Receptors

Question 1

Pathogenic strategies to avoid immediate destruction by macrophages and neutrophils

Answer 1

Macrophages and neutrophils have receptors able to recognize pathogens. But pathogens can also avoid this recognition.
For example:
- Streptococcus pneumonie has a thick coat, that is not recognized by any phagocytic cell, so it can cause pneumonia.
- Mycobacterium tuberculosis can cause tuberculosis because inside the macrophages they can be resistant to the acidic environment.

Question 2

Pathogen recognition and tissue damage initiate an inflammatory response

Answer 2

The interaction between the pathogen and tissue activates the macrophages to release cytokines and chemokines (which are important for chemotaxis), that set up inflammation in the tissue, attract monocytes and neutrophils to the site of infection, and allow plasma proteins to enter the tissue from the blood.
Inflammation is initiated within hours of infection. Macrophages are stimulated to secrete pro-inflammatory cytokines and chemokines by interactions between microbes, microbial products, and specific receptors.

Question 3

The role and characteristics of inflammation

Answer 3

Role:
1) Deliver additional effector molecules and cells from the blood into the site of infection, increasing the destruction of the pathogen;
2) Induce local blood clotting, providing a physical barrier to the spread of the infection in the bloodstream.
3) Promote repair of the injured tissue.
Characteristics of the inflammation site, activated by the cytokines:
- Redness and heat:
Cytokines produced by macrophages cause dilation of local small blood vessels, leading to an increased blood flow.
Leukocytes move to the periphery of the blood as a result of increased expression of adhesion molecules by endothelium.
- Swelling and pain:
Increased permeability and leukocytes extravasate at the site of infection, and blood clotting occurs in the microvessels.
Monocytes can differentiate between cytokines through some stimulating factors, such as GM-CSF recognizing macrophages, and GM-CSF+IL-4 recognizing dendritic cells.

Question 4

Inflammatory mediators

Answer 4

• Initially released by macrophages due to the recognition of pathogens,
  later neutrophils and other white blood cells.
• Macrophages and neutrophils secrete lipid mediators,
  followed by chemokines and cytokines, TNFα
• C5a increases vascular permeability and induces the expression of certain adhesion molecules on the endothelium, also activates local mast cells.
• Mast cells release their granules containing histamine, TNFα, and cathelicidins

On the phospholipid membrane, we can activate the arachidonic acid by the presence of phospholipase A2, which initiates prostaglandins (pain mediators), thromboxanes (blood clotting mediators), and leukotrienes.

Injury in blood vessels
activates two cascades of protective enzymes that reduce the spread of infection:
- Kinins - stimulate the complement system, promote localized vasodilation and increased capillary permeability, activate pain receptors, act as chemotaxis.
- Coagulation system - produces fibrin coat which prevents blood loss.
The clot physically encases the infection reducing entry into
the bloodstream.

Question 5

Toll-like receptors Intro

Answer 5

Cytokine and Chemokine production by macrophages is the result of stimulation of signaling receptors on these cells by a wide variety of pathogenic components.

• Toll-like receptors (TLR)
  Homologous to toll receptors in Drosophila melanogaster.
• The receptor protein toll was identified as a gene controlling the dorso-ventral pattering embryo of D. melanogaster.
• In the adult insect, Toll signaling induces de expression of several host-defense mechanisms, including antimicrobial peptides, and drosomycin. It is critical for defense against Gram-positive bacteria and fungi.
• In mammals, important for resistance to viral, bacterial, and fungal infection.

Question 6

TLRs

Answer 6

There are many TLRs (from 1 to 10 in humans), which become dimers when activated. Most of them will be homodimers, but TLR1:TLR2 and TLR2:TLR6 will be heterodimers. They can be found in different cells, such as monocytes, macrophages, neutrophils, NK cells, etc.
A lot more of the leukocytes will have TLRs.
These receptors can recognize nucleic acids (in RNA or DNA) (TLR-3,7,8,9), flagellin (TLR-5), LPS - cell wall of gram-negative bacteria, and lipoteichoic acids - gram-positive bacteria (TLR-4), and the heterodimers will recognize sugars.

TLRs are single when they are inactivated. They have an exterior domain.
LRR(leucine-rich repeats) creates a horse-shoe protein scaffold that is adaptable for ligand binding and recognition on both the outer and inner surfaces.
The cytokine receptor interleukin 1β (IL-1 β) has a TIR domain in its cytoplasmic tail and signals the same as that activated by some TLRs.
Dimerization occurs by ligand binding.

They can be expressed at the level of the plasma membrane, at the extracellular domain, But they can also be intracellular, and the recognition domain is found inside the vesicle. The TIR domain is facing the cytosol.
Endosomes and lysosomes will be vesicles that contain material from the extracellular face. So they can not only recognize an infection but also the presence of a virus on the outside.

There will be receptors that require co-receptors, f.ex. TLR-4 will need a co-receptor MD-2 to recognize LPS.

Dimerization:
The convex surfaces of TLR1 and TLR2 have binding sites for lipid side chains or triacyl lipopeptides.
The binding of each TLR to the same lipopeptide includes dimerization, bringing their cytoplasmic TIR domains into close proximity.

Question 7

TLR2:TLR6

Answer 7

Recognition of some ligands by the TLR2:TLR6 heterodimer, such as long-chain fatty acids and cell wall β-glucans requires an associated co-receptor- CD36, which binds long-chain fatty acids, and Dectin-1, which in turn binds β-glucans, both cooperate with TLR2 in ligand recognition.

Question 8

TLR5

Answer 8

Recognizes a highly conserved site on flagellin that is buried and inaccessible in the assembled flagellar filament. Therefore, the receptor is only activated by monomeric flagellin, which is produced by the enzymatic breakdown of flagellated bacteria in the extracellular space.
TLRs that recognize nucleic acids are transported to endosomes via the ER.

Question 9

TLR3

Answer 9

Recognizes nucleic acids in the double-stranded RNA and the genome present in viruses, so it can recognize different groups of viruses.

Question 10

TLR3,7,9

Answer 10

Are delivered from the endoplasmic reticulum to the endosome due to interaction with UNC93B1, which is composed of 12 transmembrane domains.

Question 11

TLR4

Answer 11

Senses and responds to numerous bacterial infections, binds MD-2, and is transported to the plasma membrane. It is found in macrophages, dendritic cells, eosinophils, and mast cells.
A lot of the hydrophobic pocket of the molecule will bind the MD-2, and the other portion is accessible to the horseshoe repeat.
They recognize the LPS, but not in the bacterial cell, it needs to be processed before the TLR complex can recognize it.
There is an LBP present in the blood that is able to cut the LPS, which gets delivered to CD14, which allows the binding of LPS and delivers it to MD2. The TLR4 will be sitting and waiting for the LPS to be delivered to it.

Question 12

TLRs function

Answer 12

Signaling by mammalian TLRs in various cell types induces the production of:
- Inflammatory cytokines
- Chemotactic factors
- Antimicrobial peptides
- Antiviral cytokines
- IFNα
- IFNβ
- Type I interferons

TLRs activate several different signaling pathways that activate different transcription factors:
- NFκB ->Cytokines and chemotactic factors
- Interferon regulatory factor (IRF) -> Cytokines and chemotactic factors
- Activator protein 1 (AP-1) family, such as c-Jun, through MAPKs -> Type I interferons

A single pathway can activate more than one outcome.

The TLR dimers have 4 possible molecules to bind:
- Myeloid differentiation factor 88, MyD88 Adaptor-like, TIR domain-containing adaptor-inducing IFN-β, TRIF-related adaptor molecule. (NO need to remember by names, just know that TIR can bind 4 classes of molecules). They allow us to get to the 3 activation mechanisms of the TLRs. There are TLR receptors that can bind to more than one of those molecules.

Question 13

NFkB transcription factors

Answer 13

NO NEED TO MEMORIZE, just know the general mechanism.

The most important to remember:
NFkB is always present, it’s just inhibited. Dimerization places TIR domains together, and there is a scaffold of different molecules, which results in the phosphorylation of IkB, which results in the release of NFkB, which is a free transcription factor, which transcribes cytokine genes.

In details, it can be activated as follows:
- Dimerized TLRs recruit IRAK molecules activating the E3 ubiquitin ligase TRAF-6.
- TRAF-6 and NEMO create a scaffold for activation of TAK1.
- TAK1 associates with IKK and phosphorylates IKKB, which phosphorylates IkB.
- IkB is degraded, releasing NFkB into the nucleus to induce the expression of cytokine genes.

Question 14

NEMO mutations

Answer 14

Can produce X-linked Hypohidrotic ectodermal dysplasia and immunodeficiency.

Characterized by recurrent bacterial infections.

NEMO is important during differentiation, and if people don’t have NEMO then there will be no signal for TLRs.

Question 15

NOD-like receptors

Answer 15

These receptors are located in the cytosol. They will look like the TLRs and have the LRRs (leucine-rich repeats), but for signaling they will have a CARD domain, instead of a TIR domain.
They are called NOD because they have nucleotide-binding oligomerization domains (more than 2 - dimers).
They sense cell wall peptidoglycan fragments, such as:
- NOD1- Gram-neg bacteria γ-glutamyl diaminopimelic acid (breakdown product of peptidoglycans). Expressed in epithelial cells.
- NOD2 - Muramyl dipeptide. Expressed in Paneth cells regulating the expression of defensins.

Cytoplasmic NOD proteins reside in the cytoplasm in an inactive form.
The binding of bacterial ligands to NOD proteins induces the recruitment of RIPK2, which activates TAK1, leading to NFkB activation.

Question 16

NLRs

Answer 16

The NLRs happen extracellularly, and they will oligomerize upon activation.
They sense cellular damage and stress of the host.
This happens when the cell membrane is damaged, the potassium will start leaking out of the cell because some bacteria can make a porin permeable to potassium and initiate this leakage. This means that intracellularly, there will be an imbalance of the cellular environment (from sodium-potassium interactions).
When the cell loses its positive charge, it will promote a conformational change that will initiate oligomerization of the receptors and caspase1 cleavage and activation, so that cytokines and IL can be released.
The cytokines will act on other healthy cells.
Those cytokines will induce cytoskeletal changes in neutrophils (f.ex.) and will guide them to where the infection is occurring.
This complex is called an inflammasome, so it will cause inflammation.

Gout is an autoinflammatory disease that occurs in some joints that are inflamed and painful, the reason being the uric acid, so will activate the inflammasome. Gout will provoke periodic and recurrent fever, even without the presence of a pathogen.

The alum KAI(SO4)2 is a salt that acts as an adjuvant that can activate the inflammasome.

Question 17

RIG-I-like receptors
RLRs

Answer 17

They will be widely expressed in tissues and cell types, and they will be viral sensors, as when a virus enters the cell, there will be some RNA present.
Our mRNA is capped with a 7-methylguanosine molecule, meaning that it is protected. The viral RNA doesn’t have this cap, and therefore it will be different from humans.

This receptor has CARD domains. When it binds the RNA inside the cell, it will form a supramolecular complex that will allow the release of ubiquitin.
Ubiquitin will phosphorylate and activate the transcription factors in the cytosol, like NFkB (production of cytokines) and IRF-3 (production of type 1 interferons).

Question 18

STING
Stimulator of interferon genes

Answer 18

The receptor will be present in the ER.
When we have a virus entering the cell, with a DNA genome, there will be a molecule inside the cell that will recognize the foreign DNA, and dimerize the nucleotides, so there will be heterodimers of GTP and ATP.
All those dimers will be recognized by the STING at the level of ER, and this will produce phosphorylation of the transcription factors that translocate the nucleus, bind to the promoter, and promote the transcription of the type I interferon genes.
These receptors will be sensors of intercellular infection.

Question 19

Receptors function

Answer 19

• Activation of TLRs and NLRs results in the production of cytokines and chemokines.
• Activation of TLR results in the production of essential molecules for adaptive immunity, like co-stimulatory molecules (macrophages, dendritic cells): CD80, CD86.
• All receptors will result in the production of cytokines. Chemokines are a subset of cytokines.
  These cells will be on the dendritic cells.

Normally, the dendritic cells will be located on the skin, called Langerhans cells.
Immature dendritic cells are residents of the skin that are highly phagocytic but lack the ability to activate T lymphocytes.
Activated by LPS during bacterial infection causes changes in location and behavior.
These cells express TLRs upon activation in the presence of a pathogen, resulting in the co-stimulatory molecules.
Once that happens, the dendritic cells can move via lymph to the lymph nodes, where they can interact with the T cells (part of the adaptive immunity).

Question 20

Cytokines

Answer 20

• There are a lot of cytokines in the body.
• Small proteins, about 25 kDa.
• Released by various cell types including non-immune cells
• Induce responses through binding to specific receptors:
  Autocrine (communicate to themselves);
  Paracrine (communicates to other cells in short length);
  Endocrine (communicate to other cells in long length);
  They will communicate with the brain when a pathogen enters the body.
  They can be categorized as:
• Colony-stimulating factors (granulocyte or monocyte, that have to do with the differentiation of the cell.)
• Interferons (interfere with viral infections.)
• Interleukins (plenty, will induce cytoskeletal changes in the cell so they can migrate, they can increase the adhesion molecules)
• TNF (tumor-necrosis factor important to control an infection)
• Chemokines (follow the chemotaxis)
• Unassigned

A single cell can produce more than one cytokine. For example, the macrophages will induce the production of different cytokines, which can have local effects (activation of lymphocytes, vascular endothelium, antibody production, chemotactic factors, differentiation of cells) or systemic effects (fever, mobilization of metabolites, shock).

Question 21

Chemokines

Answer 21

• Induce chemotaxis
• Stimulate signaling pathways that result in changes in cell adhesiveness and in the cytoskeleton that lead to directed migration.
• More than 50 described so far
• There are different classes of chemokines: CXC, CC, and CXXXC.
  For each class, there will be different chemokines, and they will be produced by macrophages, monocytes, mast cells, T cells, platelets, epithelial cells, keratinocytes, fibroblasts (present in connective tissue, secrete molecules that can do some damage when activated), brain cells, etc.

Question 22

Adhesion molecules important for leukocyte recruitment

Answer 22

There are three groups of adhesion molecules:
- Selectins - bind carbohydrates, and initiate leukocyte-endothelial interaction. There are two types: P-selectins and E-selectins. The ligand for this molecule will be sugar, like the fucosylated oligosaccharides on leukocytes, which will bind the endothelial cells through selectins. These molecules are only activated through chemokines, like TNFα.
- Integrins - bind cell adhesion molecules and extracellular matrix. Have a strong adhesion. They have a transmembrane domain with alpha and beta subunits. An example can be LFA-1, which is a protein on the plasma membrane, which has as ligands ICAMs. They can be expressed by neutrophils, leukocytes, dendritic cells, T cells, etc.
- Immunoglobulin superfamily - various roles in cell adhesion, ligands for integrins. An example of these are ICAMs, which are expressed on the endothelium, meaning that they will be expressed only when they receive a cytokine signal.

Phagocyte adhesion to vascular endothelium is mediated by integrins
Leukocyte adhesion deficiencies caused by defects in integrins result in recurrent infections and impaired wound healing.

The function of the cytokines will start first with the selectins, and then with the LNFs and ICAMs. This is going to give a phenomenon called “rolling lymphocytes”.
Leukocytes roll along endothelial cells at sites of inflammation looking for adhesion sites.
Cytokines convert this rolling into stable binding by triggering a change of conformation in the integrins, which enables them to bind strongly to their ligands on the endothelial cells.

The rolling is happening due to selectins which will bind oligosaccharides, so they can attach and detach, so they roll until they find the ICAMs, so the rolling interaction is converted into a stable binding because once the integrins bind the ICAMs, there is a conformational change that occurs in those molecules, so it allows them to bind strongly, so they will stay there and initiate the extravasation.
The steps for adhesion are:
- rolling adhesion,
- tight binding,
- diapedesis,
- migration

Neutropenia - diseases or medical treatments that severely reduce neutrophil numbers.
Patients are highly susceptible to deadly infection with a wide range of pathogens and commensal organisms.
Treatment consists of transfusions of neutrophil-rich blood fractions.

Question 23

Tumor necrosis factor-alpha
TNFa

Answer 23

Is a very important molecule that will activate the endothelial cells for the induction and expression of the adhesion molecules.
Macrophages will secrete TNFa, f.ex. when there is a local infection with gram-negative bacteria.
Because of the activation of the adhesion molecules, we will have the extravasation of the cell, and there will be an increased release of plasma proteins in the tissue (like the complement molecules).
At the same time, there will be local vessel occlusion.

When an animal is injected with an antibody - Anti-TNFa, it will inhibit the function of the TNFa. Then, the infection will spread through the blood to the other organs, because there will be no function of local vessel occlusion. Therefore TNFa is very important during an infection.

Normally, TNFa is expressed by the macrophages, but it can be released as a soluble cytokine. When that happens, we are going to have sepsis, meaning that the infection is spread everywhere, so a lot of macrophages will be activated, so those in the liver and spleen will secrete a lot of TNFa in the bloodstream, meaning that a lot of the endothelial cells will undergo extravasation of cells, increase the release of plasma tissue, leading to decreased blood volume, neutropenia, followed by neutrophilia. Decreased blood volume will cause a collapse of the vessels.
Also, because the occlusion will happen at the same time, the tissues are not going to get nutrition, leading to disseminated intravascular coagulation, leading to wasting and multiple organ failure.

Question 24

IL-1b, IL-6, TNF-a

Answer 24

These molecules can act on the:
- liver - synthesizes a lot of the soluble molecules of the immune system. It is going to activate the acute-phase proteins (proteins associated with the fever). They are important for activation of the complement system and opsonization. C-reactive proteins act similar to the C3b in the complement system.
- bone marrow - a progenitor in the bone marrow will be directed by the cytokines to become a monocyte. Also, neutrophils are very important for phagocytosis. This mobilization of cells will lead to leukocytosis, also leading to increased phagocytosis.
- hypothalamus (increased body temperature);
- fat (protein energy mobilization); - muscle (increased contraction results in increased body temperature),
The fever decreases viral and bacterial replication and increases antigen processing and the specific immune response. This is a systemic effect.
- dendritic cells (bridging the innate to adaptive immunity).
The dendritic cells can migrate to the lymph nodes, and activate the T cells which migrate to the site of infection again.

These molecules will act on the hypothalamus and pituitary gland, which are responsible for the fever. they do it through the activation of vasomotor center neurons. Peripheral vasoconstriction and heat production.

Question 25

Natural killers
NK-cells

Answer 25

They function in between adaptive and innate immunity. They will be very important against viruses and intracellular pathogens.
These cells will act on the host cells that will be infected. They can be found in blood, skin, liver, lung, spleen, thymus, lymph nodes, peritoneal cavity, and uterus during gestation. They are also present in several tissues.
NK kills by a mechanism that has direct interaction with the infected cell. When a natural killer recognizes an infected cell, it will release some granules containing perforin and granzyme, present in NKs. The perforin makes a pore on the infected cell, the granzyme will enter through this pore and will activate caspase which starts the apoptosis.
So Nk will activate the programmed cell death, and it will not affect other cells that are located nearby.
The effect of the Nks will depend on the balance between activating and inhibitory signals, because they don’t recognize the pathogen in itself, but recognize an infected cell.
There are multiple receptors interacting, and if there are inhibitory signals that win, then the NK cells don’t kill.
If there is a win of the activatory signal, then the NK will release the granules on the target cell, inducing apoptosis.

NK has to differentiate a self-cell from an infected self-cell called an altered self. MHC are proteins expressed at the level of the plasma membrane, that will be glycosylated. They will give us the identity of the tissues.
Some of the receptors on the NK cells recognize other receptors on the infected cells, that directly will induce the cell death.

Question 26

MHC

Answer 26

There are two types of MHC:
Class I and Class II.
They recognize the infected cell, which will be expressed in less amounts, so they can be recognized by the NK cells.
A common feature of infected cells from intracellular parasites is altered expression of MHC class I molecules because many of these pathogens have developed strategies to interfere with the ability of MHC class I molecules to capture pathogen peptides and display them to T cells.
- Some viruses inhibit all protein synthesis in their host cells so that synthesis of MHC class I proteins would be blocked in infected cells. The reduced level of MHC class I expression in infected cells would make them correspondingly less able to inhibit NK cells through their MHC-specific receptors, and they would therefore be more susceptible to being killed.
- Some viruses can selectively prevent the export of MHC class I molecules to the cell surface. This might allow the infected cell to evade recognition by cytotoxic T cells but would make them susceptible to being killed by natural killer cells.