Innate Immunity

William H. Chambers, PhDUniversity of Pittsburgh Cancer Institute and Department of Immunology University of Pittsburgh School of MedicineG.17e Hillman Cancer Center412-623-3218chamberswh@msx.upmc.edu

Phases of immune responses

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

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. TCR+ IELs

• B cell compartment, e.g. CD5+ B cells [BC-1 cells]

• No memory

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

Fluid Phase Elements of Non-adaptive Immunity

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

Cellular Elements of the Non-adaptive Immune System

• Neutrophils

• Basophils

• Eosinophils

• Macrophages

• Natural Killer Cells

Recognition Receptors in Innate and Adaptive Immune Systems

Exposure to infectious agents

Barriers to Infection

Tissue damage from infections

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/-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

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]

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]

-defensins initially described as being produced by neutrophils, alveolar macrophages and Paneth cells at the base of crypts in the intestine [11 genes]

-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]

• θ-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

-defensins act as opsonins, - and -defensins induce mast cell degranulation, -defensins induce IL8 release by epithelia, activate complement and suppress anti-inflammatory mediators

Defensins

-defensins X1-2 C X C R X2-3 C X3 E X3 G X C X3 G X5 C C X1-4

-defensins X2-10 C X G/A X C X3-4 C X9-13 C X4-7 C C Xn

θ-defensins GXCRCXCXRGXCRCXCXR

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.

Humoral elements

[Alternative] Complement Pathway

Collectins

Ficolins

-Defensins

Interferons

Characteristics of Collectins/Ficolins

Collectins Ficolins

Structure Collagen domain, C-type Collagen domain, Fibrinogen

lectin CRD, -helical -like domain, CRD, -helical

coiled-coil neck domain, coiled-coil neck domain, trimeric N-terminal cysteine rich

domain, multimericDiversity MBL, SP-A, SP-D, CL-L1 L-ficolin, H-ficolin

CL-P1, conglutinin, CL-43 M-ficolinCL-46

Production/Distribution MBL: liver L-ficolin: liverSP-A: lung [II alv.] H-ficolin: liver, lung [II alv.] SP-D: lung, GI M-ficolin: monocytes CL-L1: liverCL-P1: endothelial cells

Specificity man, glu, L-fuc, ManNAc GlcNAcGlcNAc

Complement Fixation MBL L-ficolin, H-ficolin

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.

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

MBL Recognition of Bacterial Surfaces

direct MBP, ficolin Ab

Interferons

Six classes – (5 are Type I; 1 is Type II) - multigene family [14-20 depending upon species];

produced by leukocytes and other virus-infected cells single gene [except 5 in ungulates], produced by

fibroblasts, leukocytes and other virus-infected cells produced by hematopoietic cells Identified in cattle and sheep [trophoblasts] – immune interferon/Type II, produced by hematopoietic

cells, e.g. NK cells and T cells [both CD4+ (Th1) and CD8 +]

Three Major Functions of Class I Interferons [/]

• 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

Viral interference with the IFN system. Viruses that can block the four parts of the IFN system are grouped together.

Phagocytic cells: macrophages and neutrophils

Phagocytic Cell Function

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!

Bactericidal Agents in Phagocytic Cells

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)

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

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 NFB 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 system…even found in plants, and have antimicrobial function

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 endosomes-lysosomes.

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].

TLR Expression

Receptor Cell typesTLR1 mDCs, iDCs+, mDCs+/-

TLR2 m, MDCs, iDCs+, mDCs +/-, mast cells, renal epithelial cells

TLR3 mDCsTLR4 mDCs, iDCs+, mDCs+/-, mast cells,

intestinal epithelial cells [low], renal epithelial cells, pulmonary epithelial cells, corneal epithelial cells, dermal endothelial cells

TLR5 mDCs, iDCs+, mDCs+/-, intestinal epithelial cells

TLR6 mmast cellsTLR7 mPDCsTLR8 mDCs, mast cellsTLR9 mpDCs, B cells

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 I:CTLR4 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 ProfilinTLR12 ?TLR13 ?

Immune Recognition by TLR

TLR1 – cell surfaceTLR2 – cell surfaceTLR3 – cytosolTLR4 – cell surfaceTLR5 – cell surfaceTLR6 – cell surfaceTLR7 - cytosolTLR8 - cytosolTLR9 - cytosolTLR10 – cell surfaceTLR11 – cell surfaceTLR12 - ?TLR13 - ?

LPS Binds TLR-4/MD-2/RP105 Complex Following CD14 Association

MD-2

RP105

MD-2

RP105

MD-2

RP105

MD-2

RP105

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.