Date of Award

Fall 2021

Document Type

Open Access Dissertation

Department

Biomedical Engineering

First Advisor

Mitzi Nagarkatti

Second Advisor

Prakash Nagarkatti

Abstract

Cannabinoids are group of compounds that exerts their anti-inflammatory action through activation of CB1 and CB2 receptors and other minor receptors. In the current study, we investigated the effect of Anandamide (AEA) on Acute Respiratory Distress Syndrome (ARDS) induced by exposure to Staphylococcus Enterotoxin B (SEB) intranasally. We found that C57BL/6 mice treated with AEA showed improvement in the clinical functions of the lungs, accompanied by a decrease in the infiltration of inflammatory cells in the lung tissue, and a decrease in the secretion of pro-inflammatory cytokines. Furthermore, AEA regulated the miRNA profile of the mononuclear cells in the lungs leading to significant downregulation of miRNA-23a-3p which led to upregulation of Arginase 1(Arg1) and Transforming Growth Factor 2 (TGFB2), which are markers of Myeloid Derived Suppressor Cells (MDSCs) and T regulatory cells (Tregs), respectively. Another miRNA that was downregulated after AEA treatment was miRNA-34a-5p which led to induction of FOXP3, a marker expressed by the T regulatory cells. All the miRNA markers and their target genes were validated by qRT-PCR. Additionally, flow cytometry data indicated that AEA caused a decrease in CD4+T cells, CD8+T cells, V8+ T cells, and NK+ T cells in ARDS mice. On the other hand, AEA caused induction of MDSCs, as well as T regulatory cells (CD4+FOXP3+), and Tr1 subpopulation (CD4+IL10+). Additionally, proliferation assays demonstrated that AEA-induced MDSCs were indeed highly immunosuppressive and blocked T cell proliferation in vitro.

Next, we investigated whether AEA attenuated ARDS through modulation of microbiome profile in the Gut-Lung Axis (GLA) in mice treated with SEB. AEA caused a decrease in the leakage of the gut and lungs, and decreased pathology in these organs based on histopathological analysis. Furthermore, data obtained from flowcytometry demonstrated that T cell subsets in the Mesenteric Lymph Nodes (MLN) including CD4+ T cells, CD8+ T cells, V8+ T cells, and NK+ T cells were significantly decreased in the AEA treated mice, when compared to SEB+VEH-treated mice.

In addition, Single Cell RNA sequencing (sc-RNAseq) of the cells derived from the lungs demonstrated upregulation of the gene expression of Antimicrobial Peptides in the lung epithelial cells including Tracheal anti-Microbial Peptide 1 and 2 (TAP1,TAP2), Lysozyme 2 (LYZ2), Murine Beta Defensin 2 (MBD2) , and Serum Protease Leukocyte Inhibitor (SLPI) as well as tight junctions SEB+VEH-treated mice.markers including CLDN1 and CDH1, which were all significantly increased post-AEA treatment. Also, gene expression was validated by performing quantitative RT-PCR (qRT-PCR). These data suggested that AEA was inducing the AMPs to kill pathogenic bacteria as well as stabilizing the epithelial cell functions disrupted by SEB.

Furthermore, we investigated the nature of microbial dysbiosis in the Gut-Lung Axis. The r/;esults obtained from 16sRNA sequencing on MySeq platform, demonstrated significant changes in the microbiome of the gut and lungs in the AEA-treated group of mice, when compared to SEB+VEH group of mice. Also, upon analysis using both Nephele and LDA cladogram, there was an increase in butyrate producing bacteria such as S-247 in both the gut and the lungs and Lachnospiraceae in the lungs. Furthermore, we observed that Short Chain Fatty Acids (SCFAs) especially butyrate, valeric acid, and isovaleric acid were significantly increased in the AEA-treated group mice, when compared to SEB+VEH-treated group. Furthermore, direct treatment of mice with butyrate, led to attenuation of SEB-mediated ARDS and improved the clinical lung respiratory functions as well as decreased the infiltration of the inflammatory cells.

Together, these studies demonstrate for the first time that endogenous cannabinoids play a critical role in suppressing cytokine storm and ARDS. These studies are highly significant considering that ARDS is difficult to treat as seen in COVDI-19 patients with ARDS and thus, direct use of endocannabinoids or increasing their levels in vivo with agents such as Fatty Acid Amide Hydrolase (FAAH), may constitute a novel therapeutic modality to treat ARDS.

Rights

© 2021, Muthanna Ali Sultan

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