Taken together, our results suggest that out of 7 studied extracts of novel C. sativa cultivars three were most effective down-regulating pro-inflammatory pathways and key cytokines implicated in the cytokine storm and ARDS in COVID-19. We noted that novel cannabis extracts down-regulated the levels of pro-inflammatory cytokine and interleukins, including IL-1 family, IL-23/IL-17 pathway, IL-6, IL-8, TNFα, and others that play key parts in inflammation and fibrosis. IL-1 family of interleukins is important in innate inflammation and autoimmunity [28]. IL-1α was shown to be constitutively present in numerous epithelial and mesenchymal cell types of healthy individuals, whereas IL-1β is mainly induced under disease conditions [28]. Both pro-inflammatory interleukins are upregulated in numerous inflammatory and autoinflammatory diseases and are important druggable targets. Recent studies show that levels of IL-1 were strongly elevated in individuals with COVID-19, and IL-1 levels correlated with disease severity [29]. Increased expression of IL-23/IL-17 pathway was previously correlated with pulmonary inflammation in polymicrobial sepsis [30]. While on the one hand, the IL-17 family confers protection from a variety of extracellular pathogens and was shown to drive leukocyte infiltration to facilitate clearance of infectious pathogens, aberrant IL-17 signaling can lead to excess inflammation and tissue damage and fibrosis [31], and has been implicated in ARDS, cystic fibrosis, and pulmonary fibrosis and other pathological conditions (reviewed in [31]).

Together with interleukins and TNFα genes, novel cannabis extracts regulated the expression of various other genes involved in fibrosis, including pulmonary fibrosis (PF) (Table 1). Among those were metalloproteinases (MMPs), key proteases involved in ECM remodeling [23]. MMP1, MMP2, MMP7, and MMP9 were previously reported to be upregulated in PF.

CCL2, also known as MCP-1 (Figure 1 and Table 1), which is an important hallmark of fibrosis, and has been indicated as a potential druggable anti-fibrotic target [23]. In previous studies, CCL2 was shown to promote fibroblast differentiation and facilitate their recruitment to the alveolar space, thus leading to excessive collagen deposition [32]. Besides, CCL2 promoted fibroblast survival and stimulated IL-6 production [33]. Importantly, along with CCL2, IL-1, IL-6 and TNFα also regulate fibrosis [23], and their down-regulation may be viewed as a potential anti-fibrotic effect.

Overall, extracts down-regulated many pro-fibrotic genes, such as WNT5A, iCAM and others. Previous studies have shown that in vivo inhibition of WNT-5A attenuated tissue destruction, improved lung function and restoration of alveolar epithelial cell markers expression in two animal models of COPD [24, 34]. Down-regulation of iCAM1 and iCAM5 genes is also an important finding, as the levels of iCAM1 were shown to be elevated in sera of PF patients [35], and recent studies showed that iCAM-1 inhibition reduced exacerbations of lung inflammation [36].

Interestingly, in our study, extracts #6 and #13, along with down-regulation of numerous pro-inflammatory cytokines, upregulated CXCL12. The role of CXCL12 upregulation in PF still needs to be fully established, but, based on the current knowledge, CXCL12 upregulation can be viewed as a potential PF contributor, and thus its upregulation may negate the potential benefits of cytokine down-regulation by extracts #6 and #13.

Overall, pronounced inhibition of TNFα and IL-6 is the most important finding, as these molecules are currently considered to be the key actionable targets in COVID-19 cytokine storm and ARDS. Anti-cytokine therapies are thought to be important for prevention of COVID-19 pneumonia [37], as currently there is a race to develop novel anti-cytokine storm regimens. To that effect several anti-cytokine therapies have been proposed and are now in clinical trials. These include anti-IL-6 receptor antibody tocilizumab [11, 12, 38], colchicine, an agent that can potentially influence levels of IL-6 and other cytokines [39], chloroquine [15], metronidazole [40], and statins [41], as well as melatonin as an anti-inflammatory adjuvant therapy [6]. Chloroquine has some immunomodulatory effects, potentially suppressing the production and release of TNF-α and IL-6 [15]. Colchicine has been shown to effectively suppress interleukin IL-1b, IL-18 and IL-6 in patients with acute coronary syndrome [42, 43] and is now being trialed in COVID-19 ARDS, albeit it also has very significant side effects [39]. Nonetheless, a lot of studies yielded negative or controversial results, calling for more efforts aimed at the discovery of novel anti-cytokine storm regimens.

Several rheumatological drugs are now being evaluated for therapeutic potential to tame COVID-19 pneumonia, ARDS, and prevent further complications such as PF [29]. Suppression of pro-inflammatory IL-1 family members and IL-6 has been shown to have a therapeutic effect in many inflammatory diseases, including viral infections, and has been explored as a potential therapeutic avenue in COVID-19 [44]. A recent review summarized the roles of IL-6 in COVID-19 pathogenesis and highlighted the important therapeutic potential of the IL-6 blockade in COVID-19 management [45]. Interestingly, a recent in-depth meta-analysis of 1302 COVID-19 cases showed that the level of IL-6 was 3-fold higher in patients with severe vs mild/moderate COVID-19 (p < 0.001). Furthermore, high IL-6 levels correlated with the development of severe lung damage (p = 0.001) [11].

Numerous reports based on several observational or non-placebo controlled studies in patients with severe COVID-19 and ARDS suggest the therapeutic potential of these agents, especially tocilizumab, a monoclonal antibody against IL-6 receptor (reviewed in [45]).

Tocilizumab, a humanized monoclonal antibody against the IL-6 receptor, is showing some promises, albeit it carries a hefty price tag and a lot of side effects [12, 46], and the results are not fully conclusive. Indeed, results of the COVIDOSE, low-dose tocilizumab in the treatment of Covid-19 trial, showed that low-dose tocilizumab administration led to rapid improvement in both laboratory and clinical manifestations of hyperinflammation in patients hospitalized with COVID-19 [47]. On the other hand, preliminary results of COVACTA clinical trial reported no statistical difference between tocilizumab vs placebo arm in severe COVID-19 [45]. Currently several clinical trials are ongoing to ascertain the efficacy of tocilizumab - NCT04445272, NCT04479358 (COVIDOSE-2), NCT04317092 (TOCIVID), NCT04345445, including a phase 3 RCT (NCT04412772).

In parallel, a study reported successful treatment of COVID-19 pneumonia with clazakizumab, monoclonal antibody against human IL-6 [48]. Currently, five RCTs are ongoing to ascertain the therapeutic potential of anti-IL-6 antibody in COVID-19 (NCT04381052, NCT04348500, NCT04494724, NCT04343989, NCT04343989).

TNFα not only is the main cytokine storm driver, it also was shown to mediate the transition from pulmonary inflammation to fibrosis [49]. As a pro-inflammatory cytokine, TNFα is mechanistically involved in lung and vascular tissue damage, ARDS and coagulopathy [50, 51]. Elevated levels of TNFα and other pro-inflammatory cytokines and chemokines, such as IL-6, IL-10 are risk factors for the development of severe COVID-19, and their levels are much higher in critical patients than in those with milder disease [50, 52]. Furthermore, most recent in vitro data show that TNFα facilitates SARS-CoV-2 interaction with ACE2 receptor that is a key gateway of the virus into human cells [53]. Moreover, recent clinical case reports have shown that the use of anti-TNF therapies in patients with rheumatological conditions and mild cases of COVID-19 prevented their further progression to the severe COVID-19 forms, most probably by mitigating deleterious effects of the high levels of TNFα and other cytokines that drive immunopathogenesis of COVID-19 [51]. Surprisingly, up to now, no TNFα inhibitors have been trialed for COVID-19. The expert commentary in Lancet stated that "trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed" [54]. To that effect, a recent expert review identified opportunities for the use of TNFα inhibitors in COVID-19 [29]. The first phase 2 study aimed to evaluate whether or not early administration of TNFα inhibition by infliximab in patients with severe COVID-19 will reduce disease duration and severity (NCT04425538).

While potentially effective, anti-TNFα and anti-IL-6 and other anti-inflammatory biologics are very expensive and cause an array of side effects, including malignancies and their efficacy needs to be ascertained in clinical trials. On the other hand, anti-TNFα and anti-IL-6 cannabis extracts that are generally regarded as safe (GRAS) modalities can be a useful addition to the current anti-inflammatory regimens to treat COVID-19, as well as various rheumatological diseases and conditions such as rheumatoid arthritis (RA), psoriasis and psoriatic arthritis, osteoarthritis, fibromyalgia, and others. Indeed, cultivars targeting TNFα, IL-6, IL-1β and causing concerted and significant downregulation of the rheumatoid arthritis pathway, pending thorough verification and clinical validation, may present a novel and promising natural resource for RA treatments and management of other TNFα, IL-6, IL-1β-mediated diseases. Furthermore, a recent report shows that CBD and the combination of major terpenes was far superior than dexamethasone in treating COVID-19 [55], albeit a full and final report of this study is still pending.

While potentially important, our study has limitations. It was initially based on the use of human EpiDermFT 3D tissue model which is not the closest to the lung tissues. That being said, inflammation can be effectively induced in this model and it was curbed by the application of extracts. Our data need to be further substantiated using more lung cell lines and 3D tissue models of inflammation. Importantly, extracts that curbed inflammation is 3D tissues also exerted anti-inflammatory potential in WI-38 lung fibroblasts, albeit not at the same level. In the future, it would be important to further expand the study and include analysis of the effects in lung 3D tissues. Furthermore, recent screen of the battery of high-CBD extracts identified several that inhibited COX2 and other inflammation makers in lung tissues (data not shown). Notwithstanding, our study laid a foundation for the future analysis of the anti-inflammatory potency of cannabis and its applications for COVID-19 and ARDS.