To analyze the anti-inflammatory effects of novel C. sativa lines, we used a well-established full thickness human 3D skin artificial EpiDermFTTM tissue model, whereby tissues were exposed to UV to induce inflammation and then treated with extracts of seven new cannabis lines.
Global gene expression profiling revealed that several new extracts strongly down-regulated expression of interleukins, pro-inflammatory cytokines, C-C motif chemokines and C–X–C subfamily cytokines involved in ADRS and other autoinflammatory conditions (padj<0.05) (Fig. 1 and Table 1).
Upon original screening of over 200 extracts, seven - extracts #4, #6, #8, #12, #13, #14, #15, were identified for further analysis. Application of the extracts # 4, #8, #6 and #14 down-regulated both TNFα and IL-6. Extract #13 downregulated TNFα but not IL-6. Interestingly, extract #12 upregulated the expression of IL-6 and IL-23A, pro-inflammatory chemokines, and down-regulated the expression of anti-inflammatory IL-37. Application of extract#15 did not result in any statistically significant gene expression changes (Fig. 1, Table 1).
We further explored the effects of C. sativa extracts on the levels of IL-6 protein using western immunoblotting, and found that all extracts, except #15, downregulated IL-6 (Fig. 2). Interestingly, application of extract #12 down-regulated IL-6 on the protein level, but not on the level of the transcript. This is an interesting finding that may suggest the presence of post-transcriptional regulation of IL-6 expression via small interfering RNAs and the potential effects of cannabis extracts on these processes. All extracts down-regulated an important inflammation marker, COX-2 (Fig. 2).
Along with two key regulators of cytokine storm – TNFα and IL-6, C. sativa extracts also affected the levels of other key pro-inflammatory interleukins – IL-1, IL-17, IL-23 (Fig. 1, Table 1). IL-1 family of interleukins is important in innate inflammation and autoimmunity17. 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 conditions17. 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 severety18. Here, we noted that extracts #4 and #8 down-regulated both IL-1α and IL-1β (Fig. 1, Table 1).
Extracts #4, #6, #8, #13 and #14 down-regulated, while extract #12 upregulated IL-23A, a member of the IL-12 family of cytokines with pro-inflammatory properties19. Extracts #8 and #13 downregulated IL-17C, a pro-inflammatory cytokine and a member of IL-17 family, that, together with IL-23 mediates inflammation in psoriasis, psoriatic arthritis, and ankylosing spondylitis20. Increased expression of IL-23/IL-17 pathway was previously correlated with pulmonary inflammation in polymicrobial sepsis21. 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 fibrosis22, and has been implicated in ARDS, cystic fibrosis, and pulmonary fibrosis and other pathological conditions (reviewed in 22).
Three extracts, #4, #8 and #14 downregulated the levels of Toll-like receptor 2 (TLR2). Toll-like receptor 2 (TLR2) signaling has been implicated in numerous inflammatory diseases23, including pulmonary diseases and ARDS24.
In addition, extracts #4, #8, #13 and #14 significantly down-regulated the expression of NFKB2 gene. NF-κB pathway has been often referred to as a prototypical proinflammatory signaling pathway. NF-κB is usually upregulated by IL-1 and TNFα, and play important roles in the expression of other proinflammatory genes25.
Further analysis revealed that extracts #4, #8, #13 and #14 down-regulated CCL2, also known as MCP-1 (Fig. 1 and Table 1). Along with inflammation and ARDS, CCL2 expression is an important hallmark of fibrosis, and CCL2 has been explored as a potential druggable anti-fibrotic target26. In previous studies, CCL2 was shown to promote fibroblast differentiation and facilitate their recruitment to the alveolar space, thus leading to excessive collagen deposition27. Besides, CCL2 promoted fibroblast survival and stimulated IL-6 production28.
Importantly, along with CCL2, IL-1, IL-6 and TNFα also regulate fibrosis26, and their down-regulation may be viewed as a potential anti-fibrotic effect. Together with IL-1, IL-6 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 remodelling26. MMP1, MMP2, MMP7, and MMP9 were previously reported to be upregulated in PF. In our study, several extracts down-regulated MMPs (Table1).
Extracts #4, #6, #8, #14 and #13 also down-regulated WNT2 and WNT5a. WNT signaling alterations have been linked to pathogenesis of a variety of diseases and conditions, including pulmonary fibrosis29,30. 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 COPD29,31. Furthermore, extracts also affected the levels of iCAM1 and iCAM5 genes. Levels of iCAM1 were shown to be elevated in sera of PF patients32 , and recent studies showed that iCAM-1 inhibition reduced exacerbations of lung inflammation33.
One more important pro-fibrotic protein is CXCL12, and its down-regulation was shown to dampen fibrocyte recruitment and collagen deposition34. 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.
Having seen cannabis extract-induced changes in pro-inflammatory and pro-fibrotic genes, we further conducted an in-depth analysis of the effects of the extracts on global signalome using Pathview Bioconductor platform. We found that extracts # 4, #8, #14 significantly down-regulated cytokine-cytokine receptor interaction pathway, rheumatoid arthritis pathway, chemokine signalling, Toll-like receptor signalling, JAK-STAT signalling and other pathways involved in inflammation, immunity and autoimmunity, as well as tissue remodeling and fibrosis. Contrarily, extract #12 upregulated these pathways (Table 2, Fig. 3).
Overall, our study revealed that cannabis extracts exerted different effects on the 3D tissue inflammation model - some profoundly down-regulated pro-inflammatory cytokines and pro-fibrotic molecules, some affected only several key cytokines, some did not cause any significant changes at all (extract #15), while extract #12 promoted expression of pro-inflammatory genes. This is a very important finding that shows that cannabis is non-generic, and each C. sativa line has to be thoroughly evaluated for its medicinal properties.
Taken together, our results suggest that out of 7 studied extracts of novel C. sativa lines three were most effective down-regulating pro-inflammatory pathways and key cytokines implicated in the cytokine storm and ARDS in COVID-19. Extracts #4, #8 and #14 were the most effective, causing profound and concerted down-regulation of TNFα, IL-6 and CCL2. 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 pneumonia35, 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 tocilizumab9,10,36, colchicine, an agent that can potentially influence levels of IL-6 and other cytokines37, chroloquine13, metronidazole38, and statins39, as well as melatonin as an anti-inflammatory adjuvant therapy4. Chloroquine has some immunomodulatory effects, potentially suppressing the production and release of TNF-α and IL-613. Colchicine has been shown to effectively suppress interleukin IL-1b, IL-18 and IL-6 in patients with acute coronary syndrome40,41 and is now being trialed in COVID-19 ARDS, albeit it also has very significant side effects37.
Numerous rheumatological drugs are now being evaluated for therapeutic potential to tame COVID-19 pneumonia, ARDS, and prevent further complications such as PF18. Suppression of pro-inflammatory IL-1 family members and IL-6 have 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-1942. Tocilizumab, a humanized monoclonal antibody against the interleukin-6 receptor, is showing some promise, albeit it carries a hefty price tag and a lot of side effects10,43.
TNFα not only is the main cytokine storm driver, it also was shown to mediates the transition from pulmonary inflammation to fibrosis44. Surprisingly, up to now, no TNFα inhibitors have been trialed for COVID-19. The recent expert commentary in Lancet stated that “trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed”45. While potentially effective, anti-TNFα and anti-IL-6 biologics are very expensive and cause an array of side effects, including malignancies.
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, lines targeting TNFα, IL-6, IL-1β and causing concerted and significant down-regulation 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.