Innate Immunity I and III

Question 1

Monocytes/Macrophages

Answer 1

• Phagocytosis and activation of bacteriocidal mechanisms
• Monocytes circulate through blood, then enter tissues and differentiate into macrophages 3 important functions:

1. engulf and kill invading organisms
2. help induce inflammation to trigger the immune system
3. secrete signaling proteins to recruit/activate other immune cells

• Classically Activated M1
• Alternatively Activated M2
• Macrophages express numerous PRRs to aid their role in pathogen detection – triggering of these receptors leads primarily into the production of classical activated / M1 macrophages.
• TLRs, NLRs, RLRs, CLRs.
• Complement receptors to detect Complement-bound pathogens.
• Scavenger receptors to detect oxidized/acetylated low density lipoproteins (LDL) (sign of tissue damage).
• Cytokine receptors (especially the IFNg receptor) to allow activation even in the absence of direct pathogen detection. In other words, if surrounding cells are producing IFNg (in response to infection), nearby macrophages will become activated as well. Upon their activation in tissues, macrophages are responsible for:
• Cytokine and chemokine production to initiate and regulate inflammation, and recruit other cells to the site of infection. CLASSICAL/M1
• Ingesting pathogens through phagocytosis, followed by pathogen killing via the oxidative burst within the phagosome. CLASSICAL/M1
• Clearing dead cells and tissues and initiating wound repair. ALTERNATIVE/M2

Question 2

PMNs

Answer 2

• neutrophils
• basophils
• eosinophils

aka granulocytes

Question 3

Neutrophils

Answer 3

• Phagocytosis and activation of bactericidal mechanisms
• Most abundant leukocyte in circulation and rapidly recruited to infection
• Excellent phagocytosis, express high levels of CR1 and CR3
• VERY SHORT lifespan (hours upon recruitment to tissues)
• Major component of pus upon their death

Question 4

Basophils and Eosinophils

Answer 4

•Defense against parasites: release cytotoxic granules

Question 5

Mast Cells

Answer 5

• Defense against parasites: release histamine granules
• basophils in blood, mast cells in tissue and mucous membranes
• Key roles in wound repair, immunity to parasitic worms and allergy

Question 6

Natural Killer Cells

Answer 6

• Anti-viral & anti-tumor responses by release of cytotoxic granules
• Typically held in check by normal ratio of activating and inhibitory proteins on host cell surface.
• Infection/transformation changes this ratio, makes host cell a target.
• Many virus target NK receptor proteins to avoid immune detection.

Question 7

Dendritic Cells

Answer 7

• Antigen uptake and presentation – LINKS INNATE & ADAPTIVE
• • Phagocytic cells
• Reside in tissues in an “immature” state – maturation is triggered upon pathogen recognition
• Migrate to nearest secondary lymphoid organ to present antigens to the adaptive system

Question 8

PAMPs

Answer 8

•pathogen-associated molecular patterns, or PAMPs

Question 9

PRRs

Answer 9

• pattern recognition receptors (PRRs)
• recognize PAMPs
• TLRs
• NLRs
• RLRs
• CLRs

Question 10

TLRs

Answer 10

• Toll-Like Receptors (TLRs)
• There are at least 13 different TLRs identified to date.
• These receptors function to detect different components shared by microbes that are highly distinct from host molecules, including bacterial cell wall components and pathogen-derived nucleic acids.
• They are membrane bound and are distributed both on cell surfaces as well as intracellularly in the membranes of endosomal compartments.
• Although different intracellular adaptor proteins and signaling pathways are used, the end goals are the same, control infection and buy time while the adaptive response develops:

1. Trigger the inflammatory response via NF-kB activation
2. Produce antiviral Type-I IFNs via IRFs

Question 11

NLRs

Answer 11

• NOD-Like Receptors (NLRs)
• These cytosolic receptors recognize PAMPs as well as host molecules released in the “wrong location” following cell stress or damage, such as ATP, glucose, urate, and cholesterol (Damageassociated molecular patterns; DAMPs).
• Their activation leads to formation of the inflammasome and promotes the inflammatory response.

Question 12

Inflammasome

Answer 12

• NLR stimulation by PAMPs or DAMPs activates the inflammasome, a multi-component complex to produce pro-inflammatory cytokines.
• Upon final assembly of the inflammasome, (pro) caspase-1 is cleaved into active caspase-1, which then cleaves and activates precursors of the pro-inflammatory IL-1b family (IL-1b and IL-18).

Question 13

RLRs

Answer 13

• RIG-I-Like Receptors (RLRs)
• These cytosolic receptors recognize the presence of viral RNA in the cytosol as a consequence of viral replication, leading to the production of key antiviral Type-I interferons (IFNa and IFNb).

Question 14

CLRs

Answer 14

• C-type Lectin Receptors (CLRs)
• These cell surface receptors recognize carbohydrate PAMPs (bacterial mannose and fungal dectins). Pathogen binding to lectins improves phagocytosis and inflammatory responses to those pathogens.

Question 15

The downstream consequences of PAMP recognition by PRRs…

Answer 15

1. Activate transcription of pro-inflammatory cytokines, adhesion molecules, and costimulatory molecules.
2. Activate transcription to stimulate production of Type-I IFNs (IFN a/b; primarily in response to virus detection).

*NLR stimulation by PAMPs or DAMPs activates the inflammasome, a multi-component complex to produce pro-inflammatory cytokines.

Question 16

Phagocytosis

Answer 16

•Phagocytosis is the process of ingesting extracellular items by engulfing them within the host membrane. For pathogen clearance, the phagocytosed vesicle containing the extracellular pathogen is fused with lysosomes within the cytosolic compartment that contain numerous proteases. This fusion event results in the dramatic lowering of the pH within the vesicle. Ultimately these events cause breakdown of the ingested matter and destruction of the pathogen.

Question 17

3 Professional Phagocytic Cell Types

Answer 17

• Neutrophils
• Macrophages
• Dendritic Cells

Question 18

M1 Macrophages

Answer 18

•• Classically activated macrophages (M1) are activated through pattern recognition receptors (PRRs) and/or IFNg in response to microbial products. These are proinflammatory, and secrete cytokines that stimulate the production of reactive oxygen species within phagosomes to destroy ingested microbes.

Question 19

M2 Macrophages

Answer 19

•Alternatively, activated macrophages (M2) are induced by non-inflammatory cytokines produced in the absence of a strong PRR signal. They promote fibrosis and wound repair, helping to control and resolve inflammation. These cells are key players in the resolution of the infection and in restoring tissue homeostasis.

Question 20

Cytokines

Answer 20

• In response to PAMP detection, macrophages and dendritic cells are key secretors of cytokines that signal and coordinate the immune response to the infection.
• Cytokines are soluble proteins that enable communication between leukocytes, thus many are called interleukins.
• Cytokines are secreted in small amounts and bind to very high affinity receptors on their target cells. They can function in 3 manners:
• Autocrine, acting on the cell that originally secreted the cytokine.
• Paracrine, acting on adjacent cells.
• Endocrine, acting on distant cells.

Question 21

Cytokines are critical coordinators of the innate and adaptive immune responses:

Answer 21

• These soluble mediators recruit circulating leukocytes to the site of infection.
• They activate innate cells (NK and macrophages) to become more potent killers.
• Their systemic effects include signaling the hypothalamus to induce fever, the liver to produce acute phase proteins, and the bone marrow to increase neutrophil production.
• The nature of the innate cytokine response (which cytokines are made? how much?) influences the subsequent adaptive response that develops. This is dictated by which PRRs are triggered during the initial detection of the pathogen. In other words, the type of PRRs triggered during innate recognition (dependent on the pathogen type) dictates which early inflammatory cytokines are produced, which then dictates the type of adaptive response that develops – ensuring the right adaptive response (TH1, TH2, CD8, B cell isotype) for the type of pathogen encountered (extracellular bacteria, virus, parasite).
• TNFa and IL-1 family members are the primary pro-inflammatory cytokines that activate endothelial cells to promote leukocyte recruitment to the site of infection in tissues. Systemically, these cytokines induce the fever response in the hypothalamus. TNF-a also stimulates production of acute phase proteins by the liver.

Question 22

Chemokines

Answer 22

•Chemokines are soluble mediators that control adhesion, chemotaxis and activation of many leukocyte populations. They are the major regulators of cell trafficking.

Question 23

Tumor necrosis factor a (TNFa)

Answer 23

• Pro-Inflammatory
• Macrophages, T cells
• Endothelial cells: activation (upregulate adhesion molecules to recruit other cells)
• Neutrophils: activation leading to their secretion of various effector molecules
• Hypothalamus: fever
• Liver: synthesis of acute-phase proteins

Question 24

Interleukin-1 (IL-1)

Answer 24

• Pro-Inflammatory
• Macrophages, endothelial cells
• Endothelial cells: activation (upregulate adhesion molecules to recruit other cells)
• Hypothalamus: fever
• Liver: synthesis of acute-phase proteins

Question 25

Interleukin-6 (IL-6)

Answer 25

* Pro-Inflammatory * Macrophages, endothelial cells, T cells * Liver: synthesis of acute-phase proteins

Question 26

Interleukin-12 (IL-12)

Answer 26

* Pro-Inflammatory * Dendritic cells, macrophages * NK cells and T lymphocytes: IFNg production, increased cytotoxic activity

Question 27

Interferon-g (IFNg)

Answer 27

* Pro-Inflammatory * NK cells, T lymphocytes * Macrophages: activation to become better killers of ingested microbes

Question 28

Type I IFNs (IFN-a, IFN-b)

Answer 28

* Pro-Inflammatory * IFN-a: dendritic cells, macrophages * IFN-b: fibroblasts * All cells: enhance antiviral state, increase class I MHC expression * NK cells: activation

Question 29

Interleukin-10 (IL-10)

Answer 29

* Anti-Inflammatory * Macrophages, dendritic cells, T cells * Macrophages, dendritic cells: inhibition of IL-12 production, reduced expression of costimulatory molecules and class II MHC.

Question 30

TGF-b

Answer 30

* Anti-Inflammatory * Many cell types * Many cell types: Inhibition of inflammation

Question 31

Interleukin-8 (IL-8)

Answer 31

* Migration * Macrophages * Major chemotactic signal for neutrophil migration

Question 32

Chemokines

Answer 32

* Migration * Macrophages, dendritic cells, endothelial cells, T lymphocytes, fibroblasts, platelets * Leukocytes: increased integrin affinity, chemotaxis, activation

Question 33

Innate Immune Reactions

Answer 33

1. Acute Inflammation 2. Wound Repair 3. NK Cell COntrol of Viral Infection

Question 34

Innate Immune Reactions - Acute Inflammation

Answer 34

* Many bacterial infections activate phagocytes and trigger an acute inflammatory response. This complex process can also be induced by mechanical injury (cut), physical injury (burn), or chemical injury (corrosive chemical). * The hallmarks of inflammation include heat, redness, pain, swelling, and loss of function of the inflamed area (“calor, dolor, rubor, tumor, functio laesa”). * An inflammatory response is required for effective adaptive immune responses in two key ways: * Identifying the type of pathogen (through PRRs) to dictate the type of adaptive response that develops. * Recruiting effector cells to the site of infection by upregulation of local cytokine and chemokine production to draw circulating cells to that location, and upregulation of local adhesion molecules to allow the cells to get in to the tissue. Acute inflammation is a multi-step process that rapidly delivers mediators of host defense to the site of infection. In general, PAMP detection in the tissues by resident macrophages and dendritic cells leads to the production of TNF-a and IL-1b. These diffuse to nearby vascular endothelium and cause the upregulation of selectins. Vasodilation aids this process by increasing the volume of blood through the vessel, and slowing the rate of flow.

Question 35

Innate Immune Reactions - Acute Inflammation, Inflammation Driven Leukocyte Recruitment

Answer 35

1. CELL ROLLING: Vasodilation leads to an increase in blood vessel diameter = more volume, slower blood flow. This allows circulating leukocytes to roll along the vessel wall, mediated by weak interactions between selectins and carbohydrates. (heat, redness) 2. ACTIVATION: Both the rolling leukocytes and the vascular endothelium express chemokine receptors. If phagocytic cells in the underlying tissue have detected a pathogen through their PRRs, they will secrete chemokines. These chemokines are sensed by the vascular endothelial cells, causing activation of the endothelium, and increased expression of high-affinity integrins and adhesion molecules. 3. ARREST & ADHESION: Increased expression of adhesion molecules on the endothelium allows circulating leukocytes to tightly attach to the vascular endothelium. Once arrested, the leukocytes also sense the inflammatory cytokines and/or chemokines in the local environment, leading to further upregulation of integrins and adhesion molecules on the leukocytes. 4. TRANSMIGRATION/DIAPEDESIS: Extravasation of immune cells out of the circulation and into the inflamed tissue site (neutrophils, monocytes, lymphocytes) for elimination of the pathogen, tissue repair & regeneration. (pain, swelling).

Question 36

Recruitment of circulating leukocytes to the site of infection is accomplished by adhesive interactions with the vascular endothelium, with different interactions used by different leukocytes (neutrophils, monocytes, lymphocytes). There are 3 main classes of adhesion molecules:

Answer 36

1. Selectins 2. Integrins 3. Adhesion Molecules \*Mast cells present in the skin and mucosal epithelium are also able to sense pathogenassociated molecular patterns (PAMPs) leading to their release of granules containing vasoactive amines (i.e. histamine) and prostaglandins, which increase vascular permeability and promote inflammation.

Question 37

Selectins

Answer 37

• Selectins (L-selectin, P-selectin, E-selectin) – Carbohydrate-binding proteins that mediate adhesion of leukocytes to endothelial cells by relatively weak binding; allows cells to “roll” along the vascular endothelium. Selectins can be expressed by both leukocytes and endothelial cells.

Question 38

Integrins

Answer 38

• Integrins (Mac-1, LFA-1, VLA-4) – Steady state expression on leukocytes is in a low-affinity state (weak binding); this undergoes a (reversible) conformational change to a high-affinity binding state after exposure to pro-inflammatory cytokines and/or chemokines. Integrins bind to adhesion molecules on the endothelium.

Question 39

Adhesion Molecules

Answer 39

•Adhesion molecules (ICAM-1, ICAM-2, VCAM1) – Expression on endothelial cells mediates tight binding to allow for the arrest of circulating leukocytes on the endothelium. Adhesion molecules bind to integrins expressed on circulating leukocytes.

Question 40

Innate Immune Reactions - Wound Repair

Answer 40

* Inflammation is highly beneficial for clearing pathogens that have breached barriers (for example, when the skin in cut), but the resolution of the inflammatory response, and the repair of the wound is also highly important. * During the inflammatory response, increased vascular permeability allows serum proteins access into the tissue space, including components of the Kinin, Clotting, Fibrolytic and Complement systems. * M2 macrophages are a major component of resolving inflammation and repairing tissues. Short-lived neutrophils at the site of infection undergo apoptosis after a few days, and are phagocytosed by macrophages. Macrophage uptake of dying neutrophils is a signal to begin the wound repair process, and helps to reprogram M1 “classically activated” macrophages to the M2 “alternatively activated” phenotype and function. Thus, M1 and M2 are not necessarily distinctly different macrophage populations, but rather can represent changes in the function of the macrophage over the course of time as the infection resolves and repair begins. * M2 macrophages produce TGF-b and extracellular matrix proteins to aid in tissue repair. TGF-b is a powerful stimulator of collagen production by fibroblasts. Secretion of anti-inflammatory TGF-b and IL-10 helps resolve the inflammation at the tissue site, and antagonizes the pro-inflammatory activity of remaining M1 macrophages.

Question 41

Innate Immune Reactions - NK Cell Control of Viral Infection

Answer 41

* NK cells patrol the host at a moderate state of activation and at a relatively high frequency (~10-15% of peripheral blood lymphocytes). * Innate cytokines (Type I IFNs, IL-12) are produced by macrophages and dendritic cells in response to detection of viral infection (PAMPs) and induce rapid NK cell proliferation and activation. Once, activated, NK cells have 2 primary functions: 1. Induce death of virally infected cells via secretion of lytic granules. 2. Produce IFNg to activate neighboring macrophages, allowing the macrophages to become more potent killers of their intracellular microbes. * NK cell activity is controlled by the balance of signals they receive through their multiple activating and inhibitory receptors. As they survey their environment, these NK receptors assess other host cells for signs of: * Infection or damage (NK activating) * Cellular stress related molecules (NK activating) * MHC proteins (normal expression inhibits NK, loss of expression activates NK) * Some viral proteins directly (NK activating)