Cannabis compounds have anti-inammatory activity in lung epithelial cells but pro-inammatory activity in macrophages while increasing phagocytosis in vitro

Cannabis sativa is widely used for medical purposes and has anti-inammatory activity. The purpose of this study was to examine the anti-inammatory activity of cannabis on markers of immune responses associated with Coronavirus disease 2019 (COVID-19) inammation. 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 phytocannabinoid standards that compose F CBD (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 macrophages (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 the anti-inammatory activity in alveolar epithelial cells, led to reduced phagocytosis and pro-inammatory IL secretion in macrophages in comparison to F CBD . The phytocannabinoid mixture may show superior activity versus cannabis fraction for reduction of lung inammation, yet there is a need of caution in proposing cannabis as treatment for COVID-19.


Introduction
Coronavirus disease 2019 (COVID- 19) is an acute resolved disease following infection by SARS-CoV-2 with a mortality of ~3.7%. Respiratory failure due to acute respiratory distress syndrome is the leading cause of mortality 1 . Disease progression of COVID-19 is often characterized by a two-phase immune responses. A speci c adaptive immune response is required at the rst phase to eliminate the virus and to prevent disease progression to severe stages 2 . Therefore, strategies to increase immune responses at this rst stage are critical.
The second phase is usually associated with a virally induced cytokine storm syndrome [1][2] . The cytokine storm syndrome is characterized by elevated levels of several cytokines including interleukin 6 (IL-6) and interleukin 8 (IL-8), tumor necrosis factor (TNF) and C-C Motif Chemokine Ligand 2 (CCL2) 3 . Speci c to the respiratory system, lung epithelial cells were suggested to play a crucial role in the release of several pro-in ammatory cytokines such as IL-6 and IL-8 4 .
Cannabis sativa is widely used for medical purposes worldwide. Cannabis strains produce more than 500 compounds, including phytocannabinoids, terpenes and avonoids [5][6][7] . Cannabinoids were previously suggested to be immune modulators and to change the balance between pro-and anti-in ammatory cytokines [8][9] . Cannabinoids also in uence macrophage activity. For example, Δ9-tetrahydrocannabivarin (THCV) inhibited nitrite production and interleukin 1β (IL-1β) protein levels in lipopolysaccharide activated macrophages 10 . Further, Δ9-tetrahydrocannabinol (THC) was shown to inhibit macrophages phagocytosis by 90% 11 . However, little is known regarding the effect of different cannabis compounds and their combinations on alveolar epithelial and immune cell in ammation.
Here, we identi ed cannabis compounds that have anti-in ammatory activity in lung epithelial cells, yet substantially induce polarization, phagocytosis and IL expression in macrophages in vitro.

Results
Cannabis crude extract and fractions reduce the level of IL-8 and IL-6 in lung epithelial cell model The high CBD cannabis strain Arbel was used to examine extract activity in reducing in ammation induced by TNFα 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-in ammatory activity (Fig. 1A, Supplementary Fig. S1).
F THC exhibited only low anti-in ammatory activity; however, F CBD showed considerable activity in the reduction of IL-6 and IL-8 secretion levels from lung epithelial cells, with IC 50 of 3.45 and 3.49 µg/mL, respectively (Fig. 1C,D). F CBD activity was greater than that of dexamethasone at 4 µg/mL for reducing IL-8 levels, and similar to that of the crude extract for reducing both IL-6 and IL-8 levels (Fig. 1A,B). Crude extract and F THC led to substantial cell death, whereas F CBD at 5 µg/mL was comparatively less cytotoxic (76.7% viability; Supplementary Fig. S2).
CBD (the main phytomolecule in F CBD ) alone showed a bell shaped activity curve, i.e., 3.0 µg/mL showed anti-in ammatory activity for both IL-6 and IL-8 levels, similar to F CBD at 4.1 µg/mL ( Fig. 2 A,B).
Nevertheless, higher or lower concentrations of CBD had lower and/or non-signi cant activity in reducing IL-6 and IL-8 levels ( Fig. 2 A,B). F CBD:std showed activity similar to F CBD in lung epithelial cell model Based on HPLC and GC/MS analysis, F CBD contains approximately66% phytocannabinoid by total content. The phytocannabinoid assemblage included CBD (93.5%), CBG (6.1%) and minute amount of THCV (0.4%) ( Supplementary Fig. S1). Multiple terpenes were detected in F CBD (Table 1; Supplementary   Fig. S3). The combination of phytocannabinoid standards at the ratio found in fraction F CBD (F CBD:std ) resulted in activity similar to that of the initial fractions (IC 50 of 4.1 μg/mL for IL-6 and IL-8; Fig. 1E,F). CB2 inverse agonist attenuated F CBD and F CBD:std activity in lung epithelial cell model Using CB2 receptor inverse agonists (IA) with F CBD or F CBD:std treatments led to reduced activity of the fraction and standard mix against IL-6 and IL-8 secretion in A549 cells (Fig. 3A,B). However, treatment with CB1 IA or a TRPA1 blocker did not affect F CBD or F CBD:std activity. Treatment with CB1 or CB2 IA led only to reduction in IL-6 and IL-8 levels in these cells, CB1 to a greater extent (Fig. 3A,B).
F CBD treatment lead to reduction in CCL2, CCL7, ACE2 and IL-7 gene expression in lung epithelial cell model qPCR analysis demonstrated that F CBD or F CBD:std treatments reduced the steady state level of mRNA of the pro-in ammatory cytokines CCL2 and CCL7 in TNFα treated A549 cells, determined with HPRT1 as a reference gene (Fig. 4A,B). However, the reduction in expression of the two genes was less than that of dexamethasone (Fig. 4A,B). F CBD:std treatment led to only a minor reduction in the expression level of IL-7, whereas F CBD and dexamethasone reduced IL-7 expression substantially (Fig. 4C). Moreover, F CBD or F CBD:std treatments reduced the expression level of ACE2, F CBD to a greater extent than dexamethasone or F CBD:std (Fig. 4D).
F CBD and F CBD:std attenuate macrophages polarization To examine the effect of the treatments on macrophage phagocytosis activity we incubated PMA-treated macrophages with SNP or SNPG. In the control (vehicle treated), most of the cells were non-polarized and featured a round structure ( Table 2; Fig. 6), whereas the macrophage population treated for 16 h with F CBD (7 µg/mL) consisted of ~48 % polarized cells (Table 2). Multiple silica particles and membrane pseudopods were detected in these polarized cells (Fig. 6). Likewise, treatment of the macrophage population with F CBD:std resulted in ~49 % polarized cells ( F CBD and F CBD:std attenuate expression of phagocytosis-associated receptors F CBD treatment but not F CBD:std increased expression of FcγRII and CD36, in comparison to the vehicle control ( Fig. 7A,B). Treatment with Ruxolitinib which inhibits monocyte activation 12 reduced FcγRII expression (Fig. 7A), and PA reduced expression of CD36 (Fig. 7B), in agreement with 13 . Expression of SCARB1 was reduced by F CBD and Roxulitinib but not by F CBD:std (Fig. 7C).

F CBD increase internalization of silica particles in macrophages
Based on cell analysis by Imaging Flow Cytometry for macrophages with internalized silica particles, it was found that F CBD increased the percentage of cells that internalized uorescent-labeled silica particles (SNP, Table 3; Supplementary Fig. S4). The increase in percentage of positive cells by F CBD was higher in comparison to the vehicle control also for the smaller uorescent-labeled silica particles, ENP and ENPG.
F CBD:std and CBD treatments were less effective in increasing of internalization (for SNP) or presence (ENP and ENPG) of the particles in cells in comparison to the F CBD treatment (

Discussion
We have identi ed CBD rich fraction (F CBD ) from the in orescence 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 cells 3 . In addition to CBD, F CBD contained CBG and minute amount of THCV. IC 50 of the combinations of active standards (F CBD:std ) at the relative concentrations found in the original fraction were similar to that of F CBD in the alveolar epithelial cell model.
Treatment with CBD by itself led to a reduction in IL-6 and IL-8 levels in a 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 the earlier publication suggesting that CBD has a bellshaped dose-response for anti-in ammatory activity 14 . 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 with 14 , suggesting that the addition of other phytomolecules to CBD (crude cannabis extract in the case of 14 ) prevented its bell-shaped dose-response. The CBD bell-shaped dose-response is associated with a narrow therapeutic window, which is di cult 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 signaling 15 . TRPA1 is a receptor in alveolar epithelial cells involved in the pathogenesis of several airway diseases including chronic obstructive pulmonary disease and asthma 16 . Nevertheless, co-treatment with inverse agonists for CB2 only had an effect on F CBD or F CBD:std activity. Treatment with CB1 IA or TRPA1 blocker had no effect. Hence, this anti-in ammatory activity may be mediated, at least partially, via the CB2 receptor. Nevertheless, the involvement of CB receptors in F CBD or F CBD:std activity still needs to be demonstrated and additional studies should be conducted to prove this suggested association.
In addition to reducing IL-6 and IL-8 levels, F CBD and F CBD:std reduced the expression level of CCL2 and CCL7 in alveolar epithelial cells induced with TNFα. The systemic cytokine pro les detected in severe COVID-19 patients includes increased production of in ammatory chemokines such as CCL2 17 . Moreover, CCL2 and CCL7 were shown to be abundant in bronchoalveolar uid from severe COVID-19 patients and were associated with recruitment of monocytes into the lungs 17 . Our results suggest that treatment with F CBD or F CBD:std may lead to reduced secretion of in ammatory 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 counts 18 and to restore protective immunity in patients that suffer from CD4+ T cell de ciency (e.g., as in the case of HIV infection 19 ). It was suggested that treatment against SARS-CoV-2 infection should also attempt to increase IL-7 levels 18 . 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 puri ed phytocannabinoids may have an advantage over cannabis-derived fractions for COVID-19-like in ammation. F CBD reduced the expression level of ACE2, and F CBD:std and dexamethasone also reduced its expression but to a lesser extent. The ACE-2 receptor is a part of the dual renin-angiotensin system 20 . ACE-2 was shown to be involved with SARS-CoV-2 human infection ; the ectodomain of the S protein of SARS-CoV binds to the peptidase domain of ACE2 with relatively high a nity 21 . 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 renin-angiotensin system 22 . The ability of F CBD to reduce ACE2 expression is important concerning treatment of COVID-19 patients, yet should be considered with care as the advantages and disadvantages of ACE2 expression reduction are disputed 20 .
In the rst phase of the disease, a speci c adaptive immune response is needed to eliminate the virus and to prevent disease progression to more severe stages 2 . Indeed, the dysfunction of alveolar macrophages are among the abnormal characteristics in some severe COVID-19 patients 23 , and an abundance of increased in ammatory monocyte-derived macrophages replaces tissue-resident alveolar macrophages in patients with severe disease 24 . In addition, during SARS-CoV-1 infections that provoke a disease course similar to those seen during infection with SARS-CoV-2 24 , a marked reduction in phagocytosis by macrophages was detected 25 . Also, phagocytosis was important in the antibody-mediated elimination of SARS-CoV-1 in a mouse model 26 .
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 membrane lopodia-like structures 27 . F CBD reduced expression of SCARB1; SCARB1 encodes SR-B1 that is a scavenger receptor (class B) also is responsible for phagocytosis of silica particles in macrophages 28 . However, F CBD treatment also led to an increase in FcγRII and CD36 gene expression. Phagocytosis is initiated by ligation of Fcγ receptors to IgG-opsonins on the target cell 29 , whereas CD36 expression in macrophages was shown to be involved with lung brosisin in mice 30 . Alveolar macrophages play an important role in Fc receptor-mediated responses during acute virus infections and in phagocytosis-mediated clearance of respiratory virus infections [31][32] . CD36 is an important scavenger receptor for phagocytosis of Streptococcus pneumoniae, a primary bacterial agent associated with pneumonia, which is down regulated by in uenza 33 . Indeed, F CBD led to a marked increase in the internalization of silica particles by macrophages, and in so doing, to increased levels of phagocytosis.
Possibly, the increase in macrophage polarization and phagocytosis, and the upregulation of expression of FcγRII and CD36 in these cells following F CBD treatment may facilitate phagocytosis-mediated clearance of respiratory viruses, and bene t the rst 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 infect macrophages as a result of IgG-mediated phagocytosis that requires FcγRII receptor signaling pathways 34 . Advantages and disadvantages of increasing macrophage phagocytosis activity should be carefully considered 3,35 .
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 of them such as γ-Curcumene or 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-in ammatory role by releasing proin ammatory cytokines such as IL-6, IL-8 and CCL2 3 . 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 nodes 35 . Notably, F CBD led to a marked increase of IL-8 expression levels in macrophages, IL-8 protein levels and to increase in IL-6 expression levels, above that induced by PMA 36 . These results suggest a substantial, in vitro, proin ammatory 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 and ACE2 expression in lung epithelial cells. However, treatment with F CBD containing terpenes in addition to phytocannabinoids substantially induced macrophage phagocytosis and increased their ILs levels. These results suggest a pro-in ammatory role for cannabis preparation that is higher than that of the phytocannabinoid standard mix only. The latter maintained the antiin ammatory activity in alveolar epithelial cells with relatively reduced pro-in ammatory activity in macrophages. Hence, the mix of phytocannabinoids shows superior activity versus the cannabis-derived fraction. Nevertheless, there is a need of caution in proposing cannabis treatment for COVID-19, as is presently being suggested in the media. Increase of macrophage-secreted IL-6 and IL-8 levels by cannabis-based treatment may potentially lead to worsening of the "cytokine storm" identi ed in severe COVID-19 patients. It should be stressed, in agreement with 37 , that as for now, users and healthcare personnel should avoid the use of cannabis for COVID-19 prevention or treatment.

Extract preparation
High CBD Cannabis sativa strain Arbel (IMC, Israel) in orescence was extracted using ethanol as previously described 38 and decarboxylated by heating the dried crude extract to 220 °C for 10 minutes. The dried decarboxylated extract was weighed, and then resuspended in absolute methanol (volume of solvent added according to the desired concentration) and ltered through a 0.45 μm syringe lter.

Extract fractionation
A ash chromatography apparatus equipped with a diode array detector was used to fractionize the decarboxylated crude extract. An Eco ex C-18 80g (Flash Pure, Buchi, C-18, 50 µm spherical, max. pressure 180 psi) column was used for separation, with methanol and water as the mobile phase as suggested by the manufacturer.

Chemical analyses
High performance liquid chromatography (HPLC) and gas chromatograph with mass selective detector (GCMS 8860 and GC/MSD 5977B, Agilent) analysis was carried out as previously described 38  They were allowed to attach and grow at 37 °C in air and 5% CO 2 in a humidi ed incubator overnight with complete DMEM, and then the media was replaced with serum free DMEM for on. Following, cell excitation was performed with 300 ng/mL TNFα. Treatments were performed with plant extract, fraction/compounds given together with 100 µL serum free DMEM. IL-6 and IL-8 secretion levels were analyzed after 4 h of incubation. Supernatant samples were taken and tested using IL-6 and IL-8 ELISA kits (DY206 and DY208 respectively, R&D Systems, Minneapolis, MN, USA). Dexamethasone was used as a positive control. For cell viability, an Alamar Blue (resazurin) assay was performed on each well as described previously 40 . For dose response assays, data points were connected by non-linear regression lines of the sigmoidal dose-response relation. GraphPad Prism (GraphPad Software Inc., San Diego, USA) was employed to produce dose-response curves and IC50 doses were calculated using nonlinear regression analysis.

Salinization of silicon dioxide surfaces with APTES
To prepare the silica dispersion 1 g of silica was added to 40 mL of methanol and stirred. Then, APTES (0.0045 moles) was slowly added to the solution. The reaction was carried out at ambient temperature for 45 min. After silanization, 50-100 nm or 30-70 nm particles were collected by centrifugation (9000 rpm, 10 min) washed 4 times with water, and dried at 35 °C under vacuum for 3 h 41 .
Labeling of amine functionalized silica nanoparticles with 5(6)-Carboxy uorescein and IgG Stock solutions of 1 mg of EDC were prepared separately in 1 mL of 0.1 M MES (pH 4.5-5) buffer. 100 mg of the amine functionalized silica nanoparticles were added to 600 µL of the MES buffer followed by 200 µL of the EDC. The mixture was vortexed for 10 min. Then 100 µL 5(6)-Carboxy uorescein (1mg/mL) only (for 50-100 nm SNP or 30-70 nm ENP nanoparticles) or 100 µL 5(6)-Carboxy uorescein (1mg/mL) and IgG (10mg/mL; for 30-70 nm ENPG nanoparticles) solutions were added. The nal solution was then mixed by vortex for 3 h at ambient room temperature. Subsequently, the mixture was centrifuged and rinsed with MES buffer to remove excess reactants. EDC was used as a cross-linker to chemically attach the carboxyl group of the 5 (6) Cells were analyzed by multispectral imaging ow cytometry (ImageStream markII ow cytometer; Amnis Corp, part of EMD Millipore, Seattle, WA, USA). Fluorescence intensity of the Fluorescein labeled silica beads was measured in channel 2 of the cytometer (480 nm ex, 560 nm em). A X60 magni cation was used for all samples. At least 4,000 cells were collected for each sample and data were analyzed using a dedicated image analysis software (IDEAS 6.2; AmnisCorp). Cells were gated for single cells using the area and aspect ratio features, and for focused cells using the Gradient RMS feature. Cropped cells were further eliminated by plotting the cell area of the bright eld image against the Centroid X feature (the number of pixels in the horizontal axis from the left corner of the image to the center of the cell mask).
Cells were further gated for cells that were positive (for ENP, ENPG or SNP). Because of their larger size only SNP beads could be further analyzed for beads internalization vs. those attached to the cell surface.
The was done using the intensity feature (the sum of the background − subtracted pixel values within the masked area of the image) and max pixel (the largest value of the background subtracted pixel). SNP internalization was calculated by the internalization feature, i.e. the ratio of the intensity inside the cell to the intensity of the entire cell, mapped to a log scale. To de ne the internal mask for the cell, the object mask of the bright eld image was eroded by 8 pixels. Cells with an internalization score higher than 0.33 were gated as cells with internalized SNP.

Statistical analysis
The data were processed using JMP statistical package (SAS Inc, NC, USA     Representative examples of confocal images of macrophages following treatment with FCBD (7 µg/mL) and solvent (vehicle) control. Differentiated KG1 cells were treated with FCBD or control for 16 h and then incubated with silica beads (SNP; 40 µg/mL) for 4 h. Cell were stained for F-actin (EasyProbes™ ActinRed 555 Stain, red stain), and nuclei (Hoechst, blue stain); n ≥ 5, in each biological replicate multiple cells were examined (see Table 2). Membrane lopodia-like structures are marked with white arrows.