Technical Report, pp.1-83, 2019
he immune system is a set of complex and dynamic mechanisms, that protects the
body against pathogens and maintain homeostatic balance in the presence of an
internal disruption. Besides its protective roles against pathogens, the immune system
is intertwined with the large scale systems such as the nervous system and different
types of metabolic systems and constitutes a variety of sophisticated multi-
dimensional mechanisms ranging from antibody production to tissue remodeling.
The immune system consists of two distint categories; innate immunity and adaptive
immunity which are interrelated. The innate immunity is much more rapid and
abundant whereas the adaptive immunity develops specific responses upon
encountering a pathogen in concert with the innate immunity. Additionally, the
adaptive immunity generates a memory mechanism to fight off the pathogens that have
already been encountered.
Innate immune responses are generally regulated by conserved yet limited set of
Pattern Recognition Receptors (PRRs). PRRs contain two types of receptors:
membrane bound and cytosolic receptors. While Toll-like receptors (TLRs) and C-
type lectin receptors (CLRs) are considered membrane bound, NOD-like receptors
(NLRs) and RIG-I-like receptors (RLR) are considered cytoplasmic PRRs. This thesis
involves a comprehensive set of experimental studies designed to deepen the
knowledge of NLRC4 and its activation through TLRs.
Members of the TLR family are transmembrane receptors that have critical roles in
promoting rapid and diverse pro-inflammatory responses. For instance, TLR2
recognizes microbial glycolipids and lipopeptides whereas TLR 4 recognizes
lipopolysaccharides. TLR 5, another surface TLR, recognizes bacterial flagella.
Activation of TLR signaling pathways result in the expression of a broad range of pro-
inflammatory cytokines and receptors such as IL-1, IL-6, IL-12, IL-18 and CD80,
NLRs are cytosolic PRRs that are induced by molecular patterns of pathogens or
damage associated molecular patterns (DAMPs). The NLR family consists of 22
members in humans. NLRs consist of three main domains: a C-terminal LRR domain
that sense microbial molecules or danger associated molecules, a central NACHT
domain that mediates NLR oligomerization and an N-terminal domain which activates
NLRP3 is probably the most studied and well-characterized member of the NLR
family. As a member of the NLRP subfamily, NLRP3 constitutes a PYRIN domain
and inflammasome assembly upon ligand recognition by NLRP3 requires the adaptor
protein ASC. NLRC4, on the other hand, does not have a PYRIN domain and can form
inflammasome with or without the interaction with the adaptor protein ASC. Uponxx
activation, both of the members of the NLR family are reported to form protein
complexes known as inflammasomes and this activation results in cleavage and
secretion of the cytokines IL-18 and IL-1β into their biologically active forms.
Eosinophils play critical roles in the defence againts extracellular parasites and in the
development of allergic responses. Since they develop extensive inflammatory
responses, eosinophils are not abundant in the human blood, only constituting 1-6% of
all white blood cells. And even though NLRC4 and NLRP3 are well-studied in other
contexts and white blood cells, their roles in shaping the eosinophilic functions are
Since eosinophils are sparse in the human blood, we used the human eosinophilic
leukemia cell line EoL-1 which provides a model for the investigation of eosinophilic
functions. To characterize the functions of PRRs in general and NLRs in particular,
we first analyzed the expression levels of NLRC4 and NLRP3 and the expression of
eosinophilic markers at both mRNA and protein level.
We have observed that in the eosinophil like EoL-1 cells, NLRC4 and NLRP3 are
expressed both at mRNA and protein levels. We have also observed that NLRC4
mRNA and protein levels are augmented upon induction with two signals, signal 1
being the TLR ligand and signal 2 being the NLRC4 ligand, transfected flagella.
Moreover, our western blotting results demonstrated that in EoL-1 cells NLRC4 and
NLRP3 inflammasomes were activated and caspase-1 and IL-1β are cleaved into their
active forms in EoL-1 cells upon induction with NLRC4 and NLRP3 ligands. Our
ELISA results also displayed that EoL-1 cells secreted the cytokine IL-1β at 24 hour
NLRC4 poststimulation and 10 hour NLRP3 poststimulation. EoL-1 cells also secreted
the inflammasome independent cytokine IL-6 upon TLR4 induction but stimulation
with flagella alone without transfection did not cause any IL-6 secretion in these cells.
Of note, 40% NLRC4 knockdown was sufficient to detect both biologically and
statistically meaningful decline in the secretion of IL-1β.
Furthermore, we verified that EoL-1 cells display a number of specific characteristics
of human eosinophils. They express IL-5 receptor IL-5Rα which takes part in the
survival and activation of eosinophils and the allergy related IgE low affinity receptor
FcεR2 which is expressed on eosinophils. Additionally, EoL-1 cells expressed the high
affinity Fc epsilon receptor FcεR1α and the immunoregulatory Siglec-8 protein upon
induction. Hence, it is safe to say that EoL-1 cells provide a good model for the
investigation of eosinophilic functions.
There are studies underlying the effects of PRRs on eosinophilic functions and their
involvement and relationship with the Th2 responses. In the light of this study, we
have shown the positive effects of NLR and TLR induction in promoting the
eosinophilic functions. PAM3CSK4 (TLR2 agonist) stimulation and flagella
transfection both significantly elevated the number of EoL-1 cells expressing the
eosinophilic marker IL-5Rα and low affinity Fc epsilon receptor FcεR2. Additionally,
NLRC4 activation led to an increase in the high affinity Fc epsilon receptor FcεR1α
and Siglec-8 expressing cells. Nevertheless, NLRP3 or TLR5 induction had no impact
on the expression of the above mentioned receptors.
Because eosinophils respond in extensive measures to infections or allergens, their
proliferation must be tightly regulated and limited. The functional roles of the surface
receptor IL-5Rα is of great importance in the survival of the eosinophils and their
activation. We observed a reciprocal relationship between NLRC4 and the IL-5Rα.
EoL-1 cells expressed higher levels of NLRC4 protein when cells were stimulated withxxi
IL-5 as compared to non-stimulated cells. Interestingly, percentage of IL-5Rα
expressing EoL-1 cells were higher in cells that are stimulated with PAM3CSK4 and
flagella than cells stimulated with PAM3CSK4 alone.
A few studies reported the antigen presenting capabilities of eosinophils upon
activation. Our flow cytometry results suggest that non-treated EoL-1 cells expressed
CD80 and CD86. Stimulation of cells via NLRP3 and NLRC4 axis with TLR2 agonist
caused an increase in the number of CD80 expressing cells and ATP treatment and
flagella transfection even further increased this effect.
On the basis of current data, NLRC4 could be deduced to be more closely related to
allergic responses while TLR signaling promotes more general, global immune
responses in EoL-1 cells. Taken together, our findings present potential targets for both
the further characterization of eosinophilic functions and allergic responses coupled
with a new frame of thoughts to understand the relationship between PRRs and