Download The Body`s Defenses Against Infection

Innate Immunity
Robert Binder, PhD
E1051, BSTWR
Department of Immunology
University of Pittsburgh School of Medicine
412-383 7722
[email protected]
Phases of immune responses
Innate Immunity
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Primary defenses
No evidence for clonality
Self vs. Non-self or lack-of-self recognition
No memory/No secondary response
Cellular mediators: Phagocytes (monocytes,
macrophages, neutrophils) Natural Killer cells,
dendritic cells, basophils, eosinophils
Soluble mediators: complement, MBPs, IFNs,
defensins, bacteriocidal peptides and proteins
Physical barriers: surfactant, skin,
epithelial/endothelial layers
Adaptive Immunity
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Self vs. Non-self discrimination
Fine specificity
Clonality facilitating expansion of cells
capable of specific antigen recognition
Memory
Secondary response to recall antigens
Cellular mediators: CD4 T (Thelper) lymphocytes,
CD8 (Cytotoxic T) lymphocytes, B cells
Cellular mediators: cytokines, Ig, perforin,
granzymes
Immune system
Cellular Elements of the Non-adaptive
Immune System
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Neutrophils
Basophils
Eosinophils
Macrophages
Natural Killer Cells
Dendritic cells
Recognition Receptors in Innate and Adaptive
Immune Systems
Pattern
recognition
Receptors
(PRR)
Exposure to infectious agents
Barriers to Infection
• Epithelium- physical barrier, tight junctions between
cells.
• Internal epithelium- secrete mucus containing mucins
which is in constant motion. Mucins prevent bacterial
adherence necessary for colonization and infection.
• Secretion of Microbicidal agents- lysozyme and
phospholipase A in tears and saliva, low pH of the
stomach, bile salts, lysolipids, a-defensins in small
intestine, b-defensins in respiratory and urogenital tracts
and skin and tongue, surfactants.
• Cytotoxicity by normal flora- lactic acid, antimicrobial
peptides, competition for attachment
A variety of pathogens
• An agent that breaches these barriers and
causes damage to tissues (pathology) is
called a pathogen/pathogenic microorganism.
• Viruses, bacteria, fungi, protozoa, helminths
• Immune response (innate or adaptive) to
pathogens is varied due to distinct habitats
and life cycles of the pathogen
Tissue resident immune cells
(macrophages & dendritic cells)
• Mature from monocytes that leave circulation
and migrate into tissues
• Recognize, ingest and destroy pathogens
without the aid of adaptive immunity.
• Recognition occurs through cell surface
receptors specific for Pattern Associated
Molecular Patterns (PAMPs) mannose
receptor, scavenger receptor, CD14 & Toll-like
receptors (TLRs)
• Recognition  receptor binding  phagocytosis
 death in the phagocyte
•Bound pathogen is surrounded by
phagocyte membrane.
•Internalization and enclosure into a
vesicle called phagosome.
•Phagosome fuses with lysosomes
to form phagolysosomes
•Death of pathogen by the
acidification, enzymes
Other effector mechanisms of macrophages
Respiratory burst
An increase in oxygen consumption by
macrophages
Membran associated NADPH oxidase converts
oxygen to superoxide ion: O2  O2Superoxide dismutase converst superoxide ion to
hydrogen peroxide: O2- H2O2
Hydrogen peroxide can be converted to
hypochlorite ions and hydroxyl radicals
Defensins
• Diverse group of small cationic, antimicrobial peptides found in plants,
insects, fungi, reptiles, birds and mammals
• 28-42 amino acids, cysteine rich cationic proteins with an even number of
cysteines [6-8]
• a-defensins initially described as being produced by neutrophils, alveolar
macrophages and Paneth cells at the base of crypts in the intestine [11
genes]
• b-defensins [variant cysteine spacing] made primarily by leukocytes and
epithelial cells lining the respiratory, GI and GU tracts; found in skin,
surfactant [respiratory and urogenital tracts] and serum [39 genes]
• anti-bacterial against gram positives bacteria; anti-fungal effects; activity
against enveloped viruses; also assist in killing phagocytized bacteria in
granulocytes
• have hydrophobic and positively charged domains that insert into cell
membranes, polymerize to form aggregates [pore ?] and disrupt membrane
function allowing efflux
• a-defensins act as opsonins, a- and b-defensins induce mast cell
degranulation, a-defensins induce IL8 release by epithelia, activate
complement and suppress anti-inflammatory mediators
Defensins
a-defensins X1-2 C X C R X2-3 C X3 E X3 G X C X3 G X5 C C X1-4
b-defensins X2-10 C X5-6 G/A X C X3-4 C X9-13 C X4-7 C C Xn
θ-defensins GXCRCXCXRGXCRCXCXR (only in rhesus monkeys)
The α, β and θ defensin bonding archetypes. The canonical cysteine spacing for the three classes of
defensin peptides that have been described to date with the cysteines (C) involved in the
disulphide bonds (lines) indicated.
Neutrophils
• Also referred to as Polymorphonuclear neutrophilic
leukocytes (PMNs)
• Abundant in blood and not present in normal healthy
tissue
• Short lived
• They are a major population of phagocytic cells and
have the same effector mechanisms as macrophages
• Recruited into tissues by the cytokines and chemokines
released by activated tissue macrophages
• Between the macrophages and neutrophils many
infections can be controlled outright
Pattern recognition
• Regular patterns of molecular structures
on pathogens are recognized (PAMP).
These patterns are absent in the host
• Receptors may be on the surface of
macrophages, dendritic cells and
neutrophils OR may be secreted
molecules (PRR)
PRR- Mannose-binding lectin (MBL)
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Free protein in the blood
Recognizes certain carbohydrates and their spacing.
Oligomeric structure [400-700 kDa]
Built of subunits that contain 3 identical peptide chains of
32 kDa each
• Can form oligomeric forms; Dimers and trimers are not
biologically active, and at least a tetramer is needed for
activation of complement
• MBL-pathogen complex recognized by phagocytes and
facilitates phagocytosis. (opsonization- the process of
coating a particle with a protein to facilitate
phagocytosis)
• MBL can also facilitates the initiation of the complement
pathway.
pathogen
host
PRRs- Surfactant Proteins
• Proteins produced by epithelial cells, immune cells, alveolar cells, parietal
cells [in lung = 10%; half of this is from plasma, and most of the rest is SPA,B,C,D]
• Surfactant proteins A-D described; all collectins [lectin domain/collagen
domain]. Generally exist a oligomers of trimers [“bouquet of tulips”]
• SP-A – hexamer of trimers, binds saccharides associated with lipid A in a
Ca++-dependent fashion; blocks penetration of viruses; SP-A receptor found
on macrophages
• SP-D – hexamer of trimers; binds sugars on LPS in a Ca++-dependent
fashion; blocks penetration of viruses; SP-D receptor found on
macrophages
• SP-B, -C – hydrophobic membrane proteins that are required for proper
biophysical function of the lung
• Both MBL and surfactant proteins belong to a family of collectin proteins
(have both a collagen-like and lectin domain)
The subunit structures of collectins and ficolins. The molecules are drawn
approximately to scale, except for CL-P1, which has a long α-helical coiled-coil next to
the membrane. Interruptions in the collagen structures are indicated.
The overall structures of the human collectins and L-ficolin.
PRRs- Macrophage mannose
receptor (MMR)
• Cell surface receptor (CD206)
• Calcium dependent lectin
• Recognition properties are similar to MBLbinds sugars found on bacteria and
viruses.
• Functions directly as a phagocytic receptor
PRRs- Scavenger receptors
• Cell surface phagocytic receptor 1-6
(CD204)
• Recognizes anionic polymers and
acetylated LDLs
• These structures are shielded by sialic
acid on host cells and are thus specific for
pathogenic molecules
PRRs- fMLP receptors
• Recognizes fMet-Leu-Phe the start of
many bacterial polypeptides.
• Not a phagocytic receptor
• Binding of this receptor leads to induction
of signals for induced innate immunity
PRRs- Toll-Like receptors (TLRs)
• Toll was defined as a signaling molecule in Drosophila sp.
Responsible for dorso-ventral morphogenesis via induction of
apoptosis [Nusslein-Volhard, et al. 1985]
• Toll shares homology with the IL1r cytoplasmic domain which raised
the question of whether TLRs are important in immune responses
• Toll was found to be important in activating Drosophila sp. nonadaptive immunity, i.e. production of anti-fungal and anti-bacterial
peptides
• At least 10 TLRs in man [estimated to be between 10 and 15 in most
mammals, 11-13 defined in mice]; some can dimerize and form
homo- or heterodimers
• One of the most ancient and conserved set of proteins in the
immune system…even found in plants, and have antimicrobial
function
• Engagement of TLR leads to cellular responses that deal with the
particular type of pathogen present
– dsRNA binding to TLR3 interferon production which is antiviral
– LPS binding to TLR4  TNFa, IFN production which are
directly/indirectly bacteriocidal
TIR Domains
• TLR and IL1rs form a superfamily that has a
common Toll-IL1r (TIR) domain
• 3 subgroups of TIRs
• Group 1 = receptors for interleukins that are
produced by macrophages, monocytes, and
dendritic cells
• Group 2 = classical TLRs that bind directly or
indirectly to molecules of microbial origin
• Group 3 = adaptor proteins that are exclusively
cytosolic and mediate signaling from proteins of
Groups 1 and 2
TLR Recognition
TLRs 1, 2, 4, 5 and 6 specialize in the recognition
of mainly bacterial products that are unique to
bacteria and not made by the host. Their
detection therefore affords a straightforward
self:non-self discrimination.
TLRs 3, 7, 8 and 9, in contrast, specialize in viral
detection and recognize nucleic acids, which are
not unique to the microbial world. In this case,
self:non-self discrimination is mediated not so
much by the molecular nature of the ligands as
by their accessibility to the TLRs. These TLRs
are localized to intracellular compartments and
detect viral nucleic acids in late endosomeslysosomes.
Toll-like Receptors and Their Ligands
TLR family
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
TLR10
TLR11
TLR12
TLR13
Ligands (origin)
Tri-acyl lipopeptides (bacteria, mycobacteria), Soluble factors
(Neisseria meningitides)
Lipoprotein/lipopeptides (a variety of pathogens), Peptidoglycan
(Gram-positive bacteria), Lipoteichoic acid (Gram-positive bacteria),
Lipoarabinomannan (mycobacteria), A phenol-soluble modulin
(Staphylococcus epidermidis), Glycoinositolphospholipids (Trypanosoma cruzi),
Glycolipids (Treponema maltophilum), Porins (Neisseria), Zymosan (fungi),
Atypical LPS (Leptospira interrogans), Atypical LPS (Porphyromonas
gingivalis),
Double-stranded RNA (virus), poly I:C
LPS (Gram-negative bacteria), Taxol (plant), Fusion protein (RSV), Envelope
proteins (MMTV), HSP60 (Chlamydia pneumoniae), HSP60 (host), Type III
repeat extra domain A of fibronectin (host), oligosaccharides of hyaluronic acid
(host), Polysaccharide fragments of heparan sulfate (host), Fibrinogen (host)
Flagellin (bacteria)
Di-acyl lipopeptides (mycoplasma)
Single stranded RNA, Imidazoquinoline (synthetic compounds), Loxoribine
(synthetic compounds), Bropirimine (synthetic compounds)
single stranded RNA, small synthetic compounds, (Imidazoquinoline)
Unmethylated CpG DNA (bacteria)
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Profilin
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TLR Expression
Receptor
TLR1
TLR2
TLR3
TLR4
TLR5
TLR6
TLR7
TLR8
TLR9
Cell types
mf, MDCs, iDCs+, mDCs+/mf, MDCs, iDCs+, mDCs +/-, mast cells, renal
epithelial cells
mDCs
mf, MDCs, iDCs+, mDCs+/-, mast cells,
intestinal epithelial cells [low], renal epithelial
cells, pulmonary epithelial cells, corneal
epithelial cells, dermal endothelial cells
mf, MDCs, iDCs+, mDCs+/-, intestinal
epithelial cells
mf, mast cells
mf, PDCs
mf, MDCs, mast cells
mf, pDCs, B cells
LPS, CD14 and TLR4
• Bacterial LPS is a cell
wall component of
gram negative bacteria.
Responsible for septic
shock in humans due
to the overwhelming
cytokine response
• LPS is recognized by
TLR4 in combination
with MD-2 and CD14
TLR Signal Transduction Pathway
Toll-like receptor (TLR) signaling pathway. TLRs recognize specific patterns of microbial
components. MyD88 is an essential adaptor for all TLRs and is critical to the inflammatory response.
Lipopolysaccharide (LPS)-induced activation of signaling molecules such as IRF-3, PKR, MAP
kinase, and NF-kB has been reported, indicating the presence of the MyD88-independent pathway.
TIRAP/Mal was identified as a component specifically involved in TLR4-mediated signaling.
• Activation of NFkb via TLRs leads to the
production of cytokines and chemokines
• Up-regulation of co-stimulatory molecules
necessary for adaptive responses
• The exact profile of the cytokines
produced depends on the cell type
(Macrophage, DC etc) and the TLR
stimulated
PRRs- NOD proteins
• Contain a nucleotide binding oligomerization domain
• Ligand binding domains are very similar to TLRs
• Present in the cytosol of the cell. Not on cell surface or in
intracellular vesicles
• Recognize microbial products, activate NFkb
– NOD1: glutamyl diaminopimelic acid (most bacteria)
– NOD2: muramyl dipeptide (Gram-negative bacteria)
• Activate and recruit proteases called caspases
• While NODs and TLRs recognize microbial products their
expression patterns vary somewhat
– NODs more important in epithelial cells where TLR expression is weak