Inflammation & Immune Response PDF

20/11 Recap: -​ Substrate: What the enzyme works on -​ Ligand: molecule that binds to another. Can activate or inhibit another molecule In pharmacology we don’t use ligand so much, but more agonist and antagonist -​ Agonist: ligand that binds and activates -​ Antagonist: ligand that binds...

20/11 Recap: -​ Substrate: What the enzyme works on -​ Ligand: molecule that binds to another. Can activate or inhibit another molecule In pharmacology we don’t use ligand so much, but more agonist and antagonist -​ Agonist: ligand that binds and activates -​ Antagonist: ligand that binds and inhibits Ligand = PAMP = agonist all same so no panic What characterises MAMP’s/agonists of all the sensors is that they have a returning polymer structure. So think about DNA, RNA, lipopolysaccharide… molecules that form chains that repeat each other. The way they identify and bind their molecules is by an area called the agonist binder which is rich in leucine (Leucine Rich Repeat). So all the receptors we’ll see will have this agonist binder area, which is the outer area. Then we have the transmembranal area which only has one anchoring site (unlike GPCRs which have 7 for example) and finally the TIR domain which is important for the recruitment of the adaptor protein that passes the signal until the transcription factors which activate the gene that activates inflammation. All the while there isn’t dimerization, the TIR domain is inactive. Once there is dimerization, which usually happens when a certain concentration of the agonist appears next to the cell, the signal is passed. Intracellular sensing: The first family we’ll talk about is NOD: -​ We have a receptor with a Leucine Rich Repeat (which binds the agonist) and an area for the adaptor protein recruitment. -​ They are soluble receptors found in the cytosol that identify MAMPs, agonists… -​ If a bacteria managed to enter the cytoplasm, the NOD would be the one to be there and stop it. -​ Was easy to identify them because so similar to the other receptors. The Inflammasome: A protein complex that activates inflammation. One of its component is the green spots which are the macrophages which are coloured because of their actin cytoskeleton. 3 Main Components: -​ The sensor: it has Leucine Rich Repeat, the area that binds the agonist. Very similar to NOD so called NOD-like-receptor-protein - NLRP (similar in soluble in cytoplasm and in its structure). -​ ASC: adaptor protein -​ Active component - procaspase 1. In its procaspase 1 state it isn’t a mature, active protein yet. But it has a subunit that is supposed to be activated when we want to activate inflammation. It’s called caspase 1, which is a protease - it has a protein target that it cuts/does cleavage at a very specific site. The whole point of the inflammasome is to turn inactive procaspase 1 (built from the yellow and red subunits) into active caspase 1 (so basically to release the yellow subunit which is inhibiting the red one). When there is no agonist, these 3 subunits are sitting together in the cytoplasm waiting. When a danger is detected and inflammation is needed, the inflammasome needs to bring the red subunits close enough so that the first procaspase 1 can cut the second. The chance of this meeting in the cytosol is extremely low, for a protein this size the cytosol is like the size of Israel for us. The adaptor protein binds on one side the NLRP and on the other side the procaspase-1. Why do I need active caspase-1? Its target proteins need to be cut in order to be activated. We will go over a few examples like Pro-IL-1, a protein that if released from the cell, activates inflammation. In a normal situation it undergoes transcription, translation and becomes Pro-IL-1 and just sits in the cytoplasm without being secreted out. In order for it to be secreted out, it needs to cut itself and become active IL-1; then it will be secreted and will bind to the other cells which have a receptor and will activate inflammation. The inflammasome is the main complex that activates inflammation. It has been found to also activate cell death - pyroptosis. This can be divided into necrosis and apoptosis. The main difference between these two is that in necrosis when the cell dies it has a messy death, where the cell explodes and all its contents are spilled outside causing inflammation since it comes from tissue damage. Meanwhile, apoptosis has a clean death. Necroptosis is necrosis but it is planned; basically the tissue damages itself on purpose in order to release stuff like DNA, RNA, energy… so as to activate even more inflammation. What are the agonists that activate the inflammasome? There are sterile activators and pathogenic activators as we can see. Learn a few examples of each. High cholesterol or sugar concentrations inside the cell or high ATP concentration outside the cell are also examples of inflammasome activating factors. There are many other receptors that we have that also act as sensors for these kinds of abnormalities. For example there is a receptor that identifies high levels of triglycerides. Let’s say there is a pathogen inside us and there’s also a microbe which not only isn’t a pathogen but may even be good for us. How does the immune system know in the first case to activate strong inflammation but not in the second case? At the end of the day, a bacteria is a bacteria, its walls are the same. Decisions must be made. Example: We have a bacteria E.coli that isn’t damaging our tissue, just an E.coli living alongside us. A macrophage in the intestine will detect through its receptor the components of this bacterium's cell wall, an adaptor protein will add there molecules that activate inflammation. Some of them will cause immediate inflammation and the others will undergo transcription, translation and stay in the cytosol inactively. While these aren’t activated, inflammation won’t get worse. If that E.coli suddenly starts causing damage, the inflammasome will be activated and so caspase-1 will be too, which will cut the inflammation-causing molecules that stayed inactively in the cytoplasm and will start activating them. They will be secreted and inflammation will worsen. Cytoplasmic sensors of viral RNA: RIG-I and MDA-5 The last group of sensors we talk about are cytosolic receptors that identify RNA. Why RNA? Story about the RNA Vaccine: Some scientists tried to create a vaccine against cancer. Their idea was: if we take RNA from a protein that expresses in cancer in a very high strong manner and we insert it into dendritic cells it will undergo translation inside them, a protein will be formed and the dendrites will show the T cells of this protein and they will come and attack the cancer. But it didn't work at all. This is because taking this RNA and inserting it into the cells caused inflammation and so the process didn't even get up to the point where translation occurred. They didn't understand why until they understood that RNA in the cytosol has sensors that identify modifications on the RNA being inserted, but not just any modifications… modifications that would usually be found in viruses for example. So once they understood this they inserted RNA with specific modifications so that the cytoplasmic RNA wouldn’t recognise it and initiate inflammation. -​ These sensors we’re talking about, there are two main ones: RIG-I and MDA-5. Both of them identify single strand RNA in the cytosol, the first identifies short sequences (up to 20bp) and the second identifies longer sequences. Both of them look for RNA that has a modification called triphosphate RNA - a modification our normal cells don’t have but many viruses do have (we have a cap) so inflammation is activated. -​ RIG-I and MDA-5 are receptors soluble in the cytosol that have an area that binds the RNA at this triphosphate area (the Leucine rich repeat area) and recruit adaptor proteins that recruit proteins that pass signals and activate inflammation. -​ So in order for their vaccine to work, the RNA with the triphosphate modification must be not identified so it can be translated.  Flow Chart Summary Inflammasome: -​ Cytosolic sensor (soluble receptor) that has area (Leucine Rich Repeat) that binds agonist -​ The sensor recruits adaptor protein -​ Adaptor protein recruits Pro-caspase-1 -​ Inflammasome structure is created -​ Active caspase-1 is released and cuts proteins that formed when sensors were activated -​ Inflammation is activated Designation - emphasize foreign origin We have identified the danger and now we want to recruit other cells from immune system. We want the liver to start producing antibacterial substances and that the brain will secrete neurotransmitters that will cause the adrenal gland to secrete hormones to fight and adapt against the change. For this, we need second messengers to pass the message correctly. Cytokines: -​ Small proteins ~25KDa - “hormones” of the immune system, all of them proteins -​ Can be released by almost all cells in the body, usually in response to an activating stimulus, and can affect any cell in the body -​ Induce responses through binding to specific receptors -​ Can act in three manners: -​ Autocrine: affecting the behavior of the cell that releases them -​ Paracrine: affecting the behavior of adjacent cells -​ Endocrine: affecting the behavior of distant cells There are 4 main groups of Cytokines: 1.​ Interleukin-1 (IL-1) family -​ Has 11 members (IL-1 alpha & beta, IL-18) -​ When it goes through translation, we receive a pro-protein which is inactive (pro-IL-18 etc). So in order for this cytokine to be secreted from the cell and for it to affect other cells it needs to be cut by caspase-1. So we need the inflammasome complex for this to happen. -​ IL-1 for example is essential for raising the body temperature when there is inflammation in the body, pathogenic bacteria can’t survive and T cells are more effective in killing pathogens at higher temps. We can do this via two methods; muscle shaking and in fatty tissues the mitochondria produce heat instead of ATP. Something also has to tell the body to accumulate this heat because if no one says so, we would start sweating and so we’d lose this heat we’re producing. For this, the hypothalamus in the brain, which is the thermostat of the body, is increased by IL-1β, so the body considers the new temperature it says as the temperature it must keep. 2.​ Tumor Necrosis Factor (TNS) -​ Has 17 members whose prototype is the TNS alpha. It is very infamous because it is well known for causing the inflammation in IBD diseases. Nowadays there are medicines that bind to the TNS and don’t allow it to bind to its receptor so no inflammation is created.  -​ Usually found as a homotrimer - so 3 subunits of TNS alpha bind to it and only then it can bind to the receptor. -​ When TNS is formed in a protein and forms the trimer, it isn’t immediately secreted to the outside of the cell. It is passed through the golgi and using its transmembrane area it goes into the membrane and sits and stays there. It will be activated when there is inflammation - so proteins will be released that need to cut the cytokine so it can release the outercell part. 3.​ Hematopoietin Superfamily -​ Very big family with a lot of variety of cytokines. -​ Most known are Erythropoietin, which is the cytokine that stimulates red blood cell production in the bone marrow in response to low oxygen levels. -​ Also GM-CSF : it stimulates the production and differentiation of white blood cells, specifically granulocytes (e.g., neutrophils, eosinophils) and macrophages, from hematopoietic stem cells in the bone marrow in response to infection eg. -​ IL-6 (don’t get confused, it doesn’t belong to the IL-1 family from before) stimulates the differentiation and proliferation of immune cells (e.g., B cells, T cells) and supports the acute-phase response during inflammation. 4.​ Interferon (IFN) family -​ Viral interference; very potent against viruses. They are the most important in our antiviral response since they can bind their receptors on cells and stop them from doing translation, so basically cell shutdown. -​ Type I : includes IFN-α and IFN-β, almost all cells in the body knows how to create them and is affected by them -​ Type II : IFN-γ. Produced by and affects T cells and NK cells. Of course it doesn't stop their translation activities but the opposite, increases them. The cytokines TNF-α, IL-1β and IL-6 coordinates the body’s responses to infection Chemokines: -​ A subfamily of cytokines. -​ Primarily functions in directing migration of cells and not so much in their activation, these are called “chemotactic cytokines” or “chemokines” -​ If I want to attract a cell to a certain area, I secrete chemokines which the cell has receptors for and the cell will move from the place where the chemokines concentration is low to where it's high. So if there’s a cell in the middle of a high inflammation area, the chemokines concentration in that area is high and as we get further away from it it’s low. -​ Chemokines have systematic names: CCL1, 2, … and CXCL1, 2, … (but older names sometimes used, including IL-8) How do we attract cells to an inflammation area due to a pathogenic incursion? Adhesion Molecules: their function is to adhere between cells-tissues-surfaces etc Selectins: -​ A lot of them are found on the endothelial cells that compose the blood vessels. -​ White blood cells - neutrophils have receptors - these pass through the golgi, undergo sugary modifications on the cell surface. -​ One of them is a very specific type of sialic acid called Sialyl-LewisX. It happens on many receptor types, almost all that pass through the golgi. So now the cell that has it is enveloped in sialic acid. -​ This is important because the ligand of the E-selectin is sialic acid. Rolling: This image depicts selectin-mediated rolling adhesion, a key step in leukocyte extravasation during inflammation. -​ E-selectin (on endothelial cells) binds weakly to sialyl-Lewis^x carbohydrates on leukocytes. -​ This weak, low affinity interaction allows leukocytes to "roll" along the endothelium under blood flow but without it actually fully binding and not being able to let go. -​ Rolling slows down leukocytes, enabling stronger integrin-mediated adhesion for eventual migration into tissues. -​ As the cell gets closer to an inflammation zone where there is a high concentration of cytokines; the cytokines have receptors on the endothelium and so they increase more adhesion molecules onto the cell. If we increase E-selectins for example, the cell will move even slower. -​ It also increases another adhesion molecule called ICAM. It joins the E-selectin and the closer we are to the inflammation zone the more the ICAM amount will increase. -​ This is necessary because ICAM has a receptor on the surface of the leucocyte called Integrin. Integrin on the neutrophil while ICAM is on the endothelial, and they bind with high affinity. The more integrins bind to the more ICAM, the cell will be stopped better and so the leukocyte is stuck in the blood vessel and doesn’t leave. So small recap: our cell doesn’t move freely in the blood flow, it rolls all the time (irrelevantly to inflammation or not) with the help of the bond of Sialyl-LewisX to the receptors on the leukocytes that bind to E-selectin on the blood vessels. In an inflammation zone, thanks to the inflammation activation and secretion of cytokines that bind to the endothelial of the blood vessel, which becomes more filled with selectins on the surface of the cell. And so a molecule ICAM increases and binds with high affinity to the integrins on the surface of the leukocyte. And so the leukocyte is stuck in the blood vessel. CHAT GPT EXPLANATION: Leukocytes are not entirely free-floating in the bloodstream but instead exhibit a process called rolling adhesion, even under normal physiological conditions. This rolling is mediated by weak interactions between sialyl-Lewis^X (s-Le^x), a carbohydrate expressed on the surface of leukocytes, and selectins, such as E-selectin, present on the endothelial cells lining blood vessels. These interactions are transient, allowing leukocytes to roll along the vascular endothelium without firmly adhering to it. During inflammation, the endothelial cells become activated by pro-inflammatory cytokines (e.g., TNF-α, IL-1β). This activation leads to an increased expression of selectins (like E-selectin and P-selectin) on the endothelial surface, enhancing leukocyte rolling. Furthermore, inflammation induces the expression of ICAM-1 (Intercellular Adhesion Molecule-1) on endothelial cells and activates integrins on leukocytes. Unlike the weak selectin-mediated rolling interactions, integrins bind ICAM-1 with high affinity, causing the leukocytes to stop rolling and firmly adhere to the endothelium. This firm adhesion allows the leukocytes to undergo transmigration (diapedesis) through the endothelial layer and into the inflamed tissue, where they participate in immune responses. This diapedesis works like this: the cell secretes a protease that breaks the tight junctions between two endothelial cells and through it the cell penetrates into the tissue. This is overall one of the most effective mechanisms Elimination -​ After entering tissues, many pathogens are recognized, ingested and killed by phagocytes via phagocytosis. This is done by neutrophils and macrophages. The dendritic cells act more in the showing of the antigen and not so much in the killing. -​ For phagocytosis we need receptors that will specifically bind to the bacteria. It can be a mannose receptor since many bacteria have mannose on them, it can be complement or lipid… -​ Phagocytosis knows how to ingest and kill huge things. Phagocytosis: 1.​ Recognition and Binding: Phagocytes recognize pathogens or debris through receptors (e.g., PRRs binding to PAMPs or opsonin receptors binding antibodies/complement). 2.​ Invagination and Engulfment: The plasma membrane invaginates, folding inward to surround the target particle, forming a pocket. This pocket closes and pinches off, creating a vesicle called a phagosome. 3.​ Phagosome-Lysosome Fusion: The phagosome fuses with a lysosome, forming a phagolysosome. 4.​ Degradation: Lysosomal enzymes break down the engulfed material. 5.​ Exocytosis: Debris from the digested material is expelled or presented on MHC molecules for adaptive immunity. There is another mechanism apart from phagocytosis that makes the neutrophil a great killing machine - The Microbicidal Respiratory Burst. The Microbicidal Respiratory Burst The respiratory burst is a rapid release of reactive oxygen species (ROS) in the phagosome to destroy pathogens. This process involves the assembly of a functional NADPH oxidase complex and subsequent chemical reactions to produce ROS and activate other antimicrobial mechanisms. 1.​ Pathogen Engulfment and Phagosome Formation: The phagocyte engulfs the pathogen, forming a phagosome. This vesicle contains the pathogen and will fuse with granules for digestion. Pathogens are recognized via receptors like those for bacterial peptides (e.g., fMet-Leu-Phe). 2.​ NADPH Oxidase Activation: The Rac2 protein activates the assembly of the NADPH oxidase complex on the phagosomal membrane. NADPH oxidase catalyzes the reaction: 𝑁𝐴𝐷𝑃𝐻 + 2𝑂2 - 2e → 𝑁𝐴𝐷𝑃+ + 𝐻+ + 2𝑂2 This produces superoxide anions (O₂⁻), initiating the respiratory burst.  3.​ Ion Influx and Acidification: Potassium (K⁺) and hydrogen ions (H⁺) flow into the phagosome to neutralize the charged ROS and maintain the ionic balance. This influx leads to acidification, which helps activate digestive enzymes in the vesicle. 4.​ Conversion of Superoxide to Hydrogen Peroxide (H₂O₂): Superoxide dismutase (SOD) converts superoxide (O₂⁻) into hydrogen peroxide (H₂O₂): 2𝑂2− + 2𝐻+ → 𝐻2𝑂2 + 𝑂2 Hydrogen peroxide is a potent antimicrobial agent that also contributes to the production of other reactive species. 5.​ Enzyme Activation and Pathogen Degradation: Acidification and ROS generation activate granule proteases (enzymes) within the phagosome, breaking down the pathogen. This ensures effective killing and digestion of the microbe. Key Outcomes -​ Respiratory burst generates ROS, like hydrogen peroxide, to damage and kill pathogens -​ Acidification and enzymatic activity contribute to pathogen destruction within phagosome EXPLANATION FOR BABIES: 1. The Phagocyte Eats the Germ -​ The phagocyte recognizes a bacteria using special receptors on its surface -​ It engulfs it, trapping it in a small bubble called a phagosome, inside the phagocyte. 2. Activation of NADPH Oxidase -​ The phagosome forms, and a protein complex called NADPH oxidase assembles on its membrane. -​ This complex is activated by signals like Rac2 (a small GTPase). -​ NADPH oxidase uses oxygen molecules to produce superoxide (O₂⁻), a reactive oxygen species (ROS) that’s toxic to the germ. 3. Acidification of the Phagosome -​ To balance the electric charge of the ROS, potassium ions (K⁺) and hydrogen ions (H⁺) are pumped into the phagosome. -​ This causes the phagosome to become more acidic, a process called acidification, which is crucial for activating certain enzymes that help kill the germ. 4. Conversion of Superoxide to Hydrogen Peroxide -​ Superoxide dismutase (SOD), an enzyme inside the phagosome, converts superoxide into hydrogen peroxide (H₂O₂). -​ Hydrogen peroxide is a stronger ROS that works with other molecules to kill the germ -​ Some of the hydrogen peroxide can even form hypochlorous acid (bleach), which is very effective at killing bacteria. 5. Activation of Granule Enzymes -​ The phagosome fuses with lysosomes and granules in the phagocyte. These granules release enzymes like proteases (protein-digesting enzymes). -​ Acidification activates these enzymes, which break down the germ’s proteins, lipids, and DNA into harmless pieces. 6. Killing and Cleanup -​ With the combined effects of ROS (like superoxide and hydrogen peroxide), acid, and digestive enzymes, the germ is killed and digested. -​ The phagocyte either expels the broken-down remains or uses them to signal the immune system.