It has been reported that nucleotide-binding oligomerization domain 2 (NOD2) can also function as a cytoplasmic viral pattern recognition receptor (PRR) by triggering activation of interferon regulatory factor-3 and production of interferon-β (IFN). In a murine model of human respiratory syncytial virus (RSV) that mimics virus infection in humans, IFN was induced early during infection (at 12h-2d post-infection) but its production was absent at 3d post-infection suggesting that IFN is important to restrict RSV spread during early infection and that this determines the clinical outcome of the disease.
Compared to wild type mice, NOD2-KO mice showed diminished IFN production in the lung and increased viral titer that resulted in more severe lung pathology with significant peri-bronchial lymphocytic inflammation and exudates of infiltrating neutrophils and mucus filling of the lumen (Sabbah et al. 2009).
It is well known that the immunopathogenesis of this phenomenon is characterized by an exaggerated production and secretion of pro-inflammatory cytokines which is blamed on the emergence of lethal clinical symptoms such as those that unfortunately are now associated with the present Covid-19 pandemic, especially in elderly patients (Channappanavar and Perlman 2017).
Some years ago, we demonstrated that β2-adrenergic receptor (β2-AR) stimulation could increase bone marrow derived dendritic cells (DC) or directly in vivo, the Th17 immune response following Toll-like receptor-2 (TLR2) and NOD2 activation. This cytokine bias depended on inhibition of IL-12 production while IL-6 and IL-23 were not affected. Consistently, gene expression analysis revealed that salbutamol did not affect IL-6 mRNA while both IL-12 and IL-12/IL-23 p40 mRNA were inhibited. Conversely, the expression of IL-23p19 was increased. NOD2 and TLR2 signaling was activated by muramyl dipeptide and by Pam3CysSK4, respectively. Thus, contrary to the concept that β2-AR agonists are immunosuppressive and anti-inflammatory agents, our findings suggest that in combination with TLR2 and/or NOD2 activation, stimulation of β2-AR may enhance an IL-17-type immune response at the expense of a Th1-type response (Manni et al. 2011).
Relevant to the present issue, it has been reported that TLR2 may also sense viral PRR (Henrick et al. 2016) and that IL-17 contributes to immune activation and acute pulmonary immunopathological injury in respiratory viral infection (Mukherjee et al. 2011; Van et al. 2020). Most interesting, in a model of influenza virus infection, depletion of gdT cells significantly rescued the virus-induced weight loss and improved the survival rate by decreasing IL-17A secretion and reducing immunopathological injury in the lung (Xue et al. 2017).
However, one might consider that although both are RNA viruses, the SARS-CoV2 and RSV are different viruses, more specifically SARS-CoV are enveloped positive strand RNA viruses in the family Coronaviridae while RSV is a negative-sense single-stranded RNA genome containing paramyxovirus. Therefore, at variance with the present hypothesis they might be sensed by PRR different from NOD2 and TLR2. Yet there is evidence that the SARS coronavirus spike protein may induce the innate immune response by activating TLR2 (Dosh et al. 2009). In regard to NOD2, the Middle East Respiratory Syndrome (MERS) coronavirus spike protein promote CCR2–mediated NOD2 signalling which induces production of IFN (Kim et al. 2019). Thus it seems very probable that SARS- Cov2 may also be sensed by the same PRR.
Moreover, a recent report shows that the binding of SARS-CoV2 to the TLR causes the release of pro-IL-1β which is cleaved by caspase-1, followed by inflammasome activation which is a mediator of lung inflammation, fever and fibrosis (Conti et al. 2020). The inflammasome is a multiprotein complex that plays a key role in the innate immune system, and its aberrant activation is involved in the pathogenesis of inflammatory diseases. A recent report showed that β-AR blockade resulted in an inhibition of the inflammasome activation by inducing autophagy (Wong et al. 2018).
Last but not least it has been also reported that activation of both human and murine CD8(+) T cells with β2-AR agonist decreased IFN-γ and TNF-α secretion and suppressed their cytolytic capacity in response to T-cell receptor (TCR) activation. It is well known that CD8 T cells play a crucial role in the adaptive immune response against viruses (Estrada et al. 2016).
In our study, we used salbutamol as β2-AR agonist in vitro and norepinephrine in vivo mimicking a state of increased adrenergic activity. It has been recently reported that long acting β2-AR agonists, which also mimics an increased sympathetic tone, suppress IFN in human plasmacytoid DC (pDC) which are the predominant cells producing IFN against viral infection (Kuo et al. 2018; Eisenbarth 2019). Moreover, stimulation of β2-AR in granulocyte/macrophage precursors cells has been found to increase myelopoiesis and production of pro-inflammatory cells (Vasamsetti et al 2018) which in case of respiratory viral infection may infiltrate the lung, worsening the disease. Autonomic dysfunction, especially sympathetic nerve overactivity is recognized to be a hallmark linking aging and obesity with hypertension and increased cardiovascular risk (Balasubramanian et al. 2018). In addition, adrenergic mechanisms seem to play a role in the pathogenesis of pulmonary edema, inflammation and fibrosis via stimulation od inflammatory cytokines (Rassler, 2013).
Most interesting, it has been reported that exposure to particulate matter air pollution results in a systemic release of catecholamines which in turn activate β2-AR in alveolar macrophages resulting in increased production of IL-6 and lung inflammation (Chiarella et al. 2014). This finding deserves to be taken in consideration because it might explain, at least partially, why the actual mortality rate in northern Italy is so high. In fact, the heavily industrialized plain of northern Italy, being located immediately south and east of the Alps, is poorly ventilated and air pollution is one of the highest in the world.
Taking all these considerations together, it is hypothesized that β-AR stimulation due to a pre-existing condition of adrenergic overactivity early during Covid-19 infection might result in an increased IL-17 production and as a consequence, more severe and life-threatening disease.
Schematic figure representing the possible β2-AR and IL-17 involvement in Covid-19–induced lung immunopathology. In this schematic figure I refer to pDCs as the cell type sensing the SARS-CoV2 virus by PRR such as TLRS and NOD2. However, it is well known that in the innate immune response to viruses, a similar role may be played by other types of DCs. In any case, activation of PRR induces a complex cytokine response which in turn instructs the proper adaptive immune response to fight the virus. As catecholamines inhibit IFN but potentiate IL-17 production, in presence of a balanced SNS activity, the ratio between IFN and IL-17 concentration during the immune response to the infection is higher. Being that IFN is a potent antiviral agent, this allows the development of an effective Th1 response, which eventually results in a positive outcome of the disease and recovery. On the other hand, when the SNS is overactivated the β2-AR expressed on pDCs are overstimulated and this results in a lower IFN production and higher IL-17 resulting in a lower IFN/IL-17 ratio which in turn induces an exaggerated inflammatory response leading to a life-threatening pneumonia. Abbreviations: β2-AR, β2-adrenoceptors; IFN, type 1 interferon IFN: IR, immune response; NOD2: nucleotide-binding oligomerization domain 2; pDC, plasmacytoid dendritic cells; PRR, Patter Recognition Receptors; TLRs, Toll like receptors; SNS, sympathetic nervous system.
In line with this hypothesis, most recently it has been reported, although in a relatively few patients, that both MERS-CoV and SARS-CoV2 viruses may drive a Th17 response while decreasing IL-12 and IFN-γ along with the Th1 response. This was particularly evident in patients who failed to produce type I IFN who also had a poor outcome of the disease (Faure et al. 2014; Wu et al. 2020). In addition, another study reported that decreased number of CD4+T and CD8+ T cells along with decreased IFN-γ production correlated with disease severity in SARS-CoV2 infection (Chen et al. 2020). Thus, administration of a β-AR antagonist such as propranolol, at low dosage and at the beginning of the infection, might produce a more balanced and effective anti-viral response and improve the clinical outcome of the disease. Furthermore, in case of arterial hypertension which is a common pathology in elderly patients, propranolol may exert a beneficial antihypertensive effect.
Clearly, this hypothesis needs to be substantiated by a series of studies before approaching any therapeutic trial. I would suggest to stimulate in vitro human dendritic cells or macrophages with the SARS-CoV2 virus in presence or absence of the β2-AR agonist salbutamol and/or the catecholamine norepinephrine and then measure IL-6, IL-12 and IL-23 concentration in the supernatant just as we did with NOD2 and TLR2 agonists and murine dendritic cells (Manni et al. 2011). If the results will show an IL-12 inhibition by salbutamol and no effect on IL-6 and IL-23, I would proceed by analyzing the mRNA expression of IL-6 and of the various subunits of both IL-12 and IL-23.
In case the results obtained will confirm ours (Manni et al. 2011), I would try to correlate the IFN/IL-17 concentration ratio with the disease outcome in serum of patients of the present pandemic to confirm the most recent observations (Faure et al. 2014; Wu et al. 2020). Finally, if the data obtained will further support my hypothesis I would suggest to promote a preliminary clinical trial by neutralization of IL-17A or the receptor subunit IL-17RA via monoclonal antibodies and/or administering propranolol to a selected number of Covid-19 patients at the beginning of the infection.
The patient’s enrollment should consider age and likelihood of sympathetic overactivity. However, the β-AR manipulation may eventually be beneficial only in the early phase of the disease vs later, in case of pulmonary distress, it should be taken into consideration the well-known role of β2-AR in regulating airway smooth muscle contraction. For the same reason, the dosage and administration schedule of β-adrenergic blocking drugs are markedly very crucial points in this approach.
Georges JM Maestroni, PhD – Center of Research in Medical Pharmacology, University of Insubria Varese, Italy; email: email@example.com; phones +39 333 6241316 or +39 0323 70248
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