Study: Cannabis compounds relieve Covid lung inflammation

Written by nature.com on January 18, 2022. Posted in Current News

Cannabis compounds exhibit anti-inflammatory activity in vitro in COVID-19-related inflammation in lung epithelial cells and pro-inflammatory activity in macrophages

Abstract

Cannabis sativa is widely used for medical purposes and has anti-inflammatory activity. This study intended to examine the anti-inflammatory activity of cannabis on immune response markers associated with coronavirus disease 2019 (COVID-19) inflammation. An extract fraction from C. sativa Arbel strain (F_CBD) substantially reduced (dose dependently) interleukin (IL)-6 and -8 levels in an alveolar epithelial (A549) cell line.

F_CBD contained cannabidiol (CBD), cannabigerol (CBG) and tetrahydrocannabivarin (THCV), and multiple terpenes. Treatments with F_CBD and a F_CBD formulation using phytocannabinoid standards (F_CBD:std) reduced IL-6, IL-8, C–C Motif Chemokine Ligands (CCLs) 2 and 7, and angiotensin I converting enzyme 2 (ACE2) expression in the A549 cell line. Treatment with F_CBD induced macrophage (differentiated KG1 cell line) polarization and phagocytosis in vitro, and increased CD36 and type II receptor for the Fc region of IgG (FcγRII) expression.

F_CBD treatment also substantially increased IL-6 and IL-8 expression in macrophages. F_CBD:std, while maintaining anti-inflammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-inflammatory IL secretion in macrophages in comparison to F_CBD. The phytocannabinoid formulation may show superior activity versus the cannabis-derived fraction for reduction of lung inflammation, yet there is a need of caution proposing cannabis as treatment for COVID-19.

Results

Cannabis crude extract and fractions reduce the level of IL-8 and IL-6 in lung epithelial cell model

Inflorescence extracts of the high CBD C. sativa strain Arbel were used to examine cannabis activity in reducing TNFα induced inflammation in the lung epithelial cancer cell line A549.

The crude extract led to a substantial reduction of IL-6 and IL-8 secretion levels at 5 µg/mL (Fig. 1a,b). Subsequently, high CBD (F_CBD) and high THC (F_THC) fractions were examined for their anti-inflammatory activity (Fig. 1a, Supplementary Fig. S1). F_THC exhibited only low anti-inflammatory activity; however, F_CBD showed considerable activity in the reduction of IL-6 and IL-8 secretion levels from lung epithelial cells, with an IC₅₀ of 3.45 and 3.49 µg/mL respectively (Fig. 1c,d).

F_CBD reduced IL-8 levels more than dexamethasone at 4 µg/mL, and reduced IL-6 and IL-8 to levels similar to that of the crude extract (Fig. 1a,b). The crude extract and F_THC led to substantial cell death (61 and 42 percent viability, respectively), whereas F_CBD at 5 µg/mL was comparatively less cytotoxic (76.7% viability; Supplementary Fig. S2).

Discussion

We have identified a CBD rich fraction (F_CBD) from the inflorescence extract of a high CBD cannabis strain with immune-modulation activity in alveolar epithelial and macrophage cell models. F_CBD reduced IL-8 and IL-6 secretion in alveolar epithelial cells. IL-8 is one of the cytokines that characterizes the cytokine storm in severe COVID-19 patients; IL-6 is a prominent cytokine also involved in the cytokine storm and is secreted during the disease from alveolar epithelial cells³.

In addition to CBD, F_CBD contained CBG and minute amount of THCV. The IC₅₀ of a combinations of active phytocannabinoid standards (F_CBD:std) at the relative concentrations found in F_CBD were similar to that of the original fraction in the alveolar epithelial cell model.

Treatment with CBD by itself led to a reduction in IL-6 and IL-8 levels in an inverse bell-shaped dose–response in alveolar epithelial cells; i.e., only 3 µg/mL was active whereas other CBD concentrations exhibited lower or no cell activity. These results are in line with an earlier publication suggesting that CBD has a bell-shaped dose–response for anti-inflammatory activity by Gallily et al.¹⁶.

Notably, F_CBD (i.e., combination of CBD with CBG and THCV) led to a dose-dependent response rather than a bell-shaped dose–response. These results are in accordance with¹⁶, suggesting that the addition of other phytomolecules to CBD (crude cannabis extract in the case of¹⁶) prevented its bell-shaped dose–response. The CBD bell-shaped dose–response is associated with a narrow therapeutic window, which is difficult to use effectively in clinical therapy. Therefore, the fact that F_CBD has a dose-dependent response makes it better suited than CBD for patient care.

CBD is a negative allosteric modulator of CB1 signaling¹⁷. TRPA1 is a receptor in alveolar epithelial cells involved in the pathogenesis of several airway diseases including chronic obstructive pulmonary disease and asthma¹⁸. Both TRPV1 and TRPV2 interact with phytocannabinoids, including CBD, CBG and THCV¹⁹. Also, TRPV1, TRPV2 and TRPA1 were found to be associated with pulmonary inflammation²⁰.

Nevertheless, co-treatment with CB1 IA, TRPA1 blocker or TRPV1 or TRPV2 antagonist had no substantial effect on F_CBD and F_CBD:std activity. Only co-treatment with CB2 IA affected F_CBD:std activity on IL-8 secretion. The involvement of receptors in F_CBD and F_CBD:std activity remains to be demonstrated.

In addition to reducing IL-6 and IL-8 levels, F_CBD and F_CBD:std reduced the expression levels of CCL2 and CCL7 in alveolar epithelial cells by 6 h treatment. The systemic cytokine profiles detected in severe COVID-19 patients includes increased production of inflammatory chemokines such as CCL2²¹. Moreover, CCL2 and CCL7 were shown to be abundant in bronchoalveolar fluid from severe COVID-19 patients and were associated with recruitment of monocytes into the lungs²¹.

Our results suggest that treatment with F_CBD or F_CBD:std may lead to reduced secretion of inflammatory cytokines associated with the disease, and possibly to a reduction of macrophage recruitment during the cytokine storm. However, dexamethasone was more effective than F_CBD in reducing both CCL2 and CCL7 expression.

IL-7 was shown to raise lymphocyte counts in septic patients with low absolute lymphocyte counts²² and to restore protective immunity in patients that suffer from CD4+ T cell deficiency (e.g., as in the case of HIV infection²³). It was suggested that treatment against SARS-CoV-2 infections should also attempt to increase IL-7 levels²². The fact that F_CBD:std reduced IL-7 expression only to a minor extent in comparison to dexamethasone or F_CBD suggests that using purified phytocannabinoids may have an advantage over cannabis-derived fractions for COVID-19-like inflammation.

The ACE2 receptor is a part of the dual renin-angiotensin system (RAS)²⁴. ACE2 was shown to be involved with SARS-CoV-2 human infection; the ectodomain of the S protein of SARS-CoV-1 binds to the peptidase domain of ACE2 with relatively high affinity²⁵. In cells of patients with severe symptoms of COVID-19, ACE2 was substantially upregulated 199-fold; this upregulation was suggested to be one of the factors leading to disruption of the RAS, as ACE2 is a part of the counteracting hypotensive axis of RAS.

The increase in ACE2 and other key RAS components is predicted to elevate bradykinin levels in multiple tissues, leading to increases in vascular permeability and hypotension; the latter is highly associated with severe COVID-19 patients²⁶. Indeed, a negative correlation was identified between ACE2 gene expression and COVID-19 mortality²⁷. F_CBD reduced the expression level of ACE2 at 4 and 6 h post treatment.

F_CBD:std and dexamethasone also reduced ACE2 expression but to a lesser extent. However, the ability of F_CBD to reduce ACE2 expression should be examined at both the protein and functional levels (e.g., binding of the viral protein) to fully determine the effect F_CBD may have on ACE2-related treatment of COVID-19 patients. In any case, such reduction of ACE2 expression should be considered with care as the advantages and disadvantages of this reduction are disputed²⁴.

In the first phase of the disease, a specific adaptive immune response is needed to eliminate the virus and to prevent disease progression to more severe stages². Indeed, the dysfunction of alveolar macrophages are among the abnormal characteristics in some severe COVID-19 patients²⁸, and an abundance of increased inflammatory monocyte-derived macrophages replaces tissue-resident alveolar macrophages in patients with severe disease²¹.

Additionally, during SARS-CoV-1 infections that provoke a disease course similar to those seen during infection with SARS-CoV-2²¹, a marked reduction in macrophages phagocytosis activity was detected²⁹. Also, phagocytosis was important in the antibody-mediated elimination of SARS-CoV-1 in a mouse model³⁰.

Notably, F_CBD and F_CBD:std, and CBD to a lesser extent, led to a marked increase in macrophage polarization and to cell actin remodeling that corresponds to the growth of filopodia-like membrane structures³¹. F_CBD reduced expression of SCARB1; SCARB1 encodes SR-B1 that is a scavenger receptor (class B) and is also responsible for phagocytosis of silica particles in macrophages³².

However, F_CBD treatment also led to an increase in FcγRII and CD36 gene expression. Phagocytosis is initiated by the ligation of Fcγ receptors to IgG-opsonins on the target cell³³, whereas CD36 expression in macrophages was shown to be involved with lung fibrosisin in mice³⁴. Alveolar macrophages play an important role in Fc receptor-mediated responses during acute virus infections and in phagocytosis-mediated clearance of respiratory virus infections^35,36.

CD36 is an important scavenger receptor for phagocytosis of Streptococcus pneumoniae, a primary bacterial agent associated with pneumonia, which is down regulated by influenza³⁷. Indeed, F_CBD led to a marked increase in the internalization of silica particles by macrophages, and in so doing, increased levels of phagocytosis.

Possibly, the increase in macrophage polarization and phagocytosis, and the upregulation of FcγRII and CD36 expression in these cells following F_CBD treatment may facilitate phagocytosis-mediated clearance of respiratory viruses, and benefit the first phase of the immune response to SARS-CoV-2.

However, it should be noted that macrophages themselves can be infected by the virus, as SARS-CoV-1 infects macrophages as a result of IgG-mediated phagocytosis that requires FcγRII receptor signaling pathways³⁸. Advantages and disadvantages of increasing macrophage phagocytosis activity should be carefully considered^3,21.

Notably, although F_CBD:std treatment increased macrophage polarization, it did not increase the phagocytosis-associated gene expressions, nor phagocytosis. Hence, additional active compounds in the cannabis-derived F_CBD and not in the phytocannabinoid standard mix that composed F_CBD:std are responsible for this increased gene expression and phagocytosis activity.

Indeed, F_CBD contained multiple terpenes, some including γ-Curcumene and Guaiol at considerable percentages. The presence of terpenes in F_CBD may account for the differences in activity between F_CBD and F_CBD:std.

During the second phase of COVID-19, pneumonia patients exhibit features of macrophage activation syndrome (MAS) in which macrophages play a major pro-inflammatory role by releasing pro-inflammatory cytokines such as IL-6, IL-8 and CCL2³. Moreover, subsets of macrophages in patients with COVID-19 were found to express genes associated with IL-6, whereas expression of IL-6 was again associated with severe depletion of lymphocytes from the spleen and lymph nodes²¹.

Notably, F_CBD led to a marked increase of IL-8 expression and IL-8 protein levels in macrophages. It also led to an increase in IL-6 expression levels, above that induced by PMA³⁹. These results suggest a substantial, in vitro, pro-inflammatory role for F_CBD in macrophages. However, F_CBD;std was less active in ILs induction, again demonstrating a notable difference between F_CBD and F_CBD:std, which may originate from the presence or absence, respectively, of terpenes.

To conclude, treatment with cannabis compounds CBD, CBG and THCV may have clinical value in reducing cytokine secretion in lung epithelial cells. However, treatment with F_CBD containing terpenes in addition to these phytocannabinoids substantially induced macrophage phagocytosis and increased their IL levels.

Yet, to confirm more specifically the pro-inflammatory effect of F_CBD in macrophages it is necessary to perform the same experiments on primary alveolar macrophages (e.g., from mice). Nevertheless, these results suggest a pro-inflammatory role for cannabis extract that is higher than that of the phytocannabinoid standard mix.

The latter maintained anti-inflammatory activity in the alveolar epithelial cells with relatively reduced pro-inflammatory activity in macrophages. Hence, the mix of phytocannabinoids shows superior activity versus the cannabis-derived fraction.

Although more studies are needed of cannabis treatment in COVID patients, there needs to be caution in proposing cannabis treatment for these patients, as is presently being suggested in the media.

The increase of macrophage-secreted IL-6 and IL-8 levels by cannabis-based treatment may potentially lead to a worsening of the “cytokine storm” identified in severe COVID-19 patients.

It should be stressed, in agreement with Pastor et al.⁴⁰, that for now, users and healthcare personnel should avoid the use of cannabis for COVID-19 prevention or treatment.

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