Immunomodulatory effects of 7-hydroxy frullanolide, a plant-based sesquiterpene lactone, in inhibiting immune cell responses and roles of disease and vaccination outcomes on Covid-19 mortality

Abstract

Immunomodulation is a process by which the immune response of the host is either enhanced or suppressed due to various factors which either protects or makes the host more susceptible to particular diseases. Immunomodulators are a broad class of drugs that are used either to suppress (immunosuppressants) or enhance (immunostimulants) immune responses. These have been used to combat against the dysregulated immune system, observed during tissue/organ transplantation and disorders such as rheumatoid arthritis, ulcerative colitis and cancer. Plant-based immunomodulators such as capsaicin, curcumin and resveratrol are extensively used as immunomodulators and anti-inflammatory drugs. Sesquiterpene lactones are a major class of plant-based immunomodulators that are derivatives of sesquiterpenoids and are obtained from Asteraceae. These molecules are known for its anti-inflammatory and anti-tumor effects. The objective of my first study was to understand the roles of 7-hydroxy frullanolide (7HF) on different immune cells and to decipher the underlying mechanisms of action. Primary CD4+ T cells were isolated from lymph nodes of C57BL/6 mice, followed by treatment with different doses of 7HF and in-vitro activation with plate bound (pb) αCD3 and soluble αCD28. 7HF in a dose dependent manner caused a reduction in IL2 production and inhibition of upregulation of CD69, an early activation marker in CD4+ T cells. Subsequently, it was also observed that 7HF abrogated the increase in cell size and cellular proliferation with T cell activation. Ca2+ -dependent signaling pathways are an indispensable part of CD4+ T cell activation and overall T cell responses; hence it was important to understand whether 7HF had any effects on Ca2+ levels. Surprisingly, 7HF increased intracellular Ca2+ levels, more than optimal levels, during CD4+ T cell activation at early timepoints. We functionally confirmed this observation using a specific intracellular Ca2+ chelator, BAPTA-AM which rescued cellular proliferation with 7HF treatment during CD4+ T cell activation. To understand how 7HF caused a significant increase in intracelullar Ca2+ amounts, we performed Ca2+ channel inhibitor studies to understand whether 7HF behaved as a Ca2+ channel agonist in CD4+ T cells. Additionally, 7HF also caused a significant increase in extracellular lactate amounts which was functionally confirmed using 2-Deoxy D-glucose (2DG) experiments suggesting that 7HF was working on multiple fronts to hinder T cell responses. Subsequently, we tried to understand whether there was a link between increased Ca2+ levels and lactate levels during CD4+ T cell activation. Finally, to understand whether there is a link between Ca2+ and lactate, experiments were done using BAPTA-AM that confirmed Ca2+ levels did regulate lactate levels during CD4+ T cell activation. Next, we tried to understand the effect 7HF had on other immune cells. Although 7HF had no effect on lipopolysaccharide (LPS) stimulated splenocytes which primarily activates B cells, 7HF did inhibit activation of LPS treated adherent peritoneal macrophages with respect to nitrite and IL6 amounts in a dose dependent manner. Furthermore, 7HF inhibited peritoneal macrophage activation via a Ca2+ dependent mechanism. Ca2+ channel inhibitor studies also suggested that 7HF was behaving as a Ca2+ channel agonist in peritoneal macrophages, similar to CD4+ T cells. However, 7HF had no effect on lactate levels in peritoneal macrophages as compared to CD4+ T cells. Lastly, we wanted to identify the molecular mechanisms by which 7HF was acting as a Ca2+ agonist. Thus, we performed autodocking experiments using the autodock modelling simulation software and standardized the binding of 7HF to Ca2+ channels. We initially obtained the crystal structure of the Ca2+ channel proteins, IP3R and TRPV1 and tried to dock the 3D structure of the ligands into the respective pockets of the protein molecules. We observed that 7HF was binding to both TRPV1 and IP3R Ca2+ channels with a significant binding energy when compared to the positive controls. Hence it is likely that 7HF behaved as a Ca2+ channel agonist to greatly increase Ca2+ amounts that inhibited both T cell and macrophage responses. To understand the physiological relevance of 7HF, we performed in-vivo experiments with 7HF using the Dextran sulfate sodium (DSS) induced colitis model of mice which is an animal model of autoimmune Inflammatory Bowel Disease. We used the golden standard of estimating the severity of colitis by measuring the colon length. DSS, as reported, caused a severe shortening of colon length which was rescued by 7HF in a dose dependent manner. The extent of colon damage as observed due to severe damage in the crypts and extensive immune cell infiltration by DSS was significantly reduced by 7HF. Also, 7HF rescued the loss in thymocyte and mesenteric lymph node (MLN) cell numbers due to DSS treatment. Finally, 7HF significantly reduced the amounts of proinflammatory cytokines, IFNγ and IL6 in the sera. Overall, this study clearly demonstrates the in-vivo effects of 7HF as an anti-inflammatory molecule. Both in-vitro and in-vivo studies together strengthen the observation that 7HF may have potential therapeutic applications to treat different autoimmune disorders. My next study was performed during the Covid-19 pandemic. An epidemiological study was performed during the months of April-June 2020 to understand the effects of pathogen exposure and vaccination coverage on Covid-19-induced mortality. Covid-19 deaths display a high heterogeneity across different countries and the extent of infection in a particular population might be dependent on multiple factors such as age, sex, temperature, humidity etc. This led us to perform correlation studies with 36 (out of 215) countries having more than 1000 Covid-19 deaths as of 24th June 2020. The major observations of this study are: measles, HBV and polio vaccination coverage had no correlation with Covid-19 incidences and deaths. Subsequently, flu incidences had no correlation with respect to Covid-19 incidences and deaths. However, flu deaths showed a significant negative correlation with Covid-19 mortality rate and Covid-19 deaths/million. Additionally, flu vaccination had a significant positive correlation with Covid-19 incidences, Covid-19 deaths, mortality rate and Covid-19 deaths/million. Next, Tuberculosis (TB) only negatively correlated with Covid-19 mortality rate and Covid-19 deaths/million. This trend was also holding for TB deaths. Similarly, Bacillus Calmette Guerin (BCG) coverage had a significant negative correlation with Covid-19 deaths, mortality rate and Covid-19 deaths/million suggesting its protective role. Interestingly, countries which have flu, but no BCG, vaccination show highest number of Covid-19 deaths. Countries that have high flu and BCG vaccinations demonstrate more immunomodulatory effects in determining the fate of a particular disease and its effects on the host.