User:Kinkreet/Immunology/The Immune Response

Barriers
There are barriers which keeps out or destroy pathogens before it can be reached by the immune system. This includes the normal flora of bacteria on the skin and inner linings of the body, as well as mechanical and chemical barriers.

Mechanical
All epithelial cells are joined by tight junctions, this creates an universal mechanical barrier to pathogens. The other form of mechanical barrier includes flow of fluids, mucus, saliva, tears, urine and sperm on the linings to move the pathogens along, and so there are less chance of them entering the body.

Chemical
If the pathogen manages to break pass the mechanical barriers, it will encounter chemical barriers. One such chemical barrier is sebum secreted by the sebaceous gland holocrine secretion, which contains tryglicerides, wax esters, squalene, other fatty acids, lacic acid and lysozyme. Some lipids in the sebum, such as free sphingoid bases, are thought to have antimicrobial properties; also, some derivatives of sebaceous triglycerides (e.g. sapienic acid) have antibacterial activity (it was found that sapienic acid in combination with a low concentration of ethanol, is more effective in killing methicillin-resistant Staphylococcus aureus (MRSA) than mupirocin. . In the urogenital tract, females secrete vaginal fluid which contains acids such as acetic acid, lactic acid which keeps it at pH 3.8-4.5, and this can inhibit bacterial growth; in males, semen contains spermine and zinc which can destroy some pathogens. Other chemical barriers include hydrochloric acid and proteases in the stomach; C-reactive proteins in the blood which binds to pathogens to activate complement; pattern recognition receptors (PRRs) which recognizes general patterns commonly found on pathogens but not humans. They can recognize lipopolysaccharide, lipoteichoic acid (Gram+ bacteria), lipoarabinomannan, mannose (and mannans), nucleic acids, acterial peptides (e.g. flagellin, ax21), N-Formylmethionine and other glucans; and other antimicrobial peptides such as lactoferrin, acidic proline-rich proteins, plunc, salivary mucin glycoprotein, histatin, defensin HBD1-4, and hCAP18/LL-37.

Recognition of pathogens by the innate immune system
If the pathogen manage to evade the barriers mentioned above, then the cells of the innate immune system would be first to respond. They express a collection of germ-line encoded pattern recognition receptors (PRRs), which identifies a broad range of patterns found on pathogens and not the host, such as dsRNA and lipopolysaccharides, collectively termed pathogen-associated molecular patterns (PAMPs). PRRs can also recognise damage-associated molecular patterns (DAMPs), which are released when cells are damaged, and microbe-associated molecular patterns (MAMPs). There are many types of PRRs, including NOD-like receptors (NTRs); but the most important are the Toll-like receptors (TLRs)

C-type lectins are a group of calcium-dependent mannose receptors, which recognize and bind to bacterial and viral sugars and promotes phagocytosis. Scavenger receptors recognize polyanionic ligands (e.g. Lipoteichoic acid, LTA, on the cell wall of Gram+ bacteria) and acetylated LDLs.

Receptors can recognize microbes and cause deposition of Complement factor C3 on the microbial surfaces; phagocytes then recognizes this complement factor C3 and initiate phagocytosis and possibly cell lysis.

Macrophages
Macrophages have pseudopodia.

When pathogens are phagocytosed, they travel in phagosomes, which merges with lysosomes to give phagolysosomes.

Activated macrophages, mast cells and endothelium cells secretes cytokines which attracts other cells such as neutrophils to the site of infection.

Neutrophils
Neutrophils are the first cells of the innate immunity that are recruited to the site of infection. Their role is to isolate, engulf and kill pathogens (usually bacterial and fungal) using oxidative and non-oxidative mechanisms; they make up ~90% of the granulocytes in the body. Neutrophils are characterised by a multi-lobed nucleus, primary azurophilic granule (named because it can be stained with azure dye; they contain lysozymes, defensins and hydrolytic enzymes), secondary granules and the presence of glycogen in the cytosol.

Selectin-dependent recruitment
Most of the neutrophils' life span (~5.4 days in circulation, and 1-2 days in tissues) are spent in circulation. When the endothelium cells are stimulated by histamine (from inflammatory respones) and thrombin (from coagulation cascade), they are induced to present P-selectins (SELP) on the cell surface; furthermore, TNF-α produced (primarily) by macrophages induces these endothelium cells to display E-selectins and additional P-selectins. The lectin-like domains of the E- and P-selectins binds to their ligands (siaylated carbohydrates) on the neutrophil's surface, and slows it down. Under the microscope, it'd appear that the neutrophil is tethering to the inner walls, and then rolling along the walls of the blood vessel. Different modified sialyl Lewisx expressed on the surface of monocytes and granulocytes can bind to L-, P- and E-selectins. Lectins will then bind to intracellular adhesion molecules (ICAMs) and stops the cell from moving. Chemokines are displayed on the epithelial cell wall surface, and binds to chemokine receptors on the leukocytes, at which point it starts the migration of neutrophils through the blood vessel walls, and into the tissue, where they will follow the concentration gradient set down my chemokines, and move towards the site of infection.

Other mechanisms of recruitment
Although selectins are the major mechanism of how leukocytes are recruited to the site of infection, other molecules can also perform this task; these include α4 integrin (also known as CD49D and ITGA4), CD44, vascular adhesion protein 1 (also known as AOC3) and, at lower shear stress, β2 integrin (also known as CD18 and ITGB2)

Concentration Gradient
Neutrophils are recruited to the site of infection by following a concentration gradient of chemokines. These chemokines can be the ones the host immune cells produce, or it can be cell content from damaged tissues, and also bacterial products.

Recent studies in zebrafish models identified a concentration gradient of hydrogen peroxide (H2O2) being produced during cell damage. Hydrogen peroxide was thought to have a direct antimicrobial activity, but the study suggests that is acts in developing a gradient for neutrophils to follow. Although the mechanism is unknown, dual oxidase (Duox) is activated upon cell damage, and this produces a H2O2 gradient with H2O2 diffusing in a paracrine fashion.

Cellular components released from damaged cells are thought to be able to bind to Toll-like receptors (TLRs) and also activate intracellular molecules, such as nucleotide-binding-domain and leucine-rich-repeat-containing family proteins (NLRs)

Activation
Once they are themselves activated, will releases more cytokines, which amplifies to signal for more neutrophils to come. Neutrophils attack pathogens using three methods: phagocytosis (ingestion), release of soluble anti-microbials (including granule proteins), and generation of neutrophil extracellular traps (NETs).

Eosinophils
Eosinophils make up ~2-5% of all granulocytes, and are involved in the cytotoxic response for larger pathogens such as parasites and worms. Eosinophils flatten over the pathogen, and releasing its crystalloid granules (acidic) over it.

Basophils
Basophils do not perform any cytotoxic tasks, but releases mediates to provoke inflammation.

Mast Cells
Mast cells exist in the tissue near capillaries, and recognizes allergens and release mediators of inflammation, especially for vascular permeability and orchestrate allergic responses.

Killing by Innate Immunity
Neutrophil extracellular traps

Phagocytosis
When the pathogen is phagocytosed, the phagosome undergoes acidification until it reaches a pH of less than 4; under this condition, most bacteria cannot reproduce or survive. Furthermore, respiratory burst (oxygen burst) is induced using enzymes, producing many oxidative moeities such as superoxide anion, peroxide, hydroxyl radical and hypochlorite, oxidising the bacteria and catabolising it.

The phagosome will then fuse with the lysosome, and the enzymes, free radicals and anti-microbial peptides in the lysosome will attack the pathogen.

Resistance
Some pathogens, such as Mycobacteria, can evade phagocytosis. Others, such as the parasite Leishmania, can use phagocytosis as a means of gaining entry into the cell.

Respiratory Burst
NADPH oxidase contains 6 subunits, two of which lies on the phagosome membrane, and when activated, converts O2 to O2- (superoxide anion); cytosolic superoxide dismutase (SOD) then converts O2- to H2O2; other peroxidases, iron and enzymes can convert O2- to hypochlorite ions, hydroxyl radicals and other highly reactive species that can kill the pathogen.

Anti-microbial peptides
Anti-microbial peptides can be stored in lysosomes, but also released on various epithelial surfaces. The peptides neutralises a patch of outer membrane and inserts itself into the patch. It can also bind to the divalent cation binding site of LPS of Gram- bacteria, and disrupts the membrane. They may aggregate in the membrane to form a pore, causing an efflux of solutes and possibly lysis of the cell; or the peptides bind to the membrane, flip-floping across. Once it has gained entry to the cytosol, it can migrate and bind to important proteins, disrupting their functions.

A subset of B cells (CD5+) found mostly in the skin produce IgM with unknown specificity; the CD5+ B cells undergo limited antibody rearrangement and thus not considered part of the adaptive response.

γδT cells recognises ligands directly or through MHC class 1b including HSP, nucleotides, phospholipids. γδT cells undergo minimal rearragement of the TCR and is found in mucosal epithlia and skin.

Natural Killer Cells non-specifically clear out abnormal cells such as tumour cells and virus-infected cells.

Eosinophils and basophils kill parasites and larger pathogens.

Inflammation
Humoral immunity describes immunity mediated by soluble components in the blood. When a pathogen is recognised by the resident effector cells (usually macrophages and sometimes dendritic cells), the effector cells will release cytokines. Some cytokines promotes coagulation to form a temporary barrier at the wound, others repair the wounded tissues; there are a specific type of cytokines called chemokines that attract other cells (mainly neutrophils and monocytes) to the site of infection by them following a concentration gradient, while other cytokines induce vasodilation (increasing the permeability of the blood vessels), which allows more fluid, proteins and inflammatory cells to leave the blood and enter the tissue. Because of this movement of cells to the site of infection, it causes inflammation, characterised by swelling, pain (caused by pressure on nerves), redness and heat.

Antigen presentation
Although macrophages and some granulocytes can present antigens to T cells (usually helper T cells) to activate them, dendritic cells are the professional APC - they are specialised in this role. Immature DCs resides or are recruited to tissues, and phagocytose and macropinocytose microbes, which activates them into APCs. They migrate to lymph nodes via the afferent lymphatics, and present antigens to naive T lymphocytes. Along with a co-stimulatory signal, any T lymphocytes that recognizes these antigens undergo clonal selection and expansion, and mature to acquire effector function.

Major Histocompatibility Complex (MHC)
Major Histocompatibility Complex (MHC) are a class of molecules displayed on the cell surface of all vertebrates. There are three classes of MHC - I, II and III, all of which are polymorphic; only class I and II are involved in antigen presentation. Class I and II have similar structures but differs in subunit composition, distribution and T cell specificity. Together, the MHC locus covers >200 genes and ~7 million base pairs in humans. The MHC genes are highly polymorphic (HLA-B has 728 variations) and thus it is unlikely two people will have exactly the same alleles, which can be a problem during transplantation, because if the donor expresses a different MHC allele, the host will treat that MHC as another non-self antigen; this is known as MHC restriction. Most of the variations occur at the peptide binding cleft. All HLA alleles are co-dominant, and so both proteins encoded in both the chromosomes are expressed at the same time. Furthermore, all genes in that allele are expressed, giving rise to a huge diversity of MHC molecules. MHC Class I is displayed on almost all cells, and MHC Class II is mostly restricted to immune cells, and its expression is induced by cytokines. Red blood cells do not express (or at minimal amount) MHC moelcules, which means it can be a safe haven for pathogens such as the malaria parasite Plasmodium.

MHC Class I
MHC Class I is made up of subunits α1α2α3β2. The alpha chains are encoded by HLA-A, -B and -C, and also by pair of genes (HLA-DP, DQ and -DR), in which the other member of the pair encodes for the beta subunits.

MHC Class II
MHC Class II is made up of subunits α1α2β1β2. The subunits are encoded by the HLA-DM locus. HLA-DO genes are negative regulators of HLA-DM.

An individual can have a reprotoir of many different MHC molecules, thus different people have different susceptibility to pathogens. Class I MHC are displayed on every nucleated cell of the body (so not on erythrocytes), whereas Class II are only displayed on APCs.

The APC would process the antigen (usually mean degrading it) and bind to MHCII in MHCII-loading compartments, and is exocytosed to the cell surface to be displayed. CD4+ T cells (Helper T cells) recognizes this signal and becomes activated.

Epithelial cells are not antigen presenting cells. When an antigen is processed, it is displayed on MHCI, which signals to CD8+ T cells (Cytotoxic T cells) that the cell is infected and the T cell will kill it.

Adaptive immune response
Clonal Expansion and differentiation

B cells
B cells circulate in the lymphatic system as naive cells in the G0 (resting) phase. When its B cell receptors (BCRs) binds specifically to antigens, it initiates a signalling cascade which leads to the transcription of genes associated with B cell activation, leading to replication and antigen presentation.

Antigen presentation
Once the BCRs bind to antigen, it is internalized and either degraded or trafficked to an intracellular compartment called MIIC (maybe both). In the MIIC, newly synthesised MHCII molecules form complexes with processed antigen bound to BCRs. The MHCII-peptide complex is transported to the cell surface, where it is recognised by the TCR of TH cells. Bound TCR recruits CD3 with ITAMs to activate the downstream signal that leads to T cell activation. The activated TH cell then help the B cell to become activated by providing cytokine signals, as well as co-stimulatory signals such as CD40-CD154.

Replication
The B cell enters into the cell cycle at G1, and undergo gene activation and DNA synthesis to form lymphoblast S, which grows and divides into two mature B cells. One of the daughter cells can enter back into the cell cycle to replicate more B cells, while the other daughter cell can differentiate into effector plasma cells, or memory B cells. If the memory B cell encounters an antigen straight away, it will enter straight back into the cell cycle; otherwise, it will remain in circulation until its next encounter.

The plasma cell will produce antibodies which will bind to the antigens. Other cells, including neutrophils have receptors for those antibodies, and can bind and kill the cell which bears it.

Expansion.

Memory requires co-stimulation.

Cytotoxic T cells
Kill abnormal cells

Helper T cells
Help B cells differentiate into B cells

Regulatory T cells
Regulate

Complement
Complement system mediates inflammation, or the movement of immune cells to the site of infection.

Complement can be activated to cause cell lysis, opsonization, activation of inflammatory responses and clearance of immune complexes.

Complement can form membrane attack complexes (MACs) which directly lyse the cells.

It can bind directly to pathogens, and also used immune complexes, marking them for phagocytosis.

Complement can also act as signalling molecules, such as for extravasation and degranulation.

Inflammation Resolution
After the infection has been dealt with, the inflammatory response must be terminated. Neutrophils will no longer be recruited to the inflammation site as no new cytokines are produced; but the existing neutrophils need to be removed. Most neutrophils die by apoptosis; some at the site of inflammation, but also some migrate away from the site, against the chemokine concentration gradient. The migration against the gradient is termed 'reverse migration'.

After the neutrophil has induced apoptosis, they are phagocytosed by macrophages.

Findings from zebrafish
It was found in zebrafish models that the activation of in vivo inflammatory pathways, especially hypoxia-inducible factor, HIF, altered reverse migration.

It is thought more and more likely that there are two main types of signals that determine the behaviour of neutrophils: one to signal for survival (and thus affects the lifespan) and the other for movement (both forward and reverse)