Innate Immunity: Barriers

• Innate immunity mechanisms effectively differentiate between host cells and pathogens.
• Innate immune defenses are present in everyone and occur within minutes to hours after encountering an infectious agent.
• Adaptive immune response is needed only when innate defenses are overwhelmed, bypassed, or evaded.

Barriers

• Epithelial cells are linked by tight junctions.

Mechanical Barriers

• Skin clears pathogens via:
  • Flow of fluid.
  • Perspiration.
  • Sloughing off skin cells.
• The gastrointestinal tract clears pathogens via:
  • Flow of fluid.
  • Mucus.
  • Food.
  • Saliva.
• The respiratory tract clears pathogens via:
  • Flow of fluid and mucus.
  • Cilia.
  • Air flow.
• The urogenital tract clears pathogens via:
  • Flow of fluid.
  • Urine.
  • Mucus.
  • Sperm.
• Eyes clear pathogens via:
  • Flow of fluid.
  • Tears.

Chemical Barriers

• Skin has:
  • Sebum (fatty acids, lactic acid, lysozyme).
• The gastrointestinal tract has:
  • Acidity.
  • Enzymes (proteases).
• The respiratory tract has:
  • Lysozyme in nasal secretions.
• The urogenital tract has:
  • Acidity in vaginal secretions.
  • Spermine.
  • Zinc in semen.
• Eyes have:
  • Lysozyme in tears.
• Antimicrobial peptides (defensins) can be found in multiple areas of the body.

Microbiological Barriers

• Normal flora are present in the:
  • Skin.
  • Gastrointestinal tract.
  • Respiratory tract.
  • Urogenital tract
  • Eyes.

Chemical Barriers

• Lysozyme is found in secretions like mucus, tears, milk, and saliva.
  • It uses hydrolysis to break apart the peptidoglycan wall, causing bacterial cell lysis.

Antimicrobial Peptides

• Defensins are small, heterogeneous, cationic peptides.
  • Effective against Gram-negative/positive bacteria, enveloped viruses, and fungi.
  • They destabilize membranes and form pores in bacterial cell walls.
  • Enable proteolytic degradation of bacterial proteins.
  • Inhibit viral binding/entry and virus particle assembly.
• Defensins can act as chemical barriers secreted by epithelial cells in mucosal surfaces.
• Some antimicrobial peptides (AMPs) like cathelicidins are stored in neutrophil granules and released in response to inflammation.
  • They kill microbes extracellularly when neutrophils die during inflammation.
  • Can kill microbes intracellularly when cells like neutrophils phagocytose a pathogen.
• Defensins have multiple roles in the immune system beyond just being chemical barriers.

Phagocytosis

• Phagocytosis is a first line of defense when microbes invade tissue.
  • It involves engulfing and destroying microorganisms, particularly bacteria.
  • It plays a key role in innate immunity by recognizing, ingesting, and destroying pathogens without needing an adaptive immune response.
• Phagocytosis can occur after an antibody binds to an antigen.
  • The antibody acts as a signal for efficient phagocytosis.
• Macrophages and neutrophils are the main phagocytes in the body.

Phagocytic Cells

• Monocytes and Macrophages are part of the phagocytic cell system:
  • They are long-lived cells residing within the tissues.
  • Starts as Promonocytes (BM), then Monocytes (blood), which then become Macrophages and Macrophage-like cells (tissues)
• Neutrophils:
  • Are derived from hematopoietic precursors in the bone marrow (BM).
  • Are non-dividing, short-lived cells, and are the dominant white blood cell (WBC) in blood.

Phagocytosis

• Phagocytosis starts when a pattern-recognition-receptor (PRR) binds to a microbe or debris. Opsonins created by other cells can also bond to microbes.
• The PRR can bind to:
  • A microbe.
  • Debris.
  • An opsonin which is a soluble and secreted PRR that enhances phagocytosis effectiveness.
• The opsonin coats a microbe, and the phagocyte has receptors for the parts of that opsonin.
• PRRs that trigger phagocytosis include:
  • Mannose receptor, bacterial, fungal, parasitic cell walls.
  • SR-A, SR-B LPS lipoteichoic acid - mostly bacteria.
  • Calreticulin – binds to particular opsonins-collectins, ficolin - mostly carbohydrate groups on bacteria.
  • CR3, CR1 – binds to complement opsonins, good for bacterial cell walls.
  • Different Fc receptors are able to bind to almost anything.
• The microbe is engulfed when the PRR receptors signal the cell membrane to approach, coat, and surround areas where the receptor is bound including:
  • Forming a phagosome mediated by intracellular signalling events.
  • Actin polymerization.
  • PI3 kinase.
• Microbe killing occurs when phagosomes fuse with lysosomes, as well as (in neutrophils) primary and secondary granules.
  • Phagosomes contain many molecules that are effective at cellular killing which includes: - Reactive oxygen species. - "Pore”-forming proteins or peptides. - Hydrolytic enzymes. - pH changes causing an acidic environment from the lysozyme.
• Microbe remnants are either digested/used or excreted from the phagocyte.
• Microbe killing can also occur when:
  • After the microbe has been phagocytosed, the phagosome will dock with a lysosome and/or neutrophil granules.
  • Lysosomes pretty much break down anything (acid hydrolases).
  • The low pH of a lysosome is unpleasant for many bacteria.
  • A complex becomes associated with the membrane of the phagolysosome. - Which then requires a large amount of oxygen (respiratory burst). - If a particle is too large to phagocytose, macrophages will surround it and “place” their NADPH oxidases close to it to try to kill it.
  • Macrophages can kill cells by inducing the synthesis of nitric oxide at high concentrations.
  • Neutrophils have a multitude of pore-forming molecules within their granules that will fuse with the phagosome.
• Neutrophils contain potent granules:
  • Defensins are rich in cysteine. - Form voltage-dependent pores in bacteria permeable to water. - Cause lysis.
  • Cathepsin is a type of protease.
  • Cathelicidins are pore-forming molecules. - Cause lysis with a multitude of different structures.
  • Lysozyme is glycoside hydrolase. - It doesn't require an acidic pH to function. - Is found in glandular secretions. - Effective at killing gram positive bacteria.
  • Lactoferrin is iron-binding protein effecting iron metabolism in microbes.
• Neutrophils can lyse and release DNA into the extracellular fluid:
  • Which becomes a Neutrophil Extracellular Trap (NET) that is then able to perform the following: - NETs are “sticky” so that they are able to trap most bacteria in the chromatin. - Histones are toxic to many bacteria. - Granule contents will remain near the NETs and help with killing bacteria – even after the neutrophil itself is dead.

Phagocytosis Takeaways

• Recognize a "target" for phagocytosis and what an opsonin is and its role.
• How phagocytes kill and the role of lysosomes, free radicals, and anti-microbial peptides.
• How a NET can add to host protection beyond phagocytosis.

Pattern Recognition

• Pattern Recognition is an evolutionarily conserved mechanism that recognizes signs of common microbial infection, physiological stress, other damage.
• Recognition is immediate and activates the inmate and adaptive immune response.
• Pattern Recognition Receptors (PRRs) are found on phagocytes and elicits responses that are elicited in response to:
  • Pathogen-Associated Molecular Patterns (PAMPs).
  • Danger Associated Molecular Patterns (DAMPs).

PRRs:

• Lectins.
• Toll-like receptors.
• Nod-like receptors. Elicit responses such as:
  • Phagocytosis.
  • Cytokine secretion.

Toll-Like Receptors

• A family of 10 cell membrane receptors with variable specificity for a range of pathogens.
• Ligands include:
  • LPS.
  • dsRNA.
  • ssRNA.
  • DNA.
  • Flagellin.
• Cytokines secreted in response to TLR activation by:
  • Cytokines (IL-1β, IL-6, CXCL8, IL-12, TNFα).
    • Cytokines are small protein messengers, secreted by cells including leukocytes, that can do the following: - Influence the differentiation, activate/inactivate the activity, or increase/decrease the production of stem/hematopoietic cells.
  • Interferons - Interferon (IFN) alpha, beta, and lambda (IFNa, IFNb, IFNI). - Autocrine and paracrine signaling molecules are effective in activating macrophages, NK cells, and inducing an antiviral state.
• MyD88 is an essential adaptor in TLR signaling.
  • Patients with MyD88 deficiency experience have frequent and severe bacterial infections. - Antiviral responses generally unaffected.
  • Patients with constitutively active MyD88 causes blood disorders and cancers. - Overproduction or dysregulation of IgM - Causes B cell lymphoma, marginal cell lymphoma.

Nod-Like Receptors

• A Family of intracellular receptors are triggered inside of of the cytoplasm and are able to detect products that derived from the phagocytosed pathogens.
• Also recognizes DAMPs associated with cellular stress and activates expression of inflammatory cytokines.

Acute Inflammation

• First requires the alteration of vascular caliber - vasodilation.
  • Can lead to increases in blood flow at the capillary bed due to arteriolar dilation, dilation of precapillary sphincters via: - Nitric oxide and histamine. - Various prostaglandins (PGI2, PGE2, PGD2). - Platelet activating factor (at low concentrations) can cause vasoconstriction (higher concentrations). - Complement C5a and C3a stimulate histamine release. - Low concentrations of Nitric oxide is a potent vasodilator. - Able to destroy microbes and host cells, is a free radical. - Produced by an inducible nitric.
• Arterioles and pre-capillary sphincters dilate to vastly increase blood flow in inflamed tissue.
• Vasodilation and fluid loss lead to slower blood flow.
  • This is called vascular congestion, which helps with the margination of leukocytes.
• Second requires an enhancement of vascular permeability and has the following results:
  • Capillaries and venules become more "leaky” with the release of a number of mediators. - Histamine and serotonin links inflammation and blood clotting. - Prostaglandins (PGD2 and PGE2). - Leukotrienes (LTC4, LTD4, LTE4). - Platelet activating factor. - C3a and C5a. - Bradykinin.
  • A wide variety of proteins and mediators can enter the interstitial space from the bloodstream.
• Increased vascular permeability is due to contraction of endothelial cells:
  • Occurs in venules and this is short-lived.
• Endothelial damage:
  • Can be caused by trauma, burns, or microbial damage.
  • Leukocyte-mediated damage often is longer-lived.
• Increased transcytosis can occur through:
  • Pinocytosis.
  • Receptor-mediated endocytosis.
• Interstitial fluid accumulation increases pressure in the interstitial space with increased lymphatic drainage.
• Excess fluid, microbes, debris, and leukocytes all migrate into the lymph inflaming lymphatic vessels (lymphangiitis).

Leukocyte Migration

• Neutrophils, monocytes, eosinophils, and basophils will all migrate from the circulation into inflamed tissue.
• The steps for Leukocyte Migration include:
  • Margination.
  • Rolling is mediated by binding of selectins and cellular adhesion molecules to their respective ligands on leukocytes.
  • Adhesion.
  • Diapedesis.
  • Chemotaxis occurs from injury or infection sites.
• Cytokines are messangers secreted from leukocytes.
• Chemokine is a structurally-related family of small cytokines, which are able to:
  • Bind to cell surface receptors
  • Leukocytes can move along the chemokine concentration gradient and adhesion of leukocytes for the purposes of differentiation, inflammation and migration.
• CXC chemokines attract neutrophils, are angiogenic, and are very similar in structure.

Adhesion

• Leukocytes can migrate towards the vessel wall from the following:
  • Rolling - formation & dissociation of adhesion. - Activation by chemokines, which are presented on endothelial cells, is required for cells. - Integrins.
• Increased ability to migrate to a target from histamines and thrombins:
  • Rolling to cells with selectin expression by endothelial cells.
  • Activate TNF & IL1 due to ICAM expression by endothelial cells.
  • Chemoekine release has integrin affinity.

Chemotactic Agents

• All is created in higher concentrations at damage/pathogen sites.
  • Leukotriene B4 release
  • Bacterial products containing N-formyl-methionine and the product of activates complement (C5a).
  • Release of Chemokines (IL-8, RANTES, eotaxin) allows leukocytes to "follow the breadcrumbs" to a concentration gradient.