Innate Immunity

A/Prof Glen Scholz

glenms@unimelb.edu.au

18 July 2018
 Chronic Periodontitis

Polymicrobial
Synergy and
Dysbiosis

Hajishengallis G, Nature Reviews Immunology (2015) 15:30-44

 Outline of Today’s Lecture

1. Overview of innate immunity

2. Complement system

3. Key cells of the innate immune system

4. Pathogen recognition by innate immune cells

5. Signalling by pathogen recognition receptors

6. Anti-microbial peptides

7. Inflammatory cytokines and chemokines

Text book: Janeway’s Immunobiology (Kenneth Murphy, Paul Travers and Mark Walport, Garland Science)
3
Evolution of the Immune System

Saleh M, Immunological Reviews (2011) 243:235-46 4

 Overview of Innate Immunity

• Responsible for protecting us from the vast majority of microbes

we encounter on a daily basis

• Responds rapidly to microbes (immediately to a few hours)

it leads to adaptive immune response

• Limited repertoire of germline-encoded, invariant immune

receptors (NB: contrasts with T cell and B cell receptors)

• Response is not specific for a particular microbe

▪ Responding to a broad pathogen class

• No immunological memory
encounter with the pathogen for the second time doesnt lead to a faster respons

5
 Overview of Innate Immunity

• Consists of:
▪ Physical barriers (e.g. mucins, saliva, tears, cells)
▪ Chemical barriers (e.g. anti-microbial peptides, enzymes, pH)
▪ Complement proteins provide immediate protection against infection

▪ Immune cells (e.g. neutrophils, macrophages, dendritic cells)

Innate immunity has 4 main objectives

• 1st objective: Prevent the entry of pathogens into tissues

1st is try to keep the pathogen outside the body

• 2nd objective: Contain and kill pathogens that get into tissues
even if it gets in, you dont want it to get in the circulation which can lead to systemic
infection
• 3rd objective: Trigger the adaptive immune response
Try bring in B and T cells to try kill the pathogen

• 4th objective: Resolve inflammation, restore homeostasis

you need to turn off the inflammatory response
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Phases of the Immune Response

7
Complement Proteins
this is the reasoin why the innate immune system can respond
so quickly, theses proteins are already in the circulation ready to
respond
 Complement System

• Discovered on the basis of its ability to

complement the antibacterial activity of
antibodies

• Bacterial components activate a cascade of

proteolytic reactions leading to the coating of
bacteria with complement fragments
normally there is a complement protein which will bind to the
antigen and it will activate proteolytic enzymes and proteins
• Binding of antibodies to bacteria can also
trigger complement activation

• Opsonise bacteria to promote phagocytosis

• Trigger inflammation

• Form a membrane-attack complex (MAC)

9
 Complement System

Lectin = a carbohydrate-
binding protein.

C3 convertase = C4b + C2a Binds covalently to the surface of pathogens

C3 convertase is an enzyme
which acts on inactive protiens
and activates it and initiate
complement fragments

Remember C3a, C5a and

C3b

10
 Complement System

Inflammatory
response

allows antibodies to get

into the site of infection.
you need weaken junction
so that immune cells
can get out of the blood
vessels and fight the
infection

11
 Complement System

these molecules build

a massive hole in the
bacteria or the virus
which disrupts the
Membrane Attack homeostasis of the cell
and allows it to die
Complex

12
Key cells of the innate immune system
 Key Cells of the Innate Immune System
• Keratinocytes (specialised epithelial cells): these are the first cells that are going
to come in contact with the pathogen
▪ E.g. Oral cavity, skin, etc.
• Mucosal epithelial cells: the cells play a barrier function
▪ Orogastrointestinal tract, respiratory tract, urogenital tract
these are innate immune system
cells
• Neutrophils
• Macrophages
• Dendritic cells

14
 Keratinocytes and Mucosal Epithelial Cells
in chronic periodontitis, the cells release factors which break
down the tight junctions and can gain entry
• Form a physical barrier (e.g. via
tight junctions) to prevent
microbial entry

• Release anti-microbial peptides

(see later)

• Mucosal epithelial cells produce

mucins (prevent microbes from
reaching the cells, trap
microbes, aid in the flushing
away of microbes)

• Function as immune sentinels

can sense presence of pathogens

• Release inflammatory mediators

(e.g. cytokines and chemokines) Hooper LV, Nature Reviews Microbiology
(2009) 7: 367-374

15
 Keratinocytes and Mucosal Epithelial Cells
there is a
breakdown of
the barrier
though which
the pathogens
can enter the
body, leading to
a robust
inflammatory
response. with a
certain level of
inflammation,
there is tissue
damage. if there
is chronic
inflammation,
there is chronic
in homeostasis, there is breakdown of
very little inflammation the tissue and
so ensure that bacteria chronic tissue
are kept out of the body destruction

Fritz JH et al., Trends in Immunology (2008) 29: 41-49 16

 Neutrophils

• First immune cells recruited to site of injury/infection

• Half-life of only a few hours (6-8 hours)

• But produced at a rate of >5 x 1010 per day!

Neutrophils provide 98% of the protection in out
• Potent anti-microbial activity: everday lives. it gets rid of 98% of the
pathogens, other cells help out with the other
▪ Highly phagocytic 2%. these are crucial as they have a vast range
of antimicrobial functions

▪ Generate reactive species (e.g. ROS, HOCl, chloramines)

ROS - reactive
▪ Produce lysozyme (damages bacterial cell wall) oxygen species - kill
bacteria

▪ Produce matrix metalloproteinases (e.g. MMP8)

▪ Produce inflammatory cytokines and chemokines
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Neutrophils

these ROS are potent in

killing bacteria and
viruses

Nathan C, Nature Reviews Immunology (2006) 6: 173-182

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 Macrophages
• Arise from blood monocytes that have migrated into the underlying tissue
In the bone marrow, macrophages go to the site of infection and they become a macrophage.

• Tissue-resident macrophages (homeostasis), and recruited macrophages

(inflammation) TRM - clean up the dead cells that are present in the circulation.
RM - they enter the blood during and inflammatory response and they are coming in,
in response to infection
• Resident and recruited macrophages are phenotypically different

Gordon S, Nature Reviews Immunology (2003) 3: 23-35 19

 Macrophages

• Highly phagocytic

• Kill phagocytosed microbes through the production of ROS, and

degradative enzymes (e.g. cathepsins)
remember these 4 cytokines
• Release various cytokines (e.g. IL-1b, TNF, IL-6, and IL-12), and
chemokines (e.g. CXCL8, CCL2)
CXCL8 = IL8

• Present microbe-derived antigens (via MHC II molecules) to T cells

(to trigger Adaptive Immunity)

20
Macrophages

cricual role in
gingival health
and plays a role
in the bacteria
that colonise the
root of the tooth

21
 Macrophages

• Highly phagocytic

• Kill phagocytosed microbes through the production of ROS, and

degradative enzymes (e.g. cathepsins)

• Release various cytokines (e.g. IL-1b, TNF, IL-6, and IL-12), and
chemokines (e.g. CXCL8, CCL2)

• Present microbe-derived antigens (via MHC II molecules) to T cells

(to trigger Adaptive Immunity)
these foreign antingens are loaded on to the MHC class II

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 Adaptive immune response is
activated by these DCs. DCs
Dendritic Cells present the antigen to adaptive
immune cells
• Arise from myeloid and lymphoid
progenitor cells

• Resident under most surface

epithelia, and found in most solid
organs

• Specialised to capture, process and

present antigens (foreign and self) to
naive T cells (prime immunity or
induce tolerance)
Self leads to tolerance, foreign leads to immunity
• Up-regulate MHC and co-stimulatory
molecule (e.g. CD86) surface
expression in response to various
stimuli (e.g. microbial products,
cytokines, immune complexes)
T cell requires multiple messages to become activated.
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How Does the Innate Immune System
Detect Pathogens?

What are you

looking at?
 Pattern Recognition Receptors

Drosophila Toll mutant

Christiane Nusslein-Volhard
(1995 Nobel Prize
in Physiology or Medicine)

Jules Hoffmann
(2011 Nobel Prize
in Physiology or Medicine) Lemaitre B et al., Cell (1996) 86: 973-873
25
 Pattern Recognition Receptors (PRRs)

• Recognise structural components (i.e. PAMPs) that are broadly

shared by pathogens (e.g. lipopolysaccharide; LPS)

• PAMP = Pathogen Associated Molecular Pattern

• Alternative names:
▪ MAMP = Microbe Associated Molecular Pattern
▪ DAMP = Danger Associated Molecular Pattern

• Localisation: Extracellular, Cell-surface, Endosomes/phagosomes,

cytoplasm

• Main PRR families:

▪ Toll-like receptors
▪ NOD-like receptors
▪ RIG-I-like receptors
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Pattern Recognition Receptors (PRRs)

LPS

TLR4 recognises
gram negative
bacteria as it
recognises LPS

27
 Pattern Recognition Receptors (PRRs)

• Recognise structural components (i.e. PAMPs) that are broadly

shared by pathogens (e.g. lipopolysaccharide; LPS)

• PAMP = Pathogen Associated Molecular Pattern

• Alternative names:
▪ MAMP = Microbe Associated Molecular Pattern
▪ DAMP = Danger Associated Molecular Pattern

• Localisation: extracellular, cell-surface, endosomes/phagosomes,

cytoplasm

• Main families:
▪ Toll-like receptors (TLRs)
▪ NOD-like receptors (NLRs)
▪ RIG-I-like receptors (RLRs)
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 Toll-Like Receptors (TLRs)

• Ten human TLRs (>200 in sea

urchin)

• Cell-surface TLRs: TLR1, TLR2,

TLR4, TLR5, TLR6 and TLR10

• Endo/phagosomal TLRs: TLR3,

TLR7, TLR8 and TLR9 these only recognise nuclei acids.
Only when nucleic acid is engulfed
Iwasaki A et al., Science (2010) 327: 291-295

• Structural organisation: leucine-rich

▪ Ectodomain containing Leucine- repeats

Rich Repeats (PAMP recognition)

▪ Transmembrane region
▪ Cytoplasmic domain (activate
signalling pathways)
Leulier F et al., Nature Reviews Genetics (2008) 9: 165-178 29
 NOD-Like Receptors (NLRs)
• Cytosolic receptors (>20 members)

• Contain a nucleotide oligomerisation domain (NOD or NACHT domain),

and leucine-rich repeats
• May also contain a
caspase activation and
recruitment domain
(CARD), and/or a pyrin
domain

• NOD1 and NOD2

recognise specific
components of
bacterial
peptidoglycans (e.g.
muramyl dipeptide)
Magalhaes JG et al., Current Opinion in Immunology (2011) 23: 29-34
Peptidoglycan is on gram-ve and +ve bacteria and can
recognise these bacteria 30
 Pattern Recognition Receptors and their Ligands

remember
what
these
recognise

Damgaard RB, Discovery Medicine (2011) 31

Cell Signalling by Pattern Recognition
Receptors
 Signalling by Toll-Like Receptors

• Broadly divided into two

pathways:
▪ MyD88-dependent pathway
antimicrobial pathway
▪ TRIF-dependent pathway
antiviral pathway
• All TLRs, except TLR3, use the
MyD88 pathway
• TLR3 uses the TRIF pathway
• TLR4 uses both pathways
• Activate various transcription
factors, including NF-kB, AP-1,
and IRF3 master regulator of immunity
pathway
• Induce expression of cytokines,
chemokines, and type I
interferons
Mogensen T H, Clinical Microbiology Reviews (2009) 22:240-273 33
 Signalling by NOD-Like Receptors
can distinguish between gram -ve and +ve bacteria and use the Ng-kB pathway to express cytokines and
chemokines
• NOD1 and NOD2 form multi-
protein signalling complexes

• Initiate signalling by activating

the protein kinase, RIP2
(Receptor-Interacting Protein 2)

• Activate various transcription

factors, including NF-kB, AP-1,
and IRF7

• Induce expression of cytokines

and chemokines (and type I
interferons)

Correa RG et al., Bioscience Reports (2012) 32, 597-608

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 Signalling by NOD-Like Receptors

• Other NLRs are activated by

diverse stimuli (e.g. toxins,
bacterial proteins, ds DNA,
particulate matter)

• Activation triggers formation

of an “Inflammasome”

• Inflammasome = multi-protein
complex consisting of a NLR
(e.g. NLRP3), ASC (an adaptor
protein), and Caspase-1 (a
protease) It is made as an inactive
version, the pro-IL-1B
needs to be cleaved into
• Activated Caspase-1 cleaves IL-1B to be activated IL-1b
pro-IL-1b to generate mature A highly potent inflammatory cytokine.
IL-1b, which is then released
Koizumi Y, Cellular Microbiology (2012) 14:149-54 35
Signalling by Pattern Recognition Receptors

Jeong E, Yonsei Medical Journal (2011) 36

Key Effectors Molecules Produced
by Innate Immune Cells
 Anti-Microbial Peptides (AMPs)

• Effective against bacteria, viruses, fungi, and parasites

• Generally small (12-50 amino acids), cationic, and amphipathic

• Contain at least 2 positive charges (e.g. lysine and arginine)

• Insert into the anionic cell wall and phospholipid membranes of

microbes

• Cause disruption of cell integrity, resulting in killing

• Two major classes:

▪ Defensins (e.g. b-Defensin-2)

▪ Cathelicidins (e.g. LL-37)

• Some are produced constitutively, while others are induced in

response to infection
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Anti-Microbial Peptides (AMPs)

Guani-Guerra E et al., Clinical Immunology (2010) 135:1-11

39
 Anti-Microbial Peptides (AMPs)

• Effective against bacteria, viruses, fungi, and parasites

• Generally small (12-50 amino acids), cationic, and amphipathic

• Contain at least 2 positive charges (e.g. lysine and arginine)

• Insert into the anionic cell wall and phospholipid membranes of

microbes

• Cause disruption of cell integrity, resulting in killing

• Two major classes:

▪ Defensins (e.g. b-Defensin-2)

▪ Cathelicidins (e.g. LL-37)

• Some are produced constitutively, while others are induced in

response to infection
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 Inflammatory Cytokines
• Have inflammatory or anti-inflammatory properties (e.g. TNF versus IL-10)

• Diverse biological actions:

remember the functions of the
▪ Endothelial cell activation (e.g. TNF) cytokines

▪ Macrophage activation (e.g. IL-1b)

▪ T-cell differentiation, activation (e.g. IL-12, IL-23)
▪ B-cell proliferation, differentiation (e.g. IL-6, IL-13, IL-21)
▪ Macrophage recruitment (e.g. CCL2)
▪ Dendritic cell proliferation, differentiation (GM-CSF)
▪ Neutrophil proliferation, differentiation (e.g. G-CSF)
▪ Natural killer cell activation (e.g. IL-12)
▪ T-cell recruitment (e.g. CXCL9, CXCL10)
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Inflammatory Cytokines
these act locally and also at a
physiological level

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 Immunology Lecture Series

Lecture 4: Introduction to Immunology (3:00 pm, 16 July 2018) ✓

Lecture 5: Innate Immunity (8:00 am, 18 July 2018) ✓

Lecture 7: Adaptive Immunity (3:00 pm, 23 July 2018)

Lecture 8: Periodontal Inflammation (10:00 am, 25 July 2018)

Lecture 12: Innate Immune Response in Periodontitis (9:00 am, 15 Aug 2018)

Lecture 13: Adaptive Immune Response in Periodontitis (12:00 pm, 17 Aug 2018)

Lecture 4: Immune Subversion by Bacterial Pathogens in Periodontitis (9:00 am, 10 Sep 2018)

Lecture 5: Vaccination in Disease Control (9:00 am, 19 Sep 2018)

Lecture 11: Revision (9:00 am, 2 Oct 2018)