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Immunology Dr.

Leitenberg 1/08/14 2-3pm Note Taker #10 The Innate Immune System (contd) Slide 30: So there are a bunch of different kinds of receptors and we are going to learn about a few of them today. So on this cartoon, it is showing you a macrophage with a few different things that are abbreviated. Some of these you can kind of figure out for yourself. So a mannose receptor is a receptor that recognizes the carbohydrate mannose. A glucan receptor is a receptor for a different carbohydrate, these are carbohydrates that tend to be expressed by yeast and certain bacteria. There are different scavenger receptors and these tend to recognize different microbial patterns that are associated with bacteria. One thing you can think of is these receptors can be thought of as innate immune receptors. So without recognizing the pathogen directly, they recognize the complement which is bound to the pathogen which in turn activated the cell. And the final thing that I want to talk more about later are these TLR receptors. What TLR stands for are toll-like receptors. And there are a bunch of toll like receptors that I am going to tell you about in a second that are a good paradigm for how these receptors recognize a different molecular patterns expressed by pathogens. So I am going to talk about them in more detail later. Slide 31: So this is a table showing you again just some of the receptors that I talked about on the previous slide. I am not going to read through them all again but I did want to point out that they tend to recognize different microbial components typically expressed by microbes but not our own eukaryotic cells. So it is a way for these cells to tell the difference between a microbe that could be infectious and our own cell tissues. Slide 32: So we are going to talk about toll-like receptors the most and this is an image from the cover of a journal showing a fruit fly just covered in fungus. This is because the fruit fly has a mutation in a molecule that is important in signaling from toll-like receptors. This is an example of how the innate immune system was actually first identified. That was through fruit flies. This is an example how in science you dont always know what the result of your experiments is going to be. Now toll-like receptors is becoming a huge field and you may remember that last year or the year before the guys that discovered toll-like receptors won the Nobel Prize. They are the best example of how the innate immune system differentiates dangerous stuff from nondangerous stuff. Slide 33: So toll-lie receptors differentiate different molecular patterns and we refer to these patterns as PAMPs. That stands for pathogen associated molecular patterns. And they recognize different things, for example carbohydrates with gram negative bacteria from carbohydrates with gram positive bacteria, and fungi. They recognize proteins that are in flagella. They actually recognize the nucleic acids that recognize bacterial nucleic acids and viral nucleic acids. And one

of the things that you can realize from this cartoon is that these receptors can be expressed on the membrane facing outside the cell. They can also be expressed inside the cell on a membrane. So imagine that you have a cell that has phagocytized a microbe. They can get activated after that phagocytic event by toll-like receptors that are inside the cell like this. So lets say for example the cell is infected by a virus and the virus is inside the cell replicating. The viral obvious is using viral RNA and the toll-like receptors can recognize the viral RNA and activate that cell to mount an antiviral immune response. Slide 34: So this is just an example of some of the different toll-like receptors. There are actually more of these now that have been described. And these are some of the different ligands that these toll-like receptors recognize. DO NOT memorize each toll-like receptor and each ligand. I just wanted to show you that they recognize different ligands that are expressed by different pathogens. Slide 35: And so they toll-like receptors recognize many different pathogen associated molecular patterns that are associated with different kinds of pathogens. So this is a complicated slide and I dont want you to get bogged down by the details. I just wanted to get across that this stuff happens when the PAMPs interact with the toll-like receptors which induces a cascade of biochemical signaling events that regulates gene transcription in the nucleus. This is the important part of this. So recognition of PAMPs by a toll-like receptor induces a cascade of biochemical signaling events that regulates transcription factors which regulate genes in the nucleus. And one thing that I do want you to know is that one of the transcription factors to regulate this cascade of inflammatory responses is the transcription factor NFKB. So we are going to come back to that a few more times today. Slide 36: So this is just another example of that kind of thing. So these are other toll-like receptors that induce slightly different signaling events. The end result is the same which is the up regulation of transcription events that leads the nucleus to make genes that are involved with infectious disease protection. So lets pretend that the name of this gene is tumor necrosis factor . Here it is telling you the name of the gene and actually several different genes that include the interferon genes. These are examples of cytokines. Cytokines are just molecules that are made by cells. And these particular cytokines are involved with an immune response. So interferons are particularly involved in protecting against viral infections. And that is why I am showing this in this picture because these are toll-like receptors that are recognizing viral nucleic acids. That is leading to up regulation of transcription of viral cytokine mechanisms. I am now going to go through a couple other different kinds of pattern recognition receptors. So receptors that recognize PAMPs are called pattern recognition receptors. Slide 37: So another one of these guys are called NOD like receptors. And basically the principle is the same. They recognize relatively conserved molecules made by microbes that activate gene transcription because of activation of transcription factors in this case it is also NFKB. One of the reasons that I include this slide is because when people do whole genome wide association studies and look for changes associated with increased or decreased likelihood of developing diseases. Molecules like this come out in a genome wide association screening. For example if we have a bunch of people who has Crohns disease a large percentage of these patients have

mutations that affect NOD-like receptors. The idea is that if you have altered NOD-like receptors you may be more likely to develop inflammatory bowel disease. So I am linking these molecules with human disease. Slide 38: So far everything that I have talked about has depended on pathogens. So in this top panel you should be able to recognize that that is a tonsil but it is not a normal tonsil. So this is somebody with tonsillitis that has pus in his tonsil and a bacterial infection. So that would be a classic strep throat presentation. So that is bacteria that activates pattern recognition receptors which activated an inflammatory response which is why the tonsils are big, red, and raised. But on the top right, this is someone who has a sprained ankle. There is still an inflammatory response. It hurts, it is red, it is swollen, but it doesnt look like it broke the skin. There is no reason to think that we have developed that kind of response because of some kind of bacterial infection, but we still get an inflammatory response. What about the one in the bottom left? That is arthritis. Again, there is no obvious pathogen involved. Or the final one? This is atherosclerosis and there is again no obvious pathogen involved. So this question is how you get an inflammatory response without the presence of pathogens. Slide 39: So it is really the same as everything that we have talked about before except that instead of PAMPs, these are patterns that are released from damaged cells. These are called damaged associated molecular patterns (DAMPs). They also alert innate immune cells that there is something going on that they need to be aware of. So these are molecules released upon tissue injury but they are usually not released during normal cell death. They are only released during abnormal or necrotic cell death. Slide 40: There are a bunch of different examples of these molecules which are referred to as DAMPs. And some example of these molecules include uric acid. Uric acid is normally only in the cytoplasm and would not be in the extracellular space, but if you break open a cell, the uric acid leaks out. And that can cause an inflammatory response. There are a bunch of other examples as well like nuclear proteins and stress-related proteins that are not usually in the extracellular space but are released after tissue damage. And the thing that is interesting about this is that those same pattern recognition receptors that I have alr eady talked about like TLRs and NOD-like receptors, they will also recognize these things. So the same receptors recognize both PAMPs and DAMPs. So it is confusing but it is also handy because it does the same thing and still initiates an inflammatory response. This is just another example of these called inflammasome. This receptor contains a domain called a pyrin. So one of the hallmarks of an immune response if a fever. So activation of pyrin receptors activate a fever response. And the reason they do this is these receptors activate a cascade yet again which results in the secretion of proteins that promote fever. The main protein that does this is interlukin 1 (IL1). IL1 are the cytokines made by immune cells that induce a fever response. So this is a protein secreted by innate immune cells upon recognition by pattern recognition receptors. (Break because microphone stopped working. So this is an example of an immune response. If you have destruction of a cell membrane then you will have exposure of pattern recognition receptors to things that arent normally present in intact cells. So these molecules will activated these pattern recognition receptors that can induce an inflammatory response. One of the main

reasons I include this slide is that these receptors are involved with a couple of the cytokines that we have talked about already. So these are inflammatory responses that can be induced not only by pathogens but also by sterile tissue diseases. Slide 41: So they are some examples of sterile tissue diseases. There is gout that is induced by uric acid crystal build-up which leads to inflammation. There is also periodic fever syndromes that are genetic diseases. There are a bunch of different ones, but one of them called Familial Mediterranean Fever is associated with an inappropriate activation of that pyrin piece of the molecule. So these are released all the time and you have episodic fevers. Other types of sterile tissue diseases include atherosclerosis and heart disease, some autoimmune diseases and traumatic tissue injury. Slide 42: And this is a nice overall cartoon that shows us the big picture. You have two major ways of activating these immune cells. There are PAMPs which are sometimes called the stranger model and DAMPs which are sometimes called the danger model. And with both cases they act on these pattern recognition receptors which activate innate immune cells to make cytokines that mediate an innate immune response. Now one of the things that happens later is that it also results in the activation of an adaptive immune response. We will talk about this more in later lectures. So this is the central thing that activates immunity. Slide 43: So one thing I want you guys to think about, but that I am not going to spend time right now discussing is: Why doesnt the immune system typically generate an inflammatory response to commensal bacteria? So we have all of these bugs in our gut and on our skin, but we are not all walking around inflamed. And I want you to remember that the bacteria still have PAMPs that all bacteria have. So if you think about that, it is a really important question. You need to understand how we are able to tolerate and not generate an inflammatory response to some things but to generate an inflammatory response to others. We have all this bacteria in our gut and if we generated an inflammatory response, we would all have inflammatory bowel disease when only few of us do. That is a complicated question and there is not only one right answer. But it is something to think about though the rest of the course. Slide 44: So what are the major innate immune cells that are a part of acute inflammation? So we have talked about these already, they are the neutrophils, macrophages, and dendritic cells. One way to think about this is the neutrophils are cells that are mostly in the blood stream, but are rapidly recruited to peripheral sites upon tissue damage or infection. Whereas macrophages and dendritic cells are constitutively present in tissues already. So the macrophages and dendritic cells are constantly alerting the body that something is going on. And the neutrophils come in a little bit later and consequently activate these cells. So lets go through how that works. Slide 45: So here is a macrophage and a dendritic cell and they are continuously present in tissues. You can think of them are serving a surveillance function. All these cells originally developed in the bone marrow and the migrated from the blood into the tissue where they can live for a very long time. They have receptors that recognize PAMPs and DAMPs. Slide 46: So macrophages again are continuously present and one of the things that they are really good at is engulfing pathogens. They also kill them and secrete cytokines. They usually

stay where they are and help to contain the infection. The cytokines that they make serve to attract other leukocytes. Dendritic cells are also similar, they are phagocytic when they are in the tissue. They also make cytokines. The biggest difference between them and macrophages is that they are more prone to leave the site once they get activated and migrate into secondary lymphoid organs like lymph nodes and activate an adaptive immune response. Slide 47: So first we are going to talk about macrophages. Macrophages recognize and engulf pathogens and also release these soluble factors. There are really three main classes of soluble factors that you should be aware of. One are cytokines that we have talked about already. These include IL1, IL6 and TNF. These are molecules that serve to activate neighboring cells but they also go out and activate a systemic inflammatory response. Another class of molecules that these cells can make are a class called chemokines. These molecules serve a particular function and allow the cells that they interact with to be attracted to a particular site for the chemokine to invade. So they serve a chemo-attractive function. One particular chemokine is IL8 also known as CXCL8. And the third thing that these things make are not proteins but instead are lipid mediators like prostaglandins and leukotrienes. These also provoke the early effects of inflammation such as vasodilation. These act on neighboring cells to produce the initial effects of inflammation such as redness, pain, and swelling. Slide 48: So here is a cartoon to show you in a little bit more detail some of the major molecules. And you guys SHOULD know what these things are. So we talked about IL1 already. One of the biggest things about it is that is promotes fever but also acts locally to allow cells to be recruited into the site of inflammation. TNF basically does the same type of thing by activating vascular epithelium and increasing vascular permeability which allows complements and other cells to get in which initiates that inflammatory response. IL6 also has some local effects but also has some systemic effects like all of these guys that produce a fever. IL6 is particularly famous for inducing an acute phase protein response. Examples of acute proteins are fibrinogen, CRP, and MBL which are made by liver cells as a consequence of systemic release of IL6 bind these local inflammatory cells and induces a systemic inflammatory response. People use these acute phase proteins clinically as markers of acute inflammation. So they are using CRP to look at your risk of atherosclerosis from a blood test. Now we just talked about IL8 as a chemokine. And IL12 is another cytokine that is produced early on that acts to activate other immune cells, primarily natural killer cells. And we will learn more about that later. Slide 49: So this is another important slide. You should the names of these cytokines. These are the same ones in table form which says the same thing that I just told you. Slide 50: This is a cartoon of acute phase proteins. So these are made as a consequence of IL6 production in hepatocytes and these molecules play a role in various parts of the inflammatory immune response. Some of it has to do with activating complements as shown here in the cartoon. Just to add a little bit of complexity. These a molecule primarily made by macrophages and we made it sound like there is only one type of cell. Slide 51: Actually there are different kinds of cells that can make different kinds of cytokines. As a precursor to later lectures, there are two types of macrophages. There are classically

activated macrophages (M1) which is what I have been talking about. The cells that make all of the cytokines that you need to learn on slide 48 and 49. These classical macrophages do everything I said they did but are also associated with developing a certain arm of the adaptive immune system called Th1 effect immune responses which are really good at killing bacterial cells. So there is another class of macrophages called alternative macrophages (M2). They are also immunologically important and are associated with the arm of the adaptive immune response referred to as Th2 cells. These macrophages are activated by cytokines that we havent learned about yet like IL4 with as associated with tissue repair and blood vessel development. So even though we say these immune responses do the same thing all of the time, I want to show you that there are more subtleties than that. The immune responses can be regulated by their micro-environment to do different things. And these differences are important in how these cells protect us from infectious diseases. This was in Science a few years ago because when these macrophages get around adipocytes, they alter the metabolism of adipocytes to make them more fat producing cells. Whereas the classical macrophages modulate the adipocytes so that they are more fat burners. Some people think that the regulation of these inflammatory responses regulate why some people get obese and others dont. We will get back to the Th1 and Th2 cells in later lectures. Slide 52: There are also different kinds of dendritic cells which gets even more complicated. There are lots of different kinds but we are only going to talk about two kinds. There are conventional dendritic cells and plasmocytoid dendritic cells. Conventional cells have long dendritic processes and lots of arms. The idea is that the arms can take up a lot of space in the tissue and sample lots of micro-environments. And by sampling and engaging with these pattern recognition receptors, they make cytokines that initiate the inflammatory response. They will also be migrating to lymph nodes to serve antigen presenting cells to activate T lymphocytes. These are really important in activating an adaptive immune response. Plasmocytoid dendritic cells make large amounts of Type I interferons. They are a very rare cell subset but they make the majority of Type I interferons ( and ) in our bodies. This is an antiviral response so when these cells are exposed to viral nucleic acids, they get signaled to make lots of Type I interferons very early on after viral infections. Slide 53: So this is a summary slide of Type I interferons. So one big thing they do is induce resistance to viral replication in all cells. So when cells interact with these interferons, they interfere with viral replication. When it was first discovered, they thought this was going to cure viral infections. It turned out to be wrong but they do play an important role in viral immune response. The other way that these cells play a role in defense is that they increase the expression of MHC class I on antigen presenting cells. Now we havent talk about what this means yet but this is showing you an important link between the innate immune system and the adaptive immune system. The other thing that these cells can do is activate natural killer cells to kill virally infected cells. Slide 54: So now we are going to summarize the steps of local inflammation and bring everything together. So we talked about local inflammation with an example of when you get a splinter. S you break the integrity of the barrier tissue, usually the skin. Then you release

microbes into the extracellular space. So the microbes engage with the pattern recognition receptors on resident cells like macrophages. These macrophages make cytokines and chemokines that act on neighboring vascular epithelial cells to make them both leaky to allow fluid and molecules to get in but also sticky to allow molecules that are migrating through the blood stream to get into that cell. And so you get recruitment of all of these cells into this site as a consequence of this initial reaction and they help to contain that infection. Slide 55: And this recruitment response is important to contain cells at the local site to prevent to infection from becoming systemic. When you have a systemic infection, particularly bacterial, that is was causes sepsis and what kills you. The idea is that you want to keep these things contained. Slide 56: The answer to the question on slide 56 is D) IL-12. That is the one that is involved with the Th1 response. Slide 57: One of the main cells that is initially recruited are these neutrophils. Neutrophils are made all of the time in the bone marrow and circulate in the peripheral blood. When there is an infection, the bone marrow will make many extra neutrophils to put into the blood AND the neutrophils that are already in the blood will migrate from the blood into the infected tissue. Slide 58: There is a classical sequence of events that allows the neutrophils to move from the blood stream into the tissue site of inflammation. There is a rolling step, where the neutrophils recognize certain things expressed on the epithelium. They are not stopped but they are slowing down. As they roll down the epithelium, they engage with other molecules that activate these neutrophils which then makes them stop and adhere to the surface of the epithelium. The final thing that happens is the neutrophil will migrate between the endothelial cells into the site of inflammation in the tissue (transendothelial migration). So these steps are all regulates by chemokines and chemoattractants and adhesion molecules that are all made at the site of inflammation. Slide 59: So these are really cool because they recruit cells to the highest concentration of chemoattractant. So the cells have receptors that recognize the chemokines and move up the gradient to the highest concentration. Slide 60: This is to get you guys to think about how cells know where to go. One way to think about chemokines is that there are like an area code for leukocyte trafficking. So we have a white blood cell with different chemokine receptors. So in the example the cell will move to the left because of which receptors it is expressing. So you regulate where cells go in the body by which chemokines you produce and which receptors are expressed. Slide 61: So there are different classes of chemokines but there are also adhesion molecules. And different receptors are expressed on different types of white blood cells. These things vary depending on the state of activation. So this is a complicated slide, obviously DONT MEMORIZE this. It is just showing that there are a bunch of chemokines and receptors that regulate where cells go. And clinically this is very important if you want to develop therapies in order to prevent cells from going to different spots. For example you could try to cure MS by

preventing cells that cause MS form going to the brain. So they have developed agents that actually do that. These could alos be used to develop therapies to inject cells to go to tumor cells and kill them in someone with cancer, These cells need to be able to identify where these tumor cells are and how to get there. And if we understood that better, we could develop cells to target these cancer cells. People are starting to do that experimentally right now. Slide 62: So this is again a paradigm for how this works. You have different steps of the leukocytes going through the blood. They roll and stop and adhere and then finally migrate through the epithelial cells. Slide 63: And they do all of this by expressing receptors for in this example IL-8 which we talked about. And these receptors will engage with the IL-8 that is being expressed on the surface and allow these cells to stop rolling and adhere to the vascular epithelium. Slide 64: Movie on leukocyte extravasation. So there are really three classes of molecules. The selectins, integrins, and chemokines which are all in the outline. Slide 65: Movie of neutrophil chemotaxis. This is a movie of a white blood cell hunting and killing a bacteria. Slide 66: So the question is: What happens when the neutrophil catches the microbe? It is the same old story that it engages with these pattern recognition receptors that facilitates phagocytosis and killing of the microbe. Slide 67: There are a variety of ways that this works and this is a cartoon showing you some of them. So they internalize the microbe and then the granules of the neutrophils acts as lysosomes to release toxins that will degrade the cell membrane and lyse the cell. There are also changes in the pH that play a role in the degradation. So there are a lot of things that allow the neutrophil to kill the microbe. Slide 68: This is a list of enzymes and molecules that are used to kill the microbe. All of these things play an important role in the neutrophil. These are also used to try to remove nutrients from the microbe. Slide 69: So there are a variety of different clinical deficiencies that are associated with leukocytes. You dont need to memorize those right now but you will need to memorize them by the end of the course. So this is an explanation of how certain diseases are developed from leukocyte modifications. Slide 70: Macrophages and neutrophils are important. They are important because they clear extracellular pathogens by phagocytosis. They produce chemokines and cytokines for leukocyte recruitment. And their function is to contain the infection. They also play a role in initiating adaptive immunity.

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