CAM101: Innate Immunity - Cell Types

• CAM101 covers Innate Immunity and Inflammation.
• Guna Karupiah is the Associate Professor for this course.
• Karupiah can be reached at [email protected].
• This course is part of the College of Health and Medicine.

Haematopoiesis, Leukocyte Differentiation, and Types

• Hematopoiesis is the process of blood cell formation, leading to various leukocyte types.
• Lymphoid and Myeloid are two major lineages produced via haematopoiesis.
• Lymphoid lineage includes innate lymphoid cells (ILC), B cells, T cells, plasma cells, effector T cells, and NK cells.
• Myeloid lineage includes monocytes, neutrophils, eosinophils, basophils, macrophages, dendritic cells, and mast cells.
• Hematopoietic stem cells differentiate into common lymphoid precursors and common myeloid precursors.

Cells of the Innate Immune System Functions

• Dendritic cells are the most important antigen-presenting cells, activating T cells and initiating adaptive immune responses.
• Natural killer cells kill tumor cells and virus-infected cells.
• Polymorphonuclear neutrophils (PMN) are the most abundant cells that mediate the earliest phases of inflammation.
• Eosinophils are involved in parasite killing and immunopathology associated with asthma.
• Mast cells are in the skin, mucosal epithelium, and release histamine, activating endothelium and increasing blood vessel permeability, resulting in oedema.
• Monocytes are inflammatory, and the source of tissue-resident macrophages & dendritic cells.
• Macrophages phagocytose dead cells and microorganisms. They also stimulate new blood vessel growth through angiogenesis. Macrophages promote fibrosis through collagen-rich ECM synthesis and wound healing.
• Basophils have similar functions to mast cells.

Innate Lymphoid Cells (ILCs)

• NK cells have perforin and granzyme and TNF, and IFNy. They provide immunity to viruses and cancer and are involved in chronic inflammation.
• ILC1 cells produce TNF and IFNy which provide immunity to intracellular bacteria and protozoa, and are involved in chronic inflammation.
• ILC2 cells produce IL-13, IL-5 and IL-9 provide immunity to helminths, asthma and allergic diseases, and metabolic homeostasis.
• ILC3 cells produce TNF, IFNy, IL-22, and GM-CSF. They help with lymphoid tissue development, intestinal homeostasis, immunity to extracellular bacteria, and chronic inflammation.

Three Lines of Defence

• First line of defence is anatomical (physical) barriers, mechanical barriers, chemical barriers, biological barriers, and cellular components.
• Second line of defence involves innate immunity via complement and interferon responses.
• Third line of defence is adaptive immunity.

Innate Immune System

• The innate immune system is an intrinsic response that is not learned.
• It generates responses rapidly within minutes.
• No memory is generated.
• It does not refine or improve over time.
• It exhibits some selectivity, recognizing lipopolysaccharide (LPS) from gram-negative bacteria as foreign pathogen-associated molecular patterns (PAMPs) using the same set of receptors on different innate cells.

Physical, Chemical, Biological and Cellular Barriers

• Physical barriers prevent infection.
• Chemical barriers involve killing microbes by locally produced antibiotics like defensins and cathelicidins.
• Biological barriers include the microbiota.

Chemical Barriers

• Acid, such as stomach acid with low pH, kills many types of microorganisms.
• Natural antibodies like IgM are directed against highly conserved epitopes with low affinity, with production driven by microbiota.
• Complement is part of the chemical barriers.
• Defensins are small cysteine-rich cationic proteins that form pores and have anti-bacterial, anti-fungal, and anti-viral properties.
• Peptide antibiotics, like Cathelicidins, are stored in lysosomes of macrophages and polymorphonuclear leukocytes (PMNs), acting against invasive bacterial infection.
• Lactoferrin transports iron and has anti-bacterial and anti-fungal properties. Found in milk, saliva, tears, and nasal secretions.
• Enzymes like lysozyme break down ingested microbes.

Complement System

• Consists of around 30 plasma proteins present in blood plasma and interstitial tissues.
• Is activated by pathogens.
• Involves proteolytic cascades, inactive precursor enzyme is altered into an active protease that cleaves and induces the proteolytic activity of the next complement protein in the cascade.
• Triggers enhancement of phagocytosis via opsonisation, inflammation, and lysis of microbes.

Three Pathways of Complement Activation

• Alternative pathway involves C3b binding to activating surfaces like microbial cell walls.
• Classical pathway is activated by C1 binding to antigen-antibody complexes, requires antibody production by the adaptive immune system
• Lectin pathway involves lectins binding to carbohydrates on microbes
• All three pathways lead to formation of C3a, C3b, C5a, and C5b.
• Steps and outcomes are the same in all three pathways.
• C3b binds to the microbial surface or the antibody in the classical pathway, it becomes part of the enzyme that cleaves C5 and starts the later stages of complement activation.
• C5b starts the late steps of complement activation, leading to C5a production.
• C3a and C5a are chemo-attractants causing inflammation and trigger histamine release by mast cells.
• C3b binding promotes phagocytosis
• C5b forms a complex with C6, C7, C8, and C9 to create the membrane attack complex (MAC), creating pores for water and ions to move freely, which lyses the microbe.

Macrophages (Mononuclear Phagocytes)

• Macrophages are usually non-circulating, are tissue-bound, and trap antigens at sites of infection, deriving from circulating monocytes.
• Macrophages associated with a slight increase in numbers during acute inflammation.
• They are involved in phagocytosis, wound healing, and inflammation.
• Some are tissue resident, living for years.
• They produce high levels of cytokines and are present in normal tissues.
• They have many mature forms, including alveolar macrophages (lung), microglial cells (brain), Kupffer cells (liver), and sinusoidal macrophages (spleen).

Neutrophils

• Neutrophils are the most numerous white blood cell in the blood.
• Count is between 3.7-5.1 x 103 Cells/mm3 equating to 50-70% of total leukocytes
• They are associated with a rapid increase in numbers during acute inflammation.
• They are involved in phagocytosis.
• They secrete high levels of lysosomal enzymes.
• Are short-lived with a lifespan of 1-2 days in tissues.
• They are present in the blood and migrate to sites of inflammation.
• Dead neutrophils make up pus.

Phagocytosis

• Phagocytosis protects against invading microorganisms.
• Neutrophils and macrophages are primary types of phagocytes.
• Phagocytosis steps are chemotaxis, adherence, ingestion, digestion, and killing.
• Chemotaxis is the movement of phagocytes to the site of damage due to a chemical stimulus.
• Adherence is the attachment of a phagocyte to a microbe.
• Ingestion forms a phagolysosome.
• Digestion uses lysosomes to degrade microbial material.
• Killing eliminates the microbe.
• Oxygen-dependent oxidative burst includes NADPH-dependent oxidases and oxygen radicals, Hypocholrous acid and Nitric oxide.
• Chronic granulomatous disease (CGD) is a rare genetic disease that predisposes individuals to recurrent life-threatening infections mostly caused by catalase-positive bacteria and fungi, and exuberant granuloma formation due to defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

Opsonisation

• Opsonisation enhances the targeting of particles like microbes for destruction by phagocytes.
• Acts as a means of identifying the invading particle to the phagocyte.
• Opsonins include antibodies (IgG, IgE), complement (e.g. C3b), and antibody + complement.
• C3b is a potent opsonin in tagging pathogens and immune complexes (antigen-antibody).
• Opsonisation is augmented when the degree of binding of phagocyte to microbe is enhanced

Pattern Recognition Receptors (PRR)

• Pattern Recognition Receptors (PRR) are an early warning system for the Innate Immunity
• Toll-like receptors (TLR) includes molecules (proteins, lipids), nucleic acids of microorganisms.
• Can be extracellular, cytosolic or endosomal
• TLRs are a group of receptors present on antigen presenting cells and other cells binding to common microbial components, present on cell surface, cytosol and endosomes which alert the immune system to microbial threats
• Damage-associated molecular patterns (DAMPs/Alarmins) include host proteins, DNA and RNA
• High-mobility group box 1 (HMGB1) is a major mediator of endotoxic shock, and trigger inflammation

Recognition Receptors (PRR)

• One type of PRR can recognise PAMPs on different types of microorganisms
• One cell type can have many different types of receptors

Acute Inflammation

• Inflammation (or the inflammatory response) is the body's natural response to harmful stimuli.
• It is part of the innate immune response and the initial vascular response.
• A localised response to a stimulus such as an Infection, damaged cells, trauma, irritation, or allergy.
• Response continues only for as long as the threat exists
• Gives no direct protection against subsequent attack by the same agent
• Alerts the adaptive immune system and contributes to its activation

Acute Inflammation Characterisation

• Characterised by increased blood flow
• Leakiness of capillaries, bringing in fluid, nutrients, anti-microbial proteins and phagocytic cells into the tissue to fight infection or trauma
• Redness, swelling, heat, pain, and sometimes loss of function
• Heat, redness, and swelling result from the dilation and increased permeability of blood vessels
• Migration of inflammatory cells into tissue, their local effector actions, and swelling contributes to the pain, causing pressure on the skin and other tissue
• Pain is also from stimulation of sensory pain receptors linked to sensory neurons
• Loss of function may result from inflammation where an inflamed joint may not move properly
• Inflammation allows specialised cells of the innate immune system called dendritic cells to take pathogen or pathogen-derived material to lymph nodes via the lymphatic vessels to initiate adaptive immunity if infectious agents are present

Purpose of the Inflammatory Response

• Functions are to contain the offending agents and limit their effects
• To neutralise and dispose of pathogens and cellular debris
• To set the stage to promote healing and repair and stimulate/enhance the adaptive immune response

Sequence of Events in Inflammatory Response

• Vasodilation and increased permeability of blood vessels.
• Inflammatory chemicals are released by damaged tissue and mast cells due to an infected splinter, complement activation produce C3a and C5a, which bind to mast cells and release histamine and other chemicals
• Histamine release induces vasodilation, leakiness of capillaries, and margination (migration of leukocytes towards the blood vessel wall)
• Phagocyte emigration involves:
  • Neutrophil diapedesis (movement out of blood vessel).
  • Neutrophils undergo chemotaxis towards bacteria (in response to chemokines).
  • Phagocytosis of the bacteria.
• Tissue repair involves removal of debris and wound healing.
• C3a and C5a are complement proteins and Chemokines which provide the chemotactic signal for leukocytes to migrate towards the site of inflammatory stimulus.

Fever vs Inflammation

• Heat in inflammation is due to localised increase in blood flow and enhanced local metabolic activity.
• Fever is a systemic response caused by immune response.
• Pyrogens released by leukocytes act on the hypothalamus to reset body temperature.
• Fever assists in defence, because microorganisms are less able to replicate.
• Can be harmful if >40 degrees for extended time.

Lymphocyte Recirculation and Trafficking

• Lymphocytes recirculate all the time, irrespective of infection.
• Every minute, 5 x 10^6 lymphocytes leave the blood & enter secondary lymphoid tissues.
• Lymphocytes leave the blood & enter lymph nodes, where they can be activated by pathogens in the afferent lymph draining from a site of infection.

Leukocyte Migration (Trafficking)

• Occurs between primary and secondary lymphoid organs.
• Travels from blood to lymph nodes (naïve).
• Travels from blood to tissues.
• Occurs from sites of infection to lymphoid organs.
• Travels from lymphoid organs to peripheral tissues.
• Travels via recirculation (Lymphocytes – lymphatics to blood).
• Occurs within secondary lymphoid organs.

Molecules involved in leukocyte migration

• Includes soluble factors and their receptors.
• Chemokine and chemokine receptors.
• Adhesion molecules includes Selectins, Integrins, and Immunoglobulin (Ig) superfamily members.

Chemokines and Chemotaxis

• Chemokines comprises a large family of low molecular weight proteins secreted by various cell types.
• They transduce signals by binding to specific receptors on cells.
• Chemotaxis is movement in response to chemical signal.
• They are classified according to positioning of cysteine (C) residue - CC, CXC, CX3C, C.
• Chemokine receptors (e.g. CCR, CXCR, CX3CR) bind respective chemokines.
• Chemokine receptor activation in leukocytes results in alteration of integrin structure to lead to increased affinity of integrin for its ligand and integrin clustering.
• Conformational changes result in stable binding of leukocyte to the vascular endothelium.

Homeostatic Chemokines

• Homeostatic chemokines are constitutively produced in certain tissues and responsible for basal leukocyte migration.

Inflammatory Chemokines

• Inflammatory chemokines are formed under pathological conditions actively participate in the inflammatory response attracting immune cells to the site of inflammation.

Selectins

• Are glycoproteins (C-type lectin) and bind to carbohydrate determinants on selectin ligands.
• L selectins are expressed by leukocytes. They interact with ligands on vascular endothelial cells.
• P and E selectins are expressed by endothelial cells; ligands expressed on leukocytes.
• Help bind ligands with low affinity and facilitate the slow down of leukocytes flowing freely in blood allowing leukocytes to roll along vascular endothelium
• P-selectin is on activated endothelium and platelets and has PSGL-1, sialyl-Lewisx as a ligand
• E-selectin is on activated endothelium and has Sialyl-Lewisx as a ligand

Integrins

• Heterodimer molecules – made up of α and β chains
• They are transformed from an inactive to active state following activation.
• Integrins mediate stronger adhesion of leukocytes to vascular endothelium and their interaction with ligands of the Ig gene superfamily.

Immunoglobulin (Ig) Superfamily

• Family of proteins with a globular structural motif called an Ig domain.
• Includes intracellular adhesion molecule 1 and 2 (ICAM-1 and -2) and vascular cell adhesion molecule -1 (VCAM-1)
• Play important roles in cell-cell adhesion and the interaction of lymphocytes with antigen presenting cells.