Innate Immunity

Question 1

Give examples of soluble innate immune molecules

Answer 1

• Enzymes- lysozymes
  
  • disrupt bacterial cell walls; in tears and blood
• Antimicrobial peptides
  
  • disrupt the microbial membrane
• Collectins, ficolins and pentraxins
  
  • ​bind to pathogens and target them for phagocytosis and to activate the complement cascade
• Complement components
  
  • lyse baceteria opsonise bacteria and induce inflammation

Question 2

What is the action of Lysozymes in innate immunity?

• where are they secreted from?

Answer 2

• secreted by phagocytes and paneth cells from the SI
• cleaves the bond between alternating sugars that make up the peptidoglycan layer of bacteria
  
  • most effective in gram-positive bacteria as there is no LPS outer-membrane to overcome
• cleavage of these bonds allows phospholipase A2 to disrupt the exposed phospholipid cell membrane of the bacteria

Question 3

What are the three families of Antimicrobial peptides?

• how do they work?

Answer 3

• Histatins
  
  • Produced in the oral cavity. Active against pathogenic fungi, e.g. Candida albicans
• Cathelicidins
  
  • LL-37 broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria
• Defensins
  
  • Two classes – α, β defensins
    
    • important in the new born
• cover epithelial surfaces and found inf saliva
• constitutively secreted by neutrophils, epithelial cell and paneth cells in the crypts of the small intestine
• kill bacteria in minutes by disrupting the membrane
• also attack fungi and virus by inhibiting DNA and RNA synthesis

Question 4

What are defensins and how do they work in innate immunity?

Answer 4

• 35-40 aa amphipathic peptide (both hydrophobic and hydrophilic regions on their cell surface)
  
  • ​inserts into the membrane of the microbe - causing pores to form.
  • allows movement of sugars and fluids into the cell –> cell lysis
• they disrupt the microbial membranes but that of the host

Question 5

What are collectins ficolins and pentraxins?

Answer 5

• Collectins have globular lectin like heads that bind bacterial cell surface sugars. Sialic acid on host cells hides mannose antigens.
• Ficolins (have a Fibrinogen like domain rather than a lectin like head) recognise acylated compounds (COCH3) such as n-acetylglucosamine, a monosaccharide found in bacterial cell wall
• Pentraxins are cyclic multimeric proteins in the plasma. C-reactive protein (CRP) is used as a clinical measure of inflammation – CRP binds to phosphocholine on bacterial surfaces
• they are soluble pattern recognition receptors
• they are as opsonins that bind to pathogens and infected cells targeting hem for phagocytosis
• these all activate the complement cascade through the lectin pathway

Question 6

Where are Pentraxins released from and five an overview of how they work

Answer 6

• binds to one end of the pathogen then the other to CD89 on phagocytes.
• released by hepatocytes, monocytes, macrophages, DC’s epithelial cells and endothelial cells

Question 7

What are the three ways to activate the complement system?

Answer 7

• Classical pathway: antigen-antibody complexes
• Lectin pathway: mannose-binding lectin or ficolin binds to carbohydrate on pathogen surface
• Alternative pathway
  
  • pathogen binds to the pathogen surface
• they all converge onto C3 –> MAC (membrane attack complex)

Question 8

Explain what the complement system is

Answer 8

• Series of over 30 proteins that constantly circulate in blood and fluids that bathe the body tissues
• When they detect the presence of foreign material, they initiate a cascade of reactions that amplify the signal - works with hosts defences to generate inflammation and rapidly remove the pathogen
• Most made by the liver but also produced by monocytes, macrophages and epithelial cells of the intestine and urinary tract
• circulate as a pro-form in the blood
• on activation, they split into small and large fragments triggering an amplification cascade
• normally ‘a’ is the small fragment except c2a

Question 9

Describe activation of the classical pathway

Answer 9

• Classical pathway initiated by activation of C1
• C1 is a complex made up of
  
  • C1q, C1r, C1s - C1q dominants the complex as a large 18 polypeptide collagen-like triple-helical structures
  • stabilised by Ca2+ ions
• Activation occurs when C1 binds to at least two FC domains on antibodies bound on bacteria
  
  • this causes a conformational change that exposes the C1q binding site
• IgM is the most efficient at activating complement as it has 5 Fc domains. IgG1 and IgG3, and to a lesser extent IgG2 can also activate complement when close together bound to antigen

Question 10

Explain why serum IgM doesn’t activate the complement system

Answer 10

• the binding site isn’t exposed until it finds to a bacteria and undergoes a conformational change to expose it’s binding site for C1q
• it maintains a planar conformation in serum

Question 11

How does Amplification occur in the Classical Complement Pathway?

Answer 11

• Binding C1q with the Fc domain causes a conformational change in C1r
• C1s is cleaved and can activate C2 and C4 splitting into their large and small fragments
• C3 convertase (C4b2a) can then activate over 200 C3 molecules producing a massive amplification of the signal
• C4b, C2a and C3b fragments form the C5 convertase that activates C5 leading to the membrane attack complex

Question 12

Explain the Lectin Complement Pathway

Answer 12

• Antibody independent, activated by ficolins and mannose-binding lectin (MBL) - these are structurally similar to C1q
• MBL binds mannose residues on carbohydrates and glycoproteins on bacteria and some viruses
  
  • activates similar downstream mechanism to the classical pathway
• Upon binding MBL forms a complex with MASP-1 and MASP-2 (serine proteases) - these are structurally similar to C1r and C1s
• this active complex cleaves C2 and C4 leading to the C3 convertase complex (C4bC2a)
  
  • amplification cascade of C3
  • C5 convertase (C4bC2aC3b)

Question 13

Explain the Alternative Complement Pathway

Answer 13

• in the body C3 spontaneous hydrolyses into C3a and C3b
• C3b binds to a cell membrane and factor B, making it susceptible to cleavage by factor D to Bb
• C3bBb has a half-life of 5 min, unless it binds the serum protein properdin, which extends it half-life to 30 min by protecting it from proteases
  
  • properdin released by monocytes, macrophages etc. (cells that can recognise infection)
• C3b,Bb can hydrolyse more C3 creating more C3b which can amplify the signal

Question 14

What is the role of C3a and C5a?

Answer 14

• act as peptide mediators of inflammation
• promote phagocyte recruitment

Question 15

What is the role of C3b?

Answer 15

• Binds to complement receptors on phagocytes
• stimulates opsonization of pathogens and removal of immune complexes

Question 16

Explain the Terminal Complement Components and MAC

Answer 16

• C5 convertase cleaves C5
• C5b combines with C6-C9 to form
  
  • C5b6789 (MAC) Membrane attack complex
• this is a ring-like structure that forms pores that insert into the membranes of cells
  
  • allows diffusion of ions, small molecules and water –> cell lysis
• Human cells are not affected as they have soluble and cell surface-associated proteins that prevent MAC formation

Question 17

What is Hereditary Angioedema?

How is it treated?

Answer 17

• C1 inhibitor deficiency
• this allows the classical complement cascade to be easily activated but can be treated with an injection of C1 inhibitor
  
  • this causes swelling and inflammation in the individual

Question 18

Give an overview of complement deficiencies

Answer 18

• Patients deficient of components of the complement pathway experience recurrent infections
• MBL deficiency causes serious pyogenic infections in neonates and children
• C3 deficiency is the most severe leading to successive severe infections
• Patients deficient of C8 are prone to infection with Neisseria meningitis
• C4 deficiency tend to develop systemic lupus erythmatosus

Question 19

Explain the role that complement Deficiency plays in Systemic Lupus Erythematosus (SLE)

Answer 19

• 90% of people deficient in C4 develop SLE which is an autoimmune disease
• C4 deficiency means less C3b (C4b2a is C3 convertase)
• C3b bound to immune complexes binds to CR1 on erythrocytes which transports them to phagocytes in the liver and spleen.
• Phagocytes recognise the immune complexes via their Fc receptors and engulf them

Question 20

What is the immune system?

Answer 20

• innate immune system is evolutionary conserved, and germline-encoded (transcribed directly from the genome).
• it is non-specific, recognizing pathogen-associated molecular patterns (PAMPS) which can be found across many pathogens.

Question 21

What are the Innate immune cells?

Answer 21

• Neutrophils
  
  • phagocytosis
  • produce ROS
  • antimicrobial peptides
• Macrophages
• Dendritic Cells
  
  • Plasmacytoid dendritic cells of lymphoid origin - good at producing interferon
  • Myeloid dendritic cells- antigen presentation
• Natural killer cells
  
  • Perforin and Granzyme,

Question 22

Explain Phagocyte recruitment

Answer 22

• tisssue resident cells release cytokines
• cytokines dilate local blood vessels
• chemokines attract monocytes and neutrophils to the infection
• Cell adhesion molecules (ICAM-1 and VCAM-1) are upregulated on the endothelium which binds to integrins on the leukocytes allowing them to roll
• the vascular endothelium becomes more permeable, they are able to move through the endothelium to the site of infection in the tissue

Question 23

How does Phagocytosis occur?

• what cells, factors, receptors and complements are involved in this process?

Answer 23

• Phagocytosis carried out by Neutrophils, Dendritic cells and Macrophages (neutrophils that have entered the tissue)
• Opsonins: C3b and Collectins and antibodies also help in this
• C-type-lectin receptors (Dectin-1 & mannose receptor) help phagocytose bacteria. Mannose Receptor binds mannose and fructose residues of glycans (polysaccharides).
• The complement receptor CR1 binds to C3b
• Scavenger receptors bind to lipids recognize bacteria, viruses and apoptotic cells.

Question 24

What are Macrophage and Neutrophil products/ mechanism that facilitate phagocytosis?

Answer 24

• They have an overall bacteriocidal environment
• low pH = ~3.5-4.0
• toxic oxygen-derived products: O₂^-, H₂O₂, singlet oxygen ¹O₂^., hydroxyl radical ^.OH and hyphoalite OCl^-
• toxic NO
• Antimicrobial peptides
  
  • Macrophages: cathelicidin, macrophage elastase-derived peptide
  • Neutrophil: alpha & beta-defensins, cathelicidin, azurocidin, BPI, lactoferrin: binds Fe2+ which is needed for bacterial growth
• Lyzozymes and acid hydrolases

Question 25

What are Neutrophil Extracellular Traps (NETs)?

Answer 25

* when activated some neutrophils undergo a special form of cell death termed 'NETosis' * during NETosis nuclear chromatin is released from cells trapping microorganism this aiding phagocytosis

Question 26

What are the five main families of Pattern Recognition Receptors (PRRs)

Answer 26

* **C type lectin receptors** (CLRs) * **Toll-like receptors** (TLRs) * **NOD-like receptors** (NLRs) * **Rig-I like receptors** (RLRs) * **Cytosolic DNA sensors** (CDS) * they recognise conserved structures so are able to recognise a broad range of pathogens * they recognise pathogen-associated molecular patterns **PAMPs** * **​**so innate immunity must focus on highly conserved and essential components of microbes (cell wall structures; nucleic acids) * these cells can also recognise Damage associated molecular patterns, which are molecules released from necrotic cells

Question 27

Give an overview of C type Lectin receptors (CLRs)

Answer 27

* they recognise and bind to carbohydrates in a calcium-dependent manner * Type I CLRs assist with antigen uptake by phagocytes * Type II CLRs are involved in fungal recognition * Mannose-binding lectin is a soluble CLR

Question 28

Give an overview of Toll-like receptors (TLRs) - structure and signalling

Answer 28

* Extracellular section: * LRR (leucine-rich repeats) domain - site of pathogen binding when it binds to the PAMPs or DAMPs it causes a conformational change in the TIR domain * Cytosolic side: * TIR-domain (toll interleukin one receptor homology domain)- conserved stretch of ~200 amino acids * this is where signalling is triggered * they are able to pair together to form dimers * changes the signalling going on downstream * this also changes the ligands they are able to bind to * humans have 10 TLRs

Question 29

What do the 10 TLRs recognise? - where are they located?

Answer 29

* TLR- 1,2,4,5,6 are all found on the plasma membrane (mainly bacteria) * TLR-2,6 recognise: diacyl lipopeptides * TLR-2,1 recognise triacly lipopeptides * TLR-5 recognise flagillen * TLR-4 works wiht **co-factor MD-2** to recognise LPS * TLR-3,7,8,9 are found within the endosome and recognise nucleic acid structures (mainly viral) * TLR-3: dsRNA * TLR-7: ssRNA * TLR-8: ssRNA * TLR-9: CpG DNA * TLR-10 is found in the endosome and recognise dsRNA

Question 30

Explain the TLR signalling Cascade

Answer 30

* TLR signalling induces genes that function in host defense: * Pro-inflammatory & anti-inflammatory cytokines * MHC & co-stimulatory molecules * antimicrobial peptides & complement components to drive the complement cascade * they use TLR adapter proteins at the top of the cascade, which affects the signalling response * Myd88: drives production of AP-1 which produce pro-inflammatory cytokines like IL-1, IL-6 and TNF * Mal * TRIF: drive production of Type 1 IFN * TRAM

Question 31

What would happen if there was a MyD88 gain of function mutation? - In which condition can this be found?

Answer 31

* **Waldenström macroglobulinemia -(rare type of non-Hodgkin lymphoma)** * MyD88 mutation is present in 90% of patients causing cell growth and survival * B cells make large amounts of IgM that can cause excess bleeding, vision problems and headaches * Lymphoma cells proliferating in the bone marrow cause anaemia, neutropenia and thrombocytopenia

Question 32

What happens if you are deficient in MyD88

Answer 32

* At an increased risk from life-threatening recurring pyogenic bacterial infections * however, you are otherwise healthy and your immune function improves with age

Question 33

Which TLR is linked with immunodeficiency and what occurs when you are deficient in this TLR?

Answer 33

* **TLR-3** * They are more susceptible to Herpes Simplex Encephalitis * inflammation of the brain due to the herpes simplex virus (HSV-1) which is a double-stranded DNA virus * when replicating in the cytoplasm HSV-1 produces double-stranded RNA which TLR-3 recognises * defects in other signalling molecules involved in the TLR signalling pathway have also been associated with HSE

Question 34

Which TLRs are activated in these Infections HIV Sepsis Tuberculosis

Answer 34

* HIV: TLR8 * Sepsis: TLR2 and 4 * Tuberculosis: TLR2 and 4

Question 35

Which TLRs are activated in these Inflammatory diseases Systemic Lupus Erythematosus Alzheimer's Disease Atherosclerosis

Answer 35

* Systemic Lupus Erythematosus: TLR7, 8, 9 * Alzheimer's Disease: TLR2,4 * Atherosclerosis: TLR2,4

Question 36

What TLR agonists are there and what are they used for?

Answer 36

* Infection- genital warts (TLR7 ligand - Aldara) * Cancer - Melanoma (TLR7 ligand - Aldara) * Allergy- Ragweed pollen (TLR9) (low level activation) * Vaccine adjuvant (added to stimulate a bigger immune response)

Question 37

Give an overview of Nuceltoide-binding Leucine-Rich (NLRs)

Answer 37

* they are cytoplasmic pattern recognition molecules with two major groups * NLRCs: can be 1 or 2 (NOD1/ NOD2) * Caspase recruitment domain (CARD) * NLRPs * Pyrin domain

Question 38

What is the role of NLRCs - NOD1 - NOD2 (mutations)

Answer 38

* there is NLRC1/2 (NOD1/2) * they have a leucine-rich domain that can bind to **peptidoglycan** which is present on the cell membrane of most bacteria * NOD1 binds _γ-glutamyl diaminopimelic acid (iE-DAP)_ (*Mainly Gm-ve Bacteria)* * NOD2 binds _muramyl dipeptide_ *(both Gram+ve and Gram-ve bacteria)* * *​*gain of function mutation linked to early-onset **sarcoidosis** where granulomas develop in the organs of the body * loss of function mutation is associated with susceptibility to **Crohn's disease**

Question 39

What is the role of NLRPs?

Answer 39

* NLRP3 (NALP3) * activated by cellular stress; K+ efflux, ATP, ROS and lysosomal damage * NLRP3 combine with ASC and Caspase-1 to form the inflammasome * **Inflammasome formation and activation is essential for the maturation and secretion of IL-1 and IL-18** * they are able to sense danger * uric acid crystals (gout) * Asbestos * Amyloid beta (Alz) * Islet amyloid polypeptide (T2DM) * Hemozoin (malaria)

Question 40

Review the role of the Inflammasome n the cleavage of pro-IL-1 and pro-IL-18

Answer 40

Question 41

What happens in a gain in function mutation of NLRP3? treatment?

Answer 41

_this causes Cryopyrin-Associated Periodic Syndromes (CAPS) -_ Caused by rare mutations in exon 3 of NLRP3 gene causing overproduction of IL-1 * **_Muckle wells syndrome_** (Prevalence unknown) * Can occur spontaneously or be triggered by cold, heat, fatigue, or other stresses. * Symptoms of fever, rash, arthralgia, conjunctivitis, uveitis, sensorineural deafness, and potentially life-threatening amyloidosis * **_Familial cold autoinflammatory syndrome_** (1: 1000000) * Triggered by exposure to cold * Symptoms of fever urticarial rash with headache, arthralgia, and sometimes conjunctivitis * can be treated with *_Anakinra (IL-1RA)receptor agonist_*

Question 42

Give an overview of RIG-I-like receptors (RLRs) - mutations?

Answer 42

* RIG-I and MDA5 are sensors of cytoplasmic RNA, a replication intermediate for viruses. They signal to induce pro-inflammatory cytokines and IFN. * **RIG-I**: binds to ssRNA containing 5'-triphosphate * **MDA5**: preferentially recognises long dsRNA * important for picornavirus detection * mutations are rare but have been associated with IFN related disease SLE and Aicardi- Goutières syndrome

Question 43

Give an overview of Cytosolic DNA sensors - examples - mutations?

Answer 43

* **STING** * Stimulator of interferon genes **_(STING)-associated vasculopathy with onset in infancy (SAVI)_** * is an autoinflammatory disease caused by **gain-of-function mutations in the gene that codes for STING.** Patients _produce too much type 1 IFN_ causing abnormal inflammation throughout the body, especially in the skin, blood vessels, and lungs.

Question 44

How can the acute phase response be used clinically?

Answer 44

* Raised **erythrocyte sedimentation rate (ESR)** and **C-reactive protein (CRP)** are characteristic of acute phase response and are used clinically to detect inflammation * hence these can be measured in serum