1. “MECHANISM OF DECTIN MEDIATED
ANTIFUNGAL IMMUNITY”
A DISSERTATION THESIS
SUBMITTED FOR
THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR
THE AWARD OF THE DEGREE OF
MASTER OF SCIENCE IN
(INTEGRATED BIOTECHNOLOGY)
MEDICAL BIOTECHNOLOGY
OF
SARDAR PATEL UNIVERSITY
NOVEMBER’2013
SUBMITTED BY:
KRUMALI CHOKSHI,
ARIBAS
UNDER THE GUIDANCE OF
DR. REENA RAJPUT
ASSISTANT PROFESSOR
AT
INDIAN INSTITUTE OF ADVANCED RESEARCH
GANDHINAGAR- 382007
ASHOK & RITA PATEL INSTITUTE OF INTEGRATED STUDY &
RESEARCH IN BIOTECHNOLOGY AND ALLIED SCIENCES (ARIBAS)
NEW VALLABH VIDHYANAGAR
2.
3. UNDERTAKING
I, Krumali Chokshi, of Ashok and Rita Patel Institute of Integrated
Study and Research in Biotechnology & Allied Sciences, New Vallabh
Vidyanagar hereby undertake that the work presented in the dissertation
project report entitled “Mechanism Of Dectin Mediated Antifungal
Immunity” comprises the results of independent and original work carried
out by me under the supervision of Dr. Reena Rajput for the partial
fulfillment of the award of the degree of M. Sc. Integrated Biotechnology
of Sardar Patel University, Vallabh Vidyanagar.
I further declare that this work did not form a part of any other work
published or unpublished.
KRUMALI. K. CHOKSHI
Date: 23 /10 /2013
4. ACKNOWLEDGEMENT
The great accomplishments of man have resulted from the
transmission of Ideas and Enthusiasm.
On the accomplishment of the present study, I would like to take this
opportunity and words of appreciation towards those, who decided their today for my
tomorrow. I deem it a proud privilege and feel immense pleasure to acknowledge all
those who are directly or indirectly involved in enlightening me with the touch of
their knowledge and extending me their unflinching support.
I bow my head in reverence at the Lotus Feet of My Almighty for guiding me
through life, for giving me strength & courage to accomplish my dreams and for
blessing me with the love, care, support of my family members, friends, teachers and
colleagues who are greatest possessions of my life.
At the outset, I take the privilege in expressing my deep sense of gratitude and
indebtedness to my Major Advisor, Dr. Reena A. Rajput , Assistant Professor at
IIAR, Gandhinagar ,for her consistent and invaluable inspiration , introspective
guidance, keen interest , critical supervision and constant encouragement throughout
the pursuit of this study. I consider myself as fortunate and greatly privileged to have
worked under her supervision and guidance.
I bow my head in respect to Dr. Nilanjan Roy, Director, Ashok & Rita Patel
Institute of Integrated Study & Research in Biotechnology and Allied Sciences
(ARIBAS) for his munificent attitude in providing the necessary facilities to carry
out the research work.
I owe my special thanks to Ms. Nilam Gori and Ms. Ekta Patel for their
technical support , unceasing help and invaluable guidance during the research .
I am immensely thankful to My Labmates , Mr. Omkar Naik , Ms. Kshama
Jain, Mr. Manthan Patel, Mr. Divyesh Patel, Mr. Sagar Gaikwad, , Ms. Dipeeka
5. Mandalia , JRFs, Department of Human Health and Disease ,Indian Institute of
Advanced Research, Koba, Gandhinagar, for their ever willing help and continuous
motivation.
My parents, Mr. Keyur Chokshi and Mrs. Rita Chokshi need a special
mention who have always stood by my side like a lighthouse for illuminating the
pathway of any success. Without their selfless love and unflinching support, I would
have not been able to achieve this stage of life.
Words are not enough to thank to my Brother Mr. Joy Chokshi & Mr.
Janmay Chokshi for providing me constant support, boosting my moral and for all
the affection and care showered upon me and also for taking care of responsibilities at
home.
I essentially want to extend my immense sense of gratitude to my uncle, Mr.
Jignesh Patel. I am indebted to my aunt Mrs. Jagruti Patel, Tanay & Himani for
their invariable encouragement and support during the trail of this work.
Words are inadequate to express my gratitude to my friends Gayatri, Sushma,
Resham ,Anuja, Nupur, Pramiti, Karishma, Tarak, Manan, Sagar & Naishal for
their valuable support and motivation.
I am indeed indebted to all my teachers and professors who have been a
blessing of Goddess saraswati upon me and have been instrumental in shaping my life
in past five and half years. It would be pleasure for me to acknowledge the staff
members of the department of Integrated biotechnology, for their kind gesture and
support throughout my tenure.
At the end, I am also greatful to all my well wishers whom I might have
failed to mention here. I would like to accolade all of them with depth of my heart.
Date: 23 /10/2013. (Krumali Chokshi)
7. MHC : Major Histocompatibility Complex
MICL : Myeloid Inhibitory C-Type Lectin-Like Receptor
MYD88 : Myeloid Differentiation Protein
NALP3 : NACHT, LRR and PYD domains-containing protein 3
NEMO : NF-κB-Essential Modulator
NFAT : Nuclear Factor Of Activated T Cells
NFκB : Nuclear Factor kappa-light-chain-Enhancer Of Activated B Cells
NK : Natural Killer Cells
NO : Nitric Oxide
NOD : Nucleotide-Binding Oligomerization Domain
PAMP’S : Pathogen-Associated Molecular Patterns
PARs : Protease Activated Receptors
PPR : Pattern Recognition Receptor
PSK : Polysaccharide K
RANTES : Regulated On Activation, Normal T Cell Expressed And Secreted
RNS : Reactive Nitrogen Species
ROS : Reactive Oxygen Species
Sh2 : Sequence homology 2
Smad : Signal Transducer Of Bone-Morphogenetic Protein
SOS : Son of Sevenless
STAT : Signal Transducer and Activator of Transcription
SYK : Spleen-Tyrosine Kinase
TC : Cytotoxic T Cells
TCR : T-cell receptor
TGF-β : Tumor Growth Factor Beta
TGF-βR : Receptor For Transforming Growth Factor-Beta
TH : T Helper cells
TIRAP : TIR-Domain-containing Adaptor Protein
TLRs : Toll-like receptors
TNFR : Tumor Necrosis Factor Receptor
TNF-α : Tumor Necrosis Factor-α
Tregs : T-regulatory cells
TRIF : TIR-Domain-containing Adaptor Protein Inducing IFN-β
Ub : Ubiquitin
8. INDEX
CONTENT OVERVIEW Page no.
I. INTRODUCTION 1
II. REVIEW OF LITERATURE 5
III. AIMS & OBJECTIVES 39
IV. MATERIALS & METHODS 40
V. RESULT AND DISCUSSION 61
VI. CONCLUSION 75
VII. SUMMARY 76
VIII. FUTURE PERSPECTIVES 78
IX. BIBLIOGRAPHY 79
X. APPENDIX 85
9. LIST OF FIGURES
Page no.
1. Host defense mechanism for Fungal Immunity 2
2. Microscopic view of Aspergillus fumigates 6
3. A.fumigatus infection leads to various pathological conditions 6
4. A.fumigatus induced immune response in ABPA 7
5. A.fumigatus induced immune response in IPA 7
6. Integration of CLR-mediated signaling directs adaptive immunity 9
7. The innate and adaptive immune response 9
8. Schematic presentation of TLR’s and signaling pathway 13
9. Classification of cells of adaptive immune system 15
10. Development of B-Cells 15
11. Differentiation of TH1 Cells 17
12. Differentiation of TH17 Cells 18
13. C-type Lectin Receptor Superfamily 20
14. Phagocytic synapse formed by interaction of β-Glucan & 20
Dectin-1 receptor
15. Activation of Dectin-1 & Dectin-2 Receptors 22
16. Dectin-1 and Dectin-2 genomic localization within the Myeloid cell 22
expressed natural killer (NK)-cell-receptor-likecC-type lectin cluster
17. Immunostimulation by fungal β-glucans 24
18. Possible fungal β-glucan mediated signal pathway 25
19. Schematic representation of inflammasome activation 27
20. Two step activation process of IL-1β 27
21. Signaling pathways involving SYK during engagement of 28
Dectin-1/ITAM receptors leading to IL-1β production
10. 22. Role of NALP3 inflammasome in activation of IL-1β 29
23. Syk signalling pathway 31
24. MAPK/ERK signalling pathway 31
25. Dectin mediated NF- κB translocation 33
26. ROS inhibition mediated by BHA 34
27. Cell signalling inhibitors along with their respective signalling pathway 35
28. Mechanism of Dectin mediated Antifungal immunity 38
29. Cell morphology of A549 cells at 10 X objective under 61
Inverted Phase contrast microscope
30. DAPI-PI staining of A549 cells 61
31. Cellular granularity of control & β-Glucan treated groups 62
32. Β-glucan mediated dectin expression 63
33. β-glucan modulates Dectin expression and Inflammatory response 64
34. Signalling inhibitors regulate Dectin expression 66
35. β-Glucan regulates Dectin expression and signalling events 68
differentially during interference with various signaling and
Oxidative pathways inhibition
36. MTT Cell viability assay 69
37. β-Glucan regulates NF-κB translocation in the nucleus 70
38. Cytokine profiling of Pro-inflammatory cytokines 71
39. NO production values for all signalling inhibitors along with curdlan 72
40. Dectin-1 mediated cellular response to fungal β-glucans 74
11. LIST OF TABLES
Page no.
1. Difference in response in immunocompetent & immunodeficient individual 8
2. List of subsets of T-cells involved in T-cell mediated immune response 16
3. Difference between Dectin-1 & dectin-2 receptor 23
4. Mastermix Preparation For cDNA 46
5. Mastermix Preparation for PCR 46
6. Primer Design 48
7. Stacking & Resolving Gel Preparation 52
8. Percentage Cell Cytotoxicity by MTT Assay 69
9. Relative fold expression of IL-1β 71
10. Relative Fold expression of TNF-α 71
11. Standard values For NO assay 72
12. Nitric Oxide Production at 490 nm 72
12. 1
1. INTRODUCTION:
ANTIFUNGAL IMMUNITY
Many fungal species (such as Aspergillus, Candida) are opportunistic pathogens which
may cause disease when there is a supression in immune status or if the physical
barriers of the host are breached (Brown 2011). The innate immune system plays an
obligatory role in antifungal immunity owing to the ability of pattern recognition
receptors (PRRs) on phagocytes to recognize fungal pathogen-associated molecular
patterns(PAMP’s), which are major constituents of the fungal cell wall. PRRs,
including Toll-like receptors (TLRs), C-type lectin receptors and the galectin family
proteins, activate antifungal immune responses in a rapid, conserved manner (Regina &
Ronen, 2012). A coordinated host immune response is fundamental for successful
elimination of an invading fungal microbe. A panel of C-type lectin receptors
expressed on antigen-presenting dendritic cells enables recognition of fungal cell wall
carbohydrates and tailors adaptive responses by means of the instruction of CD4+ T
helper cell fates. T helper cell type 1 and IL-17-producing T helper cell responses are
crucial in anti-fungal immunity and facilitate phagocytic clearance of fungal
encounters. Strikingly, different classes of fungi trigger diverse sets of receptors to
evoke a pathogen-specific T helper response (Wevers et. al. 2013).
HOST DEFENSE MECHANISM FOR FUNGAL INFECTION
The virulence of Aspergillus fumigatus is multifactorial and is combined with both the
immune status of the patient and the biological characteristics of the fungus (Latge
2010).
Upon β-glucan binding, Dectin-1 recruits Syk to the ITAM, following the activation of
the CARD9–BCL10–MALT1 (CBM) complex[Figure 1].
(i) ROS production is also induced in a Syk-dependent manner, resulting in fungal
killing in inflammasomes and the activation of caspase-1.
(ii) Raf-1 is activated in a Syk-independent manner.
13. 2
Upon α-mannan binding, Dectin-2 recruits Syk to the ITAM of the FcRγ chain and
activates the CBM complex downstream. TLR-2 ligands (e.g. proteoglycans) are also
expressed in fungal cell walls and the TLR2–TLR6 complex activates the CBM
complex through activation of MyD88.
Figure 1: Host defense mechanism for Fungal immunity
NF-κB activation occurs by two ways:
i. The CBM complex activates NF-κB to activate cytokine genes including pro-
IL-1β, IL-23 and IL-12.
ii. Raf-1 also promotes NF-κB activation by enhancing phosphorylation of p65.
MAPKs such as p38 and ERK are also activated in a Syk- and Myd88-dependent
manner, although this activation is not required for the cytokine production. IL-23 and
IL-1β promote the differentiation of TH17 cells, and IL-17A from TH 17 cells recruits
neutrophils to the inflammatory sites and activates T cells and B cells, contributing to
the eradication of fungi. A small amount of IL-12 is also produced through activation
of NF-κB and induces TH 1 cell differentiation. IFN-γ from these cells activates
14. 3
macrophages, contributing to fungal eradication. Dectins also stimulate production of
other cytokines, such as TNF, IL-2, IL-6 and IL-10. (Saijo & Iwakura 2011).
CURRENT TREND IN SIGNALLING PATHWAYS FROM
DECTIN-1
Dectin-1 was the first PRR outside of the TLR family that was found to be capable of
inducing its own intracellular signals (Reid et al 2009). Signalling from this receptor
following ligand binding is mediated through the cytoplasmic ITAM-like motif that
becomes phosphorylated by Src family kinases, providing a docking site for Spleen
tyrosine kinase (Syk). Despite involving both SH2 domains of Syk, only the membrane
proximal tyrosine of Dectin-1 was found to be required for signalling (Rogers et al
2005).
For Dectin-1, like most other myeloid expressed activation receptors, Syk is a pivotal
kinase mediating many of the receptor's downstream cellular responses, such as
cytokine production and induction of the respiratory burst while the components of the
signalling pathway have yet to be fully elucidated, CARD9, which assembles with
BCL10 and MALT1, has been identified as an essential downstream adaptor linking
Syk-coupled receptors to the canonical NF-κB pathway.Dectin-1 can also induce the
non-canonical NF-κB pathway, being the first PRR shown to do so, and can activate
Nuclear factor of activated T-cells (NFAT), implicating these transcription factors in
innate antimicrobial immunity, although the involvement of Syk in this response has
not been established (Goodridge et al 2006). There is also evidence of Syk-dependent,
but CARD9-independent, pathways, such as those leading to the induction of ERK, a
MAP kinase regulating the Dectin-1-mediated production of cytokines, particularly IL-
10 and IL-2.
Dectin-1 can also induce intracellular signalling through Syk-independent pathways.
Phagocytosis in macrophages, for eg, does not require Syk, although this response still
involves the ITAM-like motif of the receptor. These pathways are still largely
uncharacterised, but Dectin-1 was recently found to induce a Syk-independent pathway
involving the serine–threonine kinase Raf-1 (Gringhuis et al 2009). This pathway was
15. 4
shown to integrate with the Syk pathway, at the level of NF-κB, and to be involved in
controlling Dectin-1 mediated cytokine production.
SIGNIFICANCE OF DECTIN MEDIATED ANTIFUNGAL
IMMUNITY
Fungal invasion in humans is associated with a wide variety of diseases, ranging from
benign colonization, superficial skin infections and allergy to life-threatening systemic
mycoses. The diagnosis and treatment of these infections remain difficult and
unreliable leading to high morbidity and mortality. Even exposure to fungal spores is
ubiquitous which is acquired through the respiratory tract .The contributions of innate
and adaptive immunity to the protection of these immuno competent host from
invasive A. fumigatus infections are complex and incompletely defined. The study
aims to develop an understanding of the fungal recognition by the C type lectins,
including dectin 1 and 2 which have been poorly defined. The study thus plans to
develop an understanding of the immune activation by β-glucan and simultaneous
activation of the inflammasome and T cell polarization. Deciphering this pathway will
help pinpoint the important events during fungal infection that can be utilized to design
a strategy to inhibit these infections(Taylor et al 2006).
16. 5
2. REVIEW OF LITERATURE
2.1 Aspergillus fumigatus
Aspergillus fumigatus is a saprophytic fungus that plays an essential role in
recycling environmental carbon and nitrogen .It sporulates abundantly, with every
conidial head producing thousands of conidia[Figure 2]. The conidia released into the
atmosphere have a diameter small enough (2 to 3 mm) to reach the lung alveoli . Once
the conidia are in the air, their small size makes them buoyant, tending to keep them
airborne both indoors and outdoors. This disease occurs predominantly in the lungs.
(Paul Latge 1999). Owing to their small size, A. fumigatus conidia can bypass
mucociliary clearance mechanisms and are inhaled into terminal airways and
phagocytosed by alveolar macrophages. Conidia are killed in a phagocyte oxidase-
dependent manner. Neutrophils recruited to the site of infection form a second line of
defense against germinating conidia (Hohl et al 2005). It is intriguing that we all inhale
A. fumigatus conidia but only some people develop pathological responses to this
fungus. Differences in make-up of multiple PRRs and cytokine genes in the
propagation of inflammatory responses are involved in overall risks for allergic
responses to fungi (Chaudhary & Marr 2011).
In the hypersensitive host, conidia inhaled primarily into the bronchial tree can
initiate an allergic response culminating in Allergic Bronchopulmonary Aspergillosis
(ABPA). In the immune-compromised host, conidia inhaled mainly into the lung
alveoli can cause a life-threatening fungal infection termed Invasive Pulmonary
Aspergillosis (IPA) [Figure 3]. In both cases, the initial point of contact between the
fungus and the host is a monolayer of lung epithelial cells (Osherov 2012).
17. 6
. Figure 2: Microscopic view of Figure 3: A.fumigatus infection leads to various
A. fumigatus patholoical conditions.
2.1.1 ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS
(ABPA)
ABPA is a complex hypersensitivity response to A. fumigatus in atopic patients
with CF or asthma. ABPA is defined by a constellation of clinical, laboratory, and
radiographic criteria that include active asthma, serum eosinophilia, an elevated total
IgE level, fleeting pulmonary parenchymal opacities, bronchiectasis, and evidence for
sensitization to Aspergillus fumigatus by skin testing (Knutsen & Slavin 2011). The
pathophysiology of ABPA is allergic in nature, characterized by activation of
eosinophils and elaboration of IgE. Immune suppression therefore is the mainstay of
treatment (Patterson & Strek 2010).
In ABPA, inhaled conidia initiate an exaggerated TH2 mediated inflammatory
response. Epithelial cells release large quantities of proinflammatory cytokines, growth
factors & chemokines, amplifying the influx of T-cells, eosinophils, basophils, and
other inflammatory cells, following activation by T-cell cytokines. This inflammation
leads to the associated pathological features of airway hyperresponsiveness,
hyperplasia/metaplasia of goblet cells and subepithelial fibrosis [Figure 4] (Osherov
2012).
18. 7
Figure 4: A.fumigatus induced immune response in ABPA
2.1.2 INVASIVE PULMONARY ASPERGILLOSIS (IPA)
IPA occurs predominantly in immunocompromised hosts, with increasing numbers of
cases of invasive aspergillosis among patients with chronic obstructive pulmonary
disease (COPD) being reported.
The alveolar membrane is made up of three layers : The inner layer contains two types
of epithelial cells:
(1) Type I cells, are extremely thin (<0.5µ), enabling rapid gas exchange.
(2) Type II cells, whose primary roles are to secrete surfactant proteins and
differentiate into type I cells.
Figure 5: A.fumigatus induced immune response in IPA
Adapted from: Nir Osherov 2012
Adapted from: Nir Osherov 2012
19. 8
The major epithelial cell types of the alveolus are the type I & type II
pneumocytes. Binding of surfactant proteins to A. fumigatus conidia enhances
phagocytosis & killing by neutrophils & alveolar macrophages (Osherov 2012) .In
addition, type II cells secrete cytokines, chemokines, and antimicrobial peptides in
response to pathogens (Herzog et al.,2008).
The alveolar epithelial cells are attached to a thin basal membrane layer
composed of CT proteins including laminin & fibronectin. Attached to the under
surface of the basal membrane is a single layer of capillary endothelial cells, that line
the blood vessels coming into contact with the alveoli. In IPA, due to inhaled conidia’s
small size (2-3µ),they enter the alveoli & proceed to germinate. The entire alveolar
tissue is in most places no wider than 1 micron, so that a fungal germ-tube measuring
only 2–3 microns long can easily traverse it and enter the bloodstream [Figure 5]
(Osherov 2012).
Table 1: Difference in response in immunocompetent & immunodeficient individual.
2.2 Cellular And Effector Components Of Immune System:
The ability to recognize pathogens through PRRs allows numerous antimicrobial
effector mechanisms to be activated by the innate immune response. These responses
lead to the killing of pathogens through the production of effector molecules with
direct microbicidal activities, including the membrane attack complex of complement,
Response In Immuno
Competent Individuals
Response in Immuno
Deficient Individuals
• Resident alveolar macrophages
normally ingest and destroy inhaled
conidia
• Innate immune defenses are lacking or
dysfunctional
• If the fungal inoculum is large, they
secrete chemokines to recruit
circulating neutrophils
• If the fungal inoculum is large, it leads to
fungal growth through the alveolar wall into
the surrounding blood vessels
• Destroy both conidia and growing
hyphae.
• Causes circulatory obstruction and
subsequent tissue necrosis
20. 9
a variety of antimicrobial peptides, & the caustic reactive oxygen and reactive nitrogen
intermediates generated within phagocytic cells[Figure 6] (Liaskou et al 2012).
Figure 6: Integration of CLR- mediated signalling directs adaptive immunity
2.2.1 Innate Immunity:
Innate immunity consists of defense against infection that is ready for instantaneous
activation prior to attack by pathogen. Listed below are the major components of innate
immunity[Figure 7]:
2.2.1.1 ANATOMICAL BARRIERS:
The most obvious components of innate immunity are the external barriers to microbial
invasion-skin and mucosal membrane, which comprises of the mucosal epithelia that
line the respiratory, gastrointestinal, and urogenital tracts. Skin with epithelia provides
a barrier that protects the inner domains of the body from the outer world. The
respiratory tract has cilia, which because of its synchronous movement, propels mucus-
entrapped microbes from the tract.
Figure 7: The innate & adaptive immune system
21. 10
Innate immune response consists of soluble factors, such as complement proteins, and
diverse cellular components including granulocytes, mast cells, macrophages, dendritic
cells and NK cells. The adaptive immune response consists of antibodies, B cells, and
CD4+
and CD8+
T lymphocytes. NKT cells and γδT cells are CTL that straddle the
interface of innate and adaptive immunity.(Evaggelia et al 2012).
2.2.1.2 CELLS:
The cells of innate immunity are majorly phagocytic or Antigen Presenting Cells
(APCs). It mainly consists of Macrophages , Dendritic cells (DCs), Natural Killer (NK)
cells and Neutrophils.
Alveolar macrophages are the major resident leukocytes in the lung and
provide an early line of defense against inhaled conidia that have reached the alveoli.
(Leavy et al 2011). They can quickly adhere to and ingest conidia entering the alveolar
space. Phagocytosis and the secretion of proinflammatory cytokines by them help to
eliminate conidia and restrict the initial spread of microorganisms in the alveoli. They
are sufficient to overcome small inocula of Aspergillus conidia (Sivertsen et al 2003).
Recruited neutrophils were initially thought to act exclusively on hyphae
while resident alveolar macrophages killed resting and swollen conidia. In spite of the
fact that neutrophils remain responsible primarily for hyphal killing, they have been
shown to have an fundamental role in killing germinating conidia (Bonnett et al 2006).
They bind and internalize swollen conidia to trigger respiratory burst and
degranulation, and release lactoferrin from their secondary granules as a part of their
degranulation when interacting with Aspergillus conidia(Levitz & Diamond
1985).GCSF & IFN-γ enhance the neutrophil oxidative response and their ability to kill
hyphae.
Peripheral blood monocytes(PBMC) are a heterogeneous population of
myeloid cells that contain the precursors of tissue macrophage and dendritic cells in
inflamed tissues. Upon interaction with Aspergillus conidia, monocytes ingest and kill
conidia and this killing can be enhanced in the presence of granulocyte-macrophage
colony-stimulating factor, IFN-γ, and fungicidal drugs(Chiller et al 2001).
Epithelial cells are found to be involved in the recognition of Aspergillus
species & has received less attention, although these cells are clearly the first cells to
22. 11
encounter the inhaled organism. The ingestion of fungal elements were found by
ciliated airway epithelial cell. Similarly, human nasal ciliated epithelial cells
phagocytose and kill conidia in vitro. A human alveolar epithelial cell line (A549) can
bind both Aspergillus conidia and hyphae, ingest conidia, and generate IL-6 and
CXCL8 in response to them (Zhang et al 2005). Recent studies of a human bronchial
epithelial cell line (BEAS-2B) have also demonstrated a time-dependent synthesis of
CXCL8 in response to germinated Aspergillus elements (swollen conidia, hyphae, or
both) but not resting conidia . Interestingly, the epithelial release of CXCL8 was
dependent on NF-κB activation but was independent of the TLR-MyD88 pathway,
indicating redundant pathways for epithelial recognition and responses to Aspergillus
species.
Dendritic Cells (DC) provide a broader link between Innate and Adaptive
immunity by interacting with both TH and TC cells. Upon encounter with antigen, DCs
process the antigen and express it with either MHC Class I or MHC Class II,
depending on the antigen. DCs then migrate to lymph nodes, where they present
antigen to TH cells, to elicit either TH1 or TH2 response. Apart from this, DCs can also
mount a direct attack on pathogens they detect.(Ganguly et al 2013)
Natural Killer cells provide a first line of defense against many viral
infections. Some viral infections are probably cleared by innate immune cells such as
NK cells without the aid of adaptive immune cells. Despite lacking antigen specificity,
NK cells play crucial role in innate immune mediated response against viral mediator.
2.2.1.3 PATTERN RECOGNITION RECEPTORS
In order to protect against infection, one of the first things the body must do is the
detection of the presence of microorganisms. These unique molecules are present on
cell wall of bacteria called PAMPs. Three main features of PAMPs are: (i) they are
usually expressed by microbes and not by host cells, (ii) they show little variation
among microorganisms of a given class, and (iii) their expression is essential for the
survival of the microbes. To recognize PAMPs, various body cells have a variety of
germline-encoded receptors called PRRs capable of binding specifically to conserved
portions of these molecules (Zipfel & Felix 2005). Cells that typically have PRR
include macrophages, dendritic cells, endothelial cells, mucosal epithelial cells, and
23. 12
lymphocytes. Many PRR’s are located on the surface of these cells where they can
interact with PAMPs on the surface of microbes. Others PRRs are found within the
phagolysosomes of phagocytes where they can interact with PAMPs located within
microbes that have been phagocytosed. Some PRRs are found in the cytosol of the cell.
There are two functionally different major classes of pattern-recognition receptors:
a) endocytic pattern-recognition receptors
b) signaling pattern-recognition receptors
2.2.1.3.1 Endocytic Pattern-Recognition Receptors
Endocytic pattern-recognition receptors are found on the surface of phagocytes and
promote the attachment of microorganisms to phagocytes leading to their subsequent
engulfment and destruction. They include:
o Mannose receptors(dectin2)
o Scavenger receptors
o Opsonin receptors
o N-formyl Met receptors
2.2.1.3.2 Signaling Pattern-Recognition Receptors
Signaling pattern-recognition receptors bind a number of microbial molecules: LPS,
peptidoglycan, teichoic acids, flagellin, pilin, unmethylated cytosine-guanine
dinucleotide or CpG sequences from bacterial and viral genomes; lipoteichoic acid,
glycolipids, and zymosan from fungi; double-stranded viral RNA, and certain single-
stranded viral RNAs. Binding of microbial PAMPs to their PRRs initiates signaling
which promotes the synthesis and secretion of intracellular regulatory molecules such
as cytokines which are crucial for initiating innate immunity and adaptive immunity.
o NODs (nucleotide-binding oligomerization domain)
o CARD-containing proteins
o Toll-Like Receptors (TLRs): The best understood family of PRRs are the Toll-
like receptors (TLRs). Toll-like receptors (TLRs) are evolutionary conserved
transmembrane proteins that recognize a unique pattern of molecules derived
from pathogens or damaged cells, triggering robust but defined innate immune
responses via activation of NFқB, MAP kinases, and IRFs that control the
transcription of genes encoding type I IFN & other inflammatory cytokines.
24. 13
Twelve members were identified in mammals. TLRs together with IL-1
receptors constitute a superfamily based on the homology of the cytoplasmic
region. Within the TIR domain, the similarities map at the level of three
conserved boxes that are essential for signalling (Akira and Takeda, 2004). In
addition to the intracellular signalling domain, TLRs possess an extracellular
region containing 19-25 tandem copies of LRRs and TLRs. This region is not
present in IL-1R and forms a surface suited for PAMP recognition (Akira and
Takeda, 2004).
Figure 8: Schematic presentation of TLR’s & signalling pathway.
Each TLR displays a differential expression pattern, intracellular
localization & signalling pathway, resulting in distinct immune responses
.TLRs recognize a bewildering range of microbial ligands, such as bacterial and
fungal cell wall components, bacterial lipoproteins, highly conserved microbial
proteins and bacterial and viral nucleic acids[Figure 8]. The molecular basis of
such diverse ligand binding remains poorly understood, although the
elucidation of several recent structures of ligand–receptor complexes suggests
that not all TLRs use the same ligand-binding interface(Akira et al 2006).
On ligand binding, TLRs initiate downstream signalling responses by
recruiting a TIR domain-containing adaptor such as Myeloid Differentiation
primary-response protein 88(MyD88), TIR-domain-containing adaptor
25. 14
protein(TIRAP) and TIR-domain-containing adaptor protein inducing IFN-
β(TRIF) (Akira and Takeda, 2004). TLR-adaptor interaction favours the
formation of a complex involving IL-1R-associated kinase (IRAK) and TNF
receptor-associated factor 6 (TRAF6). This ultimately results in activation of
NF-κB & MAPKs for induction of proinflammatory cytokines (Akira et al.,
2006). Thus, MyD88 and TRIF are responsible for the activation of signalling
pathways downstream of TLRs leading to the induction of pro inflammatory
cytokines and type I IFNs (Akira et al., 2006).
2.2.1.4 Mediators: The mediators such as Reactive oxygen species (ROS) and
Reactive nitrogen species (RNS) play a role in oxidative burst of innate immune
system. The oxidative arm of innate immune cells mainly employs ROS and RNS.
Inducible Nitric Oxide Synthetase (iNOS) helps in RNS production. The ROS include
superoxide ion, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). The RNS
include nitrous and nitric oxide. Together they form phagocytic barrier.
2.2.1.5 Cytokines and chemokines: After recognition and processing of foreign
antigen, innate immune cells secrete specific cytokines and chemokines. Chemokines
help in chemotaxis and extravasation of adaptive immune cells and neutrophils.
Specific cytokines lead to specific type of adaptive immune response which is desired.
These responses depend on type of infection. For example, IL-12 initiates the
differentiation of TH1 cells that are characterized by high production of IFN-γ and are
obligatory for clearing intracellular pathogens. After activation, APCs also secrete
some pro-inflammatory cytokines like, TNF-α, IL 6, IL1-β, IL 8 and IFN-α.
2.2.2 Adaptive immunity:
Adaptive immunity comes into play after innate immune cell activation. It is more
specific and requires longer time for activation than innate immunity. Listed below is
the Components of Adaptive immunity[Figure 9].
2.2.2.1 CELLS:
Adaptive immune cells are mainly of lymphoid origin.
26. 15
Figure 9: Classification of cells of adaptive immune system.
2.2.2.1.1 B lymphocytes:
B lymphocytes mature in bone marrow and express membrane bound
immunoglobulins that serve as receptor for antigen. When naive B cell encounters an
antigen, it begins to divide rapidly, differentiating into effector cells called plasma cells
and memory cells. Plasma cells produce antibody whereas memory B-cells also
express the same membrane-bound antibody as that of naive B-cell [Figure 10].
Memory B-cells have longer life span.
Figure 10: Development of B-Cells
27. 16
2.2.2.1.2 T Lymphocytes:
There are three well defines sub-populations of T cells, T helper (TH, CD4), Cytotoxic
T-cells (TC, CD8) and Regulatory T-cells (Treg, CD25). T-cells develop in thymus
through a process of positive and negative selection. Inactive T cells are called as
Naive T-cells. They express T-cell Receptor (TCR) on its surface, which recognize
expressed antigen on MHC on APC. This leads to T-cell activation, which also
involves active role of some costimulatory molecules. Naive T-cells, after activation
can switch to one of the TH1, TH2, TH17 or Treg, depending on the type of antigen
expressed on APC and cytokines produced (Veldhoen et.al. 2008). Viral infection
strongly switches T-cell class to TH1 whereas antibody production requires class switch
to TH2. TH17 cells play a role in auto-immune disorders (Korn et.al. 2009). TH1
response then leads to CTL generation. CTL are involved in killing the infected cells.
Treg are desired in auto-immune disorders to keep check on auto-reactive CTLs. A
distinct feature of T-cell is production of memory cells that can give rise to effector
cells after re exposure to antigen.
Type
Cytokine
Stimulus
Master
Transcription
Factor
Effector
Cytokine(s)
Main Target
Cells
Effector
Targets/Functions
Pathological
Effects
TH1 IL-12 & IL-2 T-bet IFN-γ & TNF-β
Macrophages,
dendritic cells
Intracellular pathogens
Autoimmunity;
cell-mediated
allergies
TH17
TGF-β plus
IL-6
Inhibited
by retinoic acid
RORγt
IL-1, IL-6 &
TNF-α
Neutrophils
Extracellular bacteria
and fungi
mediates inflammation
Autoimmune
diseases
Treg
TGF-β minus IL-6
Stimulated
by retinoic
acid and IL-2
Foxp3 IL-10 & TGF-β
all the other
types of T
cells
Immunosuppression;
anti-inflammatory
None
Table 2: List of subsets of T-cells involved in T-cell mediated immune response
28. 17
2.2.2.1.2.1 Differentiation of T Cells
TH1 Helper cells
TH1 cells produce the cytokine IFN-γ and are involved in activation of macrophages
and other cell types for clearance of intracellular pathogens. TH1 cells help B cells to
produce IgG2 type of antibodies (Awasthi & Kuchroo 2009). The cytokines IFN-γ &
IL-12 and the member of the T-box family of transcription factors T-bet, are crucial
regulators of TH 1 cell differentiation.
Figure 11: Differentiation of TH 1 cells
Upon antigen recognition, the differentiation of TH1 cells starts with the
induction of T-bet in response to IFN-γ signalling. In turn, T-bet triggers the
production of IFN-γ and induces the expression of the β2 subunit of IL-12 receptor,
conferring IL-12 responsiveness .This latter cytokine acts through signal transducer
and activator of transcription 4 (STAT4) to amplify the production of IFN-γ and to
fully establish the TH1 lineage. IL-12 also increases the expression of the receptor for
IL-18, another STAT4 dependent inducer of IFN-γ (Murphy and Reiner, 2002).
Terminally differentiated TH1 cells produce vast amounts of IFN-γ in response to TCR
triggering[Figure 11].
During microbial infections, the initiation of TH1 responses is subjected to the
activation of the innate immune system. It is proposed that in the context of antigen
presentation, pathogen-activated cells such as NK cells might initially secrete IFN-γ to
uncommitted TH1 cells to promote T-bet expression .The process of TH1 development
29. 18
continues with the supply of IL-12 and IL-18 by pathogen-activated macrophages and
DCs, which results in the generation of mature IFN-γ-producing CD4+T cells (Murphy
and Reiner, 2002) Notably, various types of TLR ligands trigger the production of IL-
12 by DCs and are involved in priming of TH1 cells (Medzhitov, 2007).
TH17 Helper cells
TH17 helper cells mediate host immunity against extracellular bacteria and fungi. It is
triggered by IL-6 and TGF-β. Its main effector cytokines are IL-6, IL-1, and TNF-α.
The main TH17 effector cells are neutrophils as well as IgM/IgA B cells, and IL-17
CD4 T cells.
The key TH17 transcription factors are STAT3 and RORg. TNF-α can activate
neutrophils to kill extracellular bacteria and fungi. Besides, IL-6 can upregulate the
complement system to directly kill extracellular bacteria and fungi. TH17
overactivation against autoantigen will cause type III immune complex and
complement-mediated hypersensitivity. TH17 is a recently described subset of
CD4+cells that produces the cytokines IL-17A, IL-17F, IL-21 and IL-22(Harrington et
al 2005).
Figure 12: Differentiation of Th17 Cells
Upon antigen encounter, the TH17 differentiation process is initiated by the
synchronized effect of IL-6 and TGF-β on naïve T cells In presence of TGF-β, IL-6
30. 19
acts via STAT3 to direct lineage commitment and regulate the expression of RORγt,
RORα and probably BATF. The TH17 differentiation process results in the production
of IL-17 and subsequent upregulation of the IL-23 receptor, which confers IL-23
responsiveness (Nurieva et al 2007). Although dispensable for the initial differentiation
of TH17 cells, IL-23 is essential to sustain and expand committed TH17 cells. This
cytokine promotes high levels of IL-17 production by activated CD4+T cells and is
critical for the development of TH17 cells in vivo[Figure 12]. Moreover, the acquisition
of TH17 pathogenic function associated with autoimmunity in the central nervous
system is dependent on IL-23 (Langrish et al 2005).
Regulatory T cells (Tregs)
Regulatory T cells (Tregs), formerly known as suppressor T cells, are a subpopulation
of T cells which modulate the immune system, maintain tolerance to self-antigens, and
abrogate autoimmune disease. Treg cells constitutively express dozens of receptors,
such as IL-2R (CD25), GITR, TCR, TGF-βR, TLRs and receptors for S1P. T
regulatory cells are a component of the immune system that suppress immune
responses of other cells. This is an important "self-check" built into the immune system
to prevent excessive reactions. Regulatory T cells come in many forms with the
mostwell-understood being those that express CD4, CD25, and Foxp3
((CD4+
CD25+
regulatory T cells).These cells are involved in shutting down immune
responses after they have successfully eliminated invading organisms, and also in
preventing autoimmunity(Hori et al 2005).
2.2.2.2 Cytokines:
T-cells and B-cells, after activation, produce variety of cytokines. Cytokine milieu
depends on type of response desired. For instance, production of IL-4 mainly leads to
TH2 response, whereas in presence of IL-6 (pro inflammatory) and TGF-β, it leads to
TH17 switch. IL-10 is involved in Treg generation. Activated T-cells secrete IFN-γ,
which has a distinct role in anti-tumor effects of immune system. IFN-γ and IL-4 are
antagonistic in action. IL-17 and IL-22 are involved in TH17 proliferation.
31. 20
3. C- TYPE LECTIN RECEPTORS
A family of receptors, C-type lectins are a diverse group of proteins which were
originally defined by their ability to recognize carbohydrate structures (Drickamer
1988). C-type lectins execute both immune and non-immune functions. Whereas some
recognize endogenous ligands to facilitate adhesion between cells, adhesion of cells to
extracellular matrix and other non-enzymatic functions, others may act as PRRs. C-
type lectins consist of a distinct protein fold, termed the carbohydrate recognition
domain (CRD), which is generated through disulphide bridges between conserved
cysteine residues (Drickamer et al 2005). This family has been divided into 17 groups
based on the organization of their CRDs, and can be functionally defined as either
classical or non-classical [Figure 13](Zelensky et al 2005).
Figure 13: C-type Lectin Receptor Figure 14: Phagocytic synapse formed by
Superfamily interaction of β-Glucan &
Dectin-1 receptor
3.1 DECTIN
Dectin is a member of CLR receptor family and based on their structural differences as
well as different ligand binding properties ,they are of two types:
32. 21
3.1.1 DECTIN-1
Dectin-1 is a transmembrane protein containing an ITAM-like motif in its intracellular
tail (which is involved in cellular activation) and single C-type lectin like domain ,
CRD in the extracellular region (which recognized ß-glucans and endogenous ligand
on T cells) (Drummond et al 2011). Dectin-1 (also known as CLEC7A) is a pattern-
recognition receptor expressed by myeloid phagocytes that detects β-glucans in fungal
cell walls (Goodridge et al 2009) and triggers direct cellular antimicrobial activity,
including phagocytosis and production of reactive oxygen species(ROS)[Figure
14] Expression is primarily found on myeloid Dendritic cells, monocytes, macrophages
and B cells(Taylor et al 2002). CLEC7A has been shown to recognize several fungal
species including Aspergillus, Saccharomyces, Candida, Pneumocystis, Coccidiodes,
Penicillum and other. Recognition of these organism triggers many protective
pathways, such as fungal uptake by phagocytosis and killing via respiratory burst.
Activation signalization via Dectin-1 also triggers expression of many protecting
antifungal cytokines/chemokines (TNF, CXCL2, IL-1β, IL-1α, CCL3, GM-CSF, G-
CSF and IL-6) and development of TH17 through activation of Syk-CARD9 signalling
pathway. Dectin-1 is one member of a cluster of NK-cell-receptor-like C-type lectins
found in the NK complex on chromosome 12 in humans (chromosome 6 in mice).
These Dectin-1 cluster of receptors includes include MICL, CLEC1, CLEC2, LOX1,
CLEC12B & CLEC9A form part of the Group V C-type lectin-like receptors(Brown
2006).
Dectin-1 engages both particulate β-glucans and soluble β-glucan polymers, but
only particulate β-glucans activate phagocytosis and inflammatory responses. The
phosphatases CD45 and CD148 regulate signal transduction by the dectin-1 hemi-
immunoreceptor tyrosine-based activation motif (hemITAM) and must be isolated
from the clustered receptors by the formation of a synapse to permit productive dectin-
1 signalling. Soluble β-glucans bind with high affinity to dectin-1 but do not form
synapses and therefore fail to trigger dectin 1-mediated responses (Underhill et al
2012).The activation of Dectin-1 & Dectin-2 leads to a sequential activation of
tyrosine kinase Syk, the CBM complex and IKK. Furthermore Syk mediates IKK
phosphorylation, whereas CARD9 controls NEMO poly ubiquitination [Figure15].
33. 22
Figure 15 : Activation of the Dectin-1 & Dectin-2 Receptor
3.1.2 DECTIN-2
Another cluster of receptors is the Dectin-2 family of C-type lectins. These
receptors are clustered in the Telomeric region of the NKC, in close proximity to the
Dectin-1 family and also appear to have diverse functions in both immunity and
homeostasis. The Dectin-2 gene family includes BDCA-2, DCAR, DCIR, Dectin-2,
Clecsf8 and Mincle, and form a cluster (red square) in the Telomeric region of the
NKC, close to the Dectin-1 cluster (blue square), on mouse chromosome 6 and human
chromosome 12[Figure 16](Graham & Brown 2009).
Figure 16 : Dectin-1 and Dectin-2 genomic localization within the myeloid-cell-
expressed natural killer (NK)-cell-receptor-like C-type lectin cluster
Dectin-2 is a C-type lectin receptor specific for carbohydrates with a ‘complex
mannose-like’ structure and as such is capable of recognizing carbohydrates on
pathogens such as fungi and mycobacterium. These CTLRs possess a single
34. 23
extracellular conserved C-type lectin-like domain and are capable of mediating
intracellular signalling either directly, through integral signalling domains, or
indirectly, by associating with signalling adaptor molecules. These receptors recognize
a diverse range of endogenous and exogenous ligands, and can function as PRR for
several classes of pathogens including fungi, bacteria and parasites, driving both innate
and adaptive immunity(Kerscher et al 2013) .Dectin-2 lacks an immunoreceptor
tyrosine-based activation motif (ITAM) in its cytoplasmic domain(Kanazawa et al
2010).
Official
name
Alternative
name
Cell
expression
Exogenous
ligands
Endogenous
ligands
Functions
CLEC7A Dectin 1 Myeloid cells,
B cells, T cell
subsets,
Eosinophils,
Mast cells
Β-glucan,
mycobacterium
ligands
T cells Activation of
innate immune
cells, apoptic
cells
CLEC6A,
CLECSF10
Dectin 2 Mature DCs,
Macrophages,
Inflammatory
monocytes
Fungal
α- Mannose
T cells NLRP3
inflammasome
activation,
processing of
pro-IL-1β
Table 3: Difference between Dectin-1 & dectin-2 receptor
4. β- Glucan as a potent target for anti-fungal immunity
Non-cellulosic β-glucans are recognized as potent immunological stimulators in
humans and some are now used clinically in China and Japan. Structurally, they have a
linear backbone of D-glucose in β-1,3 linkage with side branches in β-1,6 linkage at
various intervals. β-glucans are not synthesized by humans, so these compounds are
recognized by our immune systems as non-self molecules, inducing both innate and
adaptive immune responses (Brown & Gordon 2005).The noteworthy “natural” β-
glucans are Lentinans, Schizophyllan, PSK(Krestin)
35. 24
Consequently, fungal β-glucans probably act as PAMPs and are recognized by
appropriate cell-surface receptors, initiating immune responses. In humans, a number
of such receptors have been identified. These are dectin-1, complement receptor 3
(CR3), scavenger receptors, lactosylceramide (LacCer), and the toll-like receptor
(TLR).[Figure 17]. Evidence suggests that dectin-1 is most important in the activation
of innate immune responses in macrophages, as blocking with an anti-dectin-1
antibody and knockout of the dectin-1 gene resulted in the abolition of all macrophage-
mediated responses (Marakalala et al 2011). Dectin-1 binds specifically to β-(1/3)-
glucans, but only those consisting of at least 10-mer oligosaccharides.
Figure 17: Immunostimulation by fungal β-glucans
4.1 Curdlan
In curdlan unlike some β-glucans, no side-chain substitution occurs, hence it contains
only linear β-(1/3)-glucosidic linkages (McIntosh et
al.2005). Curdlan is obtained from Alcaligenes
faecalis(bacterial). In its natural state, curdlan is poorly
crystalline and is found as a granule, much like that of starch. The granule is insoluble
in distilled water, but dissolves easily in a dilute alkali solution, due to the ionization of
hydrogen bonds, and forms a gel when it is heated above 54° C.
36. 25
4.2 Mechanism of Fungal β-glucan binding to Dectin -1 receptor
Binding of Fungal β-glucan (Curdlan) with the ligand activates several signalling
pathways to promote innate immune responses through activation of phagocytosis,
ROS production, and induction of inflammatory cytokines (Grunebach et al.2002). The
cytoplasmic domain of dectin-1 has an immunoreceptor tyrosine-based activation motif
(ITAM) to activate a tyrosine kinase, which in turn stimulates ROS production but not
phagocytosis (Goodridge et al. 2011). Activation of this tyrosine kinase also induces
synthesis of TNF-α, and IL-2, IL-10, IL-12.
Many pathways have now been identified as being involved in dectin-1 downstream
signaling:
Figure 18 : Possible fungal β-glucan mediated signal pathway
First, some evidence suggests it might act synergistically with TLR to produce
strong inflammatory responses by stimulating cytokines such as TNF-α, IL-2 and IL-12
(Gantner et al.2003).Although both dectin-1 and TLRs are activated by β-glucan
(Rogers et al 2005), the TLR ligand is not yet known. Recent evidence also showed
that Curdlan induced production of TNF and IL-12 and IFN-γ was not affected by a
deficiency of MyD88, indicating its effect is independent of the TLR pathway (Saijo et
al.2007). This needs to be determined, and it could be that this synergistic effect arises
because phosphorylated dectin-1 can form a complex with TLR (Brown 2006).
Moreover, another pathway independent of TLR is mediated via spleen tyrosine
kinase (Syk) to produce other cytokines, including the macrophage inflammatory
protein-2(MIP2, CXC2) and IL-2 and IL-10 in mice DC cells (Rogers et al.2005).
37. 26
After binding to the ligand, dectin-1 is phosphorylated by a non-receptor tyrosine
kinase Src. Syk is then activated, which in turn activates the CBM complex. This
complex mediates the induction of cytokines like nuclear factor (NF)-kB and IL
production [Figure 18].
Furthermore, phagocytosis in macrophages is another signalling pathway
activated by the dectin-1 receptor (Brown 2006) which seems to be independent of any
involvement by either TLR or Syk. The dectin-1 cytoplasmic domain has three
consecutive acidic amino acids signaling phagocytosis and activation of phagocyte
lysosomal endosomes (Lee et al. 2011).
5. IL-1β Activation mediated by Caspase1 and inflammasome
5.1 IL-1β
Interleukin-1 beta also acknowledged as catabolin, is a cytokine protein that in humans
is encoded by the IL-1B gene. IL-1β precursor is cleaved by caspase 1 (interleukin 1
beta convertase). Cytosolic thiol protease cleaves the product to form mature IL-1β. IL-
1β is a member of the interleukin 1 family of cytokines & is produced by activated
macrophages as a proprotein, which is proteolytically processed to its active form
by caspase1 (CASP1/ICE).. Its expression is induced by transcription factor NF-
κB after exposure of innate immune cells to alarmins.
IL-1β can mediate inflammatory responses by supporting T-cell survival,
upregulation of the IL-2 receptor on lymphocytes, enhancing antibody production of B
cells and by promoting B-cell proliferation and T-helper 17 cell differentiation
(Lamkanfi et al 2009). The main sources of IL-1β are blood monocytes, tissue
macrophages and dendritic cells and, to a lesser extent, B lymphocytes and natural
killer cells. Secretion of IL-1β from these cells requires a two-step activation process:
(1) NF-κB-mediated transcriptional upregulation of pro-IL-1β, via Toll-like
receptors,and
(2) caspase-1-driven conversion of pro IL-1β into its active form.
38. 27
5.1.1 NF-κB-mediated transcriptional upregulation
(a)In resting dendritic cells, caspase-8 is in complex with MALT1. A second pool of
MALT1 interacts with Bcl-10 . (b)After stimulation of dectin-1 with fungi or
mycobacteria, engagement of Syk triggers activation of NF-κB and transcription of the
gene encoding pro-IL-1β via the CBM complex.
Figure 19 : Schematic representation of inflammasome activation
A subset of pathogens, such as Aspergillus fumigatus, stimulate the production of
bioactive IL-1β mainly via a noncanonical inflammasome composed of CARD9, Bcl-
10, MALT1, caspase-8 and ASC. (c) Other pathogens, such as M tuberculosis or M
bovis bacillus Calmette-Guérin, engage both the classical NLRP3 inflammasome as
well as the noncanonical caspase-8 inflammasome, leading to the secretion of IL-1β
[Figure 19]
5.1.2 Conversion mediated by Caspase-1:
Caspase-1 activity is controlled by a cytosolic multi-protein complex or activation
scaffold, also known as the inflammasome (Martinon et al 2002).
39. 28
Figure 20: Two step activation process of IL-1β
The inflammasome consists of a nucleotide-binding domain like receptor (NLR), one
or more adaptor proteins and caspase-1. Several NLRs have been identified so far ,the
best-characterized one is the NLR protein 3(NLRP3)[Figure 20].
5.2 .INFLAMMASOME
The inflammasome is a multiprotein oligomer consisting of Caspase 1, PYCARD,
NALP and sometimes caspase 5. It is expressed in myeloid cells and is a component of
the innate immune system. Chiefly, the inflammasome promotes the maturation of
inflammatory cytokines IL-1β and IL-18(Martinon et al 2012) The inflammasome is
responsible for activation of inflammatory processes(Mariathasan et al 2004) and has
been shown to induce cell pyroptosis, a process of programmed cell death distinct
from apoptosis(Fink et al 2005).
Figure 21: Signaling pathways involving SYK during engagement
of Dectin-1/ITAM receptors leading to IL-1β production.
C-type lectin receptor (CLR) family members contain ITAM-like motif
(hemITAM) in their cytoplasmic domain, such as Dectin-1, or are coupled with ITAM-
containing adaptor molecules, such as FcγR and DAP12. In the case of Dectin-1
engagement, SYK is recruited to activate three downstream pathways leading to IL-1β
production. This includes the recruitment of a CBM complex scaffold which activates
40. 29
NFκB-dependent gene transcription to induce pro-inflammatory factors, including pro-
IL-1β and the formation of inflammasome complexes consisting of either NLRP3,
caspase-1, and ASC (known as the classic, CARD9-independent inflammasome) or
CARD9, Bcl-10, MALT1, caspase-8 and ASC (known as the non-classic, CARD9-
dependent inflammasome), which mediate subsequent processing of pro-IL-1β into
mature IL-1β. (Tan et al 2013).
A subset of NLRs named NLRP1 were able to assemble and oligomerize into a
common structure which collectively activated the caspase-1 cascade, thereby leading
to the production of pro-inflammatory cytokines especially IL-1B and IL-18 [Figure
21]. This NLRP1 multi-molecular complex was coined the “inflammasome” & several
other inflammasomes were discovered, two of which are also NLR subsets—NLRP3
and NLRC4 (Hornung et al 2009).
5.2.1 NLRP3 inflammasome
It is known to be the biggest inflammasome of all, covering about 2 um in diameter.
NLRP3 contains a PYD domain like NLRP1 and thus activates caspase-1, using its
PYD to recruit ASC. It forms only one oligomer per cell, and its oligomer is made of
seven NLRP3 molecules. The NLRP3 inflammasome consists of NLRP3, ASC, and
caspase-1. This complex functions as an upstream activator of NF-κB signaling, and it
plays a role in the regulation of inflammation, the immune response, and apoptosis
[Figure 22].
Figure 22: Role of NALP3 inflammasome in activation of IL-1β
41. 30
5.2.2 Inflammatory Cascade
Analogous to the apoptosome, which activates apoptotic cascades, the inflammasome
activates an inflammatory cascade. Once active, the inflammasome binds to pro-
caspase-1 (the precursor molecule of caspase-1), either homotypically via its
own caspase activation and recruitment domain (CARD) or via the CARD of the
adaptor protein ASC which it binds to during inflammasome formation. In its full form,
the inflammasome appositions together many p45 pro-caspase-1 molecules, inducing
their autocatalytic cleavage into p20 and p10 subunits.(Yamin et al 1996) .Caspase-
1 then assembles into its active form consisting of two heterodimers with a p20 and
p10 subunit each. Once active, it can then carry out a variety of processes in response
to the initial inflammatory signal. These include the proteolytic cleavage of pro-IL-1B
at Asp116 into IL1β,(Martinon et al 2002) cleavage of pro-IL-18 into IL-18 to
induce IFN-γ secretion and natural killer cell activation,(Gu Y et al 1997) cleavage and
inactivation of IL-33,(Cayrol et al 2009) DNA fragmentation and cell pore
formation,(Fink et al 2006) inhibition of glycolytic enzymes, and secretion of tissue-
repair mediators such as pro-IL-1α(Keller et al 2008) .Additionally, AIM2 contains a
HIN200 domain which senses and binds foreign cytoplasmic dsDNA and activates NF-
κB, a role that is crucial in bacterial and viral infection.(Fernandes et al 2009).
6. CELL SIGNALLING MEDIATORS
6.1 SYK(Spleen Tyrosine Kinase )
SYK, along with Zap-70, is a member of the Syk family of tyrosine kinases. These
non-receptor cytoplasmic tyrosine kinases share a characteristic dual SH2
domain separated by a linker domain. There is expression of Syk in a variety of tissues.
Within B cells , Syk transmit signals from the B-Cell receptor(Chan et al 1992). Syk
plays a similar role in transmitting signals from a variety of cell surface receptors
including CD74, Fc Receptor, and integrins. Sensing of various SCPs by Dectin-1 and
Dectin-2 recruits, Syk adaptor which further recruits PLCγ and activate cascade of
signaling for induction of inflammatory cytokines via NF-κB and MAP kinases [Figure
23].
42. 31
Figure 23: Syk signalling pathway Figure 24:MAPK/ERK signalling
pathway
6.2 MAPK/ERK pathway
The MAPK/ERK pathway is a chain of proteins in the cell that communicates a signal
from a receptor on the surface of the cell to the DNA in the nucleus of the cell. The
signal starts when a signaling molecule binds to the receptor on the cell surface and
ends when the DNA in the nucleus expresses a protein and produces some change in
the cell, such as cell division. The pathway includes many proteins,
including MAPK ,originally called ERK which communicate by adding phosphate
groups to a neighboring protein, which acts as an "on" or "off" switch. This pathway is
known as the Ras-Raf-MEK-ERK pathway. At minimum, four distinct
MAPK signaling modules exist in mammalian cells. Most simplified MAPK
signaling cascade is with RAS-RAF-MEK-ERK pathway. Binding of growth
factors (for eg: β-glucans, α-mannans) to their respective receptor tyrosine kinase
receptor (RTKs), such as MET, triggers receptor dimerization and subsequent
autophosphorylation of tyrosine residues present on the internal portion of the
receptors. This activates RTKs and allows them to bind SH2 domains of proteins, such
as GRB2. This complex then brings the cytosolic protein SOS into close proximity
of RAS (HRAS, NRAS or KRAS) on the plasma membrane, and catalyzes the
conversion of the inactive GDP-bound RAS into active GTP-bound RAS.
Active RAS initiates the signaling cascade by phosphorylating RAF MKKK
43. 32
(cRAF1, ARAF orBRAF),which in turn phosphorylates
MEK MKKs (MEK1 and MEK2). Activated MEKs phosphorylate the extracellular
signal-regulated kinases ERK MAPKs (ERK1 and ERK2), which then translocate into
the nucleus and phosphorylate transcription factors or other specific substrates [Figure
24] (Downward 2003).
6.3 ROS (Reactive Oxygen species)
Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen.
For eg. Oxygen ions and peroxides. ROS form as a natural byproduct of the normal
metabolism of oxygen and have important roles in cell signaling and homeostasis
During times of environmental stress (e.g., UV or heat exposure), ROS levels can
increase dramatically which results in significant damage to cell structures.
Cumulatively, this is known as oxidative stress. ROS are also generated by exogenous
sources such as ionizing radiation.
β-glucan binding to Dectin receptor initiates syk signalling pathway which in turn
increases ROS production in the cells. Cells may die further due to oxidative stress.
6.4 NF-κB
NF-κB is a protein complex that controls the transcription of DNA. NF-κB is found in
almost all animal cell types and is involved in cellular responses to stimuli such as
stress, cytokines, free radicals, ultraviolet irradiation etc. NF-κB plays a key role in
regulating the immune response. Incorrect regulation of NF-κB has been linked to
cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and
improper immune system activation. Hence ,NF-κB is a protein responsible for
cytokine production and cell survival.
Dectin-1 binds to β-glucan and activates syk pathway. Syk activates CBM
complex & this activated complex controls NF-κB activation and subsequent
expression of cytokines/chemokines, like TNF-α, IL-1β, IL-10, and IL-6 [Figure 25].
44. 33
Figure 25 :Dectin mediated NF-κB translocation
6.5 Caspases
Caspases are essential in cells for apoptosis, or programmed cell death,
in development and most other stages of adult life, and have been termed "executioner"
proteins for their roles in the cell. Some caspases are also required in the immune
system for the maturation of lymphocytes. Failure of apoptosis is one of the main
contributions to tumour development and autoimmune diseases.
Dectin binding to it ligand initiates formation of Caspase 1+ Nlrp3+ ASC complex
which in turn activates caspase-1 and this will cleave pro-IL1β and IL-1β is secreted.
7. CELL SIGNALLING INHIBITORS
Several of the PRR (pattern recognition receptors) signaling cascades culminate in the
activation of NF-κB transcription factor and /or MAPK. The inhibitors can be used to
dissect these signaling pathway [Figure 27].
7,1 MNS (3,4-methylenedioxy-β-nitrostyrene)
MNS is a selective inhibitor of c-Src and Syk tyrosine kinases. MNS is also known as
3,4-methylenedioxy-β-nitrostyrene is a cell-permeable cytotoxic β-nitrostyrene
derivative which functions as a tyrosine kinase inhibitor that supresses human platelet
aggregation. In addition, platelets previously aggregated by ADP, U46619,
45. 34
arachidonic acid, collagen, thrombin, ionophore A23187, and PDBu can be
disaggregated by MNS. The mechanism responsible for this inhibition is observed via
prevention of glycoprotein IIb/IIIa activation.
7.2 BHA (Butylated hydroxyanisole )
BHA is a known ROS-inhibitor. Treatment with BHA inhibited GM-CSF and M-CSF-
induced superoxide production. Additional experiments demonstrated that ROS plays a
key role in the differentiation of M2 macrophages, and BHA blocks differentiation of
M2 macrophages by inhibiting NADPH oxidase-mediated superoxide production,
which is the main non-mitochondrial source of ROS.
Figure 26: ROS inhibition mediated by BHA.
BHA blocks GM-CSF or M-CSF- induced ROS generation and the second phase of
ERK activation, and further blocks the polarization to M2 macrophages. The effect of
BHA is specific for M2 cells, since the inhibition effect of BHA is overcome during
M1 but not M2 polarization. Tumor-associated macrophages (TAMs), which are
responsible for tumor-promoting activities, are alternatively activated, or M2-like
macrophages. Blocking the function of M2 cells/TAMs inhibits tumorigenesis [Figure
26].
7.3 NAC(N-acetyl-L-cysteine)
NAC is commonly used to identify and test ROS (reactive oxygen species) inducers,
and to inhibit ROS. In the present study, inhibition of proteasome inhibitors identified
as a novel activity of NAC. NAC and catalase, another known scavenger of ROS,
46. 35
similarly inhibited ROS levels and apoptosis associated with H2O2. However, only
NAC, was able to prevent effects linked to proteasome inhibition, such as protein
stabilization, apoptosis and accumulation of ubiquitin conjugates. These observations
suggest that NAC has a dual activity as an inhibitor of ROS and proteasome inhibitors.
NAC is the first known compound that directly interacts with and antagonizes the
activity of proteasome inhibitors. NAC is utilized as an antioxidant to demonstrate
ROS involvement in drug-induced apoptosis. (Halasi et al 2013).
7.4 YVMD (AC-YVBD-cmk)
It is a caspase-1 inhibitor and possess neuro protective activity. Hence there will not be
any further signaling to inflammosome, if caspase-1 is inhibited, & Il-1β activation will
not occur and these cytoKines will not release in the assay.AC-YVBD-cmk is also
found to have anti-apoptopic functions.
Figure 27: Cell signalling inhibitors along with their respective signalling
pathway.
7.5 SB202190 (SB)
MAP Kinase Inhibitor - p38/RK MAP Kinase Inhibitor - Autophagy inducer.
47. 36
SB202190, a close relative of SB203580, is widely used to assess the physiological
roles of p38α and p38β MAPKs.Recent studies have identified other protein kinases,
including GAK, CK1 and RIP2, that are potently inhibited by SB202190 (as well as
SB203580).
Further, SB202190 was shown to induce autophagic vacuoles through cross-inhibition
of the PI3K/mTOR pathway .
7.6 PD0325901 (PD)
Reprogramming Enhancer - MEK Inhibitor
PD0325901 is a synthetic organic molecule that selectively binds to and inhibits
mitogen-activated protein kinase kinase (MEK). The MEK/ERK signaling pathway
plays an important role in the self-renewing state of ES cells. Inhibition of this pathway
7.7 SP600125 (SP)
MAP Kinase Inhibitor - Autophagy Inhibitor - JNK inhibitor.
SP600125 is a potent, cell-permeable, selective and reversible inhibitor of c-Jun N-
terminal kinase (JNK) .It inhibits in a dose-dependent manner the phosphorylation of
JNK. JNK is a member of the mitogen-activated protein kinase (MAPK) family and
plays an essential role in TLR mediated inflammatory responses.Inhibition of JNK
activity by SP600125 is usually associated with downregulation of Beclin-1 and
reduced autophagy combined with the inhibition of other signaling pathways has been
shown to improve the efficiency of iPS cell generation. Combination of PD0325901
with a TGF-β receptor inhibitor was found to improve the reprogramming of OSKM-
infected human fibroblasts.
48. 37
HYPOTHESIS
Immune response to A. fumigatus have been attributed to recognition by PRRs
including Dectins, but the studies do not highlight the role of PRRs in the detail.
Binding of β-glucans to dectin-1 activates NF-κB signaling pathways via Syk-CARD9
(caspase recruitment domain 9) and Raf-1 , respectively, which coordinate the
transcription of innate response genes like p65 and c-REL which induces pro- IL-1β
synthesis required for mounting varied T cell responses depending on the
corresponding cytokines released. Hence Syk and CARD9 deficient mice fail to mount
a TH1 and TH17 response and are more susceptible to infection. Dectin-1 and the
consequence of fungal binding might allow the engagement of other signaling
receptors we thus hypothesize that Dectin-1 activates Syk and CARD9 in the response
to A. fumigatus challenge.
Dectin-1 mediates the activation of NLRP inflammasome which is an important event
in activation of IL-1β. But the mechanism by which the immune system regulates IL-
1β production after A. fumigatus recognition is unclear. We hypothesize that dectin-1
by activation of NLRP inflammasome could be controlling IL-1β production as a
defense strategy[Figure 28].
PRR triggers distinct signaling pathways that induce the expression of specific
cytokines which determine T cell polarization fates. Dectin-1 deficient mice display
increased susceptibility to other fungal infections and has been shown to activate
adaptive immune responses resulting in induction of antigen specific Th1 and Th17
cells and suppression of regulatory T cells (Treg). However with respect to β-glucan i.e
A. fumigatus antigen no reports indicate the activation of adaptive immune response
with respect to dectin-1. We thus propose to test the hypothesis that dectin-1 is the key
regulators of the Th response in A. fumigatus infection which could be regulated
through NLRP inflammosome and the IL-1β and by blocking the dectin-1/Syk/CARD9
expression by antibodies or siRNAs tilts the bias towards the Treg cell response
49. 38
contributing to immunotolerance in case of hyperactivity leading to collateral tissue
damage.
Figure 28: Mechanism of Dectin mediated Antifungal immunity
KEY QUESTIONS:
1. Does β- glucan induce Dectin mediated immune response in epithelial cell lines?
2. Does β- glucan induce activation of inflammatory cytokines?
3. Can the signaling inhibitors of kinases and oxidative pathways be utilized to regulate
β-glucan mediated inflammation?
50. 39
3. AIMS & OBJECTIVES
1. To study Aspergillus fumigatus/ β-glucans induced Dectins mediated
immune response in epithelial cell lines.
2. Profiling cytokines including IL-1β during β-glucan induction.
3. To delineate the signaling events dring β-glucan induction.
51. 40
4. MATERIALS AND METHODS:
The mentioned Ligand and inhibitors was purchased from Sigma and were used for
stimulation of A549 (Human Lung Epithelial) cell line.
4.1 Cell culture:
Materials
Fetal bovine serum (FBS) 0.25% trypsin-EDTA solution
10,000 IU/ml penicillin DMEM media
10000 μg/ml streptomycin 0.4% Trypan blue dye
25μg/ml Amphotericin B 10% DMSO
PBS Ethanol
Method
4.1.1 Cell Lines:
Cell line was procured from National Center for Cell Science (NCCS), Pune,
India. Following is the cell line which was used.
A549 (Adenocarcinomic Human Alveolar Basal Epithelial cell line) - Adherent
4.1.2 Cell Cultures conditions:
A549 cell line was cultured in DMEM; containing 10% heat inactivated fetal bovine
serum (FBS), 10,000 IU/ml penicillin, 10000 μg/ml streptomycin and 25μg/ml
Amphotericin B (complete DMEM) at 37°C in humidified 5% CO2 and 95% air. The
cell lines were maintained by routine sub- culturing in 25cm2
(T 25 flask) and 75cm2
(T
75 flask) tissue culture flasks.
4.1.3 Cryopreservation of the cell lines:
Cell lines were stored at -80°C (upto 6 months) in cryovials containing 10%
DMSO in 90% FBS as cryo-protective solution. Cells were collected after
centrifugation in the form of pellet, resuspended in 1 ml preservative solution and
kept in cryocooler (filled with isopropanol) and cooler was kept at -80°C.
Cryocooler prevent damage to cells by sudden heat shock of -80°C as such the
cryocooler reduce the temperature 1°C per minute.
4.1.4 Cell Revival:
1. Cells were preserved in cryovials and were incubated at 37°C for 2 minutes (by gentle
shaking in waterbath).
52. 41
2. The thawed cells were added in the 15 ml falcon tube, add 5 ml complete media.
3. The cells were pellet down by centrifugation at 300-400g for 7 minutes at 20ᴼ
C and
pellet was resuspend in 1ml media and transferred it into T 25 culture flask.
4. 4ml of cDMEM was added and later Incubated in CO2 incubator at 37°C. Media was
changed after 10-12hr.
4.1.5 Subculture of attached cell lines (A549) (T 25 flask to T75 flask):
Method:
1. The culture media from the flasks containing monolayer culture (70-80% confluence)
was carefully decanted.
2. The monolayer was washed out with 1ml PBS and 1ml of 0.25% trypsin-EDTA
solution (not more than 30 seconds) was used to remove dead cells and debris
simultaneously.
3. The cells were dislodged by tapping or by trypsinisation, using 1ml of 0.25% trypsin-
EDTA solution and incubated at 37ºC for 3-5 min.
4. The flasks were removed from the incubator and gently tapped to detach all the
adhering cells. The cell detachment was confirmed by observing under an inverted
microscope.
5. Once the cells were completely detached from the flasks, 3ml of complete medium
was added and mixed well to stop further enzymatic activity.
6. This cell suspension was centrifuged at 300-400g for 6 minutes at 20ºC to pellet down
the cells. Supernatant was decanted and pellet was resuspended in 1 ml of complete
media and the cells were counted.
7. Cells were split at 1:8 ratios and were seeded in 75mm2
flask and 25mm2
flask
respectively and incubated in CO2 incubator at 37°C until flasks attained 70-80%
confluence.
4.1.6 Cell Counting:
1. In the sterile eppendorf 90µl complete media and 10µl of cell suspension was taken and
mixed well. (10 time dilution to get the count 50-70 cells per square).
2. To get the ratio of dead and live cells 0.4% Trypan blue dye was used. The dead cells
uptake the trypan blue while live cells reject the uptake of the dye. Hence under
microscope dead cells expressed blue in colour while live cells remain colourless.
53. 42
3. Neubauer’s chamber was used to count the cells. Clean chamber and coverslip with
lens paper with a little ethanol.
4. With the coverslip on the chamber quickly touch the chamber inlet groove with the
tip of the pipette making sure there is liquid at the tip. Hold the pipette with one hand
and use the upper surface of the index finger of your other hand to guide the tip; the
pipette should be held at 45° angle to the chamber. The chamber should fill very
quickly with liquid by capillary action.
5. The cells were visually checked to make sure about dilution and adequate mixing.
Scan square subdivisions left to right, up to down and count if they are touching the
left or top line of each square. The cells with an X for subdivision 1 were not
counted. The cells in the bottom row was also not considered.
6. The cells in all of 4 corner squares were counted.
7. The whole chamber has 9 squares. The 4 corner squares have 4 X 4 subdivisions. The
center square has 5 x 5 subdivisions which are further divided into 4 x 4. Each square
is 1mm2 and the chamber depth is 0.1mm; therefore the volume overlying each
square is 0.1mm3
(or 0.0001ml = 0.1μl). Calculate the average number of cells per
square (total cells counted/No. of squares used) and multiply by 104
and the dilution
factor to cells per ml.
8. (No. of cells counted/No. of squares used) X (final volume/volume of suspention
taken) X 104
= No. of cells/ml.
4.2 Semi-quantitative Reverse Transcription Polymerase Chain
Reaction (sqRT-PCR)
1. A549 cells (2x106
) were cultured in 12-well plated and pre-treated with MNS(10 µM,
Syk Tyrosine kinase inhibitor), BHA(10µM, ROS inhibitor), NAC(10µM, ROS
54. 43
inhibitor), YVMD(10µM, Caspase 1 inhibitor), SB(SB203587, 10µM, p38 MAPK
inhibitor), PD(P20181, 10µM, ERK MAPK inhibitor), SP(S5567, 10µM, JNK MAPK
inhibitor), Curcumin(1µM, NF-kB Inhibitor) for 2 hours, followed by stimulation of
Curdlan (particulate,1 µg/µl, Dectin-1 ligand) and LPS(10 µg/µl, TLR-4 ligand)
2. At the end of 4 hours, RNA isolation was done with TRIzol reagent and Semi-
quantitative RT-PCR was carried out.
4.2.1 Semi-quantitative RT-PCR to check the mRNA expression:
Principle:
Semi-quantitative RT-PCR reveals changes in the mRNA levels of any proteins
affected during experimental conditions. The total RNA is reverse transcribed to
generate cDNA which are amplified using specific primers to reveal any functional
changes.
Materials
Curdlan (1µg/µl) Chloroform
LPS (10 µg/µl)
MNS(10µM)
NAC(10µM)
Isopropanol
DNA Loading dye(6X)
Phenol
YVMD(10µM)
SB(10µM)
Ethidium bromide (0.1µg/ml)
Trizol
PD(10µM)
SP(10µM)
Curcumin(10µM)
RNase free water
1.5% Agarose gel
Method:
1. A549 cells were used to check the change in expression of Dectin-1 after Curdlan
stimulation. Cytokine profile was assessed for expression of IL-10, IL-23, IL-17, IL-1β
and TNF-α.
2. A549 cells were stimulated with Curdlan, LPS, MNS, NAC, YVMD, SB, PD, SP,
Curcumin for 4 hours, washed and protocol for RNA isolation was then followed.
55. 44
4.2.2 Isolation of RNA:
Principle: RNA extraction with TRIzol is a common method of total RNA
extraction from cells based on the research of Chomczynski PSN 1987. The main
components of TRIzol are guanidinium isothiocyanate (powerful protein denaturant)
that inactivates RNases and acidic phenol/chloroform. Low pH is crucial since at
neutral pH DNA not RNA partitions into the aqueous phase. RNA is stable in TRIzol
which deactivates only RNases. Hence TRIzol is mainly used to lyse cells without
changing the integrity of RNA. After addition of TRIzol, incubation at room
temperature allows complete dissociation of nucleoprotein complexes. Isopropanol
(IPA) acts as precipitant that precipitates RNA in the suspension. Precipitation with
ethanol removes DNA from the interphase, and an additional precipitation with IPA
removes proteins from the organic phase. Total RNA extracted by TRIzol Reagent is
free from the contamination of protein and DNA.
Method:
1. The media was removed from the plates and each well washed with PBS.
2. 1 ml of TRIzol was added in each sample, and the cells were lysed directly on the
culture dish. After addition of the reagent, the cell lysate was passed several times
through a pipette to form homogenous lysate.
3. The lysate was transferred in microfuge tube and kept at room temperature for 5
minutes, for complete dissociation of nucleoprotein complexes.
4. 0.25 ml of chloroform was added to each tube and the samples were shaken vigorously
and allowed to stand for 2-15 minutes at room temperature till the layers are seperated.
5. TRIzol phases after chloroform addition
TOP - colourless aqueous phase (RNA)
MIDDLE - interphase (DNA)
BOTTOM - red (organic) phenol-chloroform phase (proteins & lipids)
6. The resulting mixture was centrifuged at 12,000 x g for 15 minutes at 4°C.
7. The aqueous phase was transferred to a fresh tube and 0. 5ml of Isopropanol( IPA) was
added and mixed gently by inverting 2-3 times and allowed the sample to stand for 5-
10 minutes at room temperature.
56. 45
8. The mixture was again centrifuged at 12,000 x g for 10 minutes at 4°C. The RNA
forms a pellet on the side and bottom of the tube which was washed in 75% ethanol
(0.2 ml).
9. The samples were centrifuged at 7,500 x g for 5 minutes at 4°C; the supernatant was
decanted and the pellet was air dried for 5-10 minutes.
10. RNA pellet was re-suspended in 20µl of RNase free water and heated on thermo
mixture at 55°C for 10 minutes for complete dissolution. Pure RNA should have an
A260/A280 ratio of ≥ 1.7
4.2.3 Estimation of RNA:
1. First the zero reading was set by measuring absorbance in Nano-spectrophotometer of
1µl RNase free water as blank.
2. The sample was mixed with the help of pipette as well as vortex and 1µl of sample
was placed to cuvettes and absorbance of sample was taken.
3. Note down the reading for concentration and A260/A280 ratio.
4.2.4 cDNA synthesis:
Principle:
Gene expression analysis has become an indispensable tool. Researchers are always
keen to find out whether their gene of interest is expressing or not. For this, the mRNA
(messenger RNA) is located and quantified in the given sample. mRNAs carry the
information coded by DNA and, thus, further gets translated to produce respective
proteins. RNAs are very unstable and fragile, and are very likely to degrade by the
omnipresent RNases. The biological informations encoded in mRNA are stored in
more stable form of nucleic acid, i.e. cDNA, prepared from RNA. This conversion is
brought about by reverse transcriptase. Reverse transcriptase is a RNA-
dependent DNA polymerase. Using mRNA as a template, reverse
transcriptase produces its complementary DNA based on the pairing of RNA base
pairs. The reverse transcriptase used (commonly used- Moloney
Murine Leukemia Virus (MMLV) RT) display terminal transferase activity on reaching
at the end of the RNA template. It adds 3-5 residues (usually dC) to the 3′-terminal of
the cDNA. A random primer cocktail is used to produce cDNA from the RNAs. The
cDNAs produced are not full length. Random primer is extremely useful if production
57. 46
of the shorter cDNA fragments is desirable. Its use increases the probability of
converting the entire 5′-end of the mRNA into the cDNA. In case of long
mRNAs, reverse transcriptase is usually not able to reach the 5′-end. Therefore,
random primer proves to be extremely advantageous.
Method:
1. RNA (2 µg) of each sample were transferred to the 0.2µl of microcentrifuge
tubes. Master Mix for cDNA synthesis was prepared.
Table 4:Mastermix Preparation For cDNA
2. RNAse free water was added to the master mixture accordingly so as to make the
total volume to 20 µl and mixed by vortexing.
3. Master mixture was added to tubes -containing RNA (2 µl) to make total volume of
20 µl and given a short spin.
4. The tubes were put in PCR machine at 25°C for 10 mins, 42°C for 1 hour and 10 min
at 60°C to stop the reaction. cDNA is ready for use in PCR reaction.
4.2.5 PCR Reaction:
The generated cDNA was used for PCR analyses to assess mRNA expression and -
actin gene was used as an internal control. Preparation of master mixture:
Components Amount (for 10µl reaction)
EmeraldAmp GT PCR MASTERMIX(2X
PREMIX)
4.0 µl
RNase free water 3.0 µl
Forward primer 1.0 µl
Reverse primer 7.7 µl
Table 5: Mastermix Preparation for PCR
Component Amount (for 20 µl reaction)
5X RT Buffer (Reaction Buffer) 4 µl
10mM dNTPs (deoxynucleotide triphosphate) 1.25µl
Random Primer 0.5µl
MMLV reverse transcriptase (20U/µl) 0.5 µl
RNase free Water 11.75 µl
58. 47
1. Above mixture was vortexed. 1 µl of cDNA prepared for each sample was transferred
to 0.2 ml PCR tubes and master mix was added to each tube.
2. PCR reaction was performed in gradient thermocycler and the PCR conditions are
mentioned below for 35 cycles:
Denaturation step at 94°C for 1 minute
Annealing step for 30 sec at (Tm-50
C) of respective Primers
Extension step at 72°C for 30 seconds.
4.2.6 Primer designing:
Gene sequence of gene of interest was retrieved from National Center of Bio-
Informatics (NCBI) www.ncbi.nlm.nih.gov. Using the application of IDT
(INTEGRATED DNA TECHNOLOGIES) i.e. Primer quest , primers were obtained.
By analyzing different criteria such as %GC, secondary structure formation, self
ligation, melting temperature (Tm) the best primers were selected and the obtained
sequences were BLAST to check the similarity to other organism or other proteins.
They were cross checked by Sigma primer calculator & Primer express software.
(Table 6)
4.2.7 DNA gel electrophoresis:
The PCR products were electrophoresed on 1.0% Agarose gel and visualized by EtBr
staining.
Preparation of agarose gel:
1. 1.0% agarose gel was prepared in 1X TBE buffer by dissolving 1g of agarose powder
and 2 µl of Ethidium bromide (0.1µg/ml) was added.
2. Gel was casted in the gel caster and combs were adjusted.
3. 10µl PCR products were loaded with 2 µl of DNA loading dye (6x) and immediately
resolved on gel at 100V for 45 mins.
4. The gel was then visualized in BioRad gel documentation system and analyzed with
Fusion as well as ImageJ software.
59. 48
Table 6: Use of Specific Primers used for Amplification
S.N PRIMER
PRIMER
LENGTH
ANNEALING
TEMPERATURE
Tm(o
C)
PRODUCT
LENGTH
PRIMER SEQUENCE
1.
β actin
(NM_001101.3)
Forward 20
59o
C
63.9
214 bp
5’-GGACTTCGAGCAAGAGATGG-3’
Reverse 20 64 5’-AGCACTGTGTTGCGTACAG-3’
2.
Dectin 1
( XM_005253468.1)
Forward 23
51o
C
55.3
130 bp
5’- GGAAGCAACACATTGGAGAATGG-3’
Reverse 21 52.4 5’- AGAACCCCTGTGGTTTTGACA-3’
3.
Dectin 2
(NM_001007033.1
)
Forward 24
63o
C
67.4
113 bp
5’-GGCTGTCTGAACTACACTCATATC-3’
Reverse 22 67.4 5’-GCAACTGGAACCAAATGAACTTC -3’
4.
IL 10
(NM_000572.2)
Forward 18
52 o
C
56
131 bp
5’-ATGCCCCAAGCTGAGAAC -3’
Reverse 21 57.9 5’-GCCTTGCTCTTGTTTTCACAG -3’
5.
IL-1β
(NM_000576.2)
Forward 23
47 o
C
51.7
132 bp
5’-ATGATGGCTTATTACAGTGGCAA -3’
Reverse 20 53.8 5’-GTCGGAGATTCGTAGCTGGA-3’
6.
TNF α
(NM_000594.3)
Forward 18
51 o
C
56
139 bp
5’-ACTTTGGAGTGATCGGCC -3’
Reverse 20 57.3 5’-GCTTGAGGGTTTGCTACAAC -3’
60. 49
4.3 Enzyme-Linked Immunosorbent Assay (ELISA):
ELISA is a technique used mainly to detect the presence of an antibody or an antigen
in a sample. Sandwich type of ELISA is used for the detection of TNFα, IL-1β and IL-
10. For the detection of TNFα, IL-1β and IL-10, A549 were plated on 24well plates
and stimulated with Curdlan / LPS for 24 hours. Supernatent was collected and stored
in -20°C. For the detection of TNFα, IL-1β and IL-10. ELISA was performed by
following protocol:
Principle:
Antibody-sandwich ELISAs may be the most useful of the immunosorbent assays for
detecting antigen because they are frequently between 2 and 5 times more sensitive
than those in which antigen is directly bound to the solid phase. To detect antigen, the
wells of microtiter plates are coated with specific (capture) antibody followed by
incubation with test solutions containing antigen. Unbound antigen is washed out and
an antigen-specific antibody conjugated to enzyme (i.e., developing reagent) is added,
followed by incubation. Enzyme labeled antibody can be produced in the same animal
that produced passively adsorbed antibody, or from a different species immunized with
the same antigen that is captured. Unbound conjugate is washed out and substrate is
added. After incubation, the degree of substrate hydrolysis is measured. The amount of
substrate hydrolyzed is proportional to the amount of antigen in the test solution.
Materials
Capture antibody HRP conjugate- secondary antibody
1X PBST TMB substrate
Blocking buffer Stop solution
Primary antibody
Method:
1. Coating of capture antibody:
ELISA plates were coated with 100 μl/well of capture antibody in coating buffer. Plate
was incubated at 4°C overnight and washed thrice with PBST.
2. Blocking to reduce the non specific binding:
Non-specific protein-binding sites were blocked by addition of 200μl blocking buffer,
by incubating plate at 37°C for 2h and the plate was washed thrice with 1X PBST.
61. 50
3. Incubation with Primary antibody:
100μl of diluted primary antibody (2μg per ml) was added to each well and incubated
for 2 hr at 37°C and the plate was washed thrice.
4. Incubation with collected supernatant:
100μl of collected supernatant was added to each well and incubated for 2 hr at room
temperature and the plate was washed thrice.
5. Incubation with secondary antibody:
100μl of diluted secondary antibody (2μg per ml)was added to each well along with
Avidin biotin HRP conjugate and plate was incubated for 2 hr at room temperature
followed by washing of the plate thrice.
6. Incubation with TMB substrate:
100μl of 1x TMB substrate was added and incubated for 15-30 min.The reaction was
stopped by addition of 50μl stop reagent and the absorbance was read at 450 nm.
4.4 WESTERN BLOTTING
Western blotting was introduced by Towbin et al in 1979 and is now a routine technique
for protein analysis. Western blotting, also called protein blotting or immunoblotting,
uses antibodies to identify specific protein targets bound to a membrane; the specificity
of the antibody-antigen interaction enables a target protein to be identified in the midst of
a complex protein mixture. Western blotting can produce qualitative and semi-
quantitative data on a protein of interest.
Principle: The first step in a Western blotting procedure is to separate the proteins in a
sample by size using denaturing gel electrophoresis (i.e.SDS-PAGE). Alternatively,
proteins can be separated by their isoelectric point (pI) using isoelectric focusing (IEF).
After electrophoresis, the separated proteins are transferred, or "blotted", onto a solid
support matrix, which is generally a nitrocellulose or polyvinylidene difluoride (PVDF)
membrane.
In most cases, the membrane must be blocked to prevent nonspecific binding of the
antibody probes to the membrane surface, and the transferred protein is then complexed
with an antibody and detection probe (e.g. enzyme, fluorophore, isotope). An appropriate
method is then used to detect the localized probe to document the position and relative
62. 51
abundance of the target protein.In addition to the challenges of immunodetection in the
protein blotting workflow, the transfer of proteins from a gel matrix to a membrane is a
potential hurdle. The best results depend on the nature of the gel, the molecular weight of
the proteins being transferred, the type of membrane and transfer buffers used and the
transfer method.
4.4.1 Cell Lysate preparation
The main principle of sample preparation, however, is to ensure that the sample is in
the best possible condition and level of purity required for the selected analysis.
FOR MONOLAYER CELLS
1.The cell culture plate was placed on ice and media was aspirated. The monolayer cells
were rinsed for 3-4 times with PBS. On the final rinse, aspirate as much PBS as
possible and 5 ml of ice-cold PBS containing 0.5 mM EDTA was added.
2.The adherent cells were scrapped off from the plate using a cold plastic cell
scraper(SPL lifesciences), then the cell suspension was transferred into a pre-cooled
microfuge tube.
3.The cells were centrifuged at 1,250 rpm for 7 minutes at 4°C and supernatant was
aspirated.
4.50 µl of lysis buffer( 2 fold volume) with protease inhibitor, PMSF (1µM) was added
to the pellet.
5. Constant agitation was maintained for 30 minutes at 4° C.
6.The cells were centrifuged at 13,000 g for 10 minutes 4°C.
7. The tubes was gently removed from the centrifuge and was placed on ice, the
supernatant was aspirated and placed in a fresh tube kept on ice, and the pellet was
discarded.
4.4.2 Determination of protein concentration
BRADFORD ASSAY
The Bradford assay is a protein determination method that involves the binding of
Coomassie1 Brilliant Blue G-250 dye to proteins (Marion1976). The dye exists in three
forms: cationic (red), neutral (green), and anionic (blue) (Compton and Jones 1985).
Under acidic conditions, the dye is predominantly in the doubly protonated red cationic
form (Amax= 470 nm). However, when the dye binds to protein, it is converted to a
63. 52
stable unprotonated blue form (Amax= 595 nm) (Reisner et al. 1975, Fazekes de St.
Groth et al. 1963, Sedmack and Grossberg 1977). It is this blue protein-dye form that
is detected at 595 nm in the assay using a spectrophotometer or microplate reader.
Cation Neutral form Anion
470 nm (red) 650 nm (green) 595 nm (blue)
Perform a Bradford assay to standardize assay at same concentration. Bovine serum
albumin (BSA) is a frequently-used protein standard.
Method:
1. 1µl of samples containing inhibitor was added to microtiter plate and 200 µl of 1X
Bradford reagent was mixed to each well.
2. The microtiter plate was shaked for five minutes at room temperature.
3. OD was observed at 595 nm on Microplate reader(Biorad).
4. The concentration of each sample was determined by calculating it by RT/g, where
R- Required protein concentration
T- Total protein concentration (i.e –30 µl)
g- Given protein concentration.
5. The final volume of samples was made to 30 µl with lysis buffer .
6. The samples were heated with dry plate for 5 minutes at 100°C.
7. Once the concentration of protein is determined, the samples were kept at -20°C or -
80°C for later use or for loading onto a gel.
4.4.3 GEL PREPARATION:
Add 1 ml Isobutanol on top of the gel to remove bubbles.
Table 7: Stacking & Resolving Gel Preparation
64. 53
1. After preparing the 10% stacking gel solution, the rack was assembled for gel
solidification (Tip: 10% AP and TEMED solidify the solution; therefore, both gels can
be prepared at the same time, if the above mentioned reagents are not added until the
end).
2. Stacking gel solution was added carefully until the level is equal to the bar holding the
glass plates. Water was added to the top. Wait for 15–30 minutes until the gel turning
solidified.
3. Overlay the stacking gel with the separating gel, after removing the water.
4. The comb was inserted, ensuring that there were no air bubbles.
5. Wait until the gel is solidified.
4.4.4 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-
PAGE)
1. The running buffer was poured into the electrophorator.
2. The gel was placed inside the electrophorator and was connected to a power supply.
3. Make sure buffer covers the gel completely, and remove the comb carefully.
4. The marker (6 μL) was loaded followed by samples (30 μL) in to each well.
5. The gel was runned with low voltage (40 V) for separating gel; higher voltage (140 V)
was used for stacking gel .
6. The gel was runned for approximately 1 hour, or until the dye front runs off the
bottom of the gel
4.4.5 WESTERN TRANSFER METHOD
Wet Electroblotting (Tank Transfer)
1. Filter sheets were cut to fit the measurement of the gel, and one polyvinylidene
fluoride (PVDF) membrane with the same dimensions was also incise.
2. The sponge and filter paper were allowed to get wet in transfer buffer, and wet the
PDVF membrane in methanol.
65. 54
3. Glass plates were seperated and the gel was retrieved. Create a transfer sandwich as
follows:
Ensure there are no air bubbles between the gel and
PVDF membrane, and squeeze out extra liquid
4. Relocate the sandwich to the transfer apparatus, which
should be placed on ice to maintain 4°C. Add transfer
buffer to the apparatus, and ensure that the sandwich is covered with the buffer. Place
electrodes on top of the sandwich, ensuring that the PVDF membrane is between the
gel and a positive electrode.Transfer for 90 minutes at 40
C
4.4.6 BLOCKING & ANTIBODY INCUBATION
1. The membrane was allowed to blocked with 5% skim milk in TBST for 1 hour.
2. Primary antibody(1:500) in 5% bovine serum albumin (BSA) was added and
incubated overnight in 4°C on a shaker .
3. The membrane was washed with TBST for 10 minutes. This was repeated thrice (All
washing and antibody incubation steps should be done on a shaker at room temperature
to ensure even agitation).
4. Secondary antibody(1:2000) in 5% skim milk in TBST, was added and incubated for 1
hour.
5. The membrane was finally washed with TBST for 10 minutes. This step was repeated
thrice.
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4.4.7 STRIPPING / RE-PROBING WESTERN BLOT
1. The filters were shaked in 2M Glycine (pH 2.2) for 20-30 minutes.
2. The filters were washed in 1x PBS + 0.1% Tween 20 (several changes)for thrice.
3. Re-block with 1x PBS + 5% non-fat dry milk + 0.1% Tween 20. This filter was later
used for the next antibody.
4.4.8 DETECTION OF PROTEINS
1. The membrane was incubated with 1 ml Novex ECL, HRP Chemiluminescent
Substrate Reagent for five minutes at room temperature in Dark condition (Ensure that
ECL covers the top and bottom of the membrane).
2. The excess developing solution was drained off from the membrane (do not let dry),&
was wrapped in plastic wrap and exposed to x-ray film. An initial 10-second exposure
indicated the proper exposure time. The result was visualized in the dark room and a
photocopy of band on a X-ray film was created.
4.5 NITRIC OXIDE MEASUREMENT:
Nitric Oxide is a known mediator for inflammation. It has been shown to induce
downstream inflammatory cascades in epithelial cells. It has been reported to induce the
MAP Kinase pathway, NFκB translocation and activation of pro-inflammatory
cytokines from A549 cells.
Principle:
This assay determines nitric oxide concentrations based on the enzymatic conversion of
nitrate to nitrite by nitrate reductase. The reaction is followed by colorimetric detection
of nitrite as an azo dye product of the Griess Reaction. The assay kit possesses two
reagents R1 and R2. R1 is 1% solution of sulfanilamide in 5% phosphoric acid and R2 is
0.1% solution of N-(1-napthyl)ethylene-diamine in H2O. The Griess Reaction is based
on the two step diazotization reaction in which acidified NO2
-
produces a nitro sating
agent, which reacts with sulfanilic acid to produce the diazonium ion. This ion is then
coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo-derivative
which absorbs light at 540-570 nm.
67. 56
Materials
Griess reagent
Method:
1. A549 cells (2x105
) were cultured in 24 wells plate and challenged with MNS(10 µM,
Syk Tyrosine kinase inhibitor), BHA(10µM, ROS inhibitor), NAC(10µM, ROS
inhibitor), YVMD(10µM, Caspase 1 inhibitor), SB(SB203587, 10µM, p38 MAPK
inhibitor), PD(P20181, 10µM, ERK MAPK inhibitor), SP(S5567, 10µM, JNK
inhibitor), Curcumin(1µM, NF-kB Inhibitor) for 2 hours, followed by stimulation of
Curdlan (particulate,3 µg/µl, Dectin-1 ligand) and LPS(10 µg/µl, TLR-4 ligand) for 24
hours in colourless media.
2. Supernatants were collected and Griess reagent(1:1) was added to each supernatant
followed by incubation of 10 minutes.
3. Intensity of purple colour was measured at 490 nm on UV-Vis spectrophotometer.
4.6 MTT CELL VIABILITY ASSAY
MTT Cell Proliferation Assay provides a simple method for determination of cell
number using standard microplate absorbance readers. Determination of cell growth
rates is widely used in the testing of drug action, cytotoxic agents and screening other
biologically active compounds. Several methods can be used for such determinations,
but indirect approaches using fluorescent or chromogenic indicators provide the most
rapid and large scale assays. The MTT assay involves the conversion of the water
soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to an
insoluble formazan.2-4 The formazan is then solubilized, and the concentration
determined by optical density at 570 nm. The result is a sensitive assay with excellent
linearity up to approximately 106
cells per well.
principle :
MTT can be used to safely assess cell proliferation, cell viability, and/or cytotoxicity.
MTT is added directly to the culture medium and is reduced by metabolically active
cells to insoluble purple formazan dye crystals. The absorbance of the sample is read
directly in the wells at an optimal wavelength of 570 nm, but any filter that absorbs
68. 57
between 550 and 600 nm may be used. Tetrazolium compound MTT changes to from
yellow to purple.
Method:
1. For adherent cells, the medium was removed and replaced with 100 µL of fresh
culture medium.
2. 10 µL of the 5 mg/ml MTT stock solution was added to each well. A negative control
of 10 µL of the MTT stock solution added to 100 µL of medium alone was included.
3. Incubate at 37°C for 2 hours. At high cell densities (>100,000 cells per well) the
incubation time can be shortened to 1 hour.
4. 100 µL of the 0.04 M MTT Solubilizing solution(acidic propanol+10% SDS)was
added to each well and mixed thoroughly using the pipette.
5. The microplate was incubated at 37°C for 15 minutes in a humidified chamber. Longer
incubations will decrease the sensitivity of the assay.
6. Each sample was mixed again using a pipette and absorbance at 570 nm was measured
Calculation:
4.7 IMMUNOSTAINING
Principle:
Immunohistochemistry (IHC) is a wide-used biological technique that visualize
distribution and localization of specific antigen or cellular components in separated
tissues, or cells. Immunohistochemistry provides in situ information which promises a
more convincing experimental result.
Major components in a complete immunohistochemistry experiment:
1)Primary antibody binds to specific antigen;
2)The antibody-antigen complex is formed by incubation with a secondary, enzyme-
conjugated, antibody;
3)With presence of substrate and chromogen, the enzyme catalyzes to generate colored
deposits at the sites of antibody-antigen binding.
Materials:
% Cytotoxicity = Experimental OD – Lysis Control OD
Control OD- Lysis Control OD
