extracellular nucleotides

expression and function in alternatively activated human microglia

P2Y12 receptors in primary microglia activate nuclear factor of activated T-cell signaling to induce C-C chemokine 3 expression

P2Y(12) antagonism results in altered interactions between platelets and regulatory T cells during sepsis

P2Y12 receptor mediates microglial activation via RhoA/ ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine

ADP acting on P2Y13 receptors is a negative feedback pathway for ATP release from human red blood cells

The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8

A selective high-affinity antagonist of the P2Y14 receptor inhibits UDP-glucose-stimulated chemotaxis of human neutrophils

P2Y(14) receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages

TNAP, TrAP, ecto-purinergic signaling, and bone remodeling

Purinergic signalling in the musculoskeletal system

Tissue-nonspecific alkaline phosphatase regulates purinergic transmission in the central nervous system during development and disease

History of ectonucleotidases and their role in purinergic signaling

Medicinal chemistry of adenosine, P2Y and P2X receptors

P2X receptors

Allosteric modulation of ATP-gated P2X receptor channels

Activation and regulation of purinergic P2X receptor channels

P2Y2 receptor desensitization on single endothelial cells

A(2B), and A(3) receptors expressed in Chinese hamster ovary Purinergic Signalling cells all mediate the phosphorylation of extracellular-regulated kinase 1/2

Internalization and desensitization of adenosine receptors

Molecular physiology of P2X receptors

Update of P2X receptor properties and their pharmacology: IUPHAR review 30

Natural compounds with P2X7 receptor-modulating properties

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

Consideration of pannexin 1 channels in COVID-19 pathology and treatment

Cathelicidin LL-37 affects surface and intracellular toll-like receptor expression in tissue mast cells

Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation

Nucleotide signalling during inflammation

Purinergic signaling, DAMPs, and inflammation

DAMPs from cell death to new life

Immune mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia

Update of P2Y receptor pharmacology: IUPHAR Review 27

Identification of ectonucleotidases CD39 and CD73 in innate protection during acute lung injury

Toll-like receptor-mediated activation of CD39 internalization in BMDCs leads to extracellular ATP accumulation and facilitates P2X7 receptor activation

P2X ion channel receptors and inflammation

Rossato M (2020) A rationale for targeting the P2X7 receptor in coronavirus disease 19

The P2X7 receptor in inflammatory diseases: angel or demon?

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Agonist-induced internalization of the P2Y2 receptor

Sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy

Immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach

The compensatory antiinflammatory response syndrome (CARS) in critically ill patients

Sir Isaac Newton, sepsis, SIRS, and CARS

Safdar N (2021) Prevalence and outcomes of coinfection and superinfection with SARS-CoV-2 and other pathogens: a systematic review and meta-analysis

CD25+CD4+ regulatory T cells suppress CD4+ T cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice

Clinical and immunological features of severe and moderate coronavirus disease 2019

Th17 and Treg cells function in SARS-CoV2 patients compared with healthy controls

The laboratory tests and host immunity of COVID-19 patients with different severity of illness

Imbalance of regulatory and cytotoxic SARS-CoV-2-reactive CD4(+) T cells in COVID-19

Systemic blockade of P2X7 receptor protects against sepsis-induced intestinal barrier disruption

Blockage of P2X7 attenuates acute lung injury in mice by inhibiting NLRP3 inflammasome

Deletion of P2X7 attenuates hyperoxiainduced acute lung injury via inflammasome suppression

The potential involvement of P2X7 receptor in COVID-19 pathogenesis: a new therapeutic target?

Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology

The protective effect of lidocaine on septic rats via the inhibition of high mobility group box 1 expression and NF-κB activation

Lidocaine protects against renal and hepatic dysfunction in septic rats via downregulation of Toll-like receptor 4

Local anesthetics reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis

Lidocaine reduces neutrophil recruitment by abolishing chemokine-induced arrest and transendothelial migration in septic patients

Targeting potential drivers of COVID-19: neutrophil extracellular traps

Radic M (2020) Neutrophilia and NETopathy as key pathologic drivers of progressive lung impairment in patients with COVID-19

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Neutrophils and neutrophil extracellular traps drive necroinflammation in COVID-19

Effects of lidocaine on regulatory T cells in atopic dermatitis

Lidocaine increases the anti-inflammatory cytokine IL-10 following mechanical ventilation in healthy mice

Intravenous lidocaine decreases tumor necrosis factor alpha expression both locally and systemically in pigs undergoing lung resection surgery

Molecular mechanisms of action of systemic lidocaine in acute and chronic pain: a narrative review

Pharmacokinetics and pharmacodynamics of lignocaine: a review

Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

Ligand-based design and synthesis of novel sodium channel blockers from a combined phenytoin-lidocaine pharmacophore

Intrathecal lidocaine pretreatment attenuates immediate neuropathic pain by modulating Nav1.3 expression and decreasing spinal microglial activation

Fast-onset lidocaine block of rat NaV1.4 channels suggests involvement of a second highaffinity open state

Potent inactivationdependent inhibition of adult and neonatal NaV1.5 channels by lidocaine and levobupivacaine

Lidocaine reduces the transition to slow inactivation in Na(v)1.7 voltage-gated sodium channels

Antinociceptive activities of lidocaine and the nav1.8 blocker a803467 in diabetic rats

Effect of verapamil and lidocaine on TRPM and NaV1.9 gene expressions in renal ischemia-reperfusion

Nebulized lidocaine ameliorates allergic airway inflammation via downregulation of TLR2

The inhibition of the N-methyl-D-aspartate receptor channel by local anesthetics in mouse CA1 pyramidal neurons

The role of voltage-gated sodium channels in pain signaling

Voltage-gated sodium channel Nav1.7 maintains the membrane potential and regulates the activation and chemokine-induced migration of a monocyte-derived dendritic cell subset

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations

Sodium channel Nav1.3 is expressed by polymorphonuclear neutrophils during mouse heart and kidney ischemia in vivo and regulates adhesion, transmigration, and chemotaxis of human and mouse neutrophils in vitro

A voltage-gated sodium channel is essential for the positive selection of CD4(+) T cells

HYP-17, a novel voltage-gated sodium channel blocker, relieves inflammatory and neuropathic pain in rats

A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat

Additive antinociceptive effects of the selective Nav1.8 blocker A-803467 and selective TRPV1 antagonists in rat inflammatory and neuropathic pain models

Selective Na(V)1.7 Antagonists with long residence time show improved efficacy against inflammatory and neuropathic pain

Voltage-gated sodium channel inhibitor reduces atherosclerosis by modulating monocyte/ macrophage subsets and suppressing macrophage proliferation

Effects of bupivacaine and tetrodotoxin on carrageenan-induced hind paw inflammation in rats (Part 1): hyperalgesia, edema, and systemic cytokines

Effects of bupivacaine and tetrodotoxin on carrageenan-induced hind paw inflammation in rats (Part 2): cytokines and p38 mitogen-activated protein kinases in dorsal root ganglia and spinal cord

Voltage-gated sodium channel blockers: new perspectives in the treatment of neuropathic pain

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

IL-6 and IL-10 as predictors of disease severity in COVID-19 patients: results from meta-analysis and regression

Circulating levels of interleukin-6 and Interleukin-10, but not tumor necrosis factor-alpha, as potential biomarkers of severity and mortality for COVID-19: systematic review with meta-analysis

Clinical features of severe pediatric patients with coronavirus disease 2019 in Wuhan: a single center's observational study

Interleukin-1β inhibits voltage-gated sodium currents in a time-and dose-dependent manner in cortical neurons

Interleukin-6 inhibits voltage-gated sodium channel activity of cultured rat spinal cord neurons

Interleukin-10 down-regulates voltage gated sodium channels in rat dorsal root ganglion neurons

ATP-P2X7 receptor modulates axon initial segment composition and function in physiological conditions and brain injury

Inhibition of P2X7 receptor ameliorates nuclear factor-kappa B mediated neuroinflammation induced by status epilepticus in rat hippocampus

P2X7 receptor positively regulates MyD88-dependent NF-κB activation

The role of N-methyl-D-aspartate(NMDA) receptors in pain: a review

Blockade of N-methyl-Daspartate receptor prevents hypoxic neuronal death and cytokine release

N-methyl-Daspartate receptor antagonist therapy suppresses colon motility and inflammatory activation six days after the onset of experimental colitis in rats

N-methyl-D-aspartate receptor antagonist MK-801 suppresses glial pro-inflammatory cytokine expression in morphinetolerant rats

NMDA-receptor antagonists block B-cell function but foster IL-10 production in BCR/CD40-activated B cells

Local anaesthetics have different mechanisms and sites of action at the recombinant N-methyl-D-aspartate(NMDA) receptors

Local anaesthetics inhibit signalling of human NMDA receptors recombinantly expressed in Xenopus laevis oocytes: role of protein kinase C

Immunosuppression by N-methyl-D-aspartate receptor antagonists is mediated through inhibition of Kv1.3 and KCa3.1 channels in T cells

A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA-and hypoxia/hypoglycemiainduced cell death

Physiological and pathological functions of P2X7 receptor in the spinal cord

Physiological roles and potential therapeutic applications of the P2X7 receptor in inflammation and pain

T cell trafficking through lymphatic vessels

Taking the lymphatic route: dendritic cell migration to draining lymph nodes

Transient migration of large numbers of CD14(++) CD16(+) monocytes to the draining lymph node after onset of inflammation

Lymph node subcapsular sinus macrophages as the frontline of lymphatic immune defense

The lymph node neutrophil

Mast cell activation and migration to lymph nodes during induction of an immune response in mice

Lymph node trafficking and antigen presentation by endobronchial eosinophils

Cutting edge: basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3, but infectioninduced Th2 immunity can develop without basophil lymph node recruitment or IL-3

Morphological heterogeneity of endothelium

Dermal capillary clearance: physiology and modeling

Lymphatic vessel network structure and physiology

Comparison of intradermal and subcutaneous injections in lymphatic mapping

Intradermal pathways. In: Spencer RP (ed) New procedures in nuclear medicine

Pharmacokinetics of lidocaine and its active metabolite, monoethylglycinexylidide, after intravenous administration of lidocaine to awake and isoflurane-anesthetized cats

Efficacy of lidocaine for pain control in subcutaneous infiltration during liposuction

Pharmacokinetics, lymph node uptake, and mechanistic PK model of near-infrared dye-labeled bevacizumab after IV and SC administration in mice

The lymphatic system plays a major role in the intravenous and subcutaneous pharmacokinetics of trastuzumab in rats

Docetaxel accumulates in lymphatic circulation following subcutaneous delivery compared to intravenous delivery in rats

Efficacy and safety of CE-224,535, an antagonist of P2X7 receptor, in treatment of patients with rheumatoid arthritis inadequately controlled by methotrexate

Clinical evaluation of the efficacy of the P2X7 purinergic receptor antagonist AZD9056 on the signs and symptoms of rheumatoid arthritis in patients with active disease despite treatment with methotrexate or sulphasalazine

Recent advances in CNS P2X7 physiology and pharmacology: focus on neuropsychiatric disorders

Clinical pharmacokinetics, pharmacodynamics, safety, and tolerability of JNJ-54175446, a brain permeable P2X7 antagonist, in a randomised singleascending dose study in healthy participants

Safety and efficacy of an oral inhibitor of the purinergic receptor P2X7 in adult patients with moderately to severely active Crohn's disease: a randomized placebo-controlled, double-blind, phase iia study

Particle uptake by Peyer's patches: a pathway for drug and vaccine delivery

Targeting immunomodulatory agents to the gut-associated lymphoid tissue

Lipid-based delivery systems and intestinal lymphatic drug transport: a mechanistic update

Population pharmacokinetics of lidocaine administered during and after cardiac surgery

Successful management of intractable epilepsy with lidocaine tapes and continuous subcutaneous lidocaine infusion

Acute respiratory distress syndrome: the Berlin Definition

Serum SP-A and KL-6 levels can predict the improvement and deterioration of patients with interstitial pneumonia with autoimmune features

The coronavirus is most deadly if you are older and male -new data reveal the risks

Prognostic factors for severity and mortality in patients infected with COVID-19: A systematic review

Lidocaine to reduce the severity of covid-19 cases

Impact of intravenous lidocaine on clinical outcomes of patients with ARDS during COVID-19 pandemia (LidoCovid): a structured summary of a study protocol for a randomised controlled trial

Lung transplantation for patients with severe COVID-19

Lidocaine: the origin of a modern local anesthetic

Topical lidocaine for neuropathic pain in adults

Perioperative use of intravenous lidocaine

Lidocaine attenuates proinflammatory cytokine production induced by extracellular adenosine triphosphate in cultured rat microglia

Lidocaine attenuates lipopolysaccharide-induced inflammatory responses in microglia

Effect of inhaled and intravenous lidocaine on inflammatory reaction in endotoxaemic rats

The protective effect of lidocaine on lipopolysaccharide-induced acute lung injury in rats through NF-κB and p38 MAPK signaling pathway and excessive inflammatory responses

Lidocaine attenuates lipopolysaccharide-induced acute lung injury through inhibiting NF-kappaB activation

Surfactant lavage with lidocaine improves pulmonary function in piglets after HCl-induced acute lung injury

Lidocaine attenuates acute lung injury induced by a combination of phospholipase A2 and trypsin

Analgesic and anti-inflammatory effects of lignocaine-prilocaine(EMLA) cream in human burn injury

Long-term care policy after Covid-19 -solving the nursing home crisis

The permeability of human oral mucosa and skin to water

Non-invasive systemic drug delivery through mucosal routes

Lidocaine base and hydrochloride

Estimating the maximal potential for intestinal lymphatic transport of lipophilic drug molecules

Lipids and lipidbased formulations: optimizing the oral delivery of lipophilic drugs

Lingual lymph nodes: anatomy, clinical considerations, and oncological significance

Anatomic-histologic study of the floor of the mouth: the lingual lymph nodes

Lymphatic architecture of the oral region -beneath the buccal mucosa

Lymphatics of the floor of the mouth and neck: anatomical studies related to contralateral drainage pathways

The effects of some permeability enhancers on the percutaneous absorption of lidocaine

Highthroughput screening of potential skin penetration-enhancers using stratum corneum lipid liposomes: preliminary evaluation for different concentrations of ethanol

Advanced drug delivery to the lymphatic system: lipid-based nanoformulations

Nano-sized drug delivery systems for lymphatic delivery

Recent advances in lymphatic targeted drug delivery system for tumor metastasis

Liposomal delivery systems for intestinal lymphatic drug transport

Djo Hasan, MD, PhD is a neurointensivist (ret.) with extensive experience in treating mechanically ventilated patients with ARDS at the Erasmus University H o s p i t a l R o t t e r d a m , T h e Netherlands and immune therapy at the Cell Therapy Clinic in Duderstadt, Germany. He is currently involved in the development of lidocaine as a