| dc.description.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. | en_US |
