key: cord-0918982-1acmlq2r
authors: Mohammed, Amira; Alghetaa, Hasan; Zhou, Juhua; Chatterjee, Saurabh; Nagarkatti, Prakash; Nagarkatti, Mitzi
title: Protective Effects of Δ9‐Tetrahydrocannabinol Against Enterotoxin‐induced Acute Respiratory Distress Syndrome is Mediated by Modulation of Microbiota
date: 2020-08-04
journal: Br J Pharmacol
DOI: 10.1111/bph.15226
sha: b719eea719391e12fa1777178036bb03d94e7422
doc_id: 918982
cord_uid: 1acmlq2r

BACKGROUND: Staphylococcal enterotoxin‐B (SEB) is one of the most potent bacterial superantigens that exerts profound toxic effects by inducing cytokine storm. When SEB is inhaled, it can cause Acute Respiratory Distress Syndrome (ARDS), which is often fatal and currently there are no effective treatment modalities. EXPERIMENTAL APPROACH: We used mouse model of SEB‐mediated ARDS to test the efficacy of Δ9‐tetrahydrocannabinol (THC). These mice were monitored for lung inflammation, alterations in gut and lung microbiota and production of short‐chain fatty acids (SCFA). Gene dysregulation of lung epithelial cells was studied by transcriptome arrays. Fecal microbiota transplantation (FMT) was performed to confirm the role of microbiota in suppressing ARDS. KEY RESULTS: While SEB triggered ARDS and 100% mortality in mice, THC protected the mice from fatality effects. Pyrosequencing analysis revealed that THC caused significant and similar alterations in microbiota in the lungs and gut of mice exposed to SEB. THC significantly increased the abundance of beneficial bacterial species, Ruminococcus gnavus, but decreased pathogenic microbiota, Akkermansia muciniphila. FMT confirmed that THC‐mediated reversal of microbial dysbiosis played crucial role in attenuation of SEB‐mediated ARDS. THC treatment also led to increase in SCFA, of which propionic acid was found to inhibit the inflammatory response. Transcriptome array showed that THC up‐regulated several genes like lysozyme‐1&2, β‐defensin‐2, claudin, zonula‐1, occludin‐1, Mucin2 and Muc5b while downregulating β‐defensin‐1. CONCLUSIONS: Current study demonstrates for the first time that THC attenuates SEB‐mediated ARDS and toxicity by altering the microbiota in the lungs and the gut as well as promoting anti‐microbial and anti‐inflammatory pathways.

This article is protected by copyright. All rights reserved.

Acute respiratory distress syndrome (ARDS), is triggered by a variety of etiologic agents and SEB remain one of them. Interestingly, patients who develop the severe form of novel coronavirus disease 2019 were found to exhibit ARDS, cytokine storm, and respiratory failure (Henry & Lippi, 2020) . The incidence of ARDS in the Unites States is 78.9 per 100,000 persons/year and the mortality rate is 38.5% (Matthay et al., 2019) . With the ongoing COVID-19 pandemic, this incidence is likely to increase further. SEB is capable of inducing fatal ARDS in nonhuman primates following airborne exposure, thereby suggesting that it can also be used as biological warfare agent ( (Madsen, 2001; Mattix, Hunt, Wilhelmsen, Johnson, & Baze, 1995) . The ability of SEB to cause acute ARDS may be related to the fact that it acts as a super-antigen by activating a large proportion of T cells expressing certain V -specific T cell receptors, which leads to cytokine storm and consequent injury to various organs, including the lungs (Saeed et al., 2012) . It is difficult to treat ARDS and thus far there are no pharmacological agents that can protect the host from SEB-mediated toxicity (Nanchal & Truwit, 2018; Rubenfeld & Herridge, 2007) . Recent studies from our laboratory demonstrated that exposure to two small doses of SEB in C3H mice can trigger acute ARDS, which if left untreated, leads to 100% mortality Mohammed et al., 2020) . Interestingly, we were able to show that administration of THC prior to SEB challenge prevents such toxicity and mortality by suppressing the cytokine storm and enhancing immunosuppressive cytokines such as IL-10 as well as induction of Tregs (Rao, Nagarkatti, & Nagarkatti, 2015) .

Currently, there are no FDA-approved drugs to treat ARDS. Pharmacological agents such as corticosteroids and neuromuscular blocking agents (NMBAs) may be beneficial for ARDS patients but the mortality rate remains high (Mokra, Mikolka, Kosutova, & Mokry, 2019) . Δ9-Tetrahydrocannabinol (THC) is a cannabinoid component found in marijuana (cannabis sativa L.). THC acts through cannabinoid receptors, CB1 and CB2 that are expressed on immune cells (Mohammed et al., 2020; Nagarkatti, Pandey, Rieder, Hegde, & Nagarkatti, 2009) . THC is well characterized to exhibit anti-inflammatory properties. THC uses multiple pathways to mediate immunosuppression including: 1) induction of Tregs and MDSCs (Hegde, Nagarkatti, & Nagarkatti, 2010; Pandey, Hegde, Nagarkatti, & Nagarkatti, 2011; Sido, Nagarkatti, & Nagarkatti, 2015) , 2) apoptosis in activated T cells and dendritic cells (McKallip, Lombard, Martin, Nagarkatti, & Nagarkatti, 2002; Rieder, Chauhan, Singh, Nagarkatti, & Nagarkatti, 2010) , 3) switch from Th1 to Th2 differentiation (Yang et al., 2014) , 4) inhibition of cytokine production by upregulation of suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ (Sido, Jackson, Nagarkatti, & Nagarkatti, 2016) . We have also shown that THC triggers epigenetic changes including alterations in the expression of miRNA (Al-Ghezi, Miranda, Nagarkatti, & Nagarkatti, 2019) , and histone modifications (Yang et al., 2014) . For example, in SEB-induced mouse model of ARDS, our previous studies showed that THC could modulate the expression of miR17-92 cluster and suppress SEB-induced ARDS (Rao et al., 2015) .

Recent studies have shown that gut microbiome may play a critical role in disease pathogenesis involving other organs besides the gut (Das & Nair, 2019) . This stems from the fact that the gut microbiota produce short chain fatty acids (SCFA) that can travel systemically and cause immunomodulatory changes at distant sites (Dopkins, Nagarkatti, & Nagarkatti, 2018) . A recent study from our laboratory suggested that cannabinoids can trigger dybiosis that is beneficial to the host. For example, we found that THC and Cannabidiol can reverse the dysbiosis triggered in the gut during an experimental Multiple Sclerosis model and that the efficacy of these cannabinoids to attenuate this autoimmune disease depended on their effect on microbiota (Al-Ghezi, Busbee, .

This article is protected by copyright. All rights reserved. and infiltration of mononuclear cells into the lungs, there are no studies to test if SEB can also alter the microbiota in the lungs and the gut, and how such dysbiosis can impact the lung inflammation and ARDS. Additionally, while we have shown previously that THC can attenuate SEB-mediated ARDS, whether this results from THC-mediated dysbiosis in the lungs, has not been previously investigated.

In this study, we comprehensively investigated the effects of THC treatment on lung and gut microbiome, induction of anti-microbial enzymes and peptides, and nature of immune response induced during SEB-induced ARDS mice. Excitingly, our results showed that SEB caused marked but similar alterations in microbiota found in the gut and lungs in ARDS-induced mice.

Moreover, THC treatment led to reversal of such dysbiosis and importantly Fecal Microbiota Transplantation (FMT) experiments showed that the ability of THC to attenuate SEB-mediated ARDS resulted from THC-induced alterations in the microbiota. The current study provides evidence for the first time that the lung and gut microbiota may play a critical role in SEBmediated pathogenesis of ARDS and thus, agents that can reverse such dysbiosis may be helpful to treat ARDS.

Mice: C3H/HeJ ( RRID:IMSR_JAX:000659) mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). Female mice, 8-10 weeks of age, were used throughout the studies.

Animals were housed in specific pathogen-free conditions, and all experiments were approved by Institutional Animal Care and Use Committee (AUP2363). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals McGrath, Drummond, McLachlan, Kilkenny, & Wainwright, 2010) . Animals grouping and housing: All delivered mice were kept for one week as acclimatization period prior to performing any experiments. Animals were housed in maximum of 5 mice per cage under 12 light/12 dark cycle at a temperature of ∼18-23 °C and 40-60% humidity. Food and water were available ad libitum. To minimize the microbiome variations from cage/rack to cage/rack due to managerial and housekeeping effects, experimental mice were selected randomly from different cages to house them in one new cage with a maximum number of 5 mice/cage and then each cage was blindly assigned for different treatments or kept as control group according to the experimental design. The person who carried out the experiments was not blinded because of injection of SEB, vehicle and THC at different times but most data analysis and experiments were blinded to avoid any bias.

Induction of ARDS: SEB was purchased from Toxin Technologies (Sarasota, FL, USA), and used in the induction of ARDS as a 'Dual Dose' as described previously (Huzella, Buckley, Alves, Stiles, & Krakauer, 2009 ). Briefly, 25 µL of SEB at a concentration of 0.2 µg/µL (5 g total/mouse) was intranasal administered into each C3H/HeJ mouse with a micropipette. After 2 h, the same mouse received an intraperitoneal injection of 100 µL of SEB at a concentration of 0.02 µg/µL (2 g total/mouse) Treatment of THC: THC was dissolved in ethanol and then diluted in 1× PBS. Each mouse was treated intraperitoneally with THC that was given at 20mg/kg, i.p immediately after first SEB exposure. Second and third doses of THC were given i.p at 10mg/kg after 24 and 48 hours after the first dose of THC. Disease progress in mice was assessed by the evaluation of cytokine levels in the serum, and histopathology on the third day after SEB administration.

Fecal contents were collected for 16S rRNA pyrosequencing for microbiome metagenomics analysis and short-chain fatty acids (SCFAs) production evaluation.

Collection of lung tissues: Lung tissues were removed from mice after anesthetization. Small portions of lung tissues were used in the isolation of bacterial DNA and histopathology, as described (Elliott, Nagarkatti, & Nagarkatti, 2016) . Lung tissues were also smashed using a Stomacher 80 Biomaster lab blender (Metrohm USA, Riverview, FL) in 10 mL of RPMI-1640 medium, lysed with Red Blood Lysing Buffer (Sigma-Aldrich, St. Louis, MO) to remove red blood cells, washed with PBS, filtered and resuspend in PBS supplemented with 5% FCS.

Then, lung-associated mononuclear cells (MNCs) were isolated from lung cell suspensions in Ficoll-Histopaque®-1077 (Sigma-Aldrich, St Louis, MO, USA) at a 1:1 ratio under 2000rpm centrifugation for 15 min, as described . The lung-associated mononuclear cells were stored immediately in TRIzol reagent at -80°C freezer for RNA sequencing. The portions of lung-associated mononuclear cells were used in the measurement of proton efflux rate (PER).

Enzyme-linked immunosorbent assay (ELISA) of cytokines: Serum samples were used in the measurement of cytokines including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), chemokine (C-C motif) ligand 5 (CCL5) , monocyte chemoattractant protein-1 (MCP-1/CCL2), and transforming growth factor beta (TGF-β), by means of ELISA according to the manufacture's protocol. All ELISA kits of mouse cytokines were purchased from Biolegend (San Diego, CA).

Histopathology of lung tissues: Lung tissues from mice were fixed in 4% paraformaldehyde solution, dehydrated in alcohol, and embedded in paraffin for microtome section preparation. The sections with 5 µm thick were stained with hematoxylin and eosin (H&E) and examined for inflammatory cell infiltrates under Leica fluorescence microscope system. Immunofluorescence staining of lung tissues: Immunofluorescence staining of lung tissues was carried out as described earlier (Alharris et al., 2018; Sarkar et al., 2019) . Briefly, the sections of lung tissues were deparaffinized according to the standard protocol and treated with the antigen-retrieval solution from Abcam (Cambridge, MA) for antigen retrieval. The Analysis of transcriptome: Lung epithelial cells were prepared according to the procedures described by the other investigators (Chapman et al., 2011; Vaughan et al., 2015) . Briefly, lung samples were collected from individual mice and injected with 1ml of dispase down trachea.

Then, the trachea was tied using string, and the lungs rinsed with cold PBS. To minimize contamination with bronchial epithelial cells, each lung lobe was cut from the main stem bronchi. The proximal-most ¼ of each lobe surrounding the bronchi was cut and the lobes were placed into a 50ml tube containing dispase and rocked at room temperature for 45 mins. Cell

Isolation: In a cell culture hood, 10ml of sort buffer and 50U/ml of DNAse were added to dissociate the lung tissue. The content was passed through 100µm, 70µm, and 40µm cell strainers over 50ml tubes. The filtered suspension was transferred into a 15mL tube, spun for 5 min at 550g at 4°C to pellet cells. The cells were resuspended in 10ml of sort buffer and left to recover shaking for at least one hour at 37°C. Then to increase the lung epithelial cells enrichment and get rid of any non-epithelial cells, CD326 antibody conjugated with PE Fluor chrome (clone: G8.8, RRID:AB_1134172) Biolegend -USA was used to positively purify the cell suspension by using magnetic microbeads (Stem Cell Technology -USA) according to manufacturer's protocol. The cells were kept in Qiazol (Qiagen, Valencia, CA) for 5 minutes at room temperature to break down the cellular membrane and used in the isolation of total RNA samples according to the manufacturer's manual of miRNeasy Mini kit (Qiagen, Valencia, CA). GeneChip WT plus reagent kit (Affymetrix, Santa Clara, CA) was used in the synthesis of fragmented single-stranded cDNAs (ss-cDNAs) from the total RNA samples with primers containing a T7 promoter sequence. The second-strand cDNAs were prepared from the first-stranded cDNAs using PCR. Next, the complimentary RNAs (cRNAs) were synthesized and amplified using T7 RNA polymerase according to Eberwine method (Borner, Smida, Hollt, Schraven, & Kraus, 2009; Van Gelder et al., 1990) , and purified by the magnetic microbeads (Affymetrix, Santa Clara, CA). The purified cRNAs were used to synthesize the second-cycle of ss-cDNAs using the 2 nd -cycle primers. Then, the second-cycle of ss-cDNAs were purified by the microbeads, and fragmented by uracil-DNA glycosylase and apurinic/apyrimidinic endonuclease 1 at the unnatural dUTP residues. The fragmented ss-cDNAs were labeled with the labeling master mix, and hybridized in the GeneChip Hybrydization Oven 645 (Affymetrix, Santa Clara, CA) at 45˚C for 16 h with rotation at speed of 60 rpm using ClariomD chip (Affymetrix, Santa Clara, CA). The Hybridization chips were washed and stained at room temperature for 2 h using GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA). Lastly, the chips were analyzed using GeneChip Scanner (Affymetrix, Santa Clara, CA) to profile the mouse transcriptome arrays (MTAs). Transcriptome analysis console (TAC) was used to analyze and interpret the MTA data for specific gene expression profiles. The dysregulation of several specific genes was validated by real-time PCR.

To validate the genes of interest, complementary DNA (cDNA) was prepared from isolated RNA with miScript II RT kit (Qiagen, USA), and qRT-PCR (Bio-Rad, USA) was used to This article is protected by copyright. All rights reserved.

determine expression levels. The data were expressed as fold change by comparing the experimental groups to naïve controls (fold mean of the controls), and considering the naïve expression as 1. PCR reactions were performed using miScript II primer assay (Qiagen, USA).

To quantitate gene expression, custom primers were designed and purchased from Integrated Pyrosequencing of microbiome: Lung tissues and colon samples were used in pyrosequencing for microbiome analysis, as described previously (Cox et al., 2017) .

Briefly, the extraction of genomic DNA from lung tissues and colon samples was carried (Weber et al., 2018) .

Colonic materials were collected from 8 donor mice in each group and used in fecal microbiota transplant, as described (Al-Ghezi, Busbee, et al., 2019) . Recipient mice were extensively treated with different antibiotics (i.e. Abx cocktail) for 4 weeks in drinking water to ensure the complete depletion of endogenous microbiome as described previously (Chevalier et al., 2015; Grivennikov et al., 2012) . The Abx cocktail was composed of 250 mg/ml bacitracin, 170 µg/ml gentamycin, 125 µg/ml ciprofloxacin, 100 µg/ml neomycin, 100 µg/ml metronidazole, 100 µg/ml ceftazidime, 100 U/ml penicillin, 50 µg/ml streptomycin and 50 µg/ml vancomycin.

This cocktail was replaced with freshly prepared cocktail every 5 days during the antibiotic treatment period. The complete depletion of endogenous microbiome in the gut of recipient mice was confirmed by PCR validation and bacteriological culture. Fecal pellets were collected aseptically under anaerobic conditions by enriching the chamber environment with special gas mixture (Praxair, Columbia, SC) from all recipient mice three days before the end of antibiotics treatment for the confirmation of complete depletion of endogenous microbiome in the gut.

Recipient mice received oral administration of normalized amount of fecal materials in PBS from each group of mice twice using tuberculin syringes in the anaerobic chamber to protect collected gut anaerobic bacteria from free oxygen during fecal microbiota transplant.

Immediately after FMT, the recipient mice received the first intranasal administration of SEB.

After 24 h, the recipient mice received the second intraperitoneal injection of SEB, as described above. Disease progress in mice was assessed by the evaluation of mouse survival for 30 days. On the third day after SEB administration, 5 mice form each group were sacrificed for the preparation of lung-associated mononuclear cells for flow cytometry analysis, and for the collection of colon contents for 16S rRNA pyrosequencing analysis. Generator software (Agilent) was used to interpret the acquired data and calculate PER in the lung-associated mononuclear cells and purified T cells (Neamah et al., 2019) .

The experimental design and analysis of this study complies with BJP's recommendations and requirements (Curtis et al., 2018) . Power analysis was carried out (a=0.05 and 1-b=0.80) to estimate the number of animals used in this study, which yielded a minimum sample size of 5 mice per group. Statistical analysis was undertaken only for studies where each group size was at least n=5. All in vitro studies were carried out in triplicate. All in vivo studies were performed with at least 5 mice in each group. Statistical analysis was performed using the data generated for individual mice and not using replicates as independent values. All outliers were included in data analysis and presentation.

GraphPad (RRID:SCR_002798) Prism 8.0 software was used in the statistical analysis.

Student's t-test was used to compare two groups, whereas multiple comparisons were made using one-way ANOVA, followed by post hoc analysis using Tukey's method. (Segata et al., 2011) .

Our previous study demonstrated that treatment of mice with THC prior to SEB exposure, attenuates lung injury through miRNA 17-92 cluster in ARDS mouse model (Rao et al., 2015) .

Because SEB is a superantigen that triggers acute inflammation, in the current study, we tested if treatment with THC after SEB sensitization would attenuate lung inflammation.

Histopathological analysis demonstrated that THC could attenuate acute lung injury, reduce congestion and minimize fibrin in mice with ARDS which was accompanied by a decrease in the infiltration of the immune cells in the lung tissues when compared to vehicle controls ( Fig.1A) . THC also significantly decreased the serum levels of pro-inflammatory cytokines including TNF-α, IFN-γ, CCL5 and MCP-1, but increased the serum levels of antiinflammatory cytokines such as TGF-β in ARDS mice when compared to vehicle control group (Fig.1B) . THC significantly decreased the number of mononuclear cells isolated from the lungs from ARDS mice (Fig.1C) . THC also significantly decreased the numbers of T cells, CD4+ T cells and CD8+ T cells in the lungs from ARDS mice (Fig 1D) . In addition, THC significantly decreased the proton efflux rate (PER) in the mononuclear cells isolated from the lungs from ARDS mice, suggesting that THC may suppress glycolysis in cell metabolism (Fig. 1E) . These data together demonstrated that THC attenuates SEB-mediated inflammation in the lungs.

We were interested in the understanding of functions of THC in the regulation of microbial dysbiosis in ARDS. Our pyrosequencing analysis demonstrated that THC dramatically altered microbiota in the lung tissues as shown in both the PCoA beta diversity plot (revealing the clustering patterns) and cladogram (displaying potential microbial biomarkers) from linear discriminant analysis effect size (LEfSe) analysis ( Fig. 2A) . THC also considerably changed microbiota in the colon (Fig. 2B ). The microbial species-level analysis in the lungs revealed that THC significantly increased the species of Ruminococcus gnavus, but significantly decreased the species of Bacteriodies acidifaciens and Akkermansia muciniphila in the lungs from ARDS mice (Fig. 2C) . Similarly, THC significantly increased the species of Ruminococcus gnavus, but significantly decreased the species of Akkermansia muciniphila in the colon from ARDS mice (Fig. 3D ). In addition, our analysis also showed that THC altered the microbiota in the blood from ARDS mice (Fig. S1A ). Similar to the lungs and colon, THC significantly increased the species of Ruminococcus gnavus, but significantly decreased the species of Akkermansia muciniphila in the blood from ARDS mice (Figs. S1B and S2). The results demonstrated that THC could significantly increase the species of Ruminococcus gnavus, but significantly decrease the species of Akkermansia muciniphila systemically following SEB exposure.

in the mucus which is a protective layer in many lining tissues in the body (Graziani et al., 2016) . Thus, we examined mucin expression in the lung by immunofluorescence staining. We found that THC significantly inhibited the expression of mucin MUC5ac (Fig. 2E ), but significantly increased the expression mucin MUC5b (Fig. 2F ). It has been reported that MUC5ac is associated with inflammation and airway obstruction (Bonser & Erle, 2017) ,

whereas MUC5b plays a role in mucociliary clearance, controlling infections in the airways and maintaining immune homeostasis in mouse lungs (Roy et al., 2014) . Additionally, THC significantly inhibited Evans blue extravasation in the lungs and FITC-dextran levels in the serum thereby suggesting that THC was blocking the vascular leak induced by SEB (Fig. 2G) .

Similarly, THC significantly inhibited the concentrations of lipopolysaccharides in the BALF and serum of SEB exposed mice (Fig. 2H) .

Because lung epithelial cells play a crucial role in barrier integrity and many other functions, we next examined the expression of genes in these cells from ARDS mice after THC treatment.

To that end, we performed transcriptome array analysis using total RNAs isolated from the lung epithelial cells. Data obtained from the transcriptome array analysis showed altered expression of a large number of genes in the epithelial cells after THC treatment (Fig. 3) .

Specifically, the scatter plot analysis demonstrated dysregulation of 1553 genes in the epithelial cells from ARDS mice after THC treatment (Fig. 3A) . The 29 most significantly dysregulated genes induced by THC were shown in the Heat map (Fig. 3B) . Among 29 dysregulated genes, several genes were particularly related to antimicrobial enzymes, antimicrobial peptides, tight junction proteins and mucins (Fig. 3B) . The up-regulation of antimicrobial enzyme genes including lysozyme 1 ((LYZ1) and LYZ2 and antimicrobial peptide gene, beta-defensin 2 (defb2), and the downregulation of antimicrobial peptide gene beta-defensin 1 (defb1) were validated by real-time PCR (Fig 3C) . Tight junction protein genes including claudin, zonula-1(ZO-1) and occludin-1 were up-regulated after THC treatment (Fig 3D) . Mucin2 and Muc5b genes were also up-regulated significantly in the lung epithelial cells from ARDS mice after THC treatment (Fig 3E) . It has been reported that the epithelial tissue secretes different antimicrobial peptides to fight against different microorganisms (Gordon, Romanowski, & McDermott, 2005) . These results suggested that THC may regulate the pathogenic microbiota promoted by SEB through induction of antimicrobial peptides.

FMT has been shown to replenish bacterial balance (Tauxe et al., 2015) . Thus, FMT was used to test the role of THC-induced microbial dysbiosis in the attenuation of ARDS. To that end, colonic material was obtained from different treatment groups including -controls, THC, SEB+Veh, and SEB+THC-treated group and transplanted into recipient mice that had been treated with antibiotics for 4 weeks and then exposed to SEB. The fact that this antibiotic treatment was effective in depletion of microbes has been shown in supplemental Fig S3. Survival data showed that all SEB-challenged mice that received FMT from SEB+Veh or vehicle alone group, died within 10 days, whereas recipient mice that received FMT from SEB+THC group showed 80% survival for more than 30 days (Fig. 4A) . Also, 60% of recipient mice that received FMT from THC alone treated group survived SEB challenge. These data suggested that the microbiota from SEB+THC group had protective effect on SEB-mediated pathogenesis. After FMT, colonic material was further examined for microbiota and we found that different treatment groups showed distinct clusters and composites of microbiota as shown in both the PCoA beta diversity plot and cladogram from LEfSe analysis ( Fig. 4B and Fig. 4C ).

Linear discriminant analysis (LDA) showed that Ruminococcus gnavus was seen only in SEB+THC group and was increased by about 3 fold compared to SEB+Veh group ( Fig 4D) consistent with the previous observation seen without fecal transfer (Fig 2C) . We wish to clarify that Fig 4D shows LEfSe analysis based on the fold change (log differences) between SEB+Veh vs SEB+THC groups. In order to compare these two groups to find unique bacteria in each group, the results are depicted on the X-axis as LDA scores in opposite directions. This however doesn't mean that those depicted on the minus side are down-regulated. It shows that all those bacteria were uniquely expressed in that group and which were not expressed in the other group, with fold change. Thus, Proteobacteria phylum significantly decreased while Firmicutes phylum was significantly increased in mice that received FMT from SEB+THC group when compared to mice that received FMT from SEB+Veh group (Fig. 4D and Fig. 4E ).

Next, we tested if FMT could also induce alterations in immune cells in the lungs of recipient mice. After the FMT of colonic materials from SEB+THC-treated mice, the percentages of both CD4+ and CD8+ T cells significantly decreased in the lungs of recipient mice when compared recipient mice that received FMT from SEB+Veh treated group (Fig. 4F and Fig.   4G ). In contrast, in the mice that received FMT from SEB+THC-treated group, the percentages of immunosuppressive cells such as regulatory T cells (Tregs) and myeloid derived suppresser cells (MDSCs) significantly increased in the lungs when compared to controls ( Fig. 4H and   Fig. 4I ). These data together suggested that THC-induced microbial dysbiosis was playing a beneficial role in suppressing SEB-induced ARDS pathogenesis.

Because of the positive role played by THC-mediated microbial dysbiosis, we next measured short chain fatty acids (SCFAs) know to play anti-inflammatory role. Thus, we quantified the concentrations of SCFSs in the colon flush from SEB+THC treated mice and our data showed that the concentrations of propionic acid, butyric acid and acetic acid significantly increased in these mice when compared to the controls (Fig. 5A ). To further elucidate the role of SCFAs in inflammation, we used in vitro cultures of T cells activated with SEB in the presence of propionic acid (PA). We found that in such cultures, addition of PA significantly inhibited the production of pro-inflammatory cytokines including IFN-γ, IL-17 and IL-1β, but significantly increased the production of anti-inflammatory cytokines such as IL-10 ( Fig.   5B ). Flow cytometric analysis also showed that THC decreased the percentage and numbers of CD3+CD4+, CD3+CD8+, NK1.1+ and IFN-+ cells in these cultures (Supplemental Fig   S4) . PA also significantly inhibited the proton efflux rate in T cells after SEB activation (Fig.   5C ). These data suggested that short-chain fatty acids such as PA suppressed the proinflammatory responses and thus may play a role in the control of ARDS pathogenesis.

infection. The enterotoxin produced by these bacteria, SEB, acts as a superantigen thereby activating a large proportion of T cells expressing certain Vβ specificities leading to cytokine storm and severe lung injury. Previous studies have shown that pro-inflammatory cytokines such as IL-1β, IL-6, IL-8 and TNF-α and inflammatory mononuclear cells are significantly increased in both ARDS patients and mice exposed to SEB Badamjav et al., 2020; Johnson & Matthay, 2010; Mohammed et al., 2020) . The current therapeutic approaches are inadequate because of which this devastating illness leads to high mortality rates, to the tune of 30-40% (Zambon & Vincent, 2008) . Because a significant proportion of COVID-19 patients develop ARDS, this incidence is further likely to increase. While microbiota found in the gut have been known to play a critical role in regulating inflammation, the role of resident microbiota during ARDS induced by SEB has not been investigated previously. In the current study we make several important observations: 1) THC when administered after SEB exposure, can attenuate SEB-mediated ARDS. 2) Exposure to SEB induces dysbiosis in the lungs and gut 3) THC triggers similar dysbiosis in the lungs and gut and THC-mediated attenuation of ARDS depends on alterations in the microbiota 4) FMT studies demonstrate that THC-mediated alterations in the lung microbiota may play a beneficial role in attenuating ARDS.

Studies from our laboratory and elsewhere have shown that THC acts as a potent antiinflammatory agent (Nagarkatti et al., 2009) . For instance, THC could significantly decrease the levels of pro-inflammatory cytokines such as IFN-γ and TNF-α, but increase antiinflammatory cytokines such as IL-10 and TGF-β (Hernandez-Cervantes, Mendez-Diaz, Prospero-Garcia, & Morales-Montor, 2017; Zgair et al., 2017) . Furthermore, THC has been shown to decrease Th1 cells while promoting Tregs and MDSCs (Eisenstein & Meissler, 2015; Hegde et al., 2010; Mohammed et al., 2020; Nagarkatti et al., 2009) . These studies are consistent with our current findings THC significantly inhibited infiltration of mononuclear cells in the lung, decreased serum levels of pro-inflammatory cytokines including TNF-α, IFNγ, CCL5 and MCP-1, while increasing anti-inflammatory cytokines TGF-β, decreased the number of CD3, CD4+ and CD8+ T cells, and suppressed the proton efflux rate in the lung-associated mononuclear cells (Fig. 1) . Inflammation in the lung also triggers structural remodeling of the airway involving epithelial cells, including increased extracellular deposition, and expansion of pro-fibrotic myofibroblast populations (Brasier, 2018) .

A few recent studies revealed that THC could alter gut microbiota in diet-induced obesity there are no studies delineating the effect of SEB or THC on lung microbiota. Thus, it was interesting to note that THC treatment in SEB-challenged mice led to similar changes in microbiota in both the gut and lungs inasmuch as we noted that THC significantly increased the abundance of bacterial species Ruminococcus gnavus in Firmicutes phylum but decreased that of Akkermansia muciniphila (Figs. 2 and S1). Ruminococcus gnavus is a human gut symbiont, and is known to play an important role in the regulation of mucin expression and glycosylation (Graziani et al., 2016) . Specifically, Ruminococcus gnavus increased the expression of glycoproteins and Muc2 in goblet cells in the colonic mucosa from mice (Graziani et al., 2016) . Mucin is one of the most important components of mucus and plays a crucial role in the defense against bacterial pneumonia, maintenance of the integrity of lung tissue and prevention of vascular leak (Johansson et al., 2011; Roy et al., 2014) . Besides,

Ruminococcus gnavus produced peptides such as ruminococcin A (RumA) and RumC, have high activity against pathogenic clostridia and exhibit other antimicrobial activities (Balty et al., 2019; Ongey et al., 2018) . In contrast, Akkermansia muciniphila is widely present in the intestines of humans and animals, and known to induce IgG1 antibodies and antigen-specific T cell responses, and thus enhancing immune responses (Ansaldo et al., 2019) . Indeed, it has been reported that Akkermansia muciniphila increased the expression of pro-inflammatory cytokines such as IFN-γ, IP-10, TNF-α, IL-6, IL-12 and IL-17 in the colon and colonic tissues from mice (Ganesh, Klopfleisch, Loh, & Blaut, 2013) . In addition, Akkermansia muciniphila has a specialized ability in mucin degradation and excessive mucin degradation may induce inflammation (Derrien, Belzer, & de Vos, 2017; Ganesh et al., 2013) . These results suggested that THC could modulate microbial dysbiosis in the lung, particularly increase the abundance of Ruminococcus gnavus, but decrease that of Akkermansia muciniphila, which were responsible for the induction of mucin expression, inhibition of the pro-inflammatory responses and suppression of pathogenic bacteria, leading to the attenuation of ARDS.

Because the epithelial cells play several important functions such as barrier protection, fluid balance, mucus and surfactant production, and repair following injury, in the current study, we investigated the effect of SEB and treatment with THC, on gene expression in lung epithelial cells. Our analysis showed that THC caused altered expression of over 1553 genes in the lung epithelial cells from ARDS mice, and particularly several dysregulated genes were related to antimicrobial enzymes, antimicrobial peptides, tight junction proteins and mucins ( Fig. 3A and   3B ). Further study revealed that THC up-regulated the gene expression of mucins such as Mucin2 and Muc5b (Fig. 3E ) and tight junction proteins such as claudin, ZO-1 and occludin-1 in the lung epithelial cells from ARDS mice (Fig. 3D ). Mucins are highly glycosylated proteins, which are the major components of the mucous on the surface of respiratory and digestive tracts and protect epithelial cells from infection, dehydration, and physical or chemical injury (Dhanisha, Guruvayoorappan, Drishya, & Abeesh, 2018; Ma, Rubin, & Voynow, 2018) . THC treatment increased the production of tight junction proteins indicating its role in protecting cell integrity (Alhamoruni, Lee, Wright, Larvin, & O'Sullivan, 2010; Gigli et al., 2017) .

Therefore, THC may increase the gene expression of mucins and tight junction proteins, and thus have protective effect during SEB-mediated ARDS. Most interestingly, our study discovered that THC up-regulated gene expression involving antimicrobial enzymes such as LYZ1 and LYZ2, and antimicrobial peptide gene defb2, but down-regulated gene expression of other antimicrobial peptides such as defb1, in the lung epithelial cells from ARDS mice (Fig.  3 ). It has been reported that the host-derived antimicrobial enzymes such as lysozyme in the mucus protect from harmful bacteria (Oliver & Wells, 2015) . There are other reports which have shown that lung injury is associated with alterations in -defensins. For example, elevated -defensin-1 protein was seen in chronic obstructive pulmonary disease (COPD) and severe asthma (Baines et al., 2015; Levy et al., 2005) . Additionally, -defensin-2 has been shown to play both an anti-inflammatory and anti-microbial role. Overexpression of -defensin-2 was shown to protect against P. aeruginosa and cecal ligation and double puncture-induced lung injury (Shu et al., 2006) . Thus, the ability of SEB to induce -defensin-2 and decrease defensin-1 correlates with the ARDS pathogenesis and in this context, it is interesting to note that THC reversed these effects. (Figs. 3 and 4) , it was expected that THC also affected the production of SCFAs. Our analysis showed that THC significantly increased the levels of propionic acid, butyric acid and acetic acid in the colon flash from ARDS mice (Fig. 5) . Furthermore, propionic acid significantly inhibited the production of pro-inflammatory cytokines but significantly increased the production of anti-inflammatory cytokines (Figs. 5). It has been reported that SCFAs could activate G-coupled-receptors, suppress histone deacetylases, affect energy metabolism, and thus regulate inflammatory responses (Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016) . Specifically, SCFAs could inhibit the production of proinflammatory cytokines, and suppress proliferation, activation and migration of immune cells (Vinolo et al., 2011) . Investigations also discovered that Ruminococcus gnavus produced propionate and propanol as the end products of metabolism (Crost et al., 2013) . These results suggested that THC may increase the abundance of bacterial species such as Ruminococcus gnavus in Firmicutes phylum, promote the production of short-chain fatty acids such as propionic acid, and thus suppress the pro-inflammatory responses, resulting in the attenuation of acute lung injury. Thus, the FMT may have worked to attenuate ARDS because of the induction of SCFA.

In summary, the major findings in the current study have been summarized in Fig 6. This study demonstrates that SEB triggers ARDS which is accompanied by lung inflammation and similar dysbiosis in the lungs and gut. This is also accompanied by decrease in SCFA, increased expression of Muc5ac, and decrease in antimicrobial peptides and tight junction proteins. This may cause increased penetrance of pathogenic bacteria and enhanced lung inflammation and mortality. Treatment with THC (SEB+THC group) leads to reversal of dysbiosis and other deleterious changes induced by SEB leading to survival of mice. FMT experiments confirm that the effect of THC is mediated through alterations in microbiota because antibiotic treated mice receiving FMT survive SEB challenge, and exhibit decreased inflammation in the lungs which is associated with induction of Tregs and MDSCs, and SCFA, which was shown in vitro to induce anti-inflammatory phenotype. Additionally, because a significant proportion of COVID-19 patients develop ARDS and cytokine storm, our studies raise a question on whether targeting cannabinoid receptors constitute a therapeutic modality to treat ARDS in such patients. The inhibitory effect of THC on the proton efflux rate (PER) on CD3+ T cells (E). Vertical bars represent data expressed as Mean±SEM with five mice in each group. p-value of <0.05 was considered statistically significant, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001. Figure 2 ; Effects of THC on the abundance of microbiota in the lung and colon from ARDS mice. Lung and colon content samples were collected from different treatment groups described in Figure 1 and microbial communities compositions were determined by 16S rRNA sequencing and analyzed by using QIIME through Nephele platform. The effects of THC on microbiota compositions in the lung tissues were shown in the PCoA beta diversit y plot (revealing the clustering patterns) on the left and cladogram (displaying potential microbial biomarkers) from linear discriminant analysis effect size (LEfSe) analysis on the right (A). Similarly, the effect of THC on microbiota compositions in the colon were displayed in the PCoA beta diversity plot on the left and cladogram from LEfSe analysis on the right (B). The effects of THC on the abundances of bacterial species including Ruminococcus gnavus, Bacteroides acidifaciens and Akkermansia muciniphila in the lung were depicted (C). The effects of THC on the abundances of bacterial species including Ruminococcus gnavus and Akkermansia muciniphila in the colon were depicted (D). The effect of THC on the expression of MUC5ac in the lung tissues was determined by immunofluorescence staining and exhibited (E). The effects of THC on the expression of MUC5b in the lung tissues was determined by immunofluorescence staining and presented (F). The effect of THC on lung vascular permeability was determined by the measurement of concentrations of Evans blue in the lung tissues, and FITC-dextran in the serum (G). The effects of THC on the concentrations of lipopolysaccharide (LPS) in the BALF and serum were shown (H). Data were presented as Mean±SEM of multiple experiments from eight mice in each group. p-value of <0.05 was considered statistically significant, * p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.

This article is protected by copyright. All rights reserved. Quantitative validations of the differential expressions of genes has been presented, including lysozyme1, lysozyme2, beta-defensin1 and beta-defensin2 (C); claudin, ZO-1 and occluding-1 (D); mucin2 and MUC5b (E). Data were presented as Mean±SEM of multiple experiments from five mice in each group. p-value of <0.05 was considered statistically significant, * p<0.05, ** p<0.01 and ***p<0.001.

Colonic material from various groups described in Figure 1were transferred into antibioticmicrobiome-depleted recipient mice that were challenged with SEB. The survival of such recipient mice was studied (A). The effects of FMT of colonic materials from mice of different treatment groups on the microbiota reconstitution in the colon of ARDS recipient mice was displayed in the PCoA beta diversity plot (B) and cladogram from LEfSe analysis (C). Linear discriminant analysis (LDA) scores were calculated for comparing differential abundance of microbiota in the colon from recipient mice after FMT of colonic materials from various groups and shown in the histogram plot (D). The effects of FMT of colonic materials from SEB+Veh and SEB+THC groups on the bacterial abundances of Firmicutes and Proteobacteria phylum in the colon from ARDS recipient mice (E). The effects of FMT of colonic materials from SEB+Veh and SEB+THC groups on the percentages of CD4+ T cells in the lungs (F), CD8+ T cells (G), CD4+Foxp3+ Treg cells (H), CD11b+Gr-1+ MDSCs (I).

Vertical bars represent data expressed as Mean±SEM from groups of 5 mice. P-value of <0.05 was considered statistically significant, ** p<0.01, and ***p<0.001. were presented as Mean±SEM from five mice or five replicates in each group. p-value of <0.05 was considered statistically significant, * p<0.05, ** p<0.01, and *** p<0.001.

This article is protected by copyright. All rights reserved. FMT experiments demonstrate such that transfer of microbiota from THC-treated mice can also cause beneficial bacteria to colonize and suppress SEB-mediated ARDS, inflammation and mortality by also inducing an anti-inflammatory state consisting of Tregs and MDSCs.

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The authors declare no conflicts of interest. 

This declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.