Mechanism of Production of Inflammatory Mediators Flashcards
What is inflammation?
- Inflammation is the initial response of the innate immune system to infection and injury
- Conserved across all metazoan organisms
- Non-specific cytotoxic response → kills pathogen infected cells and promotes healing and recovery
- Response also kills healthy cells, but collateral damage is acceptable because it is getting rid of the pathogen
- When excessive or dysregulated however, it contributes to disease.
- Can be acute or chronic
What is sterile inflammation?
- Inflammation in the absence of a pathogen
- Driven by endogenous molecules – DAMPs
- Normally retained inside cells, but can be released following injury → so shielded from the immune system until after injury.
- May resolve the initial insult, but is often a self-amplifying loop promoting disease
- Has been found to worsen non-communicable diseases (disease that occur in the absence of a pathogen) such as CVD, cancer etc…
What are the cellular effectors of innate immunity?
Circulating:
• Neutrophils → most abundant granulocyte. First innate immune cells to migrate to the area of inflammation
• Monocytes → largest. Replenish tissue macrophages
• Basophils → least common. Secrete histamine
• Eosinophils → important for parasites
• NK cells → innate-like lymphocytes. Analogous to the cytotoxic T cell.
Tissue:
• Macrophage → major phagocyte in normal tissue
• Microglia (brain specific) → resident macrophages of the brain and spinal cord, and act as the main form of active immune defence in the CNS.
• Mast cell → tissue basophil
• DC → phagocytic, but their main function is as an APC. Conventional DC main function is to phagocytose microbes to present antigen to T cells and induce an adaptive immune response. They also produce cytokines. Are seen as a bridge between the innate and adaptive immune system. Plasmacytoid DCs are the major produces of antiviral IFNs.
What is a cytokine and what are the 3 cytokine classes?
- Secreted pro or anti inflammatory mediators
- Overproduction or dysregulation is a major contributor to disease
- Around 25kDa
- Usually secreted in response to an activating stimulus
- Can act in autocrine, paracrine and endocrine manners
- Activation of PRRs induces the production of cytokines that activate the cellular arm of the innate immune system
Classes:
• Lymphokines → produced by lymphocytes, coordinate T cell responses
• ILs → produced mainly by leukocytes. Multiple inflammatory and immunomodulatory effects
• Chemokines → induce chemotaxis. Required for recruitment of immune cells to infected/injured tissue.
• The cytokines secreted by macrophages and DCs in response to PRR activation include IL-1B, IL-6, IL-12, TNFa and the chemokine CXCL8 (formerly IL-8)
The IL-1 family (main family involved in neuroinflammation):
• Contains 11 members, notably IL-1a, IL-1B and IL18
• Most produced as inactive pro-proteins that are cleaved to produce the mature cytokine → Il-1B and IL-18 produced by macrophages are cleaved by caspase 1
• IL-1 family receptors have extracellular regions composed of Ig-like domains that signal as dimers through TIR domains in their cytoplasma tails.
What are TLRs
- One of the best characterized PRRs
- Toll was first discovered in Drosophila, whereby its signaling induces expression of host defence mechanisms including the production of antimicrobial peptides such as Drosomycin.
- Production of antimicrobial peptides seem to be the earliest form of defense against infection, so receptors that signal for this have claim on being the earliest receptors dedicated to defense against infection.
- Cloning of the human homologue was peformed by Medzhitov, Preston-Hyrlburt and Janeway – but no nobel prize!
- There are 10 expressed TLR genes in humans
Describe TLR structure and function
• Sensors for microbes in the extracellular space
− Some are cell-surface receptors, and some are endosomal
− The ones that are intracellular detect pathogens that have been phagocytosed
• Single-pass transmembrane receptors comprised of 18-25 leucine-rich repeats that create a horseshoe-shaped protein scaffold for ligand binding on both the concave and convex surface.
• Activated when binding of a ligand induces them to form dimers → all TLRs have a TIR domain in their cytoplasmic tail with interacts with other TIR domains
• Activation often leads to the production of NFkB:
− Nuclear Factor Kappa-light-chain-enhancer of activated B cells (actually present everywhere)
− p65/p50 dimer retained inactive by Inhibitor of NFkB (IkB)
− Dissociation from IkB occurs after cellular stress, cytokines, TLR activation → allowing it to translocate to the nucleus.
• Activation may also result in production of interferon regulatory factor and members of the AP-1 family such as c-Jun through a signaling pathway involving MAPK. NFkB and AP-1 induce pro-inflammatory cytokines, whereas IRFs produce antiviral type 1 interferons.
Describe the signalling pathway that results in NFkB activation from TLR recognition.
- TLR signaling activated by the ligand-induced dimerization of two TLR ectodomains, which brings their cytoplasmic TIR domains close together. This allows them to interact with the TIR domains of cytoplasmic adaptor molecules, such as:
• MyD88 (myeloid differentiation factor 88)
• MAL (MyD88 adaptor-like)
• TRIF (TIR domain containing adaptor inducing IFNb)
• TRAM (TRIF related adaptor molecule) - Adaptors recruit IRAK-1 & IRAK-4, which activate the E3 ubiquitin ligase TRAF-6
• Catalyses attachment of ubiquitin to lysine 63 of TRAF-6
• NEMO is also polyubiquitinated - Ubiquitin usually targets protein for degradation, but can also have a signaling role – here it serves as a scaffold that allows recruitment of TAK1.
- TAK1 activates IKK by phosphorylation of its beta subunit.
- IKK phosphorylates IkB at serines 32 and 36. This leads to polyubiquitination of IkB at lysine 48.
- IkB is subsequently degraded by the proteasome.
- NFkB is free to migrate to the nuclease and initiate transcription of pro-inflammatory genes, eg) IL and TNF cytokines.
Describe the role of ubiquitination (inc. the proteasome) in regulating inflammatory gene expression.
• Ubiquitin is a small (76aa) protein
• The proteasome is a large protein complex that recognizes and degrades ubiquitinated proteins
− Typical proteasome is a 20S catalytic core and two 19S regulatory caps.
− The core is a large cylindrical complex of 28 subunits, arranged in 4 stacked rings of 7 subunits.it is hollow, lined by the active sites of the proteolytic subunits.
− Ubiquitination targets proteins to the 19S cap
− ATPases control the opening of the 20S core chamber, regulating protein entry.
− Inside, the protein is broken down into short peptides which are released into the cytosol
So, protein ubiquitination regulates inflammatory gene expression via multiple ways:
• Scaffold on TRAF-6 for recruitment of TAK1 and subsequent activation of IKK
• Targets IkB for degradation, enabling NFkB to migrate to the nucleus
Describe the NOD and NLRP cytoplasmic PRRs
• Mainly of the NLR family (Nucleotide-binding domain and leucine-rich repeat receptor)
• 23 members in humans
• Subfamilies can be distinguished on the basis of protein domains in their amino terminus:
− NOD → caspase recruitment domain (CARD) .
− CARD can dimerise with CARD domains on other proteins, and is involved in many intracellular pathways, including those leading to apoptosis.
− Recognise fragments of bacterial cell wall peptidoglycans → recruits CARD-containing RIPK → activates TAK1 → activates IKK → phosphorylates IkB → activates NFkB → inflammatory cytokine production.
− NLRP → pyrin domain
− Humans have 14 NLRs – best characterized is NLRP3 (or cryopyrin) which is an important sensor of cellular damage or stress.
− NLRP3 has a tripartite structure:
− PAMP/DAMP sensing C-terminal LRR
− Central nucleotide binding domain
− N-terminal effector domain (pyrin)
1. Efflux of K+ ions from damaged cells induces dissociation of the accessory proteins from NLRP3
2. Allows activation of the pyrin domain with that of the adaptor molecule apoptosis-associated speck-like protein with a caspase recruitment domain (ASC)
3. The CARD of ASC binds to the CARD domain of pro-caspase 1, facilitating production of activate caspase 1 molecule = the inflammasome.
What is meant by the priming step of IL-1 production?
- Acute brain injury such as stroke can be considered a sterile insult that induces an inflammatory response that can further exacerbate the initial injury.
- In sterile injuries, IL-1a and IL-1B have been identified as key mediators of the damaging inflammatory response
- Both produced as precursors by cells of the innate immune system in respone to PAMPs and DAMPs activating TLRs to regulate NFkB
- PAMP induced expression is the first stage in IL-1 production, and is known as the priming step.
- The precursors pro-IL1a and pro-IL1B remain associated with a primed cell until the second stage of IL-1 production, when the primed cell encounters a further stimulus → this can be another PAMP, or a DAMP in sterile inflammation.
Describe IL-1a activation
- Pro-IL-1a is biologically active and freely soluble
- Any events leading to membrane rupture or death of cells expressing pro-IL1a could result in its release and a pro-inflammatory response.
- In contrast to pro-IL-1B, pro-IL1-a can be actively transported to the nucleus of the cell, and Brough reported that this retention after cell necrosis limits IL-1a release and could potentially dampen sterile inflammatory responses.
- Although doesn’t have to be cleaved to be actived, has to be cleaved to be released → by the activity of calcium-dependent calpain proteases – these may be membrane associated, such that mature IL-1a may be formed upon release from the cell.
- Although pro-IL1a is not a substrate for caspase 1, IL-1a release is inhibited in macrophages isolated from capase 1 and NLRP3 KO mice in response to some DAMPs.
Describe IL-1B activation
• Released directly after cleavage of the proprotein by caspase 1 (inflammasome)
• As mentioned above, the best characterized inflammasome is the NLRP3 (activated by ATP, MSU, CPPD, amyloid-B, sphingosine)…. however there are others formed from other PRRs:
− NLRC4 (activated by flagellin)
− AIM2 (ds DNA sensing)
• After caspase 1 activation, IL-1b is rapidly secreted from the cell.
Inflammasome formation can be visualized in the lab:
• Prepare a virus that expresses the ASC adaptor protein fused to a fluorescent protein mCherry
• NLRP3 binds to its PAMP/DAMP, and this nucleates a reaction with ASC.
Describe the conventional pathway of protein secretion
• Proteins are co- or post-translationally translocated to the ER
− signal sequence at the N-terminus of the newly generated peptide is recognized by the signal recognition particle on the ribosome.
− The SRP bound to the ribosome and nascent protein chain then binds to the SRP receptor on the ER membrane
− Peptide chain is translocated through the protein conducting channel formed by sec61a and sec61y subunits into the ER lumen.
• One in the ER lumen, the signal is removed from the nascent peptide chain, assumes its correctly folded state, and accumulates at the ER exit site by binding to the COPII complex.
• The COPII complex facilitates budding of vesicles that then fuse with the golgi, where they undergo post-translational modifications before they are excytosed.
Describe the rescue and redirect model of IL-1 secretion.
• IL-1B is transplated on free polyribosomes associated with the cytoskeleton
• The vast majority localizes to the cytosol, however a fraction of cellular IL-B is targeted to lysosomes for degradation. This fraction can be rescued by triggering lysosome exocytosis and thus secretion if IL-1B
• Suggested autophagy may be the method that they get sequestered into vesicles → during autophagy, damaged organelles or proteins in the cytosol become enclosed in an autophagosome which fuses with lysosome, resulting in degradation of the contents.
− Indeed, LPS treatment of macrophages induces the recruitment of IL-1B to autophagosomes.
− When autophagy is inhibited, IL-1B is secreted; when activated, it is degraded.
− Thus, a fraction of IL-1B targeted for degradation can be ‘rescued’ and ‘redirected’ to the extracellular environment
Describe the protected release model of IL-1B secretion.
Microvesicles:
• Involves the shedding of IL-1B containing microvesicles that bud from the plasma membrane
• Originally observed in THP-1 cells, whereby shedding is preceded by flip of the lipid phosphatidylserine to the outer leaflet of the plasma membrane, and in astrocytes recquires the activation of sphingomyelinase.
• The IL-1B in shed microvesicles is bioactive, and may be released following contact with IL-1 receptor expressing cells
• ATP stimulation of these microvesicles can also induce the release of their contents
Exosomes:
• IL-1B can be packaged into small exosomes.
• Multiple exosomes are secreted within multi-vesicular bodies
• The secretion of these exosomes in LPS-stimulated macrophages is dependent upon ASC and NLRP3, but independent of caspase 1.
→ That fact that much of IL-1B secreted from cells is directly available, and the fact IL-1B has a short halflife in plasma, suggests that protected IL-1B is destined for signaling at sites distal to the local inflammatory lesion.
