Lecture 13 Objectives

Describe the 3 stages of the inflammation response.

1)Vasodilation and Increased Permeability of Blood Vessels 2) Phagocyte Migration and Phagocytosis 3) Tissue Repair Ex: Microbes are replicating and causing damage to host cells. Resident Macrophages able to engulf those microbes will secret signals to call for help. Damaged cells will also release chemicals. This will cause: blood vessels to dialate. Stage 1: Vasodilation is increased diameter of blood vessels initial response to injury or invasion pf pathogens, localized. --This results in the delivery of more blood and thus more white blood cells, oxygen, and nutrients., as well as clotting agents which allows a blood clot to form and prevent microbes from spreading. ( Many chemicals trigger this process:•Bradykinin•Prostaglandins•Leukotrienes•Histamine•) Some of these chemicals also increase permeability• They cause cell lining in vessels to contract and pull apart, leaving gaps in the walls through which phagocytes and clotting factors can move into damaged tissue. This will permit fluid to move into the tissue spaces which causes an accumulation of fluid or swelling. --Causes pus to form (a fluid containing dead cells, leukocytes, and pathogens) Such an isolated site of infection is called an abscess. After an hour.. Stage 2: Phagocyte Migration and Phagocytosis --Phagocytes arrives and cytokines are released. cytokines alter cellular adhesion molecules, on the lining of blood vessels which will allow Phagocytes to stick. Flow of blood gradually decreases, and in response to cytokines Phagocytes (first neutrophils, then monocytes- which become macrophages once they leave the blood) begin to stick to the inner surface of blood vessels in a process called MARGINIZATION --Phagocytes begin to squeeze between the endothelial cells to reach the damaged area. This migration OUT is called DIAPESEDIS (can take 2min) Increased permeability allows antimicrobial chemicals and clotting proteins to seep into the damaged tissue which also causes swelling pressure on nerve endings/pain. --Phagocytes begin to destroy invading microbes and cellular debris. After engulfing large numbers of microbes and damaged tissue, they die. The accumulation of damaged tissue and leukocytes forms pus. Stage 3: Tissue Repair --Tissues replace dead or damaged cells.•Involves delivery of extra nutrients and oxygen to the site via dilated blood vessels.•Ability to regenerate or repair the area totally depends on type of tissue. --Skin- high capacity for regeneration. --Cardiac- tissue cannot be repaired.

C. Diff and Fecal Transplants

1. C diff is part of the normal intestinal microbiome for some people but is kept in check by other members microbiome 2. Given antibiotic treatment (especially broad spectrum) can kill off competing microbes, allowing C diff to grow unabated 3. Symptoms include diarrhea, inflammation of the colon (pseudomembranous enterocolitis),fever, nausea, abdominal pain Cure? Stool Substitution Transplant Therapy - Repopulating the gut, not first line therapy, but consistent success - Fecal matter from a donor is transferred to the GI Tract of a patient

Other ways to prevent microbial invasion

1. Stomach acid: very low pH kills microbes• 2. Urine: acidity inhibits microbes, flushes microbes out 3. GI peristalsis: continually moves contents through GI tract 4. Saliva: helps dilute number of microbes, contains lysozyme 5. Earwax: prevent microbes from entering ear 6. Lacrimal apparatus: produce and drain away tears; washing action + tears contain lysozyme

Pathogen

A disease causing microbe if able to: 1) Gain access to the body by penetrating the surface of the skin or entering through a portal of entry. 2) Attach itself to host cells using various adhesion factors: pili, fimbriae, glycocalyx 3) Evade the body's defense mechanisms long enough to produce harmful changes

Describe the process of phagocytosis. Be sure you include (and understand) the following terms: How are Toll-Like Receptors, and Pathogen-Associated Molecular Patterns involved in this process? How are ingested pathogens destroyed once inside the immune cell?

Cells capable of phagocytosis: Neutrophils, Eosinophils, Macrophages Summary: 1) Chemotaxis: the phagocyte travels toward microbial chemical components and secretions, toward microbe 2) Phagocyte binds to microbe- adherence is facilitated by attachment of PAMPs to TLRs on the surface of phagocytes in the membrane. 3) Phagocyte extends pseudopods to surround the microbe- microbe is internalized into phagosome. Pathogens are more readily phagocytized if they are covered with opsonin- complement or antibodies (more later). There are certain proteins released by immune cells that can cover the surface of the microbe and makes it more attractive to the phagocyte. Opposite would be a capsule. 4) Phagosome fuses with lysosome (contains antimicrobial substances) >> phagolysosome 5) exocytosis Non specific chemical defenses against pathogens Toll-like receptors (TLRs) - 1. Certain responses of Innate Immune system can be activated by these proteins. Integral membrane proteins in cytoplasmic membrane of certain defensive cells like Macrophages., produced by phagocytic cells 2. TLRs Bind and recognize pathogen-associated molecular patterns (PAMPs). These are Molecular components commonly found on pathogens. A few examples: peptidoglycan, LPS, flagellin, Teichoic Acid. 3. When TLRs bind PAMPs, this initiates defensive responses. 4. When these bind, Cytokines are released. These are proteins that regulate the intensity and duration of an immune response. Immune cells communicate to each other thru Cytokines like QS.

What is the mechanism of action of MAC?

Cytolysis of microbial cells involves the Membrane Attack Complex 1) Eventually C5b, C6, and C8 bind together subsequentially and insert into the plasma membrane of the invading cell. Together C5b thru C8 make form the Membrane Attack Complex. The MAC creates a hole on a pathogen's cell membrane and makes transmembrane channels, allowing for flow of extracellular fluid into the pathogen and burst the cell. Plasma membranes of the host cell contain proteins that protect against lysis by preventing the MAC proteins from attaching to their surfaces. Gram Neg bacteria are more susceptible to cytolysis they only have 1 or few layers of peptidoglycan to protect plasma membrane

Describe the 3 lines of defense that protect our body.

First line: Intact skin Mucous membranes and their secretions Normal microbiota Second line: --Phagocytes: such as neutrophils, eosinophils, dendritic cells, and macrophages --Inflammation --Fever --Antimicrobial substances Third line: --Specialized lymphocytes: T cells and B cells --Antibodies

[Connection - L4] Which bacteria (Gram positive or Gram negative) is MAC more effective against? HINT: MAC must reach the cell membrane in order to damage it -which cell wall makes this easier?

Gram negatives are more susceptible to osmotic pressures because because they only have one or few layers of peptidoglycan. MAC must reach the cell wall in order to damage it.

More on Innate Immune System

Includes physical, chemical, and cellular barriers: 1. Physical barriers include skin and mucous membranes: line gastrointestinal, respiratory, urinary 2.Chemical barriers include stomach acidity pH (prevents growth), secreted anti-microbial peptides: Example lysozyme: inhibits in generic way 3. Cellular barriers include macrophages, neutrophils

Be able to explain which part of the innate immune response result in each of the symptoms of inflammation (redness, warmth, swelling, pain).

Increased blood flow results in redness and warmth Swelling is due to accumulation of fluids. Pain is due to release of certain chemicals and pressure of edema on nerve endings. Increased permeability allows antimicrobial chemicals and clotting proteins to seep into the damaged tissue which also causes swelling pressure on nerve endings/pain. They cause cell lining in vessels to contract and pull apart, leaving gaps in the walls through which phagocytes and clotting factors can move into damaged tissue. This will permit fluid to move into the tissue spaces which causes an accumulation of fluid or swelling.

What is inflammation?

Inflammation. C5a is chemotactic, attracting phagocytes (immune cells) to the site of infection. Some activated fragments cause vasodilation --> C5a and C3a can bind to basophils and mast cells, causing the release of histamine. Function: Inflammation is in response to pathogens, heat, cut, chemicals, acids, bases: **Protective response to eliminate cause of tissue damage and destroy that agent and remove it from body or at least wall it off. Then repair the damage or replace tissue Symptoms: REDNESS, HEAT, SWELLING, PAIN, IMMOBILITY/LOSS OF FUNCTION Two Types: 1) Acute inflammation• Develops quickly and is short lived (days/weeks). Self limiting and Is typically beneficial Ex: sore throat: •Dilation and increased permeability of the blood vessels leads to more phagocytes, oxygen, antimicrobial chemicals, and nutrients to the site• Provides a way for phagocytes that are normally confined to the bloodstream to enter infected areas and Tissue repair 2) Chronic inflammation: Long-lasting. Damage to tissues can cause disease Ex: Ulcerative Colitis, Rheumatoid Arthritis

Second Line of Defense

Operates when pathogens penetrate the skin or mucous membranes 1. Composed of cells, antimicrobial chemicals, and processes 2. Many of these components are contained in or originate in the blood Blood is comprised of: PLASMA- mostly water, containing electrolytes, dissolved gasses, nutrients, complement proteins FORMED ELEMENTS: cells and cell fragments suspended in plasma. Created in bone marrow by stem cells. Process called Hematopoiesis. One cell called Pluripotent stem cell which develops into 2 other types of cells: myeloid and lymphoid stem cells. All of the formed elements are developed from these. FORMED ELEMENTS CONTAIN 1. Erythrocytes (red blood cells) : carry oxygen and carbon dioxide in blood 2. Leukocytes (white blood cells) : immune cells that defend body against invaders. 2 types a) Granulocytes and b) Agranulocytes Complement proteins and antibodies Platelets : involved in blood clotting

What is opsonization?

Opsonization or immune adherence promotes attachment of phagocyte to a microbe. This enhances phagocytosis. Coating microbes with C3b enhances phagocytosis

Functions of the Immune System

Protection from disease: 1) Prevents invasion by pathogens from entering the body 2) If they enter, it eliminates the pathogens before causing further damage 3) Preventing reinfection

Identify components of the 1st line of defense as discussed in lecture. How can resident microbiota prevent pathogenic bacteria from establishing infections? Why are fecal transplants a treatment option for C diff?

SKIN 1. Composed of two major layers --Epidermis : Outer thin layer. Little or no material between cells. Multiple layers of tightly packed cells. Few pathogens can penetrate these layers. Top layer shedding of dead skin cells removes microorganisms. Epidermal dendritic cells phagocytize pathogens --Dermis: Collagen fibers help skin resist abrasions that could introduce microorganisms 2. Skin is dry, which inhibits growth Gram + better at resisting desiccation 3. Skin has chemicals that defend against pathogens -- Antimicrobial peptides (defensins) ****Defensins can target cytoplasmic membrane and interfere with DNA and protein synthesis, folding, and cell wall synthesis --Perspiration secreted by sweat glands -- Salt inhibits growth of pathogens --Lysozyme destroys cell wall of bacteria 4. Sebum secreted by sebaceous (oil) glands --Helps keep skin pliable and less likely to break or tear --Lowers skin pH to a level inhibitory to many bacteria MUCOUS ESCALATOR 1. In Mucous membrane of trachea 2. Goblet cells produce mucus that traps invaders. This is a thick glycoprotein 3. Cells of mucous membrane are coated in cilia (Eukaryotes, short flagella). Cilia are continually beating, propelling mucus and trapped organisms away from lungs to be coughed up or swallowed. NORMAL MICROBIOTA Some scientists consider this to be part of the 1st line defense 1. Microbes in and on our bodies. 2. Considered a defense because of Microbial antagonism: Normal microbiota compete with potential pathogens 3. Activities of normal microbiota make it hard for pathogens to compete by: a) Taking up space b) Consuming nutrients c) Creating an environment unfavorable to other microorganisms For ex. vaginal Lactobacillus produces hydrogen peroxide to prevent infection by Chlamydia, Gardnerella, Candida d) Help train immune system to recognize who is innocuous/harmful e) Stimulate the body's second line of defense f) Some argue they promote overall health by providing vitamins to host

Understand the role of iron-binding proteins in preventing infection. What are siderophores?

Siderophores are structures created by some pathogenic bacteria 1. Most pathogenic bacteria require iron because its an inorganic ion = cofactor 2. In the body, most of our iron is not freely available to pathogens because it is bound to iron binding proteins such as Transferrin which stores and transports iron in the body. 3. So Some bacteria secrete high-affinity iron chelating compounds called siderophores. These bind to iron more tightly than transferrin, so they can steal iron from you by stealing it from transferrin! 4. This is an important virulence factor that allow bacteria to be pathogenic 5. But then we can produce lactoferrin, which binds even more readily than Siderophores and steals the iron back (chemical tug-of-war)

[Connection - L14] The complement system is also triggered by antibodies during the adaptive immune response. Despite this, the complement system is considered part of the innate immune system. Explain why.

The Complement System is apart of innate immunity because its not adaptable, doesnt change over a person's lifetime = Innate Immune response. It can be recruited by Adaptive IR. Which is how to they can cooperate. Complement System consists of Multiple proteins (over 30 produced in the liver) that circulate in blood serum and tissues. They exist within blood plasma. Together the proteins of the complementary system destroy microbes by cytolysis, opsinization, and in flammation, they also prevent excessive damage damage to host tissues. **Ultimate Function is to destroy microbes Proteins are inactive form until split into activated fragments (indicated by lowercase letters a and b). Complement proteins are labeled C1-C9, they are activated. The activated forms carry out destructive actions. • This Involves an enzyme cascade (one reaction triggers another, which triggers another...) This activation can occur in 3 pathways that in the end, activates C3 ** So if you start with one protein thats an active one, it splits into fragments, activates another inactive and so on. •Many functions: 1) Direct killing of Membrane attack complex (MAC): forms a hole in pathogen's membrane and ECF will enter the cell = lysis 2) Activation of phagocytes - can enhance or activate the activity of other immune cells (complement) 3) Triggers Inflammation, fever, opsonization 4) Induced by adaptive or innate immune responses 5) Can be activated in 3 ways (see next slide) 6) Considered innate because it is not adaptable- it does not change over a person's life Classical Pathway: initiated when antibodies bind to antigens (antigen bound antibody complexes.. End result is c3 a participates in inflammation, c3b in cytolysis and opinization. Alternative Pathway: Does not involve antibodies, but activated by contact between certain complement proteins and a pathogen. C3 is constantly present in the blood, combinnes with complement proteins(factor B, D, P) on the microbes surface. Complement proteins are attracted to microbial cell surface material (lipid-carbohydrate complexes of certain bacteria and fungi) Once complement proteins bind and iinteract to C3, C3a causes inflammation, C3b in cytolysis and opsinization. Lectin Pathway: When macrophages ingest bacteria, viruses, or other foreighn material by phagocytosis, they release cytokins that stimulate the liver to produce lectins, proteins that bind to carbohydrates. -Mannose Binding Lectin binds to the carbohydrate mannose. MBL binds to many pathogens because MBL molecules recognize a distinctive pattern of carbohydrates which is found in bacterial cell walls and some viruses. - Result of binding, BML functions as opsonsin to enhance phagocytosis and activates C2 and C4 which activates C3 which results in same outcomes.

Explain what the complement system is, and the 3 consequences of complement activation.

The complement system, also known as complement cascade, is a part of the immune system that enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen's cell membrane 1) Classic : Antibodies (which are part of the adaptive immune response) bind to foreign substances (called antigens). 2) Alternative: Protein C3b accumulates on the surface of a microbe 3) Lectin: (Activated by microbial endotoxin or glycoprotein).Activated by microbial polysaccharides (mannose). all 3 of these ways the cell surface of a pathogen is recognized, and this causes the activation of C3. An enzyme cascade follows. •1) Microbes burst as extracellular fluid flows in through a channel formed by the Membrane attack complex (MAC)•A bunch of C9 proteins come together to form a circular hole in the pathogen's membrane, leading to lysis! 2) Opsonization. Coating microbes with an opsonin (in this case C3b) enhances phagocytosis. 3) Inflammation. C5a is chemotactic, attracting phagocytes to the site of infection. Some activated fragments cause vasodilation --> C5a and C3a can bind to basophils and mast cells, causing the release of histamine.

Briefly, how does the immune system distinguish self from non-self?

The immune system can distinguish between self and non self. 2) It must be able to differentiate between our cells and foreign invaders. 3) Happens by recognition of molecular signatures of cell surface receptors that appear different. Example: Immune cells can recognize bacterial flagellin, recognitions of LPS lipid, certain monomers that make up capsules that bacteria produce. This is the ability to recognize 3D structures that are different from those found on our cells.

Briefly differentiate between innate immunity and adaptive immunity.

Two branches of the Immune System: Innate immunity: 1. Present at birth, prior to exposure to any infectious agent 2. There is non-specific recognition of molecular: it responds to pathogens in a generic way. It recognized common features of a pathogen Ex: Flagellin, LPS: many microbes share these. Doesn't target a strain 3. Rapid: minutes. 4. Doesn't generate immunological memory, it doesn't provide a long lasting or prevent reinfection. 5. Considered 1st and 2nd line of defense before Adaptive Immunity Adaptive Immunity: 1. Specific response to a specific microbe 2. Slower to respond 3. Does generate immunological memory 4. 3rd line of defense ** Preventing must require our immune system has to remember certain molecular patterns

[Lab 26] List and describe the 5 major classes of leukocytes as discussed in class. Identify the agranulocytes and the granulocytes, and which leukocytes are phagocytic.

WBC/ Leukocytes are divided into 2 groups: GRANULOCYTES: Contain large granules you can see in the cytoplasm that stain different colors **3 types 1. Basophils: Not phagocytic so they can't do the process of phagocytosis. They can Release substances such as histamine that are important in inflammation and allergic responses. Rarest of WBCs: .5 - 1% 2.Eosinophils: Somewhat phagocytic, can do phagosytosis. •Important for parasitic (worms) infections and allergic infections. Can't phagocytose parasitic worms (they're huge) but secrete toxins to kill them! Relativeley rare 2-4% 3. Neutrophils (polymorphonuclear leukocytes or PMNs) First cell to appear after infection, but are short-lived and Highly phagocytic and motile They can exit the blood to attack invaders and tissues by squeezing between cells that line capillaries. 60-70% AGRANULOCYTES: They have granules but you can't see them after staining -Cytoplasm appears uniform under a light microscope ***Two types 1. Lymphocytes: Most involved in adaptive immunity= Natural Killer cells, B cells, T cells. 20-25% of leukocytes are these. 2. Monocytes: Are precursors to macrophages. They Leave the blood and mature into macrophages: which are long lived phagocytic cells that devour foreign objects. These are large, horseshoe nucleus

Immunity

When microbes attack we use mechanisms of immunity 1) Immunity is resistance: the ability to ward off disease caused by microbes or their products 2) Immunity protects us against other environmental agents: pollen, animal dander, or other chemicals

Explain the benefits of fever in fighting infection.

•Fever is systemic, abnormally high body temp --Body temperature over 37°C •Results when pyrogens (majority of them are derived from bacteria or viruses, LPS) trigger the hypothalamus to increase the body's core temperature Various types of pyrogens: 1) Bacterial toxins 2) Cytoplasmic contents of bacteria released by lysis 3) Antibody-antigen complexes 4) Pyrogens released by phagocytes that have phagocytized bacteria Functions: 1) Temperature Inhibits growth of some microbes 2) Temperature increase enhances performance of phagocytes and speeds up tissues repair 3) Only beneficial to a point. If fever is too high- critical proteins are denatured. = Hallucinations, coma, death

Differentiate between: monocyte vs. macrophage, mast cell vs. basophil.

•Mast cells are similar to basophils: they have many granules rich in histamine. They play roles in inflammation and wound healing, and they have IgE receptors> play a role in allergic responses (more on this later) Mast cells differentiate in a separate lineage Mast cells are residents of connective tissue and mucosa: they do not circulate in the bloodstream like Basophils Macrophage vs Moncocyte: Macrophage: 1. Immune cell in ECF 2. 21mm 3. Phagocytose cell debris/foreign material like bacteria and virus 4.Innate immunity and adaptive immunity by presenting antigens of foreign bodies on their MHC complex Monocyte: 1. Immune cell in blood 2. 7-10 mm 3. Macrophages 4. Innate immunity by differentiating into macrophages. Adaptive by secreting cytokines and chemokines