Immune Responses in Health and Disease Cases PDF

Cases & lectures minor BMS: Immune responses in health and disease written by roosgeelen The Marketplace to Buy and Sell your Study Material At Stuvia, you will find...

Cases & lectures minor BMS: Immune responses in health and disease written by roosgeelen The Marketplace to Buy and Sell your Study Material At Stuvia, you will find the best notes, summaries, flashcards & other study material. Search for your school or uni and find the study material you need. www.stuvia.com Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Cases immune responses in health and disease Case 1: Neutrophils and macrophages in infection, inflammation and disease 1. What is the origin of immune cells in humans (focus on innate immune system)?  These cells are almost all derived from hematopoietic stem cells (HSCs) in the bone marrow of flat bones (ribs, sternum, iliac and vertebrate) which differentiate along branching lineages. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Based on their common precursors, immune cells are broadly classified as either myeloid cells (innate) , which include phagocytes and most DCs, or lymphoid cells (adaptive) , which include all lymphocytes.  Cytokine induce certain conversions  White blood cells formation = leukopoiesis This is stimulated by chemical messengers: interleukins and colony- stimulating factors (CSF) which are released by the mature WBC and supporting cells of the red bone marrow. These factors also improve the protective potency of mature leukocytes.  Hematopoietic stem cells divide into myeloid stem cells and lymphoid stem cells. Myeloid stem cells produce eosinophils, basophils and neutrophils Lymphoid stem cells produce macrophages and lymphocytes 2. What is the function of the innate immune system (broad, 4 functions)?  Two major types of reactions of the innate immune system are inflammation and antiviral defense. Inflammation is the process by which circulating leukocytes and plasma proteins are brought into sites of infection in tissues and are activated to destroy and eliminate the offending agents. Inflammation is also the major reaction to damaged or dead cells unrelated to Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material infection and to accumulations of abnormal substances in cells and tissues. Antiviral defense mechanisms prevent virus replication and promote killing of infected cells, thus eliminating reservoirs of viral infection without an inflammatory reaction (although inflammation also may contribute to defense against viruses).  4 functions of innate immune system: 1) Physical and chemical defenses at epithelial barriers: such as the skin and lining of the gastrointestinal and respiratory tracts, they block microbial entry. → Microbes are able to colonize tissues only if they are capable of crossing epithelia. If these barriers are damaged or microbes are able to penetrate them, innate and adaptive immune responses are activated to provide the next lines of defense. 2) First response to microbes → prevents, controls or eliminates the infection (inflammation, antiviral defense) 3) Recognize damaged or dead host cells → eliminates damaged cells and initiates the process of tissue repair 4) stimulate adaptive immune responses and can influence the nature of the adaptive responses to make them optimally effective against different types of microbes.  Innate immune cells express various PRR and after recognizing PAMPs and DAMPs they respond by producing inflammatory cytokines and antiviral protein and kill microbes or infected host cells 3. What are the components of the innate immune system & what are their function? (divided in circulating and cellular components, just list them)? - barrier epithelia: block the entry of microbes; - tissue-resident sentinel cells → macrophages, mast cells, and dendritic cells (DCs): detect microbes that have breached epithelia and initiate host responses; Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - white blood cells (leukocytes)→ including neutrophils, monocytes that become macrophages in tissues, natural killer (NK) cells, and other cells: enter the tissues from the blood and eliminate microbes that have invaded through epithelia and also remove damaged host cells; - Several types of plasma proteins that combat microbes within and outside the circulation. → including components of the complement system and other mediator of inflammation. Principal components of innate immunity 1) Physical and chemical barriers (epithelia and antimicrobial chemicals at epithelial surfaces) 2) phagocytic cells (neutrophils, macrophages), DCs, mast cells, NK cells and other innate lymphoid cells 3) blood proteins (components of complement system and other mediators of inflammation) 4) cytokines (cells of immune system interact with one another through these secreted proteins) → regulate and coordinate many activities of cells of innate and adaptive immunity All immune system cells secrete some cytokines and express specific signaling receptors for several cytokines 1) Epithelial Barriers Block the entry of microbes such as the skin and lining of the gastrointestinal and respiratory tracts These interfaces are lined by continuous layers of specialized epithelial cells that serve many physiologic functions, including preventing the entry of microbes. Loss of the integrity of these epithelial layers by trauma or other reasons predisposes an individual to infections. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material List leukocytes from most abundant to least abundant: (Never Let Monkeys Eat Bananas)  Volgorde: in een weefsel zijn al macrofagen aanwezig, die slaan alarm. Dan worden neutrofielen (fagocytose) aangetrokken die als eerste in actie komen en dan de monocyten die macrofagen worden (fagocytose en antigenpresentatie). ❖ Granulocytes (with lobed nuclei and membrane bound cytoplasmic granules) 1) Neutrophils Are the most numerous ones found in bloodstream their granules can take up both basic and acidic substances. Usually are circular with fine granules. Neutrophilia → too many neutrophils. Neutropenia → patient has not enough neutrophils. Banded neutrophils are young, and their nucleus is bent and oblong with constrictions which do not yet have resulted in filaments. Segmented neutrophils are old which have a threadlike constricted shape. Bacterial infection killing them Normal maturation → bone marrow Sickness → secreted CSF in blood will mature the big amount of immature neutrophils. 2) Eosinophils Circulating Telephone shaped nucleus which cannot digest bacteria. Lead the counterattack against parasitic worms thar are too large to be synthesized. Have slightly larger granulocytes than neutrophils. Important in allergic diseases/reactions as well as parasitic infections. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Like neutrophils, eosinophils do phagocytosis and migrate into surrounding tissue since their granules are lysosome like and filled with digestive enzymes. However, they lack enzymes that specifically digest bacteria 3) Basophils primarily in circulation the rarest white blood cells. They contain large histamine containing granules and histamine is an inflammatory chemical that acts a vasodilator (makes blood vessels dilate) and attract other white blood cells to the inflamed site). They play an important role in hypersensitivity reactions, and they are able to leave the blood stream and enter surrounding tissue. Involved in allergic reactions. They are smaller than other granulocytes ❖ Agranulocytes (WBC that lack visible granules, nuclei are typically spherical, or kidney shaped) 1) Lymphocytes Are the second most abundant types of WBC. Few are actually found in the bloodstream. They are closely associated with lymphoid tissue where they play a crucial role in the immune system. T-lymphocytes act directly against virus infected cells and tumour cells. B-lymphocytes produce antibodies (example immunocytes) → thus cellular and humoral defence against infections. B-lymphocytes give rise to plasma cells which in return produce antibodies (immunoglobins) that are released into the blood T-lymphocytes Natural Killer cells which help in the innate immune system. 2) Monocytes Circulate in the bloodstream, but can leave it and enter tissues.= macrophages (phagocyte) Increase in monocytes = monocytosis and decrease = monocytopenia. Monocytes are crucial in the body’s defence against viruses, certain intracellular bacterial parasites, and chronic infections and crucial in activating lymphocytes to support the immune response. important in acute and chronic infections. Cells of the immune system: 1) Phagocytes Neutrophils and macrophages are cells whose primary function is to ingest and destroy microbes and remove damaged tissues Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Recruitment of cells to the sites of infection, recognition and activation by microbes, ingestion of microbes by phagocytosis and destruction of ingested microbes through secreting cytokines, phagocytes communicate with other cells to promote/regulate immune responses Neutrophils ▪ acute inflammatory reactions ▪ phagocytose microbes (especially opsonized), destroying necrotic cells in phagolysosomes ▪ secrete granule contents and extrude their nuclear content forming neutrophil extracellular traps (NETs) ➔ immobilize and kill extracellular microbes but also may damage healthy tissues ▪ first cells ▪ How many (50%-70% of white= 1. 10^11), ▪ live few hour- 5 days. ▪ How do they look under microscope. ▪ When it get older then it will be segmented ▪ Neutrophils are also called polymorphonuclear leukocytes/PMNs Monocytes ▪ Circulating and tissue resident ▪ many become macrophages when they migrate into tissues ▪ ingest microbes by phagocytosis and kill these ▪ tissue resident macrophages sense presence of microbes and respond by secreting cytokines that initiate and amplify the protective response against the microbes ▪ macrophages cleaning-up process after infection or sterile tissue injury ▪ serve as APCs displaying fragment of protein antigens to T lymphocytes Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material ▪ macrophages promote repair of damaged tissues by stimulating angiogenesis and synthesis of collagen-rich extracellular matrix (fibrosis) ▪ Macrophages having engulfed (opgeslokt) microbes can be induced by microbial molecules to undergo inflammatory form of death = Pyroptosis → results from activation of cytoplasmic enzymes complex called inflammasome ▪ Pyroptosis → leads to release of cytokines that enhance the host’s inflammatory response to infection ▪ Macrophages are activated in different ways are responsible for different functions ▪ 1). Classically activated macrophages → microbicidal → promote inflammation ▪ 2). Alternatively activated → promote tissue repair later 2) Mast cells Bone marrow derived most abundant in skin and mucosal epithelia Once activated, release many potent inflammatory mediators defending against infections by helminthic parasites or cause symptoms of allergic diseases Can be activated by microbial products binding to TLR (part of innate immunity) or special antibody-dependent mechanisms. Mast cell granules contain vasoactive amines (like histamine) causing vasodilation and increase in capillary permeability (as well as proteolytic enzymes) that can kill bacteria or inactive microbial toxins. They ALSO synthesize lipid mediators (prostaglandins) and cytokines (TNF) stimulating inflammations. 3) Basophils blood granulocytes with many structural and functional similarities to mast cells 4) Eosinophils granulocytes expressing cytoplasmic granules containing enzymes harmful to cell walls of parasites but can also damage host tissues 5) Dendritic cells Dendritic cells rapidly and efficiently detect invading microbes because of their location in tissues and their expression of numerous pattern recognition receptors for PAMPs and DAMPs. Form an important bridge between innate and adaptive immune system → they active T cells= adaptive immune system These dendritic cells respond to microbes by producing numerous cytokines to serve two main functions ▪ Initiating inflammation ▪ Stimulating adaptive immune response 6) NK cells Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Cytotoxic activity similar to CD8+ Circulate in blood and present in lymphoid tissues Effector functions: kill infected cells and produce IFN-y which activates macrophages to destroy phagocytosed microbes Recognize infected and stressed cells and respond by killing these cells and secreting macrophage activating cytokine IFN (interferon) -y. NK cells empty their contents of the cytoplasmic granules into extracellular space. NK-cell activating cytokines → IL-15 (NK cell maturation), type I interferons and IL-12 (killing functions enhancement). NK-cellen zijn werkzaam totdat het specifieke immuunsysteem in werking treedt en er CD8 cytotoxische T-cellen worden geproduceerd die de celdoding (apoptose) overnemen. De naturalkillercellen worden dan geïnactiveerd door de IL-10 (interleukine-10) die geproduceerd wordt door de CD8 T-cellen. Geïnfecteerde en beschadigde cellen herkend hij en dood hij door middel van eiwitten (perforine en granzymes) uit granule → CD8 cel gebruikt ook deze eiwitten Activeren macrofagen door IFN-y. Macrofagen produceren IL-12 De stoffen die vrij komen uit de lytische granules: Perforine= maakt kanaaltjes in membraan van cel dat gedood moet worden Granzyme= gaat de cel in en activeren caspase enzymen dit leidt tot apoptose Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 7) ILCs Innatate lymphoid cellen produce cytokines similar to those secreted by CD4+ helper T cells Rare in blood, mostly in mucosal tissues (lung, intestines) Lymphocyte like cells producing cytokines and performing functions similar to those of T-lymphocytes but DO NOT express T-cell antigen receptors (TCRs). Responses of ILCs are stimulated by cytokines produced by epithelial and other cells at sites of infections 8) Complement system The complement system consists of several plasma proteins that work together to opsonize microbes, to promote the recruitment of phagocytes to the site of infection, and in some cases to directly kill the microbes Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Complement cascade can be activated via three pathways ❖ Classical pathway - If there are antibodies bound to the microbes - Uses a plasma protein called C1q to detect antibodies bound to the surface of microbes or other structures - Antibodies are part of the adaptive immune response - When antibody binds to microorganism, you can get complement factor 1 activation - This gives you activation of C4/C2 which leads to the formation of C4b/C2b - This C4b/C2b complex has C3 convertase activity - Antibody IgG and IgM are the only one recognized in the classical pathway ❖ Alternative pathway - Is triggered when a complement protein called C3 directly recognizes certain microbial surface structures, such as bacterial LPS. - Takes place spontaneously → C3 is present in your blood - Spontaneous activation takes place on the surface of microbes - It will not take place on the surface of human cells because there are factors that inhibit it - When C3 binds to a wall of a microbe, it is cleaved to C3b - Factor B is converted to factor Bb by factor D - C3b/Bb complex is formed and has C3 convertase activity ❖ Lectin pathway - Activated by Mannose Binding Lectin protein (MBL) which binds to mannose on bacteria. - Lectin activates protein of classical pathway, but it is initiated by microbial product in absence of antibody and therefore it is a component of innate immunity. - Is triggered by a plasma protein called mannose-binding lectin (MBL), - This causes a number of steps to take place - Activating MASP-1 and MASP-2 leading to convertase of C2/C4 to C2b/C4b which is C3 convertase converting C3 into C3a and C3b.  Netto resultaat: al these systems leads to C3 convertase: splitsing van C3 in C3a en C3b. Dit kan dus niet op lichaamseigen cellen worden toegepast. Eigen cellen hebben die specifieke eiwitten of structuren niet waardoor complement systemen geactiveerd worden. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  C4b/2a in the lectin pathway**  Difference between the classical and lectin pathway: the classical pathway uses the antigen-antibody association, whereas the lectin pathway uses the MBL-mannose association.  Complement activation is inhibited by regulatory proteins present on normal host cells and absent from microbes.  How does the complement systeem works against microbes? Eliminates microbes during innate and adaptive immune response  All three pathways leads to formation of C3 convertase  C3 convertase will convert the complement factor C3 to C3a + C3b  This leads to three effects Formation of anaphylatoxines (C4a, C3a, C5a) → have pro-inflammatory effect. They activate phagocytes which kill microorganisms. When many of these small fragments are present, they attract cells of innate defense. Formation of C3b → leads to opsonization (this makes microbes easier to recognize) and increased phagocytosis (phagocyte has receptor for C3b). It coats microorganisms with opsonins to stimulate phagocytosis in a process which is called opsonization. Lytsche pathway will be activated → formation of membrane attack complex because C5 convertase is activated and this C5b together with C6, C7, C8 and C9 forms a tube that is in the wall of bacteria, causing osmotic lysis. begins when C3b binds to target cell’s surface and triggers the insertion of a group of complement factors (C5b, C6, C7, C8, C9) called MAC (membrane attack complex). MAC forms and stabilizes a hole in the membrane allowing massive influx of water which lyses the cell → LYTIC pathway Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material ❖ Phagocytosis  On membrane neutrophil or phagocyte there is a PRR (CLR) that recognizes e.g. sugar on surface of microorganism so you get binding with receptor  This activates the cell  Phagocyte will pull in the membrane and form a vesicle around the bound microorganism  This then goes into a phagosome  In the phagocyte are already lysosomes with enzymes ready (enzymes are in a sac and not loose in cytoplasm because otherwise it is harmful to the cell)  Phagosome (sac containing ingested microorganism) merges with lysosome = phagolysosome  Enzymes can join microorganism  Enzymes in the wall of phagolysosome are activated (= phagocyte oxidase)  Phagocyte oxidase will convert oxygen into oxygen radicals (ROS)  Oxygen radicals are harmful and contribute to microorganism destruction  You also get formation of NO for enzyme iNOS which is also harmful and also contributes to microorganism destruction Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material After digestion of pathogen, a residual body forms containing indigestible material and wastes are discharged. In lysosome low pH → easily breaks down microbial matter In lysosome contains: nitrogen, proteases (digestive enzyme)  So the bacteria are eventually broken down by oxygen and nitrogen radicals, by proteases or by bleach (hypercloride) which is also in lysosomes and can kill bacteria. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 4. What is the function of the circulating components (neutrophils and monocytes, include complement system, pentraxin)?  See previous learning goal 5. Where are the cells of the innate immune system localized in the body?  Neutrophils= only in the blood, not in the tissue (if they are in the tissue, there is an inflammation going on)  Monocytes/ Macrophages= less present in blood. Migrate in tissue to macrophages  Dendritic cells=  Mast cells= not present in blood but present in the skin and around blood vessels  NK cells= 6. How do cells of the innate immune system recognise the damaged cells or other types of danger?  The immune cells associated with the innate immune system recognize and respond to certain microbial structures.  Viruses and bacteria have certain structures, such as pili, tail, envelope, no cell nucleus, etc. Our own cells do not have this. This allows the immune system to distinguish body cells from foreign cells.  Microorganisms must be recognized so that the immune system can respond to them. This is done by: PAMPS= pathogen associated molecular pattern (structures that do not occur in host cells but do exist in microorganisms) - Double-stranded & single-stranded RNA Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - CpG rich DNA - Pili - Flagellum - Cell wall components of bacteria  Recognition of dead/ damaged cells is done by: DAMPS= damage associated molecular patterns - Nuclear protein that is activated - Stress-indicated proteins  Receptors that recognize these patterns (PAMPS & DAMPS) PRRS= pattern recognition receptors → receptors that recognize and effect these patterns (PAMPS & DAMPS) - signal receptors of the innate immune system that recognize PAMPs and DAMPs and thereby activate the immune system - Sitting on cells of innate immune system for recognition (neutrophils, monocytes, dendritic cells) - They can also be found on cells of adaptive immune system, but then it plays a role as the second signal. 1) Cell- associated: - TLR= important in defense against microbial components (bacteria and virus components). Extracellular & Endosomal Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - NLR= play an important role in inflammasome activation. Cytosolic - RLR= important for viral RNA. Cytosolic - CDS=microbial DNA. Cytosolic - CLRs= recognized sugars on the surface of microorganism. Extracellular  Membrane bound → scavenger + N-formyl receptors + TLR + CLRs  Cytosolic → NLR, RLR, CDSs  Endosomal → TLR  Depending on where PRRS (receptor) encounters the microorganism component or damaged cell, that's where they are also located. (both on membrane of cells and intracellular)  Toll like receptor: at the moment bacterial cell wall component is recognized, then bacteria is still outside the cell and then the toll like receptor is also outside the cell.  If a microorganism has entered (if it is a virus or if it has been ingested) and parts are absorbed and digested and end up in endosomes, it is useful that receptors are there. 2) Soluble: Complement Pentraxins - Several plasma proteins that recognize microbial structures and participate in innate immunity belong to the pentraxin family. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - Prominent members of this family include the short pentraxins, C-reactive protein (CRP) and serum amyloid P (SAP), and the long pentraxin PTX3. - Both CRP and SAP bind to several different species of bacteria and fungi. - The molecular ligands recognized by CRP and SAP include phosphorylcholine and phosphatidylethanolamine, respectively, which are found on bacterial membranes and become exposed on apoptotic cells. - CRP, SAP, and PTX3 all activate complement by binding C1q and initiating the classical pathway. - CPR & SAP increase in response to IL-1, IL-6 and TNF, increased synthesis by liver cells induced by these cytokines= acute phase proteins - PTX3 is produced by several cell types, including DC’s, macrophages and endothelial cells in response to TLR ligands and inflammatory cytokines such as TNF → PTX3 is stored in neutrophil granules and released as neutrophils die Collectins - Each subunit contains a collagen-like tail connected by neck region to C-type lectin head - 3 members of this family serve as soluble effector molecules in the innate immune system - 1) MBL, 2) pulmonary surfactant proteins SP-A and 3) SP-D - MBL → similar structure to C1q ➔ surface receptor = C1q receptor → mediates internalization of microbes opsonized by MBL - SP-A and SP-D → lipophilic properties in alveoli of lungs and maintain ability of alveoli to expand (through reducing surface tension of alveolar fluid) - bind to various microorganisms - act as opsonins, facilitating ingestion by alveolar macrophages - can also directly inhibit bacterial growth and may activate macrophages. Ficolins - Plasma proteins structurally similar to collectins (possessing collagen-like domain - Instead of C-type lectin domain, they have a fibrinogen-type carbohydrate recognition domain - bind several species of bacteria, opsonizing them and activating complement in a manner similar to that of MBL Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  These three homologous hexameric proteins can all initiate complement activation on binding to their ligands on cell surfaces  NLR family sensors found in different inflammasomes include NLRB, NLRC4, and at least six NLRP proteins  The formation of the inflammasome is induced either when sensor proteins in the cytosol directly recognize microbial products or when sensors detect changes in the amount of endogenous molecules or ions in the cytosol that indirectly indicate the presence of infection or cell damage. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  In response to the PAMPs or indirect signals, the sensors become able to bind other proteins  Pro-caspace-1 binds to inflammasome → autocatalytic cleavage (cleaves and self- activates) → activated caspace-1: cleaves pro-IL-1beta and pro-IL-18 → IL-1β and IL-18 stimulate inflammation. cleaves Gasdermin-D → moves to cell membrane → causes pores → swelling of cells → pyroptosis= an inflammatory form of programmed cell death of macrophages and DCs (but not of neutrophils and most other cell types) Toll like receptoren:  have similar structure, part inward through which signaling takes place 7. How do circulating cells of the innate immune system enter the site of danger? (H3)  Diapedeses= the exit of leukocytes from the bloodstream and migration to the infection (initiated by TNF and IL-1) In the first 24 hours it is mainly the neutrophils Later the monocytes Depends on chemokines that are released locally - Initially IL-8 by tissue macrophage → neutrophil receptors respond to this Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - Mcp-1 is specific to monocytes (macrophages) to attract → this is formed later If neutrophil has not been able to dissolve quickly, more cells are needed, at some point chemokines are produced that attract monocytes.  Just outside the blood vessel are tissue cells with macrophages  with microorganisms, macrophages activate the PRRs via PAMPs, resulting in the formation of cytokines and chemokines  Cytokines ensure that certain adhesion molecules (selectins) are expressed on the endothelium of a blood vessel  Chemokines bind to certain structures on the inside of the blood vessel wall  Neutrophil is in blood, blood flows more slowly due to blood vessel dilation 1) Rolling  Neutrophils with ligands for selectins attach to endothelium with selectins → this is not a strong bond, causing neutrophil to roll along the blood vessel wall  Macrophages, DCs, and other cells that encounter microbes in extravascular tissues are activated to secrete cytokines, including TNF and IL-1.  These cytokines stimulate endothelial cells lining postcapillary venules at the site of an infection to express E-selectin.  Endothelial cells also express P-selectin in response to histamine released from microbe-activated mast cells, and thrombin produced during blood coagulation, which occurs commonly in inflammatory reactions.  At these sites, blood vessels dilate, and blood flow slows.  As a result, leukocytes, being larger than red cells, tend to move away from the central axial flow and closer to the vessel lining, a process known as margination.  This allows the ligands for E- and P-selectins expressed on the microvilli of the leukocytes to bind to the selectins that have been induced on the endothelial cells. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Because the interactions of selectins with their ligands are of low affinity with a fast off-rate, they are easily disrupted by the sheer force of the flowing blood.  As a result, the selectin–selectin ligand bonds repeatedly form and break, and the leukocytes are pushed along the endothelial surface in a rolling motion. 2) Integrin activation by chemokines  Chemokines have also settled on the inside of the blood vessel wall  When neutrophil rolls along blood vessel wall, it will bind chemokine with its chemokine receptor  Chemokine binding causes a change → certain adhesion molecules on the surface of the membrane of a neutrophil will change the integrins that normally have a low affinity state to a high affinity state (by chemokine binding) 3) Stable adhesion  Integrins will bind to adhesion molecules that also have increased to increased expression on endothelium = tight binding  As a result, the rolling can no longer take place  In parallel with the activation of integrins, the expression of their ligands on the endothelial cells is upregulated by inflammatory cytokines and microbial products.  These ligands include VCAM-1, which binds the integrin VLA-4, and ICAM-1, which binds LFA-1 and MAC-1 integrins.  Thus, the leukocytes attach firmly to the endothelium, their cytoskeleton is reorganized, and they spread out on the endothelial surface. 4) Migration through endothelium  There is an increased permeability  Neutrophil migrates through the pore to the site where there is a high concentration of chemokine and then acts there  Leukocytes usually migrate out of blood vessels between endothelial cells, a process called paracellular transmigration or diapedesis, to reach extravascular tissues.  Paracellular transmigration depends on interactions of integrins on the leukocytes and their ligands on the endothelial cells, as well as the contribution of other proteins, notably CD31, which is expressed on leukocytes and endothelial cells.  This process requires a transient and reversible disruption of adherens junction proteins, primarily the VE-cadherin complex, that hold endothelial cells together.  The mechanism responsible for disruption of the VE-cadherin complex involves activation of kinases when leukocyte integrins bind ICAM-1 or VCAM- 1. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  The kinases phosphorylate the cytoplasmic tail of VE-cadherin, which leads to reversible disruption of the adherens complex.  Less often, leukocytes have been observed to move through endothelial cells rather than between them, by a poorly understood process called transcellular migration. After this repair of tissue  neutrophils are usually the most numerous type of leukocyte to accumulate in tissues within 24 to 48 hours after the onset of an infection or injury, and are then replaced by monocytes. → there are many more neutrophils in the blood, and they respond more rapidly to chemokines compared to monocytes or other leukocytes.  After entering tissues, neutrophils have short life spans before dying by apoptosis whereas monocytes survive longer and may also proliferate in the tissues.  In some inflammatory sites, neutrophils are not recruited at all, but monocytes are. → reflects variations in expression of adhesion molecules and chemokine receptors on neutrophils versus monocytes.  Neutrophils express CXCR1 and CXCR2, which bind CXCL1 and CXCL8 (IL-8), the major chemokines with ELR motifs that support neutrophil migration into tissues  Early neutrophil recruitment is a consequence of early and abundant CXCL8 production by tissue-resident macrophages and other cells in response to infections.  Classical monocytes, which are the main type of monocyte recruited to inflammatory sites, express CCR2. → This receptor binds several chemokines, the most important one for monocyte recruitment being CCL2 (MCP-1).  Thus, monocyte recruitment occurs when resident tissue cells produce CCL2 in response to infection. 8. What is the function and effect of neutrophils (include NET)?  acute inflammatory reactions  phagocytose microbes (especially opsonized), destroying necrotic cells in phagolysosomes  secrete granule contents and extrude their nuclear content forming neutrophil extracellular traps (NETs) → immobilize and kill extracellular microbes but also may damage healthy tissues In response to infections, production of neutrophils by bone marrow increases. Neutrophils ingest microbes in the circulation and rapidly enter extravascular tissues at the infection site where they phagocytose and destroy microbes. Neutrophils kill ingested microbes by phagocytosis Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Neutrophils also kill microbes by extruding their DNA and granule content which form extracellular threads on which bacteria and fungi are trapped and killed → Extruded chromatin contents, which are called neutrophil extracellular traps (NETs), are composed of strands of DNA and histones to which high concentrations of antimicrobial granule contents are bound, including lysozymes, elastase and defensins extrusion of nuclear content during NET formation leads to neutrophil cell death (NETosis) 9. What is the function and effect of macrophages? Monocytes (circulating and tissue resident)  many become macrophages when they migrate into tissue  ingest microbes by phagocytosis and kill these  tissue resident macrophages sense presence of microbes and respond by secreting cytokines that initiate and amplify the protective response against the microbes  macrophages cleaning-up process after infection or sterile tissue injur  serve as APCs displaying fragment of protein antigens to T lymphocytes  macrophages promote repair of damaged tissues by stimulating angiogenesis and synthesis of collagen-rich extracellular matrix (fibrosis) ➔ macrophages having engulfed microbes can be induced by microbial molecules to undergo inflammatory form of death = Pyroptosis ➔ results from activation of cytoplasmic enzymes complex called inflammasome Pyroptosis ➔ leads to release of cytokines that enhance the host’s inflammatory response to infection 10. Compare neutrophils and macrophages. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 11. Describe the process of acute inflammatory response over time.  The principal way by which the innate immune system deals with infections and tissue injury is to stimulate acute inflammation, which is the accumulation of leukocytes, plasma proteins, and fluid derived from the blood at an extravascular tissue site of infection or injury  Three of the most important proinflammatory cytokines of the innate immune system are TNF, IL-1 and IL-6. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 1) Injury, barrier break, microbe entry: Acute inflammatory responses begin when microbes transgress epithelial barriers or when tissue is injured 2) Microbes/injury activate sentinel cells: Then pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) activate sentinel cells, such as macrophages, dendritic cells, mast cells 3) Sentinel cells secrete inflammatory mediators: To secrete cytokines and other mediators 4) Vasodilation and increased vascular permeability; fluid and proteins enter tissues: Some of these mediators (e.g., histamine, prostaglandins) increase the permeability of capillaries 5) Complement, antibodies and antimicrobial proteins kill microbes: Leading to the entry of plasma proteins (e.g., complement proteins) into the tissues 6) Adhesion molecules and chemokines cause leukocyte migration into tissue: and others (interleukin-1, tumor necrosis factor) increase expression of endothelial adhesion molecules and chemokines that promote the movement of leukocytes from the post-capillary venules into the tissues 7) Phagocytosis and killing of microbes: where the leukocytes destroy microbes, clear damaged cells and promote more inflammation and repair. SEQUENCE OF EVENTS: Acute inflammatory responses Initiated when sentinel cells (mast cells, tissue-resident macrophages and DCs) use TLRs and cytosolic innate PRRs to sense microbes and injured cells 1. Mast cells (PAMPs and DAMPs) response is to secrete histamine and prostaglandins → vasodilation and increased capillary permeability → increased blood flow through tissues and increases movement of plasma proteins (complement proteins, pentraxins, collectins and antibodies) Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 2. Recruitment of large numbers of neutrophils, then monocytes, from blood into tissues as part of acute inflammatory response to infections and tissue injury → as more leukocytes are entering the affected tissues, local delivery of cells that can fight infections and repair tissues (TNF, IL-1, IL-6, and chemokines) 3. Postcapillary venule endothelial cells increase surface expression of adhesion molecules for leukocytes → E-selectin and ligands for integrins and ICAM-1 and VCAM-1 induced by TNF and IL-1 activation of transcription factors (including NF-kB) 4. TNF and IL-1 also stimulate various cells to secrete chemokines (CXCL8 and CCL2) binding to receptors on neutrophils and monocytes, respectively. 5. Ingestion and killing of microbes by activated phagocytes → activated neutrophils and macrophages kill phagocytosed microbes by the action of microbicidal molecules in phagolysosomes 1) ROS: process by which ROS are produces is called respiratory burst 2) NO: cytosolic enzyme iNOS absent in resting macrophages but induced by microbial products activating TLRs (especially with IFN-y) 3) Proteolytic enzymes: destroy microbes 6. Macrophages clear dead cells and secrete growth factors that promote regeneration and angiogenesis, promotion of the formation of scar tissue to replace the damaged parts Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year? Stuvia.com - The Marketplace to Buy and Sell your Study Material Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 12. How is everything down regulated?  Innate immune responses are regulated by negative feedback mechanisms that limit potential damage to tissues.  IL-10 is a cytokine that is produced by and inhibits activation of macrophages and DCs.  Negative signaling pathways block the activating signals generated by pattern recognition receptors and inflammatory cytokines.  IL-10 is a cytokine that is produced by and inhibits activation of macrophages and DCs. IL-10 inhibits the production of various inflammatory cytokines by activated macrophages and DCs, including IL-1, TNF, and IL-12. Because it is both produced by macrophages and DCs and inhibits the functions of these cells, IL-10 is an example of a negative feedback regulator. Alternatively activated macrophages make more IL-10 than classically activated macrophages. IL-10 is also produced by regulatory T cells.  Macrophages produce a natural antagonist of IL-1= IL-1 receptor antagonist (IL-1RA). that is structurally homologous to the cytokine and binds to the same receptors but is biologically inactive, so that it functions as a competitive inhibitor of IL-1. It is therefore called IL-1 receptor antagonist (IL-1RA).  There are numerous negative regulatory signaling pathways that block the activating signals generated by pattern recognition receptors and inflammatory cytokines. Suppressors of cytokine signaling (SOCS) proteins are inhibitors of JAK- STAT signaling pathways linked to cytokine receptors. TLR signaling in macrophages and DCs induces the expression of SOCS proteins, which limit responses of these cells to exogenous cytokines such as type I IFNs. Proinflammatory responses of cells to TLR signaling are negatively regulated by SHP1, an intracellular protein phosphatase that negatively regulates numerous tyrosine kinase–dependent signaling pathways in lymphocytes. There are many other examples of kinases and phosphatases that inhibit TLR, NLR, and RLR signaling and small inhibitory RNAs that inhibit production of many of the mediators of innate immunity. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Case 2: NK & innate lymphoid cells (ILCs) 1. What are the general functions, types, origins, locations of ILCs? ❖ Function - ILCs have a fundamental role in the immune system by initiating, regulating and resolving inflammation. - ILCs promote acute inflammation and innate immunity to pathogens. - ILCs promote the resolution of inflammation and tissue repair - ILCs promote chronic inflammation - ILCs limit chronic inflammation - Play a role in modulating adaptive immunity. - ILC family has a key role in mediating acute inflammation in response to infection, which is important for the control and clearance of various classes of pathogens - ILC responses are substantially altered in several disease states. - Innate lymphoid cells (ILCs) produce cytokines similar to those secreted by CD4 + helper T cells. - IL-7 main interleukin for differentiation - id2 main transcription factor for differentiation ❖ Types - ILC1 - ILC2 - ILC3 ❖ Origins Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - ILCs initially develop in the fetal (foetus) liver and later in the adult bone marrow from common lymphoid progenitors (CLPs) - All ILCs develop from CLPs, which can differentiate into NKps or CHILPs. - A common ILC precursor identified by the Id2 transcription factor differentiates into the 3 subsets of ILCs. - Differentiation of ILC from CLP required transcription factors Id2 (inhibitor of DNA binding 2), NFLI3 and Tox. - IL-7 signalling can induce NFLI3 and subsequently Id2 which support ILC progenitor generation. - CHILPs can further differentiate into LTi cells through α4β7 integrin- expressing intermediate populations, or into other ILC populations through differentiation to a PLZF-dependent ILCp - Further engagement of transcription factors, cytokines and microbial signals is critical for the development of three distinct groups of mature ILCs. - CLPs also differentiate into cells of adaptive immune system, such as T-cells and B-cells. ❖ Locations - ILCs are rare in the blood and are present mostly in tissues, especially mucosal tissues such as the lung and intestines. - They are enriched at barrier surfaces of the mammalian body, such as the skin, lung and intestine, as well as in adipose and some mucosal-associated lymphoid tissues - ILC2: Although mature ILC2s can be found in most anatomical locations, they seem to be enriched in the healthy lung, skin and adipose tissue of mice and humans. - ILC3: T-bet+ ILC3s are almost exclusively found in the skin and intestinal lamina propria, whereas LTi-like ILC3s are enriched in the intestine and lymphoid tissues. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material 2. What are the general functions, origins, locations of NKs? ❖ Functions - Have cytotoxic activity similar to that of CD8 + CTLs. - kill infected cells and produce IFN-y which activates macrophages to destroy phagocytosed microbes - NK cells also secrete IFN-γ and thus resemble ILC1s. - They are also developmentally related to ILC1s but are not considered identical because they are defined based on cytotoxic activity, not cytokine production. - NK cells do not express diverse, clonally distributed antigen receptors typical of B and T cells. Rather, they use germline DNA–encoded receptors to distinguish pathogen-infected cells from healthy cells. - They can be identified in the blood by expression of CD56 and the absence of the T cell marker CD3. - Most human blood NK cells also express CD16, an IgG Fc receptor that is involved in recognition of antibody-coated cells. - Recognize infected and stressed cells and respond by killing these cells and secreting macrophage activating cytokine IFN (interferon) -y. - NK cells empty their contents of the cytoplasmic granules into extracellular space. - NK-cell activating cytokines → IL-15 (NK cell maturation), type I interferons and IL-12 (killing functions enhancement). - ❖ Origins - NK cells originate from hematopoietic stem cells (HSC) ❖ Location  They circulate in the blood and are present in lymphoid tissues.  Unlike ILCs, which are found in peripheral tissues but are rare in the blood and lymphoid organs, NK cells constitute 5% to 20% of lymphocytes in the blood and spleen (mild).  They are rare in other lymphoid organs and in most nonlymphoid tissues but are numerous in the liver and placenta. Apoptosis 3. How are the ILC 1 activated, what do they do and how are they down regulated?  Activated: by IL-12  Function: - ILC1s promote innate immunity to intracellular pathogens by producing TNF and IFN-γ in response to DC-derived IL-12, and they subsequently promote recruitment of inflammatory myeloid cells. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Down regulated: 1) ILC1s - express transcription factor: T-bet - are responsive to IL-12 - produce IFN-γ & tumor necrosis factor (TNF). 4. How are the ILC 2 activated, what do they do and how are they down regulated?  Activated: by IL-33, IL-25 & TSLP  Function: - Rapidly respond after exposure to multicellular parasites that are typically extracellular After infection with the helminth parasites N. brasiliensis or T. muris, ILC2s produce IL-13 in response to epithelial cell–derived IL-25, IL-33 and TSLP, which increases smooth muscle contractility and mucus production from goblet cells - Resolution of inflammation in the lung. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material After viral infection in the lung, airway epithelial cells are damaged and, in conjunction (in combinatie) with resident myeloid cell populations, produce IL-33. ILC2s respond to IL-33 and produce amphiregulin, which promotes repair of the airway epithelium. - ILC2s and chronic inflammation in the lung and skin In response to infection or allergen exposure, ILC2 responses are elicited in the lung (and skin) by epithelial cell– and myeloid cell– derived IL-25, IL-33 and TSLP. ILC2 responses can be enhanced (versterkt) by basophil-derived IL-4 or mast cell-derived PGD2. Activated ILC2s can subsequently promote chronic inflammation via IL-5–dependent eosinophil recruitment, IL-13–mediated contraction of smooth muscle cells, collagen deposition, and AAMac differentiation, or by MHCII mediated enhancement of TH2 cell responses, resulting in allergy and fibrosis. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - ILC2s and metabolic homeostasis. In the intestine, ILC2s respond to epithelial cell-derived IL-33, IL-25 and vasoactive intestinal peptide (VIP) to promote IL-5– and IL-13– dependent recruitment of eosinophils and differentiation of AAMacs. → This process also occurs in adipose tissue, although the sources of IL-25 or IL-33 are less well defined. Differentiation of AAMacs or direct stimulation of adipocytes with IL-13 or methionine-enkephalin peptides (Met-enk) can promote metabolic homeostasis through a process known as beiging in the adipocytes.  Down regulation: 2) ILC2s - highly express transcription factor: GATA3 - are responsive to IL-25, IL-33 and TSLP - produce IL-4, IL-5, IL-9, IL-13 and amphiregulin. - Although GATA3 is required for the development of most ILC subsets, it is also required for the maintenance and function of mature ILC2s in humans and mice. - Location: Although mature ILC2s can be found in most anatomical locations, they seem to be enriched in the healthy lung, skin and adipose tissue of mice and humans. 5. How are the ILC 3 activated, what do they do and how are they down regulated?  Activated: by IL-1b & IL-23  Function: Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - Rapidly respond to infection of mice with either extracellular bacteria or fungi ILC3s produce IL-17 and IL-22 in response to DC-derived IL-23 and IL- 1β, which promotes innate immunity to fungi and extracellular bacteria. IL-17 and IL-22 promote neutrophil recruitment to the intestine and the production of antimicrobial peptides from intestinal epithelial cells (IEC)s. - ILC3 produce mucus production - Resolution of inflammation in lymphoid tissues and the intestine In lymphoid tissues, such as the spleen and thymus, stromal cell damage induced by viral infection or irradiation results in increased numbers of ILC3s and increased cytokine production, in response to DC-derived IL-23. → ILC3s directly promote restoration of stromal cell compartments through production of LTα1β2 and IL-22, which increase the proliferation and survival of tissue resident stromal cells. Further, ILC3s can also promote tissue repair in the thymus of mice after total body irradiation → IL-22 acts on thymic epithelial cells to promote cell survival and proliferation In the intestine, ILC3 responses can be limited by a regulatory loop whereby commensal bacteria induce intestinal epithelial cells (IEC) expression of IL-25, which acts on DCs to limit ILC3 cytokine responses in a contact dependent manner. → In contrast, upon chemical-, infection- or irradiation induced damage of the intestine, ILC3s are activated by DC-derived IL-1β, IL-23, TL1A and RA. Activation of ILC3s induces IL-22 production that directly promotes mucus production and epithelial cell repair, in part by acting directly on intestinal stem cells or progenitors Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - ILC3s, chronic inflammation and cancer in the skin, lung and intestine In patients with psoriasis and in mouse models of skin inflammation, ILC3 responses are increased, which can occur in response to DC- derived IL-23. → ILC3s largely promote skin inflammation through production of IL-22 and IL-17. ILC3s are increased in the bronchoalveolar lavage fluid of patients with asthma and in mouse models of obesity-induced asthma. In mice this occurs through activation of the NLRP3 inflammasome and macrophage (Mφ) production of IL-1β. → IL-1β activates ILC3s to produce IL-17, which directly promotes airway inflammation and hyper-responsiveness. In the intestine ILC3s can promote IL-22–dependent tumor growth, which is in part dependent upon DC-derived IL-23. ILC3s can mediate tissue inflammation in the intestine in response to DC-derived IL-23 and IL-12. This may occur through production of IL- 17 by ILC3s, production of IFN-γ following loss of RORγt in ILC3s and Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material differentiation to ex-ILC3s, or direct activation of tissue-resident ILC1s. - ILC3s can limit chronic inflammation by regulating innate and adaptive immune responses in the intestine. ILC3 responses are induced in response to myeloid cell– and DC- derived IL-1β and IL-23 after recognition of pathogenic or commensal microbes. Production of ILC3-derived LTα1β2 or LTα3 can promote IgA production by B cells indirectly by modulating stromal cell or DC responses. Production of ILC3-derived GM-CSF can influence myeloid cell homeostasis to subsequently promote Treg cell responses and tolerance to food antigens. ILC3-intrinsic MHCII can directly kill commensal bacteria-specific CD4+ T cells with the potential to cause intestinal inflammation. Production of IL-22 by ILC3s can promote antimicrobial peptide production by IECs to limit colonization with commensal bacteria, such as segmented filamentous bacteria (SFB), or it can regulate the anatomical localization of lymphoid tissue resident commensal bacteria. ILC3-derived IL-22 can induce fucosylation of IECs to promote colonization with beneficial bacteria Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Down regulation: 3) ILC3s. - express transcription factor: RORγt - are responsive to IL-1β and IL-23, - produce IL-17 and/or IL-22. - All ILC3 share a developmental requirement for RORγt - CCR6+: Fetal and adult LTi cells are members of the ILC3 group, and they develop independently of PLZF-dependent ILCps. heterogeneous in expression of CD4, express IL-22 and IL-17, and are a source of lymphotoxin (LT). - CCR6-: A subset of adult ILC3s can develop from PLZF-dependent ILCps after birth. Co-express T-bet, are heterogeneous in expression of natural cytotoxicity receptors (NCRs, such as NKp46 and NKp44), and can co-express IL-22 and IFN-γ. This T-bet+ ILC3 population requires T-bet, the presence of commensal bacteria and the Ahr for development. - Location: T-bet+ ILC3s are almost exclusively found in the skin and intestinal lamina propria, whereas LTi-like ILC3s are enriched in the intestine and lymphoid tissues. 6. How are the NKs activated, what do they do and how are they down regulated?  Activation: - Natural killer (NK) cells recognize ligands on infected cells or cells undergoing other types of stress and kill the host cells. - NK cells respond to interleukin-12 (IL-12) produced by macrophages and secrete interferon- γ (IFN-γ), which activates the macrophages to kill phagocytosed microbes. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - NK cells distinguish infected and stressed cells from healthy cells, and NK cell function is regulated by a balance between signals that are generated by activating and inhibitory receptors. - The activating receptors recognize ligands on infected and injured cells, and the inhibitory receptors recognize ligands on healthy normal cells - Stimulating cytokines: IL-12, IL-15, IL-18, and type I IFNs interferons. Each of these cytokines enhances the cytotoxic activity of NK cells, and they can stimulate IFN-γ secretion by the NK cells independent of activating receptors. IL-15 is an important growth factor for NK cells. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - NK cell activating receptors: Killer cell Ig-like receptors (KIRs)→ KIR genes are polymorphic, meaning that there are several allelic variants in the human population. As a result, one person may express different receptors than another person. Groups of KIR alleles are often inherited together from a single parent. These groups of linked genes are called KIR haplotypes. There are two major KIR haplotypes and some rarer ones. Haplotypes differ in the number of receptors encoded, and some have more or fewer activating receptors than others. Some haplotypes are associated with increased susceptibility to some disorders, including spontaneous abortion and a type of eye inflammation called uveitis. lectin-like NK cell receptor (NKG2D) → Binds class 1 MHC proteins CD16 (FcγRIIIA) → low-affinity receptor for IgG antibodies. binds to the Fc regions of certain types of antibodies called IgG1 and IgG3. During an infection, the adaptive immune system produces IgG1 and IgG3 antibodies that bind to microbial antigens expressed on the surface of infected cells, and CD16 on NK cells can bind to the Fc regions of these antibodies. As a result, CD16 generates activating signals, through its associated signaling partners, and the NK cells kill the infected cells that have been coated with antibody molecules. This process is called antibody-dependent cell-mediated cytotoxicity; it is an effector mechanism of adaptive immunity The expression of all of these NKG2D ligands is increased by cellular stress, so they are found on virally infected cells and tumor cells but not normal cells. - NK cell Inhibitory receptors: Inhibitory receptors of NK cells recognize class I MHC molecules, which are cell surface proteins normally expressed on all healthy cells. NK cells use fundamentally different types of receptors than do T cells to recognize class I MHC molecules. many viruses and other causes of cell stress lead to a loss of cell surface expression of class I MHC. → their absence is an indication of infection or damage KIRs → bind a variety of different class I MHC molecules. CD94/NKG2A heterodimer → recognizes a class I MHC molecule called HLA-E. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material NK cells → don’t attack normal healthy cells because they express MHC1 molecules. These molecules interact with inhibitory receptors on NK cells. If that interaction is there and is strong enough, NK cells will never get activated T cells → get activated because there is interaction with MHC class 1 Cell is infected by virus or tumor cell  cell should be killed by T-cell or NK cell  There is a peptide in MHC class 1 which can activate T-cells  Many viruses downregulated MHC class 1 molecules  In many virus infections you hardly have any MHC class 1 expression → T-cells will not be able to kill those cells. But the inhibitory signal disappears.  No inhibition is not enough, they always need an activating signal Activating signal NK cells  NK cells have a lot of receptors  Activating receptor → activating signal → ligands are viral proteins that can be expressed on viral infected cell. If receptor on NK cell interact with those proteins, they receive very strong activating signal. If that signal overulls inhibitory signal (for example of MHC class 1) NK cells get activated.  Inhibitory receptors → PD1, LAG3 etc.  Cytokine receptors  Activating or inhibitory receptors → KIR, NKG2, interact with MHC class 1  NK cell activation= balance between inhibitory and activating receptors  T cell → one T-cell receptor to activate, for NK cells it can be a bunch of them Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Normal cells have MHC class 1 molecules, they interact with inhibitory receptors  Normal cells don’t have lot of activating ligands of their surface  Sick cells missing self= limited amount of activating signals, but all inhibitory signals are disappeared (all MHC1 are gone). induced self= when cells do have a certain level of inhibitory molecules. It’s always the balance between activating and inhibitory.  Antibody’s can bind to cancer cell → NK cells have receptor (CD16) that can bind to FC part of antibody → NK cell get super strong activating signal and can kill. CD16 requires binding to antibody that is bound on viral infected or tumor cell  Function: - Killing infected cells - They have granules that contain proteins that mediate killing of target cells. - When NK cells are activated, granule exocytosis releases these proteins to the target cells. - NK cell granule protein: perforin → facilitates the entry of other granule proteins, called granzymes, into the cytosol of target cells. - Granzymes → are proteolytic enzymes that initiate a sequence of signaling events that cause death of the target cells by apoptosis - Activating and inhibitory NK receptors contain structural motifs in their cytoplasmic tails that engage the signaling pathways that respectively promote or inhibit target cell killing and cytokine secretion Activating receptors have immunoreceptor tyrosine-based activation motifs (ITAMs) , which contain tyrosine residues that become phosphorylated by cytoplasmic kinases after binding of ligands to the receptors Inhibitory receptors of NK cells have immunoreceptor tyrosine- based inhibition motifs (ITIMs), which engage molecules that block the signaling pathways of activating receptors What does NK cell do if it is activated? Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  NK cell cytotoxicity= Kill target cells that are sick by releasing granules with perforins= make holes in target cell and granzymes= activate caspase signalling, cells will die.  Alternative way is via dead receptors → many cells have dead receptors FAS-FASL TRAIL-TRAIL TNF-TNFR  If NK cell is activated it can express the ligands for those cells → interaction in stead of releasing granules. If dead receptors signal, they can activate caspase pathways and lead to death of target cell  Produce cytokines (helper function)→ normal blood NK cells primary produce INF-y INF-y → help to get mature dendritic cells, TH1 cells, antibody isotype switching for B cells Safety checks in immune system → make sure that only right cells response. T-cell education in thymus (positive and negative selecting) NK cells have inhibitory receptors. Those are critical to avoid and control autoreactivity. NK cell education Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - We also have NK cells that do not have so many inhibitory receptors → if NK cell mature, they acquire more inhibitory receptors and they also acquire more potent effector function (they kill more easily) - More inhibitory receptors → stronger effector function - More inhibitory receptors → also more strongly inhibited - Mature NK cells → need to interact with their inhibitory ligands. If that doesn’t happen, they become hyperresponsive= not capable of strong killing of producing lot of cytokine - If interaction does occur on healthy cells → they become licensed or educated → they change metabolically, make more granules and more cytokines → they become much more potent killers - This is a way that receptors control the activation of NK cells during maturation process. NK cells:  Function→ Killers, produce cytokines, kill tumour cells and virally infected cells.  Activation → activating ligand that comes from virus or tumour cell. Balance between activation and inhibitory signals  How do they respond to healthy cells → They do nothing to healthy cells because of the MHC class 1 & healthy cells don’t have activating ligands. What features do NK cells share with the adaptive immune system?  NK cells are innate immune cells  They have a lot of features of adaptive immune system  They can learn  Antigen specificity related to MHC class 1 or 2 expression for T cells KIR & NKG2 receptors → can see if there is a peptide (viral protein) in the MHC class 1 (we thought that they could only see if there is MHC or not, Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material because T-cell receptor binds to the peptide and KIR does not interact with the peptide.  NK cells build memory (long lived NK cells, memory NK cells) (antigen dependent memory) If pathogen is gone, most of the NK cells disappear → some long lived memory cells stay around NK cells are simple and usually can’t see MHC dependent antigen presentation → but it can occur in certain viral infections. If it occur they can also build memory  Cytokine induced memory NK cells (IL-12, IL-15 & IL-18) With those cytokines, if you activate under right conditions the NK cells, they proliferate and also form some sort of memory NK cells that are longer lived and can produce more INF-y, are better killers. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Location of NK cells → they are everywhere. Can be in any tissue in the body.  Difference between NK cells in blood and NK cells in tissue NK cell development:  NK cells come from the bone marrow  Have same progenitor as T-cells  Go through different stages  Two types NK cells CD56 bright → with lot of CD56= helper NK cells CD56 dim → low CD56= killer NK cells  Left upper grafiek: T-cells have CD3 as marker Right population → cells that have CD3 marker= T-cells NK cells have CD56 as marker Upper left population → don’t have CD3 marker, but CD56= NK cells Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Right upper grafiek Within that NK population, you can see a large population with intermediate CD56 expression= CD56 dim cells Small upper part → higher expression CD56 marker= CD56 bright cells  Right lower grafiek How it looks in blood 5% bright cells 95% NK cells are the CD56 dim subtype Bright NK cells= good in producing cytokines= helper cells Dim NK cells= good in killing In blood lot of killing NK cells In lymph nodes=more bright NK cells to help B- & T-cells → more bright NK cells (helper) and less dim NK cells There is a different between blood and tissue NK cells  Lower left part = developing NK cells in bone marrow from hematopoietic stam cell progenitor  All those CXCR= chemokine receptors  NK cells in bone marrow have different chemokine receptors then NK cells in blood or tissue  Lymphocytes always migrate because they sense chemokines via chemokine receptors  If there is a certain chemokine that can bind for example to CXCR4 → makes NK cells stay in bone marrow, because that ligand for that receptor is in the bone marrow  If NK cells start to express receptors for which the ligand is in blood (chemokine is in blood)→ they move to blood  Nk cells have different chemokine receptors → having different receptors, they can home to different tissues  The chemokine receptors determine where the NK cells go and where they stay Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  The way they move is partly mediated by how they get activated and which chemokine receptors they start to express What is the difference between NK cells and Innate Lymphoid cells (ILC)?  NK cells are a type of ILC  They have same progenitor  What happens in innate immune system (NK & ILC) is the same as what we see in adaptive immune system  ILCs → do what TH1 cells do. Produce INF-y  ILC2 → TH2 cells  Difference is the way they get activated Role of different ILCs Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Functional plasticity of NK cells and ILC  some flexibility between different types of ILC’s  an true NK cell can never become an ILC3 → it can become an ILC1 if there is enough TGF-beta  there is flexibility between ILC1 and ILC3  this is important because for a right response in tissue  in many diseases there is disbalance between right ILC subsets.  Plasticity can also be the cause of certain immune mediated diseases Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material NK/ILC in pregnancy  To get a properly functioning placenta → cells from the child need to grow into the uterus layer from the mother (see on the right)  Right picture: Lower part is endometrium Purple things are trophoblast cells (cells from child that grow into the blood vessels of the mother) → this is important. Because to get exchange of nutrients between the child and the mother, those blood vessels should open up a bit to allow more blood flow → that happens because cells from the Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material child grow into the uterus of the mother, this needs to be controlled and this is done by NK cells Two different type of NK cell function - Producing cytokines → to get those cells moving, they better not kill - Stop producing attractions and kill the ones that go too far → to Avoid overinvasion - Kill virus → Throughout pregnancy  After implantation → cytotoxicity of NK cells in uterus goes down, but production of pro inflammatory cytokines is low but they do produce factors that attract trophoblast cells NK cells Granzymes, perforines, apoptosis (pathway), Fas/FasLigand Interferons, IL-12 Activators: KIRs, NKG2D, FcgammaRIIIA Inhibitors ILC1, ILC2, ILC3, Cytokines of each subset (IFN-gamma, IL-5, IL-13, IL-22, IL-17,...) Transcription factors (T-BET, GATA3, RORgT,...) IL-10, IL-1RA, SOCs suppressors Functions of ILC (See article Sonnenberg et al) Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material Case 3: dendritic cells 1. What is the connection between the innate and adaptive immune system?  Dendritic cells  The ability of DCs to promote T lymphocyte responses after innate immune activation also makes them an important bridge between innate and adaptive immunity  DCs are tissue-resident and circulating cells that detect the presence of microbes and initiate innate immune defense reactions, and they capture microbial proteins for display to T cells to initiate adaptive immune responses.  The location of DCs in epithelia and tissues where microbes enter, their ability to capture antigens and take them to lymph nodes where naive T cells circulate, and their rapid responses to microbes all place these cells in a unique position in the immune system, serving as sentinels of infection that begin the rapid innate response but also link innate responses with the development of adaptive immune responses. 2. What is the origin and location of DC? ❖ Origin:  Derive from bone marrow precursors, both lymphoid progenitor and myloid progenitor.  Same as monocyte → monocyte and dendritic cell progenitor (MDP) ❖ Location:  Most DCs are widely distributed in lymphoid tissues, mucosal epithelium, and organ parenchyma (functional parts of an organ of structure (such as a tumour)) 3. What DC subsets are there? ❖ Classical DCs (cDCs)  Most numerous DC subset in epithelia (that interface with external environment, such as the skin and the intestinal and respiratory tracts) and lymphoid organs.  They are the DC subset that captures antigens and transports them to secondary lymphoid organs and are thus involved in antigen presentation to naive CD4 + and CD8 + T cells.  Arise from bone marrow HSCs through a developmental pathway that includes a common precursor of both monocytes and classical DCs, some of which develop into committed precursors for cDCs (called pre-cDCs).  All of these steps take place in the bone marrow.  The pre-cDCs migrate to peripheral tissues, where they mature into cDCs.  Two subsets: cDC2 (major) - most numerous subset - potent capturing exogenous antigens and inducing CD4+ T cell responses cDC1 (cross-presenting) Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material - Are especially efficient at transferring ingested antigens from vesicles into the cytosol. → this is an essential step in the process of cross-presentation, in which ingested antigens are presented on class I MHC molecules to CD8 + T cells. - Specialized to present antigens to naïve CD8+ T cells by process called cross-presentation - this subset can also present antigens to CD4+ ❖ Plasmacytoid DCs (pDCs)  Are the body’s major source of type I IFN and are thus essential for innate immune responses to viruses.  They also may capture antigens in the blood and transport them to the spleen (milt) for presentation to T cells  Are named because after activation they begin to resemble plasma cells morphologically (beginnen op plasma cellen te lijken)  They develop in the bone marrow from a precursor distinct from that for classical DCs  They are found in the blood and in small numbers in lymphoid organs. ❖ Monocyte-derived DCs (MoDCs)  Can be induced to develop from monocytes under inflammatory conditions.  Include cells with functions similar to those of cDCs but are derived from monocytes that were recruited into tissue inflammatory sites.  They express CD11c, like all DCs, and also monocyte markers such as CD11b and CCR2. ❖ Langerhans cells (dendritic cells in the skin)  These cells are related to tissue-resident macrophages and develop early in life from progenitors in the yolk sac (dooierzak voorlopers) or fetal liver and seed the skin. Their function is probably similar to that of cDC2.  They are identified by their location and morphology in the skin, the presence of tennis-racket– shaped cytoplasmic organelles called Birbeck granules, and expression of various markers  Langerhans cells may function in the context of skin infections to present antigens to and activate CD4 + T cells, or in the absence of infection, to present self-antigens to CD4 + T cells and induce tolerance to these antigens. ❖ Follicular dendritic cells (FDCs)  have a dendritic morphology but are not derived from bone marrow precursors, do not present protein antigens to T cells, and should not be confused with DCs. Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  FDCs are involved in B cell activation in the germinal centers (kiemcentra) of secondary lymphoid organs 4. How do DC mature?  Mature during migration after activation when they recognize pathogen Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  Dendritic cells ingest pathogens → CCR7  Immature dendritic cells: rest in tissues → bijv. epithelia and form here a network of cells → high phagocytosing capacity, low antigen presentation capacity, low co-stimulation.  Mature dendritic cells: are in T cell rich areas of lymph nodes and spleen.  Langerhans cell (dendritic cell in the skin) is activated the moment it sees and captured an antigen Activation by microbe entering the cell by phagocytosis or by endocytosis At the same time, microbes stimulate the production of inflammatory cytokines such as TNF and IL-1, by binding to PRRs in dendritic cells, tissues epithelial cells and macrophages → activation dendritic cells  Dendrite will migrate in the direction of lymph nodes because there are naïve T cells present to which it must present the antigen. (when it gets activated is receives CCR7 receptor)  Further maturation takes place during the migration process  Dendritic cells mature after recognition danger with PRRs Downloaded by: annaelnatzkaya | [email protected] Want to earn $1.236 Distribution of this document is illegal extra per year?  Stuvia.com - The Marketplace to Buy and Sell your Study Material  The moment a dendritic cell is in the tissue, it is not mature: High phagocytosin