The innate immune system Flashcards
The innate immune system is comprised of two lines of defence, which?
The innate immune system consists of barriers (both physiological and chemical) and cellular responses (when the barrier is breached).
The anatomical barriers make up the first line of defence. Name three key barrier organs/tissues and explain how they protect against pathogens.
Epithelial layers of the skin: physical barrier hindering pathogen entry, production of antimicrobial proteins like psoriasin.
Mucosal tissues (e.g., gastrointestinal, respiratory, and urogenital tracts): Mucus entrapment and preventing pathogen from reaching epithelium, cilia sweeping outwards (airway), low ph (urogenital) and overall production of antimicrobial like lacroferrin that limits growth of bacteria and fungi, disrupts microbial membranes and limits effectivity of some viruses.
Glandular tissues (e.g., salivary, lacrimal, and mammary glands): Flushing by secretions and mucus outwards and containing antimicrobial peptides and proteins like lysozymesthat cleaves glycosidic bonds in peptidoglycan of bacterial cells walls, leading to lysis.
What are the general characteristics of antimicrobial peptides?
The general characteristics of antimicrobial peptides are:
- They’re ancient! Very well conserved.
- Less than 100 aa long
- Positively charged and have both hydrophilic and hydrophobic regions
- Interact with acidic phospholipids in lipid bilayers and forms pores wich disrupt membranes of microbes
- The peptides enter the microbes
and exert toxic effects
For example defensins and histatins.
Which cells are involved in the cellular response of the innate immune system?
Mainly the cells of the myeloid lineage: Neutrophils, Eosinophils, Basophils, Mast cells, Monocytes, Macrophages, Dendritic cells and the innate lymphoid cells (ILCs): mainly NK cells.
How does the innate cells recognize pathogens?
Via pattern recognition receptors, that bind PAMPs, motifs present on/in groups of pathogens: bacteria, fungi, parasites and viruses.
Upon PRR-PAMP interaction, signalling pathways are activated, contributing to innate/inflammatory responses
Which cells express pattern recognition receptors?
Basically all cells express at least some PRRs. They are of course expressed by all the cells in the innate imunnity but also in cells in tissues that are more exposed to infection than others, like epithelial cells, mucosal and glandular tissues and vascular endothelial cells that line the blood vessels to name a few. Cytosolic sensors of viral nucleic acids are expressed by most if not all cells in the body, important given that most cell types are susceptible to infection with viruses.
There are several families of innate receptors, which and what are their function in brief?
– Toll like receptors (TLRs): recognize a wide variety of PAMPs and are present both on plasma membrane and intracellular membranes. They induce production of antimicrobials, antivirals,
and cytokines and the inflammatory response.
– C-type lectin receptors (CLRs): A very heterogenous group of plasma membrane PRRs (not as similar as TLRs). CLRs are expressed on many different cell types including like APCs, neutrophils and subsets of B and T cells. CLRs recognize mainly cell wall components such as sugars/polysaccharides of bacteria/fungi but also carbohydrate components of viruses, parasites and some allergens. Most CLRs initiate signalling through dimerization activated phosphorylation of tyrosine residues in their cytoplasmic domains (ITAMs) or associated signalling chains and downstream effects are similar to those of TLRs.
– RIG-I-like receptors (RLRs): Members of this family bind viral dsRNA in the cytosol with their helicase domain, assembles into a tetramer and associate with proteins on mitochondria to trigger signalling. The signalling pathway lead to the activation of NF-κB and IRF3 and IRF7 - antiviral responses.
– NOD-like receptors (NLRs): Large and diverse family of cytosolic PRRs recognising intracellular PAMPs (like breakdown products of bacteria) and DAMPs (sensing stress). Upon ligand binding they dimerize and recruit RIP2 which signals and have the same downstream effects as endosomal TLRs. Some NLRs (like ND1 and NOD2) can also trigger autophagy and some can form the inflammasone.
Remember that cells can have several PRRs, so these processes can happen simultaneously depending on the pathogen.
How does TLRs recognize PAMPs?
TLRs share a structural element called leucine-rich repeats (LRRs) which are used to recognize and bind PAMPs. Upon PAMP binding, they are induced to dimerize (ligand-induced TLR dimerization) and their cytoplasmic regions convey the signal intracellularly.
Give three examples of TLRs and what they recognize.
- TLR1 recognizes triacyl lipopeptides on mycobacteria and gram-negative bacteria
- TLR4 recognizes LPS in gram negative bacteria (plasma membrane)
TLR2 recognizes peptidoglycans on gram-positive bacteria
- TLR6 recognizes diacyl lipopolypeptides on gram positive bacteria and mycobacteria (plasma membrane)
- TLR3 recognizes viral dsRNA
- TLR7/8 recognizes viral ssRNA
- TLR9 recognizes CpG unmethylated dinucleotides on bacterial DNA and dinucleotides in some herpesviruses.
The downstream signalling of all TLRs share a key transcription factor, which and what does it do?
An important example of a shared downstream signalling component for all TLRs is the transcription factor NF-κB, which is of key importance for activating the expression of many innate and inflammatory genes.
Some pathways and components are activated only by some TLRs – specifically induced expression of genes that are important in fighting that particular type of microbe. Give one example.
One example is the expression of potent antiviral type I interferons, IFN-α and IFN-β, induced by pathways downstream of the TLRs that bind viral components.
There are two major pathways used in TLR signalling, which?
The MyD88 adaptor pathway and the TRIF pathway.
- The MyD88 pathway is used by all plasma membrane TLRs but also by some endosomal TLRs (TLR7 for example). This pathway is particularly important in defence mechanisms against extracellular pathogens.
- The TRIF pathway is commonly used by endosomal TLRs and activate transcription factors that lead to increased production of antiviral interferons.
Note: TLR4 is unique among TLRs in its ability to activate both signalling pathways, but it does so sequentially. When in the plasma membrane, it can signal through MyD88 and then TLR4 is only able to initiate the TRIF pathway from the endosomal compartment following endocytosis.
Explain the MyD88 adaptor pathway of TLR signaling in short.
MyD88 is an adaptor protein that bind the cytosolic TIR elements of dimerized TLRs. Upon TLR dimerization, MyD88 binds and initiates signalling through recruiting the IRAK1 and IRAK4 kinases, which phosphorylates components of the TAK1 complex. TAK1 phosphorylation activated MAP kinase pathways, which activate transcription factors such as AP-1 and the TAK1 phosphorylation also initates a series of events
leading to the degradation of the NFkB-inhibitor IkBNF-κB –> which releases NF-κB so it can go into the nucleus and act.
Both NF-κB and AP-1 are essential for activating key antimicrobial proteins and peptides as well as proinflammatory cytokines and chemokines that are of key importance in the innate immune response.
Explain the TRIF pathway of TLR signalling in short.
Upon endosomal TLR dimerization, the TRIF adaptor binds and recruits and activates TRAF3, which generates a scaffold that recruits and activates a kinase complex called the IKK complex which incudes TBK1 (TANK- binding kinase 1). TBK1 phosphorylates and activates IRF3 and IRF7, each of which dimerizes and enters the nucleus, inducing the transcription of the IFN-α and IFN-β genes which are crucial in the antiviral response.
Later in the response, TRIF also activates TRAF6, leading to NF-κB activation.
Does the endosomal TLRs use only one pathway for downstream signaling?
Some only use one, like TLR3 which uses TRIF, but some endosomal TLRs like TLR7, TLR8 and TLR9 all use both the MyD88 and the TRIF pathway simultaneously. In the endosome, MyD88- associated IRAK1 directly phosphorylates IRF7. This allows IRF7 dimerization, activation, nuclear translocation, and induction of IFN gene expression.
NLRs can form inflammasones, what is it?
Some NLRs contain domains that allow them to form large complexes, both by oligomerization and binding of other proteins. One example of this kind of NLR is NLRP3 which functions as a sensor for stress (either damage induced or pathogen derived) which upon activation can form inflammasones which triggers a very strong inflammatory response consisting of release of large amounts of potent cytokines.
Explain how the inflammasone is formed in short.
Inflammasone activation requires two signals:
signal 1. Binding of PAMPs to any PRR which induces synthesis of large cytokine precursors pro-IL-1β and pro-IL-18, and of the NLR NLRP3 (through NF-κB and MAPK signaling)
signal 2. Stress signals in the cell, either damage induced or pathogen derived (for example changes in ATP/glucose levels or peptidoglycan fragments or vir/bac RNA/proteins). (note: we don’t know how these components activate the NLRP3)
When the NLRP3 receives the stress signals, they are activated and change conformation which allows them to oligomerize into a round disc shape. This formation promotes assembly with the adapter protein ASC and procaspase-1 and forms the NLRP3 inflammasome. Clustering of procaspase-1 results in self cleavage which forms caspase-1. Caspase-1 then cleaves the procytokines pro-IL-1β and pro-IL-18 to their active forms IL-1 and IL-18 which are very pro-inflammatory. Caspase-1 activation can also lead to cleavage of pro-gasdermin D into its active form which produces pores in the plasma membrane, which induces the death of the activated macrophage through pyroptosis, allowing the release of the mature IL-1β and IL-18.
List the four effector responses of the innate immune system.
- Inflammatory response
- Phagocytosis
- Killing of pathogen through IFNs and antimicrobial proteins and peptides or lysis (complement system)
- Activation of the adaptive immunity
How is local inflammation induced?
- Tissue damage and pathogen entry causes surrounding cells to release chemokines and vasoactive factors that causes increased blood flow and increased capillary permeability at the area.
- The capillary endothelium expresses factors like selectins (adhesion) and integrins (extravasion) which aids in slowing down and allowing entry for immune cells through the capillaries.
- Neutrophils and other phagocytes migrate to the area, guided by chemotaxis and destroy bacteria through effector functions.
PRR signaling pathways activate the expression of a large variety of genes involved in the cellular inflammatory response against infection. These genes encode for four main categories of effector molecules. Which and what are their main function?
– Antimicrobial peptides: particularly induced by TLR and NLR signalling and their function is direct neutralisation of microbes through membrane disruption or interference with internal structures like DNA, cell wall or protein synthesis - all leading to the death of the pathogen. They also activate immune cells and modulate the immune response.
– Cytokines (inflammatory IL-1, TNF-α, and IL-6): not directly antimicrobial but activate and regulate a wide variety of cells and tissues involved in innate, inflammatory, and adaptive responses. Three of the most important cytokines are IL-1, TNF-α, and IL-6, the major proinflammatory cytokines. They act locally on blood vessels to increase vascular permeability and also on other cells, including lymphocytes, to recruit and activate them at sites of infection.
– Chemokines: molecules that attract immune cells with chemokine receptors to the site where they’re. secreted. Act on Neutrophils, basophils, immature dendritic cells and T cells.
Table 4-4 on page 303 good overview.
Give two examples of antimicrobial peptides, which cells they act on and how they act on them.
- Defensins and cathelicidin: produced by epithelia, neutrophils and NK cells and function primarily by killing pathogens directly but also through immunomodulation, by stimulating granulocytes such as mast cells to degranulate, they increase microbe clearance. They can also serve as chemoattractants and activators of cytokine production in monocytes, immature dendritic cells and T lymphocytes.
- Type I Interferons (IFN alpha and beta - main players): produced by virus-infected cells, macrophages, dendritic cells and NK cells. Induces a strong antiviral response by binding to IFNAR (IFN-alpha receptor) expressed by most cells. Genes that are turned on by IFN are known as interferon-stimulated genes (ISGs). On virus infected cells, they Inhibit virus replication (inhib of translation, transcription, assembly and through mRNA degradation). They increase expression of MHC class I proteins, making the cells better targets for T cell–mediated killing, activates NK cells regulate the activity of Macrophages and T cells.
Antimicrobial peptides are expressed both constitutively and induced, give two examples of cells/tissues of each.
Antimicrobial peptides are particularly induced by TLR and NLR signalling in mucosal and glandular epithelial cells, skin keratinocytes and NK cells.
They are constitutively expressed in skin and mucosal epithelial layers, eg defensins expressed by Paneth cells in the intestines and the cathelicidin LL-37 packaged in the granules of neutrophils ready to kill phagocytosed bacteria, fungi, viruses, and protozoan parasites.
Cytokines are the hormones of the immune system, list three main innate acting cytokines and name which cells produce them, which cells they affect and what effect they induce.
TNF-α: Produced by Monocytes, macrophages, dendritic cells, mast cells, NK cells, epithelial cells and activates cells via the trimeric TNF receptor. Effector functions:
- Activates macrophages
- Induces Activates, increases vascular permeability, fluid loss, local blood clotting of vascular endothelium cells.
- Induces acute-phase response in the liver
- Induce fever in the hypothalamus
- Cytotoxic for many tumor cells
IL-1: exist in two forms but IL-1β more common. Produced by monocytes, macrophages, dendritic cells, keratinocytes, epithelial cells,
vascular endothelial cells and have the following effector functions:
- Enhances activity of lymphocytes
- Promotes neutrophil production in the bone marrow
- Activates; increases vascular permeability of the vascular endothelium
- Induces acute-phase response in the liver
- Induce fever in the hypothalamus
IL-6: Produced by monocytes, macrophages, dendritic cells, NK cells, epithelial cells, vascular
endothelial cells. Have similar effects as IL-1 for endothelium, liver and hypothalamus but:
- regulates activity of lymphocytes
- Promotes hematopoiesis → neutrophils in the bone marrow
Phagocytosis is an key mechanism for pathogen clearance and destruction in the innate response. Define phagocytosis and mention the three main phagocytic cells.
Phagocytosis is defined as engulfment and internalization of materials such as microbes for their clearance and destruction. Monocytes, macrophages and neutrophils are the main phagocytic cells (also DCs).
How do phagocytic cells recognize microbes, triggering their
phagocytosis?
Phagocytic cells recognize PAMPs via PRRs, mainly CLRs (C-type lectin receptors) but also scavenger receptors. Most PAMPS that trigger
phagocytosis are cell wall
components, eg. the CLRs mannose receptor and DC-SIGN recognize mannan (polysaccaride with a bit of protein) present in the cell wall of bacteria, fungi and parasites and dectin-1 that recognize β-glucan in the cell wall of fungi and some bacteria. When bound,
Explain the process of phagocytosis in short.
- PAMPs bind to PRRs, mainly CLRs on phagocytic cells (mainly monocytes but also macrophages, neutrophils and DCs) This recognition activates signaling pathways that induce the polymerization of actin microfilaments, extending the phagocyte’s plasma membrane to engulf and internalize the microbes
- The pathogen is engulfed into phagosomes (endosomes resulting from phagocytosis) with a pH of ~6.2.
- The phagosomes fuses with lysosome, pH ~4.5-5 (or prepackaged granules).
- The pathogen is killed and digested by antimicrobial peptides, by low-pH activated lysosomal enzymes and
oxidative attacks by ROS and RNS. - Digestion products are released by the cell.
This process is used by dendritic cells in antigen presentation and can also activate the production of a variety of molecules that contribute in other ways to eliminating infection. Phagocytosis can also be mediated by opsonization (soluble proteins binding to microbial surfaces facilitating phagocytosis (complement system, antibodies etc.)
There are four processes of killing and degrading the pathogen after phagocytosis, which? Explain them briefly.
- Phagosomal degradation: Destruction occurs through enzyme degradation, antimicrobial proteins, and toxic effects of reactive oxygen and reactive nitrogen species (ROS and RNS).
- Autophagy: Intracellular pathogens like listeria escape phagocytosis and thus, alternative pathways are needed. Autophagy is an innate effector function activated by the NLRs NOD1 and NOD2. The process is that membrane derived from the endoplasmic reticulum envelopes the bacteria, forming an autophagosome. This vesicle then fuses with lysosomes, leading to the destruction of the pathogens in the same ways as the phagosome.
- DAMPs (danger associated molecular pattern): components of dying or damaged cells expressed on the surface of apoptitic/damaged/infected cells, ”eat me signals”, triggering PRRs and facilitating phagocytosis. Healthy cells express CD47, a ”don’t eat me signal” (but can be upregulated on tumor cells).
- Neutrophil extracellular traps (NETs)/NETosis: PRR signalling from selected PRRs activates NADPH oxidase and the generation of ROS such as superoxides which cause damage to intracellular compartments, making it possible for enzymes and other modifyers to enter the nucleus and modify and degrade chromatin/DNA and release it outside of the cell like a fishing net. The net can trap trap bacterial, fungal, and parasite cells, preventing their spread and accompanying antimicrobial proteins and enzymes also help combat the pathogen. NETosis kills the neutrophil but provide an effective defense and also release DAMPs.
- Pyroptosis: Regulated cell death induced by inflammasome activation. Useful in combating intracellular pathogens.
What are the five hallmarks of inflammation, why do they occur?
The five hallmarks of inflammation are: Redness, swelling, heat, pain and loss of function.
All of these symptoms of the inflammatory response are caused by increased vascular permeability and extravasion to the site, where more immune cells and fluids can enter (swelling, redness, heat, loss of function) and then pain is a consequence to let you know that something is happening.
Inflammation is necessary to combat infection but if persistent it can cause problems, what?
Too extreme/prolonged inflammation can cause sepsis - when the inflammation spreads to the blood and thus affect the whole body. Systemic expression of proinflammatory cytokines that can lead to septic shock which can be fatal. Therefore it’s really important that the regulatory mechanisms of the immune response works.
The major cause of sepsis from gram-negative bacteria is the cell wall component LPS, a highly potent inducer of innate immune mediators, including the proinflammatory cytokines TNF- α, IL-1β, and IL-6; chemokines; and antimicrobial components.
What is the function of fever in response to infection?
Fever is a protective response, as elevated body temperature inhibits replication of some pathogens. It’s induced by the systemic effects of the proinflammatory cytokines TNF-α, IL-2 and IL-6 which induce COX2 expression, which activates prostaglandin synthesis, as mentioned above. Prostaglandin E2 (PGE2) acts on the hypothalamus (the brain center controlling body temperature), causing fever.
One of the effector functions of the innate immune system is the activation of the adaptive immune system, explain how this works briefly.
The main antigen presenting cells are dendritic cells, which belong to the innate immune system. They also have other functions but they are the main APCs. When a dendritic cell have internalised a pathogen it’s degraded into peptides that are attached to MHC molecules and moved to the surface of the cell. Once a pathogen has been internalized, the DC looses it’s internalizing capabilities and matures into an APC, with hinger MHC expression and expression of co-receptors - all to be an effective APC. It moves into the lymphatic vessels to get to the lymph nodes where it presents the antigen to naive T cells, and through co-stimulatory cytokine signalling it “primes” the naive T cells to produce the right compounds and differentiate into the right effector subsets for the antigen specific response. Eg Th1 in response to bacteria and viruses, Th17 in response to fungi and Th2 in response to helmiths.
page 336.
Innate lymphoid cells on page 324
.
The adaptive immune system is known for its long lived memory, does the innate immune system have a memory?
Yes, to some extent. It was long believed not to exist and is still aa bit unclear, but the consensus is that the innate system can be “trained” so that the response is stonger upon repeated exposure. What has been observed is persistant/long lived epigenetic modifications leading to increased responsiveness and altered metabolic processing. We don’t know how it works.
The innate and inflammatory responses are regulated both positively and negatively. Explain the mechanisms by which the innate and inflammatory responses are positively regulated.
Positive regulatory mechanisms:
- PRRs are highly redundant, several of them recognize the same PAMP (so if one have a mutation in one, you still have others to protect you), and many of these have synergistic effects. For example, in response to yeast, signaling pathways downstream of TLR2 and the CLR dectin-1 synergize to enhance protective cytokine production.
- Positive feedback loops from cytokines that induce themselves, for example the amplification of production of IL-1β and TNF-α, two of the initial cytokines induced by PAMP or DAMP binding to PRRs. As they activate pathways similar to those downstream of TLRs, they induce more of themselves, an example of positive feedback regulation.
Explain the four mechanisms by which the innate and inflammatory responses are negatively regulated.
Negative regulatory mechanisms of innate immunity:
- Several proteins whose expression or activity is increased following PRR signaling feed back to inhibit steps in the signaling pathways downstream of the PRR. Examples include production of a short form of the adaptor MyD88 that inhibits normal MyD88 function - to activate NF-kB which in turn activates expression of proinflammatory cytokines and antimicrobial peptides, thus limiting the extent of the innate response.
- LPS-tolerance: When macrophages are continuously exposed to LPS (endotoxin) their initial production of antimicrobial and proinflammatory mediators is followed by the induction of inhibitors, including IkB (NF-kB inhibitor) and the short form of MyD88 that block the macrophages from continuing to respond to LPS. This reduces the possibility that continued exposure to LPS from a bacterial infection will cause septic shock.
- Soluble antagonists: Some cytokines induces the expression of soluble forms of their receptor, for example TNF-α and IL-1β which binds unbound fractions of the cytokines and limits other, non-target cells from being exposed. Also some cytokines can bind the same receptors as more potent ones and cause downregulation of the expression (eg IFN-β that binds IL-1β receptors, where IL-1β is more potent) which can also limit their effects.
- IL-10: Late in the macrophage response, the expression of IL-10 is induced, which inhibits the production and effects of inflammatory cytokines and promotes wound healing.
