Role of Macrophages in CBD Mediated Attenuation of SEB-Induced ARDS

Severe cases of Acute Respiratory Distress Syndrome (ARDS) and sepsis can be fatal due to pulmonary inflammation and destruction of the epithelial and endothelial cell lining. Understanding the mechanisms behind these diseases is vital to develop effective preventive and therapeutic strategies. Staphylococcus enterotoxin B (SEB)-induced ARDS mimics the cytokine storm, sepsis, and multiple organ failure presented in patients with severe COVID-19. It has been shown that the superantigen structure and sequence associated with the spike protein of SARS-CoV-2 is like that of SEB. In pre-clinical trials, treating ARDS with SEB drops survival rate to 0%. In our study, we found that Cannabidiol (CBD) administration following SEB treatment led to 100% survival indefinitely. Initial evaluation of whole single-cell sequencing data comparing lungs  with SEB induced ARDS illustrated that there was an increase in neutrophils, inflammatory macrophages, and proinflammatory cytokines (IL-1β and TNF-α) as well as a loss in lung epithelial cells. Single-cell RNA seq analysis of the toxin when treated with CBD has shown a decrease in the infiltration of inflammatory macrophage populations in the lung. Treatment with CBD also leads to increased survival probability, decreased proinflammatory cytokine production, and decreased bronchoconstriction caused by SEB. Using CCR2 KO we were able to visualize the pathogenesis of the disease in the absence of infiltrating “pro-inflammatory” macrophages. As a result, we found that disease severity was improved but not completely ameliorated. Utilizing SCID, we were able to identify whether macrophages independently of T cells would have the ability to induce ARDS caused by SEB. We found that there was an inflammatory response, however, not as severe as illustrated in the wild type. To continue to elucidate the mechanism by which CBD treatment led to amelioration of the inflammatory response, microRNA expression analysis was done that showed a significant decrease in expression of miR-124-3p and miR-298-5p in the SEB-exposed group which is directly associated with upregulation of TNF-α and IL-1β expression as well as macrophage activation gene, Cebpb. We hypothesized that CBD attenuates SEB-induced ARDS by miRNA dysregulation in lung-infiltrating cells, specifically by inducing miR-124-3p and miR-298-5pwhich downregulates Cebpb expression resulting in reduced activation of macrophages. Aim 1 will elucidate the role of resident and monocyte-derived macrophages in disease and the effect CBD on those subpopulations. Aim 2 will elucidate whether CBD affects Cebpb expression and the effects that miR-124-3p and miR-298-5p have on manifestation of disease. Aim 3 will determine the epigenetic factors regulating the expression of miR-124-3p and miR-298-5p. This study will explore CBD as a potential therapeutic for ARDS and/or sepsis-induced not only by SEB but other pathogens such as SARS-CoV-2.

Epigenetic/Transcriptomic Analysis of Multiple Causative Agents of ARDS

Multiple factors both physical and microbial can induce ARDS. Being able to determine a global mechanism for ARDS induction will be helpful in the development of treatments that would be beneficial regardless of the causative agent. While previously I have induced ARDS with the use of an enterotoxin, I would like to explore other models of ARDS specifically Streptococcus pneumoniae, MHV-3, and Poly (I:C) induced ARDS to determine if the mechanism of disease is like that of my current model. Using these models, I can perform epigenetic studies like miRNA sequencing and histone modifications to determine if there is a targetable response that is observed in ARDS induced by multiple methods.

Effects of Phytochemicals on Neutrophil NETosis induced by ARDS

One of the premier goals of my independent research is to elucidate the mechanisms of ARDS and how mechanistically different treatments might alleviate the fatal response. It is well known now that neutrophils produce extracellular traps (NETs) that heavily influence the hyperinflammatory response of viruses like SARS-CoV-2 that led to ARDS. In this study, I aim to investigate the effects of phytochemicals on neutrophil recruitment and NETosis. Utilizing the murine SEB-induced ARDS model, I will monitor neutrophil recruitment and activity via histology, flow cytometry, and cytokine production assays. This will be done in pre-clinical models treated with SEB/Cannabinoids. Next, I will focus particularly on the development of neutrophil NETs. Primary evaluations will be done using confocal imaging to visualize NET production following which, I will investigate the granule proteins, DNA, and histones produced in the lung with and without cannabinoid/SEB treatment. From the results, I would be able to infer whether cannabinoids affect NETosis and if so, the potential targets involved in regulation of NETosis.

Gut-lung axis

The gut-lung axis is a concept that has been around for decades. Multiple groups have repeatedly illustrated that there is bi-directional crosstalk between the gut and lungs that may influence the prognosis of the disease. The over misuse of antibiotics and the increase in inflammatory diseases like colitis have made this topic even more interesting. In order to determine if previous dysregulation of the gut microbiome affects ARDS. I would like to approach this project from two directions. One method will be to treat ARDS with antibiotics and expose it to the virus a week later. I would expect to see some changes in disease development and progression. Alternatively, I also plan to bacterially induce chronic colitis and then introduce the virus to determine how gut inflammation and microbe dysregulation will affect disease prognosis. This will enhance the knowledge in the field and help to inspect why ARDS responses may differ from person to person.