NF-kappaB pathway as a potential target for treatment of critical stage COVID-19 patients

Patients infected with SARS-CoV-2 show a wide spectrum of clinical manifestations ranging from mild febrile illness and cough up to acute respiratory distress syndrome, multiple organ failure and death. Data from patients with severe clinical manifestations compared to patients with mild symptoms indicate that highly dysregulated exuberant inflammatory responses correlate with severity of disease and lethality. Epithelial-immune cell interactions and elevated cytokine and chemokine levels, i.e. cytokine storm, seem to play a central role in severity and lethality in COVID-19. The present perspective places a central cellular pro-inflammatory signal pathway, NF-kappaB, in the context of recently published data for COVID-19 and provides a hypothesis for a therapeutic approach aiming at the simultaneous inhibition of whole cascades of pro-inflammatory cytokines and chemokines. The simultaneous inhibition of multiple cytokines/chemokines is expected to have much higher therapeutic potential as compared to single target approaches to prevent cascade (i.e. triggering, synergistic, and redundant) effects of multiple induced cytokines and chemokines in critical stage COVID-19 patients.


Introduction
Coronaviruses -enveloped positive-sense, single-stranded RNA viruses -are broadly distributed in humans and animals. While most human coronavirus (hCoV) infections show mild symptoms, there are highly pathogenic hCoV, including the severe acute respiratory syndrome virus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), with 10% and 37% mortality, respectively. The novel coronavirus SARS-CoV-2 with more than 30 mio infected persons and more than 940.000 deaths worldwide (https://coronavirus.jhu.edu/) September 18, 2020 has become a global pandemic with enormous medical and socio-economic burden. Patients infected with SARS-CoV-2 show a wide spectrum of clinical manifestations ranging from mild febrile illness and cough up to acute respiratory distress syndrome (ARDS), multiple organ failure, and death, i.e. a clinical picture in severe cases that is very similar to that seen in SARS-CoV and MERS-CoV infected patients. While younger individuals show predominantly mild-to-moderate clinical symptoms, elderly individuals frequently exhibit severe clinical manifestations [1][2][3][4] . Post-mortem analysis showed Diffuse Alveolar Disease with capillary congestion, cell necrosis, interstitial oedema, platelet-brin thrombi, and in ltrates of macrophages and lymphocytes 5 .
Recently, the induction of endotheliitis in various organs (including lungs but also in heart and kidney and intestine) by SARS-CoV-2 infection as a direct consequence of viral involvement and of the host in ammatory response was shown [6][7] .
SARS-CoV-2 binds with its spike (S) protein to the angiotensin-converting enzyme-related carboxypeptidase-2 (ACE-2) receptor on the host cell using the cellular serine protease TMPRSS2 for S protein priming 8 . The ACE-2 receptor is widely expressed in pulmonary and cardiovascular tissues, hematopoietic cells, including monocytes and macrophages which may explain the broad range of pulmonary and extra-pulmonary effects of SARS-CoV-2 infection including cardiac, gastrointestinal organs, and kidney affection 6 .

Cytokine & Chemokine Storm As A Hallmark Of Covid-19
The morbidity and mortality of highly pathogenic hCoV is still incompletely understood. Virus-induced cytopathic effects and viral evasion of the host immune response play a role in disease severity. However, clinical data from patients, in particular those with severe clinical manifestations indicate that highly dysregulated exuberant in ammatory and immune responses correlate with severity of disease and lethality 1,[5][6][7][9][10][11] . Signi cantly elevated cytokine and chemokine levels, i.e. cytokine storm, seem to play a central role in severity and lethality in SARS-CoV-2 infections, with elevated plasma levels of IL-1b, IL-7, IL-8, IL-9, IL-10, G-CSF, GM-CSF, IFNg, IP-10, MCP-1, MIP-1a, MIP-1b, PDGF, TNFa, and VEGF in both, ICU (Intensive care unit) patients and non-ICU patient. Signi cantly higher plasma levels of IL-2, IL-7, IL-10, G-SCF, IP-10, MCP-1, MIP-1a, and TNFa were found in patients with severe pneumonia developing ARDS and requiring ICU admission and oxygen therapy compared to non-ICU patients showing pneumonia without ADRS 1 .
Recently, immune pro ling of COVID-19 patients revealed distinct immunotypes with therapeutic implications, i.e. immunotype 1 characterized by a robust CD4 T cell activation, proliferating effector CD8 T cells was connected to severe disease, immunotype 2 with more traditional effector CD8 T cell subsets, less CD4 T cell activation and memory B cells, showed intermediate clinical outcome, and immunotype 3 with only minimal lymphocyte activation response showed the least clinical symptomatic picture 12  Interestingly, also for SARS-CoV and MERS-CoV infected patients, increased levels of pro-in ammatory cytokines in serum, including IL-1b, IL-6, IL-12, IFNg, TNFa, IL-15, IL-17 and chemokines including CCL2 (MCP-1), CXCL10 (IP-10), CXCL9 (MIG), CCL-5, IL-8 were associated with pulmonary in ammation and extensive lung damage [15][16][17] . Both, the nucleocapsid protein and the spike protein of SARS-CoV were shown to induce pro-in ammatory cytokines via activation of the NF-kB pathway 18,19 . Using comprehensive genomic analyses Smits et al showed that aged macaques have a stronger host response to virus infection compared to young macaques, with an increase in differential expression of genes associated with in ammation, with NF-kB as central player, whereas expression of type I interferon was reduced indicating a possible negative-feedback cross-talk between the pro-in ammatory NF-kB pathway and IFN-induced antiviral pathways 20 .
Furthermore, beside the three highly pathogenic hCoV, also H5N1 and certain H1N1 in uenza virus infections with high lethality in humans, showed excessive alveolar immune in ammatory in ltrates and high levels of pro-in ammatory cytokines and chemokines including IP10/CXCL10, MIG, IL-6, IL-8 and RANTES in human cell lines, mice, and macaques [21][22][23][24][25][26] and in humans infected with H1N1 27 . Taken together, these multiple reports indicate a potential common pathophysiological mechanism of highly dysregulated exuberant in ammatory reactions in response to various acute respiratory RNA virus infections.

Inhibition Of Nf-kb Can Inhibit Virus-induced Cytokine Storm
We have previously shown that elevated cytokine release of IL-a/b, IL-6, MIP-1b, RANTES and TNF-a induced by highly pathogenic avian H5N1 in uenza A virus was signi cantly reduced by application of the proteasome inhibitor VL-01 in vivo 32 . The underlying mechanism of this inhibitory effect of proteasome inhibitors is supposed to be mediated largely by the inhibition of one of the most prominent cellular transcription pathways, NF-kB. The inhibition of the nuclear translocation of the transcription factor NF-kB by proteasome inhibitors has been described [33][34][35] . It is mediated via the inhibition of the proteasomal degradation of the cytosolic inhibitor IkBa, this way keeping NF-kB sequestered by IkBa in the cytosol and thereby inhibiting the otherwise induced translocation of NF-kB to the nucleus where it would initiate the transcription of multiple pro-in ammatory proteins, such as cytokines, chemokines, adhesion molecules and growth factors (see Figure 1). Activation of the NF-kB pathway has been described for very different signal-receptor bindings, including binding of LPS to TLR4, binding of cytokines like IL-1 and TNFa to their respective receptors, or recognition of RNA viruses by Toll-like receptors, TLR7/8. Importantly, all these different signaling pathways join into a common downstream signaling sequence of phosphorylation of the cytosolic inhibitor IkBa which triggers its ubiquitination and proteasomal degradation resulting in release and translocation of NF-kB into the nucleus 35 (see Figure  1). These data suggest that interfering at these late stages (i.e. phosphorylation, ubiquitination, and/or proteasomal degradation of IkBa) of the pathway will inhibit NF-kB activation, irrespectively of the initial triggering signal. We could demonstrate the inhibitory effect of proteasome inhibitors on nuclear translocation NF-kB in various cell types such as human macrophages after stimulation with TNFa in vitro. Without stimulation of the NF-kB pathway, p65/p50 (p65 FITC stained) is sequestered in the cytosol by its inhibitor IkB. Following stimulation by TNFa, NF-kB translocates to the nucleus (shown by coinciding p65 staining and nucleus staining by DAPI). NF-kB nuclear translocation after  (Fig. 3). Treatment with proteasome inhibitor signi cantly inhibited the release of IL-1, IL-6, TNFa, MIP-1 and CXCL1 at the peak time-points in Balb/c mice after infection with the highly pathogenic avian H5N1 in uenza A virus (Fig. 3). Importantly, proteasome inhibition signi cantly decreased the release for all, early and late cytokines and chemokines, and resulted in signi cantly increased survival of mice after infection with the highly pathogenic avian H5N1 in uenza A virus 32 .
In order to investigate whether the inhibition of cytokine and chemokine release by inhibition of the nuclear translocation of NF-kB is a general mechanism, an acute lung injury (ALI) mouse model with LPS challenge was used. This model provides a rapid and strong systemic induction of pro-in ammatory cytokines and chemokines. Balb/c mice were treated i.v. with 25 mg/kg VL-01, followed by i.p. application of 20 µg LPS. Serum samples for cytokine analysis were collected before (-4hrs) LPS treatment (control) and after LPS treatment (1.5 and 3 hrs). Again distinct release patterns were found for different cytokines/chemokines, with TNFa, IL-1b, MIP-1a and MIP-1b peaking already 1.5 hrs after LPS challenge, followed by others, such as IL-6, RANTES, IL-12p40 and KC peaking 3 hrs after LPS stimulus (Fig. 4). Importantly, treatment of mice with proteasome inhibitor signi cantly reduced release of the whole panel of pro-in ammatory cytokines and chemokines. Taken together, these data generated in different models demonstrate the principal potency of proteasome inhibitors to interfere with the pro-in ammatory effects, by inhibiting the translocation of NF-kB to the nucleus. and chemokines including CCL-2, CCL-5, CXCL-1, CXCL-2, CXCL-10, correlating with increased survival. In their study four different NF-kB inhibitors, with different mechanism of inhibition, i.e. CAPE, resveratrol, Bay11-7082, and parthenolide, were used. All four inhibitors were shown to inhibit NF-kB activity, and to decrease the expression levels of pro-in ammatory cytokines and chemokines, without affecting viral titers or cell viability 36 .
Moreover, Acetylsalicylic acid (ASA) and other salicylates -in contrast to pure (COX) cyclooxygenase inhibitors, such as indomethacin -are well-known inhibitors of NF-κB activation by acting as speci c inhibitors of IKK2 -a kinase essential for phosphorylating IkB 37 . Furthermore, D,L-lysineacetylsalicylate•glycine (LASAG) a water-soluble salt of ASA (licensed as Aspirin i.v.®) was shown to decrease activation of promoter constructs of NF-κB-dependent genes for IL-6 and IL-8 and to improve the time to alleviation of in uenza symptoms in hospitalized patients in a phase II clinical trial 38  Finally, support for the role of NF-kB pathway in critical stage COVID-19 patients is provided by recent results from the RECOVERY trial. Dexamethasone was found to signi cantly reduce death in patients with severe respiratory complications of COVID-19 requiring ventilation by up to one third 43 . Dexamethasone -a broadly used glucocorticoid anti-in ammatory drug -is assumed to mediate its anti-in ammatory activity at least partially via downregulation of the NF-kB activity 44 , probably by suppression of NF-kB expression 45 and/or increased expression of IkB in the cytoplasm 46 .
All these data collectively strongly indicate that inhibition of the NF-kB signal pathway may be a promising target to control SARS-CoV-2 induced excessive immune activation associated with systemic cytokine and chemokine release, capillary leakage and multi-organ tissue damage (Figure 1).

Discussion
Reaching beyond the possibilities of currently evaluated drugs for single targets of the cytokine cascade, e.g. monoclonal antibodies against the IL-6 receptor 47-50 or IL-1 receptor antagonist 51 the inhibition of NF-kB pathway -preferably in parallel at several sensitive points (Figure 1) -could provide the unique potential to inhibit the release of multiple cytokines simultaneously, in particular strongly proin ammatory cytokines including IL-1, IL-6, TNFa and chemokines including MIP-1 and CXCL1.
Multiple approved medications with implicated NF-kB activity involving NSAIDs (e.g. acetylsalicylic acid, Aspirin), BTK inhibitors (e.g. Ibrutinib, Acalabrutinib), steroids (e.g. Dexamethasone), and proteasome inhinbitors are in wide-spread clinical use. Several registered proteasome inhibitors (Bortezomib, Car lzomib or Ixazomib) are available for treatment of oncological indications 52 . In contrast to oncological indications where eight (or more) treatment cycles are routinely applied, it seems plausible that just few applications of proteasome inhibitors will be su cient to downregulate the acute cytokine storm in COVID-19 patients.
Importantly, compared to single target approaches, a simultaneous inhibition of multiple cytokines/chemokines using (preferably a combination of) inhibitors of the NF-kB pathway, may be highly advantageous compared to single target approaches to compensate for redundant, synergistic, and triggering effects of multiple cytokines (i.e. cytokine cascade) released in critical cases of highly pathogenic hCoV infection (but also H5N1 or H1N1 infection). Whereas some clinical e cacy in COVID-19 patients has been recorded 47-51 also several notable caveats and limitations to the e cacy of singlecytokine targeting approaches have been seen and have led to the question which cytokine to target in a raging storm, calling for a systemic approach for simultaneous inhibition of multiple cytokines, including also early expressed cytokines and chemokines 53 .
In contrast to another recently suggested systemic approach for simultaneous inhibition of cytokines by JAK inhibitors 54 , NF-kB inhibition will inhibit predominantly highly pro-in ammatory cytokines and chemokines, such as TNFa, IL-1, IL-6, MCP-1, MIP-1, which are expected to be primarily involved in exuberant systemic in ammatory responses (as proven at the cellular level for COVID-19 patients by the study of Chua et al. 14 ) rather than cytokines primarily involved in antiviral responsiveness, such as IFNgwhich is primarily dependent on other pathways, i.e. JAK/STAT 20 .
Although there are still many open questions regarding e.g. which compound class -or which combination of -would be most effective, as well as the optimal timing to start treatment 53 , the potential to control the cytokine storm-induced severe lung failure and systemic organ failure by using already registered inhibitors of the centrally involved NF-kB pathway may be a real chance to get additional treatment options, hopefully decreasing the number of cases in need for arti cial ventilation, multi organ failure, and death.  Figure 1 Activation pathway of NF-B and linkage to acute respiratory RNA virus induced cytokine storm Binding of SARS-CoV-2 to its receptor, i.e. the angiotensin-converting enzyme 2 (ACE2) and the help of the cellular serine protease TMPRSS2 trigger endocytosis into the host cell. Within the endosomes, RNA from single-stranded RNA virus is generally known to activate the Toll-like receptors TLR7 and TLR8. This can lead to activation of transcription of the interferon-regulator factor (IRF) family and antiviral responses (green dotted lines). However, as a second major effect the activation of the TLR7/8 can trigger -via various intermediates -the activation of IKK (I B kinases) (grey dotted lines) resulting in phosphorylation of the cytoplasmic inhibitor factor I B triggering ubiquitination followed by its degradation by the 26S proteasome, thereby NF-B (a heterodimer complex consisting of protein subunits p50 and p65) is released from I B and can now enter the nucleus and initiate transcription of various genes coding for pro-in ammatory proteins such as cytokines, chemokines, adhesion molecules, and growth factors. Importantly, this nal sequence of NF-B activation is shared with a multiple range of cytokine receptorand Toll-like receptor mediated signal cascades, including binding of TNF or IL-1 to their receptors or binding of LPS (e.g. from secondary bacterial infections) to the TLR4. In contrast, interferon-response factor (IRF)-related responses are largely independent on NF-B translocation. Excessive cytokine release triggered by NF-B activation leads to in ammatory cell activation and in ltration, vascular leakage syndrome, nally leading to pulmonary oedema and pneumonia. This gure presents a hypothesis