Immunology Esonero 1 PDF

Immunology Esonero I  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 At the beginning of the 1900, Emil von Behring won the Nobel Prize because he was able to isolate what was called an “anti-toxin”, which means actually “antibody against a toxin”...

Immunology Esonero I  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 At the beginning of the 1900, Emil von Behring won the Nobel Prize because he was able to isolate what was called an “anti-toxin”, which means actually “antibody against a toxin”. These antitoxins were used to fight Diphtheria, a disease due to some toxins released by bacteria. Then, someone discovered the complement system, someone the blood groups and someone else the mechanisms underlying tolerance, that means that our immune system, in some way, is educated to recognise our molecules (proteins, lipids) as self and consequently not dangerous in order to switch off its function against them. Three years ago, James Allison and Tasuku Honjo won the Nobel Prize for discovering some negative regulator molecules in the immune system, that gave the idea to use them as targets in fighting cancer. They developed antibodies against these molecules, that are actually used in clinical practice for fighting lung cancer, melanoma, breast cancer. Every day the FDA (Food and Drug Administration Federal Agency) is approving these antibodies for many types of cancer. Checkpoint immunologici che quindi vanno ad inibire il sistema immunitario per permettere alle cellule tumorali di sopravvivere HISTORY OF IMMUNOLOGY Esonerato a diseas The term “immunity” comes from a Latin word, immunitas, that means “to be& exempt from”. In the XVI century it was already known this first concept of immunity: several writers in the past proved in their books that during a plague people who survived to the first encounter with the disease were able also to survive, or at least not to become ill, at the second encounter with the same pathogen. i However, our system is preserving health conditions against both external intruders and some internal molecules, called antigens, that are released or become available and they can contact our system after, for example, some pathological conditions, when cells are dying or are deeply damaged. In 1796, an English physician called Edward Jenner was laying the foundations for the birth of the real immunology. He did a very important experiment, the first control experiment of immunization. At that time the smallpox was a very dismal illness, that affected not just people but also -> livestock, destiame o such as cows. Jenner realised that smallpox in cows presented weakened symptoms compared to human beings. For this reason, he tried to inject the material taken from an infected cow in an 8- year-old boy and then, after two months, he injected the same boy with the material obtained from the human disease. As a result, the kid did not get sick: he got immunized. In this way, without knowing any molecular basis of the pathology, Jenner demonstrated that our immune system is able to react and to remember. For this reason, this experiment is considered the birth of immunology. I ↓ contactwith In 1884, Robert Koch suggested the theory of microbes. He stated that germs, invisible to naked V eye, were the real responsible for our diseases. This turned to be partially true, because nowadays we also know the importance of genetic predisposition to some diseases and DNA mutations. Immune deficiency or over activation of immune system Around the same years, in 1880, Pasteur was using bacteria, weakened in lab, to immunize animals against a viral infection, so he called this approach “vaccine” coining this word to honour Jenner I # > (vaccinus means “derived from cows” in Latin). assiour hottemperature nutrients and oxygen why he develops? defense so numerous Everywhere there are microdes. Their drear is to colonize a mammalian WHY DO WE NEED THE IMMUNE SYSTEM TO MAINTAIN THE HOMEOSTASIS AND THE HEALTHY CONDITIONS? We have many microbes around us. Indeed, humans can offer them great conditions, such as a stable temperature (37 degrees), unlimited energy and nutrients production, a way to spread in the body (through the blood circulation) and, since humans are so numerous, microbes have many places to colonize, even if someone gets sick and dies. 2  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 - HOW DO MICROBES COLONIZE HUMANS? They had to invent different strategies to colonize and enter our body. Bacteria generated distinct types of relationships: many of them are beneficial for us (for example in our mucosa or in our skin), whereas some of them are pathogenic. These last ones can be distinguished in extracellular and intracellular pathogens, based on the modality of proliferation. The extracellular pathogens are able to proliferate. even in our body fluids, in an extracellular environment. They eventually can enter, after proliferation, in the blood system and spread around in different districts and organs. These are mainly bacteria, fungi or big parasites, like worms. The intracellular pathogens need to enter in our cells to proliferate and they are not able to stay long or survive outside our cells. This is typical of viruses, but also of some bacteria and parasites. Spread and give systemic infection The process of attachment and entrance into the cells is called infection (usually this term is used with a general meaning). After infecting our cells, these viruses or bacteria can proliferate a lot and eventually kill these cells to spread in the neighbouring ones. Moreover, if they infect cells, such as blood cells, or extravasate in the blood, even these intracellular pathogens can diffuse in our body. Both extra and intracellular HOW CAN THEY DAMAGE OUR CELLS? ↓ 1 Some bacteria can produce and secrete some poisonous molecules called exotoxins. Some scarlattina examples of these toxins are the ones causing Diphtheria, Tetanus, Cholera, Yellow Fever, Scarlett Fever. What is poisonous for us is not the bacteria itself, but the toxin it secrets. They can circulate in our fluids or in our blood. 2) On the other hand, some other bacteria can contain, on the surface or inside the cell, some poisoning molecules called endotoxins. For instance, this is what triggers Sepsis (a general inflammatory disease that affects many organs), Meningitis, Pneumonia, Viral or Bacterial Dysentery. Intracellular 3) Viruses (and some bacteria) are able to kill the cells they enter. They infect a cell, start to proliferate and produce more and more virions: because of their number, they wouldn’t be able to survive inside the cell, that’s why they kill it and infect other ones. 4)The last way by which microbes can damage our cells is by activating a strong and deregulated immune response. Sometimes they activate the immune system too much, so the normal immune response actually progresses to a chronic inflammation (a condition where the immune system is always active and fighting, even when the bacteria and the viruses are no longer present, being effective also against our cells). In other cases, they can induce some reactions against our cell molecules, causing an autoimmune reaction. Finally, they can be very dangerous for immunodeficient people, who do not have a complete functioning immune system able to fight the pathogens. In this case, even not so harmful bacteria can become very dangerous. 3  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 This continuous pressure from microbes (in the air, in the water, almost everywhere) on our immune system has forced the mammalian to develop some smart approaches and strategies to defend our body. It seems a “horse race” between the pathogens and the immune system: the pathogen develops a strategy to enter our cells and the immune system has to develop something that has to block this new strategy. So, there isn’t always a “winner”, but there’s a sort of co-evolution. When the pathogen develops a new strategy, then the immune system can adapt and, in some way, escape from this strategy. The same happens for the viruses: they evolve trying to better infect our cells, because, as said before, we are a good environment to survive and proliferate in. TWO TYPES OF IMMUNE RESPONSE Until this century, there were two schools of thought: - The first one believed that the immune response against pathogens was preferentially cell- mediated. This meant that there were cells (they’re not always known) that were able to fight the infection - The other school thought that healthy conditions were maintained thanks to some soluble factors, referred as humoral immunity (humor in Latin means “fluid”). Ele Nowadays we know that both responses co-exist and both are involved in any kind of fight against pathogens. We have cells called Leukocytes (White Blood Cells), that are abundant in the blood (through which they can reach any of our tissues), but they are mostly present in tissues and in lymphoid organs, such as tonsils, spleen and thymus: organs made of lymphoid tissue, associated with the mucosa (the epithelial layer that covers gastrointestinal, respiratory and urogenital systems). On the contrary, the soluble factors, referred as humoral immunity, are mainly the antibodies. These molecules are present in the blood and other fluids and are a sort of defence against the intruders, even if they are not the only ones. The cell-mediated immunity and the humoral immunity are cross-talking, thanks to other soluble factors called cytokines. The antibodies, for example, are produced by a peculiar subpopulation of leukocytes and they are secreted when these leukocytes are activated. This activation is mainly due to a specific recognition, but even because of the presence of specific cytokines that alert these leukocytes and stimulate them to get involved in the fight producing antibodies. Recognition Activate Produce Y Leukocytes Y Antibodies I Alert from cytokines 4  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 Cell mediated A Humoral The table summarizes the main features of the two categories of the immune cells: - the ones that are involved in the innate immune response - the ones that are responsible for the adaptive immune response. INNATE IMMUNE RESPONSE Once an external pathogen has entered our body (passing through the skin), there are several methods to activate the immune response. The innate cells, also called native cells, since they’re present in every organism and they’re phylogenetically very old (some of them are even present in the fungal kingdom or in i invertebrate &m organisms). They recognise that there’s something to fight in a very unspecific and wide way. They · can recognise when our cells are morendo damaged by a bacterium, a virus or any other injurious stimulus (e.g. metabolic reasons) and are dying because the plasma membrane gets broken and leaks out its content; these released molecules, called Damage Associated Molecular Patterns (DAMPs), that are usually inside the cell, can be recognised by the innate cells. So, with the term DAMPs we consider all the molecules deriving from a damaged cell. Moreover, these innate cells can also recognise Pathogen Associated Molecular Patterns (PAMPs), that are unspecific molecules present on the surface of bacteria, virions or infected cells (after the infection). E - The native cells can activate themselves after the recognition of PAMPs and DAMPs thanks to the Pattern Recognition Molecules (PRMs), which can be soluble (when they attach the DAMPs or PAMPs, they need to be recognised by the innate cells thanks to specific receptors to activate the immune response). However, most of them are expressed by the innate leukocytes, on their surface or inside them (in the endosome or in the cytoplasm) and they can recognise different common E molecules belonging to bacteria and viruses. Since the innate cells are very abundant not only in the blood but especially in the skin and mucosae (they can actually be residential in all our tissues) and since they’re so numerous compared to the adaptive cells, they always respond with the same intensity: when they recognise, through the PRMs, the DAMPs or the PAMPs, they immediately and furiously become effectors and get involved in the fight against the pathogens. Their response is always similar, every time they encounter the same pathogen. For this reason, the innate immune response has no memory of the encountered - pathogens. This is a peculiar feature of this system, while the opposite happens in the adaptive immune response. They cannot recognise completely different from adaptive immune response I - S The innate response involves different categories of cells, tissues, molecules and processes. PROTECTION FROM THE ENVIRONMENT - The first real barrier, that can be considered as an innate immunity, are the anatomical barriers: the skin and the mucosae. They are protected by some leukocytes and they’re able, due to their intrinsic 2 features, to resist the invasion. There are also physiological barriers, such as low pH in our stomach or intestine, or the secretion of lysosomal or hydrolytic enzymes in the saliva and body fluids. 5 N° di innate cells >>> N° di adaptive cells  Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 3) The complement system is a circulating set of proteins (proteases) which is inactive in healthy conditions, but it can be activated to directly kill the pathogen by forming a pore into the surface membrane of the bacteria/virions/infected cells. The complement may be considered as a part of the innate response because it is always present and, after its activation, the cascade it starts will lead to the killing of the pathogen. 4) Other players of the innate response are the PRMs, receptors expressed on the leukocytes or soluble proteins, like the pentraxin or the C-reactive protein (its value gets high when an inflammation occurs; it is able to bind some molecules on the surface of bacteria, so it can be considered a PRM that will bind some receptors expressed on the leukocytes and activate them). Three processes are important for the innate response: - Inflammation: it involves the blood vessels. The swelling and the redness are two typical signs of inflammation, because the blood vessels increase their calibre around the infectious area. It’s important to make the leukocytes extravasate and getting outside in order to fight the intruder that has invaded the cells. The inflammatory response is mainly activated by the secretion of inflammatory cytokines, that can be secreted not only by innate cells but even by endothelial or epithelial cells that are encountering the pathogen. - Phagocytosis: the leukocytes that arrive in the infectious area are mainly able to phagocytise and directly kill the pathogens or the infected cells. It is mainly performed by these kinds of innate cells: neutrophils, macrophages or dendritic cells. Neutrophils are the scavenger cells, so their function is usually to clear the area from pathogens or damaged/dying cells. The macrophages and dendritic cells, instead, beside phagocytise molecules and material, can also present some pieces of these materials to the adaptive cells, in particular to the T- lymphocytes. So, in some way, the macrophages and dendritic cells are the bridge between the innate response and the adaptive response. - Target cell lysis: innate cells that are able to directly lyse the target (pathogen or infected cell) are again neutrophils, macrophages and natural killer cells (NK) ADAPTIVE IMMUNE RESPONSE As well as the innate cells, the adaptive cells need to recognise the pathogens to get involved in the fighting. However, there are many important differences between these two types of cells: the adaptive cells specifically recognise some unique sequences/molecules on the pathogens, the so- - called antigens (this term means “generator of antibodies”, because it was first defined as a molecule able to induce the antibodies and, therefore, the humoral response). So, the adaptive immune cells are able to specifically recognise antigens thanks to a very peculiar receptor called “antigen receptor”, in particular TCR (T cell receptor, when it’s expressed on T lymphocytes), or BCR (B cell receptor, when it’s expressed on B lymphocytes). The adaptive cells are called - lymphocytes because they are mostly abundant in the lymphoid organs. There are two big categories of lymphocytes: B and T. Each cells express numerous molecules but identical - Since each lymphocyte has only one type of receptor (present in thousands of copies) that can recognise just one specific kind of antigen, and since there are uncountable antigens, there are billions of lymphocytes, each one having a different receptor. This means that when a pathogen enters a cell, if there’s only one lymphocyte able to recognise it, this lymphocyte will need some time for the recognition and it has to replicate in order to create a real “army” against the pathogen. For ritardata this reason, the response will be for sure delayed. As said before, the innate response is immediate because of the unspecific recognition and because there are a lot of leukocytes that are able to fight any kind of pathogen. In contrast, the lymphocytes need to proliferate and so their response will come later. Moreover, during this proliferation most of 6 Ogni lymphocytes ha un solo tipo di Receptor sulla sua superficie (che riconoscono quindi tutti lo stesso antigene). Poiché ci sono una miriade di antigeni, ci saranno anche una miriade di lymphocytes ciascuno che riconosce un antigene diverso. Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 Memory cells r them will become effectors, “soldiers” against the pathogen, but some of them will become long-live cells that can remember what they have encountered the first time and so the second time they will meet the same pathogen they will immediately proliferate to fight it. Therefore, the adaptive immune response has memory of the encountered pathogens. This kind of strategy only occurs in vertebrates, and it is more complex in mammalians such as humans. lezione 1 1/10 GENERAL FEATURES OF THE ADAPTIVE IMMUNE CELLS The adaptive cells are mainly divided into two populations: B lymphocytes and T lymphocytes and each cell has a very specific receptor. Lymphocytes are characterized by five important features: garantito 1. Specificity (related to the receptors): it is warranted by the T cell receptors (TCR) and B cell receptors (BCR). As it can be observed in the image, pathogens 1, 2 and 3 express very common molecules on their surfaces, that can be recognised by PRMs or the receptors expressed by the innate leukocytes. Nevertheless, each of them has also some peculiar molecules: for example, pathogen 1 has specific molecules that are not present in pathogen 2 or 3. Each unique structure on the surface of a pathogen can be recognised by a specific adaptive immune cell presenting a specific receptor. TCR or BCR 7 Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 2. Task division: every cell has its specific job. The B cells ìproduce the antibodies, that are responsible for engaging other leukocytes to allow the clearance of the pathogens. Recognise antigen Helper ↑ The T cells, instead, can be divided into two subpopulations: cytotoxic T cells and helper T cells. The latter>produces cytokines (soluble factors that allow the communication among all the leukocytes). On the contrary, the cytotoxic T cells can directly õ kill the pathogens or the infected cells. ↳ cutoxie A big difference among the B cells and T cells is how they recognise the pathogen: they both have receptors (BCR or TCR), but the BCR is able to recognise the native circulating pathogens present in our fluids (through - a molecule on the pathogen’s surface or a soluble toxin). T cells instead need to recognise the specific molecule presented by another molecule expressed by our cells: the Major ! Histocompatibility Complex molecule (MHC). In this example, on the left, there’s a cell infected by a virus. Once the virus enters the cell, the viral proteins are uploaded on the MHC which can be recognised by the TCR. The recognition of the antigen wouldn’t have been possible without the MHC. This is why macrophages and dendritic cells, that phagocytise the pathogen and then digest it, will present the molecules of the pathogen to the T cells. This - process is called antigen presentation. 8 Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 3. Memory: it is due to the differentiation of some lymphocytes (involved in the fighting) in memory cells. This process is common in both B and T cells (the scheme down below is referred to the B cells). As it can be seen, a B cell recognises and binds, through its BCR, an antigen (toxin or molecule on the surface of a pathogen). After that, the lymphocyte becomes active and proliferates, producing identical (billions of) clones to get involved in the fighting. Since they’re B cells, some will differentiate in plasma cells that will produce antibodies*, and some will differentiate in memory cells, long-live B lymphocytes, that will be able to immediately recognise a second/third encounter with the same pathogen and so will be instantaneously engaged in this kind of proliferation to go against the pathogen. *antibodies are the soluble form of the BCR that was present on the original B cell. 4. Diversity D Each B cells and T cells express a different receptor-antigen and for this is different from all other B and T cells 5. Tolerance: since the lymphocytes, in a specific way, generate all these different receptors, it is possible that some of these receptors can also be directed to some of our proteins, because they are able to recognise uncountable antigens (indeed, in vitro, if you take blood cells and add many different antigens, even the synthetic ones, leukocytes are able to activate themselves). So, even if this is an advantage because we are certain to be protected against every pathogen, it is necessary to be sure that some of these receptors are not actually directed against our self-molecules: this is what happens during the maturation. In this process, that takes place inside the lymphoid organs, both B and T cells are tested against the self-molecules and if they have the wrong receptor (because they recognise the self-molecules), they will die. This is what is called central tolerance. 9 Fregnan Giada/Conte Laura - Lesson 1 - IMMUNOLOGY (Prof. Paola Cappello) - 05/10/2021 If some of these “wrong lymphocytes” manage to escape from this strict regulation and get outside from the central lymphoid organs, reaching the peripheral areas, there’s another mechanism that can warrant the peripheral tolerance. This is what actually maintains our homeostasis and health conditions, even having such dangerous structures patrolling our body. In conclusion, the innate and the adaptive responses are overlapping in the fighting against a pathogen. There are three different phases that are inter-connected: The first phase occurs Je when body barriers (sentinels), once they’re attacked by some external pathogens, immediately secrete alarm signals. These signals led to the second phase, engaging 1: the innate cells and all the cells of the innate immunity that secrete other cytokines and chemokines to communicate each other and with the barriers. Then, the complement activation takes place and the activated innate cells can start to phagocyte (or lyse) and kill the pathogen. All these processes happen rapidly, from hours to few days (4-96 h). Is Later, during the third phase, the delayed response by the adaptive cells occurs, because T and B cells need the antigen presentation to recognise the antigen. Moreover, they need guiding inflammation in order to extravasate and reach the site where the fight is happening. Once there, the cytotoxic T cells can directly kill the pathogen or the antibodies can be produced. However, the intervention of adaptive cells starts after at least 5 days, because they need to proliferate. Every phase is smartly regulated: even the B and T cells secrete cytokines that help the long-lasting activation of some innate cells. - From a clinical point of view, the immune system is responsible for the clearance of damaged/infected/dead cells, but it can also recognise and disrupt cancer cells. On the ! other hand, however, the immune system S Chronic inflammation · Hypersensitivity and allergy can be the cause of some pathological Distruption of cancer cells Autoimmune diseases condition, such as immunodeficiency or ·Clearance of death and damaged cells Destruction of infectious organisms Transplant rejection Immunodeficiency during transplantations (the immune system is responsible for the rejection of a transplanted organ); during hypersensitivity or allergic reactions, when our system respond in an exasperated way against innocent invaders (like food) instead of pathogens; it can induce chronic inflammation and be harmful against our cells; it can react against our cells and be responsible for the autoreactive reactions. 10  Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 INNATE IMMUNITY. ⑧ The first line of defence is represented by the anatomical barriers: the skin (2 m ) and the mucosae in the respiratory system (60 m ), the gastrointestinal system (30 m ), urogenital system… All the anatomical barriers are characterized by: - Resistance to many kinds of traumas - Elasticity, which allows contraction without provoking any kind of damage - Impermeable Autorinnovabile - Self-renewing (all the cells of the skin and mucosa proliferate a lot every day) and self- repairing in case of damage - Ability of responding to mechanical stress e - Ability of reacting to UV radiation, which is one of the most injurious stimuli for our skin (hence our skin is also made up by melatonin-producing cells that counter radiation) - Difficulty to being overcome by microbes - Involvement in our body thermo-regulation - Intrinsic anti-bacterial activity L & The anti-bacterial activity is provided by the so-called chemical barriers, which are represented by soluble factors secreted by the epithelial cells belonging to the anatomical barriers (and not only). These are very small peptides with anti-microbial activity (defensin, cathelicidin, histatin and pentraxin), but also those molecules that connect and interconnect all the immune cells (cytokines na and chemokines). When an atomical barrier is damaged by mechanical stress, metabolic stimulus or attacked by pathogens, it secrets alarmines, which are self-component of our cells such as HMGB1 (which is a protein binding the DNA), or the S100 protein (which is very abundant in the lungs and the respiratory tract). Peptides β-defensin family Disrupt microbial membranes; attract dendritic cells Cathelicidin family Disrupt microbial membranes Histatin family Disrupt microbial membranes Pentraxin family Activate phagocytosis and microbial disruption Cytokines Interleukin 1 (IL1) Triggers immune response Interleukin 6 (IL6) Triggers immune response Interleukin 10 (IL10) Dampens immune response Interleukin 25 (IL25) Trigger immune response and Interleukin 33 (IL33) favour Th2 cell activation Chemokines CXC family Attract and activate immune cells CC family Attract and activate immune cells CX C family Attract and activate immune cells Alarmins Uric acid Trigger immune response and favour Th2 cell activation High mobility group box1 (HMGB1) S100 protein All these soluble factors also give the anatomical barriers the ability to eventually immediately kill the pathogen. 1  Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 Example: THE SKIN. immature dendritic cell - sweat produces or MUCUS I impermeabilisation of the skin The skin is the most external layer of our body, which underlines the importance of the presence of a very thick stratum corneum, because it inhibits the attachment and/or penetration of any bacteria or viruses. Indeed, this layer is the first physical barrier. Underneath the stratum corneum there is the dermis, which contains sebaceous glands, stromal cells (fibroblasts), myofibroblastic cells and Stree sweat glands. The latter are very important because the sweat (as, in general, all the other body- fluids) contains many chemical substances that are able to kill the pathogens. About the immune cells (which are indicated in red in the previous image), they are more present in the tissue rather than in the blood. The most abundant immune cells in our skin are: - Langerhans cells (belonging to the dendritic cells family, which is one of the most common one and is characterized by long dendrites) - melanocytes (which protect our skin from radiations) - macrophages - neutrophils - mast cells mastrociti Lymphocytes, even though they interact later with the pathogen (because they belong to the adaptive immunity), can also circulate in our skin. 2 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 The thick corneal stratum is also necessary to protect us from mosquitoes’ bite. The female of Anopheles, for instance, is able to introduce the protozoans and, by doing so, they can transmit Malaria. Unfortunately, this kind of barriers isn’t always efficient because the mosquitoes’ proboscis is so huge and potent that it can invade the skin and even reach the capillaries and vessels. THE MUCOSA OF THE LIPS AND MOUTH. Confronting the skin and the mucosa, the latter has a much thinner corneum layer because it doesn’t have the same importance in those two structures. Inside the mucosa there are other structures that are more important than the corneum layer, such as the serous mucus glands (or salivary glands, it depends on the mucosa taken into account) because they contain the chemical substances that are able to kill the pathogens. 3 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 About the immune cells (indicated in red), there are: - the Langerhans cells - a lot of lymphocytes, however they’re not the ones belonging to the adaptive immunity (they’re called innate lymphocytes). Soluble factors produced by the mucosae. Lysozyme. The salivary glands are great producers of lysozyme (also known as muraminidase), discovered by Sir Alexander Fleming in 1922. This enzyme is able to break the peptidoglycan component of the cell wall of many bacteria, especially Gram-positive bacteria, by hydrolysing the 1-4 glycosidic bond connecting N-acetilmuramic acid and N-acetylglucosamine. Saliva is the main fluid containing the lysozyme, however it is also produced by the Paneth cells (epithelial cells present in the gastrointestinal tract) or contained inside the granules of granulocytes and monocytes (belonging to the adaptive immunity). Finally, lysozyme is also present in some epithelial tissues, tears and milk, and it’s very important during breastfeeding. Cathelicidine. These are small peptides, defined as anti-microbial because they can kill the bacteria by destroying their lipoprotein membrane even when the pathogen has been enveloped in phagosomes. In this respect, it’s important to remember what the phagocytosis process is: the external bacteria are ingulfed by the plasmatic membrane, creating a phagosome, which can fuse with the lysosome. This process provokes the destruction of the bacteria’s membrane, and so their death. danneggiare Cathelicidine must be efficient without harming our cells, that’s why they’re produced as a precursor (18 kDa), which is then cleaved into two active molecules only when they’re requested inside the cells. Both the synthesis and the cleavage are stimulated by the production of cytokines, that are released once the epithelial (or, in this case, mucosal) cells register a damage. Cytokines can alarm the neutrophiles or the other epithelial (or mucosal) cells to cleave and activate the cathelicidine. THE BRONCHIAL MUCOSA. ↓ 4 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 There’s no corneal layer, which is replaced by the mucus, produced by the serous mucus glands. The cilia on the columnar cells trap microbes in the airways. However, sometimes this mechanism isn’t enough, that’s why the mucus is so important: this active barrier traps the microbes and allows the residential innate cells to phagocyte them or kill them. The mucus contains some of the cathelicidins, lysozyme and other anti-microbial molecules. About the immune cells, the bronchial mucosa contains: - dendritic cells - innate lymphocytes - a lot of mast cells, which are the most abundant residential innate cells in the circulation, as well as the neutrophils, and they have a lot of granules containing anti-microbial substances. THE DIGESTIVE TRACT MUCOSA. The digestive tract doesn’t have ciliated cells; however, it is provided with Goblet cells, which produce the mucus (even though the production of mucus is minor than the one in the bronchial mucosa) and Paneth cells, which produce cathelicidins and so on. This structure is characterized by some invaginations, in order to increase the surface of absorption. Because of the presence of the microbiome (which may also be beneficial for the organism) in the intestine, this kind of mucosa is enriched by lymphoid tissue know as Mucosa-associated lymphoid tissue (MALT). There are also: - Macrophages - plasma cells (which are differentiated B-cells) - some B-cells - lymphocytes in the MALT 5 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 Paneth cells are involved in the production of DEFENSINS, small peptides (29-34 aa) belonging to two main families (α-defensins and β-defensins) that differ in the disulfide bonds. They’re able to directly lyse any kind of pathogen (fungi, bacteria, viruses), because they interfere with the molecules of the bacteria wall or the proteins outside of the viruses. Defensins are produced not only by Paneth cells, but also by neutrophils (also known as PMN, polymorphonuclear cells, which is an acronym used for all the granulocytes), NK cells and they’re also contained inside the granules of the CTL (cytotoxic T-cells). Because of their anti-microbial activity, defensins are also important to regulate the balance of the intestinal microbiome in order to maintain the homeostasis: the excess of the bacterial presence may damage the mucosal or epithelial cells. THE RECTAL MUCOSA. Because this type of mucosa is very close to the external environment, it has a very thin corneal stratum. Mechanically, physically and chemically speaking, this barrier should protect our inner body. It is characterized by: - lymphoid tissue - dendritic cells (Langerhans cells) 6 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 GENITAL EPITHELIUM. Comparing these two structures it is evident how the penis is characterized by a corneal layer, whereas the vagina is provided with a mucous layer. In both cases, the epithelium is enriched by residential innate cells (Langerhans cells, macrophages) and less lymphocytes than the other types of mucosae. This drawing resumes all the biological and chemical barriers that cover the outside and the inside of our body. An important component of this barrier is represented by the microbiome (human microbiome is made up by about 4,5 kg of bacteria). It is composed by more than 3000 species of bacteria. The balance between the bacteria and the human cells is about 10:1. This homeostatic N state must be maintained in order to activate the innate and adaptive cells in a proper way, without provoking an inflammatory reaction (this concept will be further explored in the next lessons). 7 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 PENTRAXIN (PRM). Pentraxin family is made up by multimeric proteins (200 aa) made up by five monomers bound together. They are able to bind some molecules on the surface of the pathogens and, because of that, they have been defined as the “ancestral antibodies”. Thanks to this bond, these proteins activate the innate response (for example, they activate the complement cascade or the phagocytosis). This big family is divided into two sub-families: - short-chain, for example the C-reactive protein (whose haematic concentration rapidly increases during an infection or inflammation process), or the serum amyloid (SAP, an abundant anti-microbial in the airways) - long-chain, for example the PTX3 Both the long and short chain pentraxins are produced not only by innate cells (monocytes, dendritic cells, macrophages), but also by the epithelial or endothelial cells. They’re considered as a class of PRR (Pattern Recognition Receptors), however they’re PRM (Pattern Recognition Molecules) because they’re not anchored on the cellular membrane, but they’re secreted. Once they’ve bound the pathogen, the pentraxins activate the innate cells thanks to specific receptors. What happens when a barrier is broken? Once the bacteria reach the lesion and start to invade the organism, the epithelial cells release alarmines or cytokines and eventually even the blood coagulation cascade occurs because some capillaries may be broken too. All these events belong to the so-called humoral response (because there is the secretion of soluble molecules) and they activate the residential innate cells which may control the invasion by releasing other factors allowing the healing of the damage, even before guarigione recruiting other cells from the blood circulation. reclutare 8 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 Simili ai mastocytes - Granulocytes —> neutrophils, basophils, eosinophils + mastocytes Agranulocytes —> lymphocytes and monocytes THE INNATE CELLS. The innate cells are the most abundant in the tissues as well as in the blood, and so they are the first ones to respond to a tissue damage. Their response is characteristically quick and potent. These cells are: mastocytes, neutrophils (granulocytes in general) and the innate lymphocytes (ILC, innate lymphoid cells). The innate cells, together with the epithelial cells, secrete the alarming signals, which affect the endothelial cells of every capillary and vessel nearby. This kind of factors (for instance, histamine which is released by mastocytes, or the serotonin) affect the permeability of the vessels: the endothelial cells, as a matter of fact, aren’t tightly adherent one to another, but they’re separated by some gaps. These gaps allow the exit of fluids, which induces the “stasis and rolling” allowing all the immune cells (neutrophils, monocytes, lymphocytes, eosinophils…) circulating in the blood, to adhere to the vessel’s wall once they’re pushed against it by the red blood cells. After adhering to the wall, these cells start to extravasate. The picture illustrates the transversal section of a vessel, showing the presence of an endothelial cell, a red blood cell. Once the alarmins are released, some gaps appear between the endothelial cells allowing the extravasation of the innate cells. The latter, of course, have to activate different pathways in order to allow this process. The extravasating cells are mostly represented by the polymorphonuclear cells and that’s not by chance: their polylobate nucleus allows them to squeeze the nucleus itself inside the gap. 9  Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 After the extravasation, a lot of innate cells are recruited in the site of damage or infection; they try to kill all the bacteria and, after 2 days, most of them die rigonfiamento provoking swelling and, in some cases, pus (which is made up by bacteria and neutrophils) is released. The extravasating cells reach the site of infection thanks to other inflammatory mediators, like the cytokines and the chemokines. These are released creating a gradient: the most abundant mediators are located close to the damage cells, whereas they’re less present nearby the vessels. Thanks to this gradient, these mediators are able to recruit the requested cells (chemoattraction). The potency and velocity of the innate response is quite efficient to rapidly kill the bacteria; however, : it’s important to consider that it’s potentially dangerous for our cells, that’s why the inflammatory response has to end very quickly. The five features associated to an inflammatory response are: - Rubor, which defines the damaged area - Calor, because of the increased blood flow (which is warm) NON SO - Tumor, which indicates the swelling fatto - Dolor, because some of the mediators act on neuronal cells - Fuctio lesa, when the inflammatory response is quite intense and long-lasting, and it means that there’s a loss of function for some cells in the tissue The table on the left indicates the average percentage of the immune cells: basophils, neutrophils, eosinophils (generally called granulocytes, or PMN) and monocytes belong to the innate immunity, whereas the lymphocytes belong to the adaptive immunity. It’s evident how the most abundant are the neutrophils and then eventually the lymphocytes. All the other ones are less abundant, however they can proliferate and become abundant when they’re needed. 10 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 no no granules - potenze BASOPHILS AND MAST CELLS. Basophils and mast cells (or mastocytes) share some features: - they both contain a lot of granules (even though the mastocytes present a much higher number of these granules) - the granules in both basophils and mast cells may be histologically studied by using the same stains !(basic solutions) It’s important to remember that the mast cells (which are mainly residential cells) are almost twice the basophils and they’re weaker. The mast cells are also characterized by very small invaginations of the membrane. Mediators released by basophils and mast cells. lezione 5 8/10 These cells release: - Pre-formed mediators present within cytoplasmatic granules: for example, the histamine and serotonin (which are able to increase the vessels’ permeability), some proteases (such as the tryptase and the chymase, which breaks the bacterial wall), or the TNF-α (Tumor necrosis factor-α) which is a cytokine that activates other innate cells. All these mediators are present in the cell even when it isn’t active, but they’re ready to be immediately used after they recognize some alarms coming from the mucosa or epithelium - Mediators synthetized after degranulation: thromboxane, prostaglandins, leukotrienes, PAF (Platelet che perde Activating Factor), Eosinophil chemotactic factor. All these factors derive from a leaking content that is usually contained in the plasmatic membrane. They’re ready to be released after few hours because it’s only necessary the activation of some enzymes which allow to release this content from the membrane and to turn it into these mediators. These mediators also affect the blood vessels, but also other innate cells (acting like the cytokines) - Mediators synthetized hours after activation: cytokines and chemokines, that recruit the proper cells needed to fight against the pathogen. They’re called “later mediators”, because they need the transcription of new genes Effect of the mediators released by basophils and mast cells: - Activation of endothelial cells - Increase of vascular permeability - Anticoagulant activity - - Attraction of neutrophils, eosinophils and monocytes (innate cells) - Increased bone marrow production of neutrophils, eosinophils and monocytes. In addition to that, basophils and eosinophils aren’t abundant in the circulation, that why the bone marrow production must be increased when these cells are required - Smooth muscle contraction - Bronchoconstriction and increase of mucus secretion, to capture and block the pathogens - Creation of a toxic environment for microbes and parasites 11 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 NEUTROPHILS. Neutrophils are similar to basophils, and they’re characterized by a polylobate nucleus. Their name refers to one of the properties of their granules: they’re histologically stained by ⑳ neutral solutions. Neutrophils contain two main types of granules: - Primary (or azurophilic) - Secondary All these granules contain lysozyme, defensins, cathepsin, some enzymes (such as the elastase, the proteinase which are able to break the bacterial wall), pentraxin, pro- Defensin (which can be then cleaved into defensin). They also include the NADH oxidase and myeloperoxidase which are crucial to produce radical oxidative species (ROS) creating a toxic environment in the autophagosome or even inside the cytoplasm of the neutrophils, in order to kill the bacteria. These ROS, thanks to the myeloperoxidase, can be associated with the chloride and create even more toxic radical species that can directly kill the bacteria. Neutrophils are more able to extravasate (the professor left two links to observe the process of extravasation: https://www.youtube.com/watch?v=B9Qi7we0Ynk https://www.sciencephoto.com/media/609709/view). Functions of the neutrophils. 1. Sentinel activity, because they’re more abundant in the blood so they’re able to monitor everything that’s happening inside the organism 2. Phagocytosis, which is a process in which the plasmatic membrane invaginates and envelops the pathogen thanks to receptors. After the phagosome is formed, it is fused with all the granules inside the neutrophil and all the content inside them start to degrade the pathogen. The - materials obtained from this process will be then discarded by exocytosis (in this way, each neutrophil is able to phagocyte a lot of pathogens, because all the material doesn’t interfere with the life of the cell). ?) The rate of the phagocytosis is drastically increased by the C3b, is a component of the complement belonging to the innate response. It can be immediately activated by the presence of the bacteria. The C3b opsonises all the surface of the bacteria and the phagosomes are provided with specific receptors for the C3b. ↳- Quella che poi vedremo nei vari pathway Opsonizzare = rendere l’antigene visibile 12 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 (there are two links to observe this process: https://www.youtube.com/watch?v=7VQU28itVVw ; https://www.youtube.com/watch?v=a1xPpsxvhVA) 3. Spiderman effect, which is due to the ability of neutrophils to release a net which can trap the bacteria. This net is made up by DNA, it directly starts from the nucleus and it can contain all the enzymes present inside the neutrophil. Thanks to this net, the bacteria may be trapped, however after this effect the neutrophil dies. After its action, the net is removed from the area, otherwise it could lead to the production of antibodies against these molecules (the innate and adaptive responses would be induced against self- components and it can provoke autoimmune diseases). (https://www.youtube.com/watch?v=TIFmtnSdolM: link to the video of this process) 4. Kamikaze effect, because after a couple of days outside of the circulation, they die together with the pathogen (their lifespan inside the circulation, on the other hand, lasts about 5-6 days) Svuotamento dei granuli ⑮ Main effects of the degranulation of the neutrophils. - Rapidly killing of invading microbes - Establishment of a toxic environment for microbes, parasites and normal and neoplastic host cells - Activation of endothelial cells: increase in the expression of adhesion molecules, WBC (white blood cells) extravasation - Chemoattraction of immune cells - Enhancement of Complex activity (Properdin, which is a component contained inside the granules and it stabilizes the components of the complement on the wall of the bacteria or parasites) - Massive death of neutrophils, dead pathogens and damaged host cells are responsible for Pus formation (because they are killed together with the pathogen) 13 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 EOSINOPHILS. They’re also granulocytes, whose granules can by histologically stained by using 0 acid solutions (they become red). Their most important characteristic is that they’re the only cells having the Major Basic Protein contained inside their granules. It’s the only enzyme able to destroy the worms’ wall. parete dei vermi (elmintil Since they’re not so abundant, when they’re needed, they have to proliferate rapidly. Their lifespan outside the circulation is shorter than the neutrophils’, as a matter of fact they live just for 6- 12 hours, eventually a day. Activities of the eosinophils: - Phagocytosis of microbes and small particles - When they degranulate, the eosinophils destroy the intruders as well damage of invaded tissue - Secretion of cytokines, enzymes and anti-microbial proteins and peptides - Antigen presentation - Anti-helminthic activity (they’re one of the few cells, if not the only ones, that are able to destroy the worms’ wall, as already mentioned) - Anti-tumor activity MONOCYTES. - Macrophages They represent 10% of our cells present in the blood, however they’re most abundant in the tissue (where they’re called macrophages). They are quite big and are characterized by a V-shaped nucleus. Their membrane is provided with a lot of receptors in order to phagocytes/kill the intruders (they’re also antigen-presenting cells). When in the tissues, some macrophages (even though they’re quite similar because they derive from the same progenitor, which are the monocytes) acquire different names: - In the epidermis -> Langerhans cells - In the lungs -> alveolar and interstitial macrophages - In the central nervous system -> microglia cells - In the bones -> osteoclasts - In the lymph organs and site of infection -> mature/immature dendritic cells (so dendritic cells and macrophages share the same bone precursor) The tissue macrophages are quite big cells (they can reach even a diameter of 80 μm) because they’re rich in internal organelles (especially the endoplasmic reticulum, the Golgi apparatus). They’re also characterized by a long life-span, in fact they’re able to live for months inside the tissue, then they’re replaced (in some cases they’re also able to proliferate). 14 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 Activities of the macrophages. - Professional phagocytosis and subsequent killing of microbes - Scavenger function: they remove old red blood cells (mainly degraded in the spleen and liver), cell debris, dead cells and extraneous particles of almost any kind - After clearing all the debris, they also secrete some factors that immediately start the tissue-repair process - Secretion of cytokines and enzymes to kill the pathogen - As the neutrophils, they produce (because they are provided with the NAD oxidase and the myeloperoxidase) reactive oxygen species and other radical species, which are toxic for the bacteria - They’re antigen-presenting cells (especially the dendritic cells), as already said The image on the left pictures the phagocytosis process. There are several types of phagocytosis, not only the receptor-mediated one, but there is also the endocytosis, the macropinocytosis (the name is based on the size of the particle that is involved). Macrophages can also trap the bacteria thanks to the invaginations of the membrane. They’re also provided with the receptor for the C3b (like the neutrophils). Once the phagosome is formed, it fuses with the granules and the lysosomes and the pathogens will be destroyed. 15 Laura Conte / Lucia Griva – Lezione 2 – Immunology (Prof. Paola Cappello) – 07/10/2021 DENDRITIC CELLS (DC). They’re very similar to the macrophages, however they’re characterized by an irregular membrane, which forms dendrites (they’re very plastic, which means that their shape can rapidly change). There are different types of DC, deriving from the monocytes (myeloid progenitors) and lymphoid progenitors. They are divided into: - Conventional dendritic cells, which are the immature ones, and they can reach the tissue thanks to the alarmins and the chemoattractant, otherwise they can be residential (e.g. the Langerhans cells in the skin). Their main function is to phagocyte everything around them (pathogens, viruses, infected cells, tumor cells, damaged cells, dying cells) and, thanks to the phagocytosis process, they differentiate into mature cells (their mature version is considered the best antigen presenting cell) - Plasmacytoid dendritic cells, which produce the interferon, IFN, type I (there are three different types), which may be IFN-α or IFN-β. These are a specific type of cytokines that are responsible to induce an efficient anti-viral response Link to see a video about the morphology of the dendritic cells: https://www.youtube.com/watch?v=w97dms9jD3I. 16 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 INNATE CELLS POPULATION PART 2 NATURAL KILLER CELLS “NK cells” means “Natural killer cells” and their function is to kill. These cells have a lot of granules inside, containing perforine and granzyme. The former is necessary to create pores in the membrane of target cells, while granzyme, which is a protease, is later used to activate other proteases, for example caspases which induce apoptosis in target cells. All the other populations of immune cells derive from, or belong to, the granulocytic/myelocytic population: they derived from a unique progenitor called the myeloid progenitor. The natural killer however, even if it participates in the innate immunity, derives from the lymphoid progenitor, just like lymphocytes, and they are morphologically very similar. Particularly, lymphocytes are quite similar to cytotoxic lymphocytes, which also have a lot of granules. Since NK cells are born to kill, they need to have some inhibitory receptors to switch off their activity; otherwise, all the weapons used by the immune system against intruders or non-self elements, can be used against our cells. Each receptor, to function, needs a ligand and, after the binding, they activate some cascade. Usually activated receptors switch on some kinase and the phosphorylation cascade leads to the activation of some transcriptional factors that by migrating into the nucleus, start the transcription of new genes. The inhibitory receptor, on the other hand, will activate some phosphatase. 1 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Education of NK cells in the bone marrow During the origin in the bone marrow the NK cells already express both activating and inhibitory receptors. The main function is indicated by which kind of receptor prevails. Usually, in a physiological state with no intruder pathogens, the inhibitory receptors prevail and the phosphatases switch off the activating receptors. The activating receptors will activate their killing function through the kinases and phosphorylation of the adaptors. In contrast, inhibitory receptors are able to maintain NK cells quiet through the activation of phosphatases, which eliminate phosphate groups from the kinases and switch off their pathways. When NKs circulate around our main tissues (or even in the blood), the inhibitory signals prevail because the inhibitory receptors are binding the MHC class I molecules. The MHC (or HLA) class I molecules are expressed by all nucleic cells: only erythrocytes don’t have the MHC class I (with some exceptions). The inhibitory receptor finds the ligands to switch off the NK function everywhere in our tissues. When the MHC class I is missing, or many other ligands for the activating receptors are exposed on the surface of the target cells, the inhibitory receptors are not activated at all and in that case, the signal coming from the activating receptors prevails. 2 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Some examples of receptors and their ligands: Important ligands: - Hemagglutinin => a viral component ->activates - MICA and MICB => they are usually exposed on our cells when metabolic stress occurs or even on neoplastic cells. They are very similar to MHC class I, but they don’t belong to the same family -> INhiD it - Molecules from the HLA complex => they are usually bound by inhibitory receptors. MHC and HLA are the same molecule, but HLA is present in humans and stands for Human Leucocyte Antigen, while MHC means Major Histocompatibility Complex and it’s usually used for mice or other mammalians. These molecules are expressed on all normal cells and even on trophoblastic ones; this is very important during pregnancy to protect the fetal growth: since the fetus carries pathogens coming from the mother and from the father, he has different HLA that can lead to an inflammatory response. However, these cells do not actually express HLA (or they express another specific kind of HLA), so they are able to switch off the activity of NK cells. Cytokines which activate NK cells Cytokines are important to regulate the functional activity of NK cells. All the cytokines in the next page are produced by macrophages or dendritic cells (after the activation), or by helper lymphocytes. IL2 is also present in lymphocytes and it’s a sort of growth factor that helps this cells to proliferate. In NK cells it also increases lytic activity and therefore degranulation. 3 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 The major important cytokine for maintenance and proliferation of NK cells is IL15. Missing self In the left picture below, it can be seen a normal cell expressing the HLA class I that is binding the inhibitory receptor à NK cells are “quiet”. A damaged or infected cell can escape from the recognition of the immune system by decreasing the expression of HLA class I. In this case, when the HLA class I is missing, the inhibitory receptor cannot signal inside, so the signal coming from the activating receptors will prevail and the NK cells will degranulate the content and kill the target cell. The activation of NK cells caused by the absence of HLA class I is a phenomenon called “missing self”. The activating receptors recognize: - Stress ligands => present in abnormal cells; - Neoplastic cells; - Cells infected by viruses => especially by Cytomegalovirus or the Herpes Virus (decrease on the expression of HLA); - Cells infected by intracellular bacteria => they cause stress in our cells; - Cells covered by antibodies: ADCC. 4 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Antibody dependent cellular cytotoxicity Another process in which the NK cells are involved is the “antibody dependent cellular cytotoxicity” or ADCC. NKs have a particular receptor, the CD16, that recognizes the constant portion of antibodies; when this bond occurs, the activating receptor is triggered and the NK cells degranulate. NK cells are the major players for ADCC, even though some macrophages are also able to do it. This important mechanism is able to kill infected cells: the antibodies involved in the antiviral response are induced and bind the surface of the infected cells leading to their death. Even if the MHC class I is expressed, NKs can kill the target through the activation mediated by the antibodies. The activating andcode inhibitory receptors function in different ways because they have a different intracytoplasmic tail. The activating ones have the ITAM motif (intracellular tyrosine activating motif) while the inhibitory ones have the ITIM motif (intracellular tyrosine-reach inhibitory motif). These are very common even in other kinds of receptors, for example the CD16 also has it. The activator motif is reaching tyrosine that can be phosphorylated and function for the attraction points of kinases. The ITIM motifs are still reaching tyrosine, but when phosphorylated they are bound by a phosphatase. 5  Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Cytokines which are produced by NK cells Globuli bianchi All leucocytes secrete cytokines. Even epithelial cells secrete cytokines, but leucocytes are the main producers. Different cytokines are able to activate different functions or different cells populations, but not at the same time: it depends on the pathogen or the damage they have to face. In case of viruses or internal bacteria infection, for example, macrophages and neutrophils are activated to phagocytes the infected cells. The NK cells and the killing lymphocytes have also to be activated and all of this is a sort of response that is activating by cytokines. Another example is: when we are infected by worms, we have to recruit eosinophil which are the only producer of major basic protein that are able to destruct the wall of the worms. To sum everything up: There are a lot of cytokines and growth factors that are mainly defined in different functions, such as the recruitment or the proliferation of immune cells, and are very important to modulate the immune response. These cytokines are usually produced after the activation of NK cells: - IL15 is important for a positive feedback mechanism: it maintain their activation and especially their proliferation. - GM-CSF is a factor that induce the bone marrow to produce neutrophils or eosinophils and E - monocytes. INF-γ, which is very active on both T cells and macrophages. - - TNF-α, which is also active in macrophages to maintain the M1 profile. This is really efficient & in phagocytosis and in oxidative burst (and so in the production of ROS) because it creates a toxic environment that kills the pathogens. With the production of these cytokines, NKs can activate different kinds of cells, the inflammatory response and hematopoiesis. NKs are also able to kill both while circulating and extravasating into the tissue. Immune System - Natural Killer Cell - YouTube Natural Killer Cells (NK-92) Explainer Video - YouTube INNATE LYMPHOID CELLS Innate Lymphoid Cells (also known as LCNK) originates from the same lymphoid precursor as the lymphocytes and both produce cytokines. Despite this similarity, ILCs don’t have specific TCRs. There are 3 families of ILC: ILC1, ILC2 and ILC3. They differentiate to each other thanks to a certain kind of microenvironment produced by specific cytokines released by damaged or infected cells. When they differentiate in ILC1, 2 or 3 they produce specific patterns of cytokines, able to activate proper innate or adaptive cells in order to fight a specific pathogen. The gastrointestinal mucosa is full of so-called “innate lymphocytes” that are promptly activated and able to induce the inflammatory response. ILCs also activate the macrophages and, based on the cytokines they produce, activate the M1 or M2 macrophages: M1 kill pathogens and phagocyte them; 6  Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 M2 participate in the end of the inflammatory response, so they secrete anti-inflammatory cytokines and growth factors to start tissue repair. They also trigger other functions from other types of cells, like the Goblet cells (which produce mucus) or Paneth cells (which produce antimicrobial peptides). Microbial signals >--- Innate lymphoid cells (ILC) ILC1, 2 and 3 differ on the microbial signal that activates the epithelial cells or the macrophages and dendritic cells residential in the tissue. ILC1 TNF induces ILC1, which produces IFN-γ; they are important for the activation of anti-viral and intracellular bacterial response. ILC2 The activation of eosinophils is induced by the ILC2 population, which will be differentiated in the presence of different cytokines. All these cytokines are produced by the epithelial cells, when the tissue needs to face big parasites like worms. Even in allergic reactions the binding with some molecules on our cells’ surface is able to induce the secretion of cytokines that activate the ILC2. The production of these specific patterns of cytokines (IL5, IL13), are also in common with TH2 cells, that are necessary to recruit eosinophils or to activate mastocytes. Allergic reactions may include sneezing or the contraction of the intestine mucosa (present in food allergies); these effects are due to the activation of mastocytes and eosinophils. ILC3 ILC3 cells are very common in the gastrointestinal epithelial gap and very similar to the TH17 population. It differentiates in the presence of the cytokine IL23, usually produced by Langerhans 7 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 cells. ILC3 produce IL17 and IL22, that are very useful for the fungal response (candida, aspergillus). Innate lymphoid cells - YouTube FIBROBLASTS Fibroblasts are abundant in the mucosae and the skin. They are used in many fields of research, such as tumor research and autoimmunity one. They produce a lot of metalloproteinases which are important in the inflammatory response because they destroy the extracellular matrix; doing so they open the way for the leucocytes that extravasate to reach the target area. Fibroblasts release also a lot of cytokines, therefore it is believed that these cells are also able to modulate and regulate the innate immune response. HEMATOPOIESIS There are 2 kinds of bone marrows: the red and the yellow one. Hematopoietic stem cells, which generate immune cells, are produced in the red bone marrow. It is characterized by a lot of capillaries (that are called sinusoids), hematopoietic stem cells, mesenchymal stem cells and adipocytes. Adipocytes, like osteoblasts, produce factors that are important to maintain the hematopoietic stem cells’ proliferation. fegorto ⑧ milza During fetal life, hematopoiesis mainly happens in the liver and very little in the spleen. From the 4th - or 5th month of pregnancy, it also starts to occur in the bone marrow. From birth on, the bone marrow is the only place in which hematopoiesis happens. After birth, the major hematopoiesis occurs in long bones, and, as the time passes by, it continuous in the ribs, sternum or vertebrae. 8 Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 From hematopoietic stem cells, we can have a myeloid precursor or a lymphoid precursor. Thanks to cytokines and other factors produced by mesenchymal stem cells, adipocytes or osteoblasts, the myeloid precursor is able to generate all the innate cells such as: erythroid progenitor => differentiates in red cells; megakaryocytes => differentiate in platelets; basophil progenitor => differentiates in granulocytes, such as basophils and mast cells; eosinophil progenitor => differentiates in eosinophils; granulocytes-monocyte progenitor => differentiates in neutrophils and monocytes o when they extravasate, monocytes can differentiate in dendritic cells or macrophages. mast cell g Erythroid progenitor The lymphoid precursor can give T progenitor or the B progenitor. The T progenitor can generate the classical T cells, the NK cells as well as innate lymphoid cells. Lymphoid progenitor I I 9 I T progenitor B progenitor T cells NK Innate B cells lymphoid cells  Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Hematopoiesis | Hematologic System Diseases | NCLEX-RN | Khan Academy - YouTube Single cell transcriptomic analysis Scientists were able to classify all the progenitors and mature cells with different morphological and functional features, thanks to the single cell transcriptomic analysis. Isolating all the cells present from the bone marrow at different ages and analyzing all their RNA molecules, scientists found out that there is a pool of stem cells which are able to give different progenitors. Each of the steps is characterized by a different profile; these progenitors can still differentiate in other cells and we know which progenitors are more similar to one other. These cells mature because of different patterns of transcription present in the nucleus or in the cytosol. However, the cells do not maintain these characteristics because they are genetically inherited, but because they have an epigenetic memory. Epigenetic modifications are reversible, therefore cells in the tissues are affected by the microenvironment: they have the potential to change à In a different contest they can change and differentiate in another type of cell; for example a macrophage can be M1 or M2 based on the cytokinetic environment; follicular lymphocytes can produce cytokines specific for TH1, but in a different cytokinetic environment they induce the production of TH2. à It’s important to remember that all immune cells are very plastic, so they can even change in the tissue based on the microenvironment. These changes are dependent on the kind of pathogens that the cells are facing. INNATE RECEPTORS Innate cells can recognize non-self elements due to the presence of specific receptors on the surface. These receptors do not recognize a specific sequence (in contrast with the adaptive response) but shared features or molecules on the pathogens’ surface. They recognize the “Pathogen Associated Molecular Patterns” or PAMPs, but they can also recognize the “Damage Associated Molecular Patterns” or DAMPs. Some of these components are only present in pathogens: Mannose or fucose residues, typical of microbes, bound to some protein; LPS or lipoteichoic acid => both are common in gram(-) and gram(+) bacteria’s wall; Double or single RNA coupled with DNA => present in our cells only when intracellular bacteria or viruses are inside them. Peptide bound to N-formyl-methionyl => this is typical of bacterial proteins, however some of our own proteins, in the mitochondria, are bound to formyl-methionine. These are part of DAMPs, because usually proteins from the mitochondria stay there and they never go out in the cytosol. When this happens, it means that the mitochondria are damaged, so the cell may be suffering. CpG sequences in the DNA => Bacteria have a lot of non-methylated CpG sequences. :[ All these molecules, belonging to PAMPs and DAMPs, are recognized by some specific receptor called Pattern Recognition Receptor (PRR) or by soluble molecules called Pattern Recognition Molecules (PRM), such as C-reactive proteins and Pentraxin. These molecules can also be produced by epithelial cells (they are called acute phase proteins) and they are part of the physiological biochemical barrier because they are able to bind bacteria on the surface and then direct these bacteria to the innate cells to be phagocyted. Since it’s soluble, Pentraxin needs to be recognized by innate cells through CD89 receptor in order to activate phagocytosis. 10  Lucia Griva/Alessia Gecchele – Lesson 3 – Immunology (Prof. Paola Cappello) – 12/10/2021 Danger signals: the alarmins - Besides the component of coagulation cascade or the complement proteins, which are induced by the inflammatory response, there are a lot of molecules that can be recognized by our innate cells and activate them, just like PAMPs. Some examples are: - ATP; - [HMG-B1]; - Lipoproteins which can ingulf microphages or mitochondrial DNA; - heat shock proteins; Receptors present on immune cells Receptors that immune cells usually present on their surface: PRRs => PRRs are the most abundant receptors. They recognize all the molecules previously mentioned. o Some PRRs are exposed on the cell surface, some are free in the cytosol and others are expressed on the endosomal membrane; Cytokine and chemokine receptors; Adhesion molecules => they allow the cell to extravasate and adhere to the tissue; Hormone receptors => the immune system, in some conditions, functions differently in men and women, because it can be modulated by sexual hormones; Antibody fragments => for complement receptor; I Scavenger Receptors => they recognize lipidic molecules, typical of bacteria or damaged cells; Esempi di PRM C-type lectin receptors => they recognize sugars on microbes, fungi, lipoproteins secreted extracellulari by stressed cells; Toll Like Receptors (TLR) => they recognize PAMPs and DAMPs; formis... Some types of scavenger, lectin and toll like receptors can also be present on the surface of macrophages and neutrophil and a lot of them can recognize all kinds of pathogens. The general function of all these re