Innate Immunity

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Innate Immunity

• William H. Chambers, PhD
• University of Pittsburgh Cancer Institute and
• Department of Immunology
• University of Pittsburgh School of Medicine
• G.17e Hillman Cancer Center
• 412-623-3218
• chamberswh_at_msx.upmc.edu

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Phases of immune responses
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Innate Immunity

• Primary defenses
• No evidence for clonality
• Self vs. Non-self or lack-of-self recognition
• No memory/No secondary response
• PMNs, NK cells, macrophages,
• complement, MBPs, IFNs, defensins,
• surfactant, skin, epithelial/endothelial
  layers

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Pre-adaptive Immunity or Innate-like Lymphocytes

• More recent articulation of immune function by
  immunologists
• Specificity based upon limited recombination
• T cell compartment, e.g. TCRgd IELs
• B cell compartment, e.g. CD5 B cells BC-1
  cells
• No memory

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Adaptive Immunity

• Self vs. Non-self discrimination
• Fine specificity
• Clonality facilitating expansion of cells
• capable of specific antigen recognition
• Memory
• Secondary response to recall antigens
• Th cells, CTLs, B cells, cytokines, Ig

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Fluid Phase Elements of Non-adaptive Immunity

• Lectin-like molecules
• Bacteriocical peptides, e.g Defensins
• Complement
• Interferons

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Cellular Elements of the Non-adaptive Immune
System

• Neutrophils
• Basophils
• Eosinophils
• Macrophages
• Natural Killer Cells

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Recognition Receptors in Innate and Adaptive
Immune Systems
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Exposure to infectious agents
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Barriers to Infection
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Tissue damage from infections
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Surfactant Proteins

• Primarily lipids in lung 90 50
  dipalmitoylphosphatidyl choline forming a
  monomolecular film at air/liquid or liquid/liquid
  interfaces
• Contains 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 SP-A,B,C,D
• Contains antibacterial enzymes, e.g. lysozyme,
  phospholipase A
• Contains histatins, histidine-rich antimicrobial
  peptides
• Cryptidins/a-defensins made by Paneth cells
• 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

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Peptide Antibiotics

• Three classes
• Linear peptides without cysteine residues
• Peptides with an even number of intra-linked
  cysteines
• Linear peptides with high proportion of 1 or 2
  amino acid residues none to date with cysteines

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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
• ?-defensins, a new family, has only been defined
  in rhesus macaques
• anti-bacterial against gram positives
  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

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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
• ?-defensins GXCRCXCXRGXCRCXCXR
• The a, ß and ? 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.

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Humoral elements

• Alternative Complement Pathway
• Collectins
• Ficolins
• b-Defensins
• Interferons

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Characteristics of Collectins/Ficolins

• Collectins Ficolins
• Structure Collagen domain, C-type Collagen
  domain, Fibrinogen
• lectin CRD, a-helical -like domain, CRD,
  a-helical
• coiled-coil neck domain, coiled-coil neck
  domain, trimeric N-terminal cysteine rich
• domain, multimeric
• Diversity MBL, SP-A, SP-D, CL-L1 L-ficolin,
  H-ficolin
• CL-P1, conglutinin, CL-43 M-ficolin
• CL-46
• Production/Distribution MBL liver L-ficolin
  liver
• SP-A lung II alv. H-ficolin liver, lung
  II alv.
• SP-D lung, GI M-ficolin monocytes
• CL-L1 liver
• CL-P1 endothelial cells
• Specificity man, glu, L-fuc, ManNAc GlcNAc
• GlcNAc
• Complement Fixation MBL L-ficolin, H-ficolin

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The subunit structures of collectins and
ficolins. The molecules are drawn approximately
to scale, except for CL-P1, which has a long
a-helical coiled-coil next to the membrane.
Interruptions in the collagen structures are
indicated.
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The overall structures of the human collectins
and L-ficolin.
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MBL/MBP

• Collectin
• 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

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MBL Recognition of Bacterial Surfaces
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Interferons

• Six classes (5 are Type I 1 is Type II)
• a - multigene family 14-20 depending upon
  species produced by leukocytes and other
  virus-infected cells
• b - single gene except 5 in ungulates,
  produced by fibroblasts, leukocytes and other
  virus-infected cells
• w - produced by hematopoietic cells
• d -
• t - Identified in cattle and sheep trophoblasts
• g immune interferon/Type II, produced by
  hematopoietic cells, e.g. NK cells and T cells
  both CD4 (Th1) and CD8

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Three Major Functions of Class I Interferons a/b

• Induce resistance to viral replication by
  interfering with protein translation
• Up-regulation of MHC Class I molecules, TAP
  proteins and components of the proteosome for
  antigen presentation
• Activation of non-adaptive effector cells, such
  as NK cells, to kill virus infected cells

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Viral interference with the IFN system. Viruses
that can block the four parts of the IFN system
are grouped together.
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Phagocytic cells macrophages and neutrophils
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Phagocytic Cell Function
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Macrophages in Tissues Regulate Migration of
Leukocytes Into Sites of Inflammation and of
Pathogens Out of Sites of Inflammation
Macrophages are in normal tissues, neutrophils
are not!
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Bactericidal Agents in Phagocytic Cells
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Recognition of Infectious Agents by Receptors in
the Non-adaptive Immune System

• Non-adaptive effectors recognize microbes via
  pattern recognition receptors
• Pattern recognition receptors bind components of
  microbes that are fundamentally different from
  those on host cells, e.g. LPS, peptidoglycan
• Oligosaccharide ligands have been identified for
  pattern recognition receptors
• Ligands are often called pathogen associated
  molecular patterns (PAMPs)

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Examples of Pattern Recognition Molecules

• fMLP receptor N-formylated peptides produced
  by bacteria, serves as a chemoattractant for
  neutrophils
• Macrophage Mannose Receptor/CD206 collectin
  family proteins that bind mannose residues on
  bacteria and viruses, e.g. HIV
• Macrophage Scavenger Receptors 1-6/CD204 MSR1
  bind anionic polymers and acetylated LDLs, and
  some structures which have lost normal expression
  of terminal sialic acid capping residues on
  oligosaccharides
• Mannose Binding Lectins serum collectins
  which recognize a particular orientation of sugar
  residues and their spacing

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Toll-like Receptors

• 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. non-adaptive 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, TLRs TLRs 11-13
  defined in mice some can dimerize and form
  homo- or heterodimers
• Data suggest a role for TLR in non-adaptive
  responses, i.e. TLR activation results in NFkB
  translocation and production of IFN, TNF and ROI
• Activation via Toll/TLRs induces production of
  IL12 and expression of co-stimulatory molecules
  by DCs
• One of the most ancient and conserved set of
  proteins in the immune systemeven found in
  plants, and have antimicrobial function

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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

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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 selfnon-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, selfnon-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 endosomes-lysosomes.

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Phylogenetic Tree of Human Toll-like Receptors
(TLRs). The phylogenetic tree was derived from
the alignment of the amino acid sequences for the
human TLR members using the neighbor joining
method Takeda 2003.
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TLR Expression

• Receptor Cell types
• TLR1 mf, MDCs, iDCs, mDCs/-
• TLR2 mf, MDCs, iDCs, mDCs /-, mast cells,
  renal epithelial cells
• TLR3 mDCs
• TLR4 mf, MDCs, iDCs, mDCs/-, mast cells,
  intestinal epithelial cells low, renal
  epithelial cells, pulmonary epithelial cells,
  corneal epithelial cells, dermal endothelial
  cells
• TLR5 mf, MDCs, iDCs, mDCs/-, intestinal
  epithelial cells
• TLR6 mf, mast cells
• TLR7 mf, PDCs
• TLR8 mf, MDCs, mast cells
• TLR9 mf, pDCs, B cells

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Toll-like Receptors and Their Ligands

• TLR family Ligands (origin)
• TLR1 Tri-acyl lipopeptides (bacteria,
  mycobacteria), Soluble factors (Neisseria
  meningitides)
• TLR2 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), HSP70
  (host)
• TLR3 Double-stranded RNA (virus), poly IC
• TLR4 LPS (Gram-negative bacteria), Taxol
  (plant), Fusion protein (RSV), Envelope proteins
  (MMTV), HSP60 (Chlamydia pneumoniae), HSP60
  (host), HSP70 (host), Type III repeat extra
  domain A of fibronectin (host), Oligosaccharides
  of hyaluronic acid (host), Polysaccharide
  fragments of heparan sulfate (host), Fibrinogen
  (host)
• TLR5 Flagellin (bacteria)
• TLR6 Di-acyl lipopeptides (mycoplasma)
• TLR7 Single stranded RNA, Imidazoquinoline
  (synthetic compounds), Loxoribine (synthetic
  compounds), Bropirimine (synthetic compounds)
• TLR8 single stranded RNA, small synthetic
  compounds, (Imidazoquinoline)
• TLR9 Unmethylated CpG DNA (bacteria)
• TLR10 ?
• TLR11 Profilin
• TLR12 ?
• TLR13 ?

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Immune Recognition by TLR
TLR1 cell surface TLR2 cell surface TLR3
cytosol TLR4 cell surface TLR5 cell
surface TLR6 cell surface TLR7 - cytosol TLR8 -
cytosol TLR9 - cytosol TLR10 cell surface TLR11
cell surface TLR12 - ? TLR13 - ?
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LPS Binds TLR-4/MD-2/RP105 Complex Following CD14
Association
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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.