Innate Immunity Flashcards
1
Q
Give examples of soluble innate immune molecules
A
-
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
2
Q
What is the action of Lysozymes in innate immunity?
- where are they secreted from?
A
- 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
3
Q
What are the three families of Antimicrobial peptides?
- how do they work?
A
-
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
- Two classes – α, β defensins
- 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
4
Q
What are defensins and how do they work in innate immunity?
A
- 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
5
Q
What are collectins ficolins and pentraxins?
A
- 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
6
Q
Where are Pentraxins released from and five an overview of how they work
A
- 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
7
Q
What are the three ways to activate the complement system?
A
- 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)
8
Q
Explain what the complement system is
A
- 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
9
Q
Describe activation of the classical pathway
A
- 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
10
Q
Explain why serum IgM doesn’t activate the complement system
A
- 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
11
Q
How does Amplification occur in the Classical Complement Pathway?
A
- 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
12
Q
Explain the Lectin Complement Pathway
A
- 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)
13
Q
Explain the Alternative Complement Pathway
A
- 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
14
Q
What is the role of C3a and C5a?
A
- act as peptide mediators of inflammation
- promote phagocyte recruitment
15
Q
What is the role of C3b?
A
- Binds to complement receptors on phagocytes
- stimulates opsonization of pathogens and removal of immune complexes
16
Q
Explain the Terminal Complement Components and MAC
A
- 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
17
Q
What is Hereditary Angioedema?
How is it treated?
A
- 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
18
Q
Give an overview of complement deficiencies
A
- 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
19
Q
Explain the role that complement Deficiency plays in Systemic Lupus Erythematosus (SLE)
A
- 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
20
Q
What is the immune system?
A
- 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.
21
Q
What are the Innate immune cells?
A
- 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,
22
Q
Explain Phagocyte recruitment
A
- 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
23
Q
How does Phagocytosis occur?
- what cells, factors, receptors and complements are involved in this process?
A
- 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.
24
Q
What are Macrophage and Neutrophil products/ mechanism that facilitate phagocytosis?
A
- 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
25
What are Neutrophil Extracellular Traps (NETs)?
* 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
26
What are the five main families of Pattern Recognition Receptors (PRRs)
* **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
27
Give an overview of C type Lectin receptors (CLRs)
* 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
28
Give an overview of Toll-like receptors (TLRs)
- structure and signalling
* 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
29
What do the 10 TLRs recognise?
- where are they located?
* 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
30
Explain the TLR signalling Cascade
* 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
31
What would happen if there was a MyD88 gain of function mutation?
- In which condition can this be found?
* **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
32
What happens if you are deficient in MyD88
* At an increased risk from life-threatening recurring pyogenic bacterial infections
* however, you are otherwise healthy and your immune function improves with age
33
Which TLR is linked with immunodeficiency and what occurs when you are deficient in this TLR?
* **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
34
Which TLRs are activated in these Infections
HIV
Sepsis
Tuberculosis
* HIV: TLR8
* Sepsis: TLR2 and 4
* Tuberculosis: TLR2 and 4
35
Which TLRs are activated in these Inflammatory diseases
Systemic Lupus Erythematosus
Alzheimer's Disease
Atherosclerosis
* Systemic Lupus Erythematosus: TLR7, 8, 9
* Alzheimer's Disease: TLR2,4
* Atherosclerosis: TLR2,4
36
What TLR agonists are there and what are they used for?
* 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)
37
Give an overview of Nuceltoide-binding Leucine-Rich (NLRs)
* 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
38
What is the role of NLRCs
- NOD1
- NOD2 (mutations)
* 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**
39
What is the role of NLRPs?
* 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)
40
Review the role of the Inflammasome n the cleavage of pro-IL-1 and pro-IL-18
41
What happens in a gain in function mutation of NLRP3?
treatment?
_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_*
42
Give an overview of RIG-I-like receptors (RLRs)
- mutations?
* 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
43
Give an overview of Cytosolic DNA sensors
- examples
- mutations?
* **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.
44
How can the acute phase response be used clinically?
* 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
