Core Concepts- Lecture 5- Phagocytes

describe the differences between neutrophils and macrophages

• macrophages- round nucleus and irregular shape and cytoskeleton

• neutrophils- multilobed nucleus with lots of chromatin and has granules

discuss macrophage and neutrophil phagocytosis and effector functions

Neutrophils

• rapid/short lived professional killers

• release antimicrobial granules enzymes and peptides to kill extracellular pathogens

• can release NETS- webs of chromatin and microbial proteins

• produces superoxide and reactive oxygen

macrophages

• cleanup/ surveillance and APC

• also efferocytosis- removing dead and dying cells

• has lysosomes- acidification by vacuolar ATPases

• generates reactive oxygen and reactive nitrogen species

• produces cytokines

• slower and long lived

describe the steps of phagocytosis- neutrophils and macrophages

1. recognition of the target via phagocytic receptors- recognise PAMPs

2. target is engulfed into a phagosome- membrane bound vesicle

3. phagosome has V-ATPases that pump H+ ions in and make it more acidic, early phagosome is Rab5+ and late is 7+ positive and V-atpases

4. phagosome fuses with the primary and secondary granules/lysosomes. lysosomes have AMP and is acidified. makes a phagolysosome

5. NADPH oxidase assembles the phagosome membrane and makes superoxide and other reactive oxygen- highly antimicrobial environment- low pH, lysosomal enzymes to kill pathogen

6. degranulation in neutrophils

discuss the neutrophils fate

• short lived and for a rapid response- after using their effector function they degranulate and die

• may die by apoptosis: regulated death and expose DAMPs to signal macrophages for clean up. non inflammatory resolution

• may die of netosis- release DNA and chromatin into the extracellular space- pro inflammatory

• necrotic/uncontrolled death- cellular contents are released and make inflammatory signalling

how are phagocytic compartments trafficked?

• RAB GTPases- determine the identity and trafficking routes

• SNAREs- mediate membrane fusion for the phagolysosome

• actin binding proteins- control movement and shape and changes of vesicles

discuss phagocytic receptors(5)

1. C type lectin- dectin-1 recognises beta glucans on fungal cell walls

2. mannose receptors- sugar on bacterial surfaces

3. scavenger receptors- bind glycoproteins on bacteria and host DAMPs- MARCO, SRA

4. complement receptors- CR3 binds fungal beta glucans or opsonised microbes

5. PRRs- TLRs- don’t activate phagocytosis but activates pro inflammatory signalling and NLRs= regulate phagosome maturations

discuss phagocytosis autophagy and xenophagy definition

• autophagy: special form where cell captures cytoplasmic to destroy organelles

• xenophagy: targets intracellular pathogens and not just exracellular microbes and makes double membrane organelles called autophagosomes

discuss xenophagy process

1. recognition- intracellular pathogen is ubiquinated

2. recruitment of adapter proteins such as p62 binds to the ubiquitinated cargo and recruits autophagosome

3. autophagosome formation- Atg5 and LC3- makes double membrane

4. fusion with the lysosome

discuss phagocyte killing mechanisms

1. pro inflammatory signalling by TNF-alpha, IL-1, IFN-gamma- enhance phagocytosis and promote lysosome maturation. induce iNOS to make nitrous oxide for antimicrobial activity and up regulate ROS

2. respiratory burst- make reactive oxygen species to kill pathogens inside the phagosome. NADPH complex made of membrane bound cytochrome b558 and cytosolic subunits p40/p47. 

describe the respiratory burst steps

• NADPH oxidase complex- composed of cytochrome b558(gp91 and Nox2) and cytosolic subunits p40/p47/p67

• activated by PRRs sensing PAMPs. G-protein coupled receptors signal activation. Rac2 GTPase critical for assembly for NADPH complex

• phagosome forms and cytosolic subunits translocate to the phagosomal membrane and NADPH catalyses to make super oxide and made into hydrogen peroxide and hypochlorite

• fuses with the lysosome and low pH acidification with ROS kill pathogen

what is efferocytosis?

process by which phagocytes recognise, engulf and digest dead or dying cells such as apoptotic neutrophils, cellular debris or infected cells

steps in efferocytosis

1. recognition of the dying cell- dying cells have eat me signals

2. phagocyte engulfs cell and makes an intracellular vesicle and then digested by lysosomal enzyme

3. cytokines that are anti inflammatory are made: IL-10 suppress pro-inflammatory responses and TGF-beta promotes tissue repair

discuss macrophage activation states

1. classic pro inflammatory activation

• M1 like

• makes TNF-alpa, IL-1beta and IL-6

• to kill microbes and promote inflammation and promote Th1 T cell responses

• use arginine to make nitric oxide via iNOS

2. alternative anti inflammatory 

• M2 like

• induced by IL-4, IL-13 and macrophages produce iL-10, TGF-beta

• repair tissues and clearance of dead cells and promote Th2 

• uses arginine to make ornithine 

name types of resident macrophages in different tissues

brain- microglia

liver- kupffer cells

lung- alveolar macrophages

skin-lanerhans cells

bone- osteoclasts

what are some things that make scientists believe innate macrophages may also have memory?

• epigenetic programming- exposure to a stimulus marks the chromatin so response genes like chemokine are more easily reactivated