Innate Immunity 2

Extravasation - step 1

- Interaction between neutrophil and selectins is weak - s-Lex = part of neutrophil that binds a specific selectin - adhesion is not very likely - rolling vs sticking more likely - neutrophil rolls along the vascular endothelial surface - blood flow continues to slow

Extravasation Details

- Tight binding: mediated by LFA (integrin) which is activated by IL-8 and ICAM-1 which is increased by TNFa - Migration is directed by chemokines (IL-8) - Neutrophils eat and die leaving behind pus

TLR4 & Pathways

- activates 2 signaling pathways ** TLR4 is involved in both the response to bacteria and viruses **TLR4 can be found both inside and outside the cell 1. TRIF-TRAM Pathway (left) Synthesis and secretion of Type I interferons (important for viral infections): IFN-alpha and IFN-beta - this pathway is activated when TLFR is located on when on endosomal membrane INSIDE 2. MyD88-IRAK4 Pathway (right) - Synthesis and secretion of TNF-alpha and other inflammatory cytokines - this pathway is activated when TLFR4 is located on a surface membrane OUTSIDE

Extravastation Continued

- due to cytokine secretion... - neutrophil w/ s-Lex - also has integrin (=LFA-1) - and chemokine receptor (CxCL8) - chemokine is being made and is attracting neutrophil - changes that result in tight binding - ICAM = adhesion molecule on the endothelium - signal from CXCL8 acts on receptor 1. Rolling adhesion 2. Tight binding 3. Diapedesis - enters between cells, breaks tight junctions 4. Migration - chemokine directs neutrophile where to go

TLR Details

- have 2 domains 1. Pathogen-Recognition Domain - horse-shoe like - outside the cell - recognizes the pathogen 2. TIR Domain (signaling) - internal

Neutrophils

- immediate innate immune response has occurred - activated complement - macrophages --> cytokines and chemokines now... - neutrophils are recruited to sites of inflammation from the bone marrow - second step in innate immune response - signal the presence of inflammation

TLR4 Signaling

- induces cytokine secretion * genetic defects can exist at key components of this signaling pathway 1. Complex of TLR4, MD2, CD14 and LPS is assembled at the macrophage surface 2. MyD88 binds TLR4 and activates IRAK4 to phosphorylate TRAF6, which leads to the phosphorylation and activation of IKK 3. IKK phosphorylates IKB, leading to its degradation and release of NFKB, which enters the nucleus 4. NFKB activates transcription of genes for inflammatory cytokines, which are synthesized in the cytoplasm and secreted via the ER

Inflammation Cascade

- inflammation recruits cells to the site - vessel dilation affects blood flow - TNFa/leukotrienes increases expression of adhesion molecules on endothelium so cells moving along can stick to endothelium (selectins) - IL8/CXC8 Attracts Neutrophils

Macrophage Actions

- ingest and kill pathogens Stages of Macrophage/Pathogen Interaction 1. Recognition of pathogen as non-self and tightly binds bacteria 2. Engulfment by macrophage (phagosytic cup and then complete engulfment --> phagosome) 3. Phagosome formed and gets internalized 4. Phagosome fuses with lysozome (=vesicle full of enzymes; marriage of phagosome and lysozome --> phagolysozome) 5. Acidification of phagolysozome - activation of enzymes 6. Release of toxic substances --> bacteria is dead!

TNFalpha - The Good

- key role in innate immunity - used to tx arthritis, inflammatory disease (i.e. Crohn's), psoriasis - multiple effects on endothelium: increase ICAM, stimulates production of proteins that increase clotting and promote vascular leakage - just the right amount locally is beneficial (local vascular leakage to control local infection)

Leukocyte Circulation

- leukocytes travel to different tissues due to ligand-receptor interactions 4 Types of Adhesion Molecules: 1. Selectins (carbohydrate binding proteins) - on cells 2. Adressins (cell specific oligosaccharides) - on leukocyte 3. Integrins (a,b heterodimers) 4. Members of the Ig gene superfamily

Activated Macrophages

- orchestrate innate immune response - secrete pro-inflammtory cytokines: 1. IL-6 2. TNF-alpha 3. IL-1beta 4. CXCL8 (aka IL8) - chemokine 5. IL-12 - all come about via LPS-mediated activation of TLR4 - cytokines = Small proteins secreted by one cell and act on a second cell via receptors; can act both locally and systemically (tell what to do) - chemokines = : like cytokines,but primary function is to attract cells to a site (tell where to go)

Specific TLR's

- recognize multiple microbial components 1. TLR4 Homodimer - recognizes LPS on gram negative bacteria - on the outside 2. TLR1:2 heterodimer - recognizes lipopeptides and GPI - surface bacterial component - on the outside 3. TLR2:6 heterodimer - recognizes lipoteichoic acid on gram positive bactera - on the outside 4. TLR3 - on the inside - recognizes double-stranded viral RNA in viruses 5. TLR9 - recognizes unmethylated CpG-rich DNA in bacteria - on the inside

Macrophage Receptors

- recognize pathogens Types 1. Complement Receptors 2. Pattern Recognition Receptors - recognize bacterial carbohydrates: mannose, & glycan ex: scavenger receptors 2a. Toll-Like Receptors (TLR) - sub-type of pattern recognition receptors - recognize bacterial products - recognize various components of bacteria

IRAK4 Deficiency: Douglas was referred to the immunology clinic when he was 6. He had suffered two episodes of pneumococcal meningitis, one causing a stroke. He had frequent ear infections, an intestinal infection at 11 months, and boils on his scalp. He had a weak febrile response to infections which were cleared with antibiotic treatment. His tonsils and LNs were slightly enlarged. He had a normal CBC, complement function and Ig levels. He had normal numbers of T and B cells. He made antibody responses to protein vaccines, but not polysaccharide vaccines.

- weak febrile is unusual - weak febrile response plus IRAK4 deficiency allows us to conclude that a consequence of activiting TLR4 receptors is: - NF-Kb (needed for a fever because it is a TF for cytokines) - on response to polysaccharide vaccines vs protein vaccines - unusual - reveals there is some overlap between innate and adaptive responses - IRAK4 is also involved in B cells

TLR's Interactions Summary

- with both extra and intracellular microbial components Outer Cell Membrane TLRs: TLR4, 1:2 - recognize molecules associated with pathogen surface Intracellular Membrane TLRs: TLR3 - recognize pathogen nucleic acid (ds RNA)

Role of Cytokines and Chemokines - Local Effects

1. Alterations in vascular endothelium (TNF-alpha, IL-1) - creates leakage 2. Influx of neutrophils and lymphocytes (IL-8/ CXCL8) - chemokines 3. Activation of Lymphocytes IL-1, IL-6 (increase antibody secretion), IL-12 (T cells and NK cells)

Role of Cytokines and Chemokines - Systemic Effects

1. Fever (IL-1, IL-6, TNF-alpha) - cytokines 2. Shock (TNF-alpha) - rapid excessive vascular leakage 3. Acute Phase Response (IL-6) - part of induced immune response ** TNF-alpha has most powerful effects on vascular epithelium

TLR4

= key component of the response to gram-negative bacteria (which make LPS) - bacterial LPS is recognized by the complex of TLR4, MD2 and CD14 - LPS binds CD14 on macrophages - TLR4 forms a complex with CD14 via MD2 --> TLR4 signal transduction

Inflammation

= the border between immediate and induced innate response - rubor (red), tumor (swollen), calor/doler (hot and painful) Cause: - dilation of capillaries - reduced blood flow - increased permeability ** all as a result of activation of macrophages

Extravastation

= the process of neutrophils leaving the blood by squeezing through the gaps between neighboring endothelial cells

How would you test for an IRAK4 deficiency? What are the primary clinical clues that would lead you to suspect the patient has a problem with TLR signaling?

??? check eBoard

3. The plasma proteins that counteract the activity of factor P by inactivating C3 convertase through the cleavage of C3b are: a. factor B and factor H b. factor H and factor I c. factor B and factor I d. decay-accelerating factor and factor H e. decay-accelerating factor and membrane cofactor protein.

B

1. Which of the following does not accurately describe complement components? a. soluble proteins b. made by the spleen c. located in extracellular spaces d. some function as proteases once activated e. activated by a cascade of enzymatic reactions.

B located in the liver

Which of the following properties is common to macrophages and neutrophils? a. life span b. anatomical location c. ability to phagocytose d. morphology e. formation of pus.

C

Which of the following TLR3 and TLR4 adaptor proteins participates in the activation pathway that culminates in the synthesis of type I interferons? a. C-reactive protein b. MyD88 c. LPS-binding protein d. TRIF and TRAM e. NFB.

D

Which of the following polymerizes to form a transmembrane channel that compromises the integrity of cell membranes? a. C5 b. C6 c. C7 d. C8 e. C9.

E

2. Which of the following is the membrane-bound form of C3 convertase of the alternative pathway of complement activation? a. iC3 b. C3a c. C3b d. iC3Bb e. C3bBb

E iC3Bb IS a convertase but it is NOT bound

TLR4 Signaling Notes

Key Players (common to TLR signaling): - MyD88, an adaptor protein, bridges signaling components; - IRAK4 - IKK - NF-kB NF-kB is a key transcription factor that induces expression of multiple pro-inflammatory cytokines. **Genetic deficiencies in IRAK4 and IKK are associated with susceptibility to bacterial infections.

Neutrophils vs Macrophages

Macrophages - Long lived macrophages are always present in tissue - Functions: phagocytosis, secretion of enzymes and cytokines, antigen processing and presentation = first cell defenders Neutrophils - Short lived and not present in healthy tissue - Functions: phagocytosis cytokine production Neutrophils predominate at sites of inflammation. - Death creates pus = 2nd responders

TNFalpha - The Bad

The systemic effects of TNFa lead to cachexia (aka shock) ...multiple organ failure...Death - systemic vascular leakage - bad!