4. Phagocytosis & Phagocytosis Maturation (LTS)

Define phagocytosis.

The cellular process for ingesting and eliminating including microorganisms, foreign material and apoptotic cells (larger than 0.5µM in diameter); part of the innate immune response and is essential for tissue homeostasis.

What is the function of phagocytosis?

• Clearance of pathogens (and present antigens).

• Tissue homeostasis and remodelling.

How was phagocytosis discovered?

Mechnikov demonstrated that amoebae feed upon fungi and bacteria.

Explain target recognition proceses.

Direct recognition of self cells from non-self cells.

• Direct recognition of a pathogen surface.

• Indirect recognition via host factors (complement or antibodies).

What is recognised in direct recognition?

Pathogen Associated Molecular Patterns (PAMPs). These are unique to the type of pathogen e.g. double-stranded RNA detected by TLRs for viruses.

How are PAMPs recognised?

By Pattern Recognition Receptors (PRRs). They are split into two categories/roles:

• Immunological Signalling

• Phagocytosis

What are the main macrophage phagocytic receptors for direct recognition?

• Mannose receptor

• Scavenger receptor

• Dectin-1 (glucan receptor)

Describe the mannose receptor and its role in direct recognition.

• C-type (calcium-dependent) lectin, recognises mannose.

• Mannoproteins are major components of most fungal cell walls and some bacteria (mTB).

• Not a dominant receptor.

Describe the scavenger receptor and its role in direct recognition.

• Part of a large family of receptors (11 mammalian classes).

• Significance in fighting infection is unclear.

• NK and T cells have numerous scavenger receptors to recognise abnormal, non-self cells.

Describe Dectin-1 and its role in direct recognition.

• Major beta-glucan receptor, recognising beta1,3- glucan, a major fungal cell component.

• Primary receptor for non-opsonic (direct) phagocytosis of most fungi.

• Pro-inflammatory signalling molecule.

How does indirect recognition occur?

Via complement or antibodies.

What are the main pathways of the complement system, and when do they all converge?

1. Classical Pathway

2. Lectin Pathway

3. Alternative Pathway

They converge at the C3 component.

What is antibody opsonisation?

Antibodies mark pathogens as foreign.

What are the classes of antibodies that most commonly trigger phagocytosis by opsonisation?

IgG

How does recognition take place in antibody opsonisation?

Via the interaction between the Fc domain (antibody “tail”) and the Fc receptors expressed on phagocytes.

• FcR binds to the Fc portion of IgG or IgA antibodies, triggering phagocytosis when they engage with multivalent antigen-antibody complexes

What is FcγRI and its role in antibody opsonisation?

It has the highest affinity for IgG antibodies among Fc gamma receptors, primarily IgG1 and IgG3.

• Binds opsonised pathogens or cells.

• Promotes phagocytosis and destruction of targets.

• Activates immune responses via ITAM signalling (cytokine release, oxidative burst).

• Facilitates antigen presentation to T cells.

Where is FcγRI expressed?

Expressed on macrophages, neutrophils, eosinophils and dendritic cells.

What is the driving force for membrane extension and particle retraction?

Actin polymerisation.

What are classified as professional phagocytes?

• Macrophages

• Neutrophils

• Monocytes

• Dendritic cells

• Osteteoclasts

  Perform phagocytosis with high efficiency, removing microorganisms and present antigens to lymphocytes.

What are the phases of phagocytosis?

1. Detection of the particle to be ingested.

2. Activation of the internalisation process.

3. Formation of a specialised vacuole (phagosome).

4. Maturation of the phagosome to fuse with lysosomes to create a phagolysosome.

5. Degradation of the ingested particle and antigen presentation.

What cells are classified as non-professional phagocytes?

• Fibroblasts

• Epithelial cells

• Endothelial cells

Perform phagocytosis with low efficiency as they cannot ingest microorganisms but eliminate dead cells.

What is phagosome maturation?

A process by which the phagosome changes its membrane's structure and the composition of its contents, transitioning from a newly-formed phagosome into a phagolysosome through a series of fusion and fission interactions with endosomes and lysosomes.

What are non-opsonic receptors?

Receptors are involved in direct recognition, identifying molecular patterns on the particle to be ingested.

• C-type lectins

• Lectin-like recognition molecules

• Scavenger receptors

What are opsonic receptors?

Receptors that detect host-derived proteins (opsonins) bound to target particles

• Fc receptors (FcR)

• Complement receptors CR)

What are some examples of opsonins?

• Antibodies

• Fibronectin

• Complement

• Milk fat globulin (lactadherin)

• Mannose-binding lectin (MBL)

How do CR function in phagocytosis?

Bind to activated complement components, such as iC3b, deposited on the particle, promoting phagocytosis.

What is the role of integrins in phagocytosis?

• Increase their affinity for their ligand after receiving an inside-out signal from other receptors.

• They also form a diffusion barrier, excluding larger molecules from the phagocytic area.

What are some characteristics of the phagolysosome?

• Highly acidic

• Contains NADPH oxidase complex for producing ROS

• Contains hydrolytic enzymes.

How is phagocytosis efficiency regulated?

• Activating stimuli such as bacterial products, cytokines, signalling pathways and inflammatory mediators.

• Cell differentiation e.g. differentiation of monocytes to macrophages.

What is the role of early endosomes in phagosome maturation?

The new phagosome combines with early endosomes in a process involving membrane fusion events regulated by the small GTPase Rab5.

This fusion is promoted by the molecule EEA1 (early endosome antigen 1)

What is the role of Rab5 in early phagosome maturation?

Rab5 is a small GTPase

• Recruits Vps34 (generates PtsIns(3)P, a signal lipid for domain proteins like EEA1, promoting the fusion of the new phagosome with early endosomes.

• Recruits Mon1.

What is EEA1 and what is its role in phagosome maturation?

EEA1 (early endosome antigen 1) functions as a bridge between early endosomes and endocytic vesicles, promoting the fusion of the new phagosome with early endosomes. It also promotes the recruitment of other proteins, such as Rab7

What happens to the phagosome as it matures beyond the early stage?

As maturation proceeds, Rab5 is lost and Rab7 appears on the membrane, recruited by the Rab5-Mon1-CcZ1 complex.

The phagosome then fuses with late endosomes

What is the role of late endosomes in phagosome maturation?

• The phagosome fuses with late endosomes, which results in the accumulation of V-ATPase molecules on the phagosome membrane. This leads to the acidification of the phagosome.

• Late endosomes also contribute lysosomal-associated membrane proteins (LAMPs) and luminal proteases (cathepsins and hydrolases).

What is V-ATPase and what role does it play in phagosome maturation?

V-ATPase is a molecule that accumulates on the phagosome membrane during maturation and is responsible for acidifying the phagosome interior by translocating protons (H+).

What are LAMPs and what role do they play in phagosome maturation?

Lysosomal-associated membrane proteins (LAMPs) are incorporated into the phagosome membrane from late endosomes and lysosomes.

What is the final step in phagosome maturation?

The phagosome fuses with lysosomes to become a phagolysosome.

What is the role of the NADPH oxidase complex in the phagolysosome?

The NADPH oxidase complex produces reactive oxygen species (ROS), such as superoxide (O2−), which are important for killing ingested microorganisms.

How are ingested microorganisms degraded in the phagolysosome?

Microorganisms are degraded by a combination of the highly acidic environment, reactive oxygen species (ROS) produced by the NADPH oxidase complex and hydrolytic enzymes such as cathepsins, proteases, lysozymes, and lipases.

What are the two main types of receptors involved in target detection during phagocytosis?

• Non-opsonic receptors: directly bind to PAMPs

• Opsonic receptors: recognise host-derived proteins (opsonins) bound to the target.

What is the second step of phagocytosis?

Activation of the internalisation process- receptors bind to their ligands and initiate signalling pathways.

What signalling events occur upon Fc receptor activation? (Internalisation Process Activation)

Clustering of Fc receptors leads to the activation of Src-family kinases and Syk, phosphorylating substrates such as PI 3-K and PLCγ.

• Conformational changes occur in the receptor complex.

• ITAM motifs found on receptor-associated proteins are phosphorylated on tyrosine residues by Src-family kinases

• Syk binds to the phosphorylated ITAMs through its SH2 domains and is activated via phosphorylation.

• Syk activation triggers multiple signalling cascades.

What signalling events occur upon activation of complement receptors such as CR3?

• Activation of CR3 involves the GTPase Rho, which promotes actin polymerisation and microtubule involvement.

• Integrins like CR3 are activated by inside-out signalling, increasing their affinity for ligands and creating a diffusion barrier

What is the third step of phagocytosis?

Formation of the phagosome- changes in the membrane and actin cytoskeleton leading to the formation of a phagocytic cup and pseudopods that surround the particle.

What is the role of Arp2/3 complex in phagocytosis?

Promotes the formation of branched actin filaments at the phagocytic cup.

• Activated by Cdc42 and PI(4,5)P2 during FcγR-mediated phagocytosis

• Activated by Rho during CR-mediated phagocytosis.

What are the roles of myosins during phagosome formation?

• Myosin II helps in squeezing the particle

• Myosin X is involved in pseudopod extension

• Myosin Ic is involved in closing the phagocytic cup

How is the phagosome formed?

Membrane protrusions (pseudopods) fuse at the distal end, creating a new vesicle called the phagosome, which contains the ingested particle.

What is the fourth step in phagocytosis?

Phagosome maturation. The newly formed phagosome undergoes a transformation to become a microbicidal phagolysosome.

What occurs during the early stage of phagosome maturation?

The phagosome fuses with early endosomes, regulated by Rab5 and EEA1. Rab5 recruits EEA1 which promotes fusion.

What occurs during the late stage of phagosome maturation?

Rab5 is lost and Rab7 appears, promoting fusion with late endosomes. V-ATPase molecules accumulate, acidifying the phagosome. LAMPs and luminal proteases are incorporated.

What is the final step of phagosome maturation?

The phagosome fuses with lysosomes to become a phagolysosome.

How is the ingested particle degraded in the phagolysosome?

The ingested particle is degraded by the acidic environment, reactive oxygen species (ROS), and hydrolytic enzymes.

How do actin and phosphatidylinositides (PtdIns) regulate phagosome maturation?

Phosphatidylinositides (PtdIns):

• PtdIns(4,5)P2: Present in early phagosomes, promotes actin polymerization.

• PtdIns(3)P: Formed by PI3K after phagosome sealing, recruits fusion proteins (EEA1); promotes actin loosening, enabling membrane fusion and phagosome maturation.

• Lipid conversions help drive maturation and fusion with lysosomes.

Actin:

• Initially forms a dense ring to support phagosome formation.

• Loosening (depolymerisation) is required after phagosome sealing to allow membrane fusion and trafficking.

• Regulated by GTPases (Rac, Cdc42) and actin-binding proteins (cofilin).

What does depolymerisation mean in phagosome maturation.

There is a drop in negative charge, recruiting Rab5 GTPase.

What does Vsp34 do?

Generates PtdIns(3)P, a signal lipid for domain proteins like EEA1.

What is the role of EEA1?

It is a tethering homodimer that facilitates EE diffusion.

What does Mon-1 recruit?

CcZ1

What recruits Rab7?

Rab5-Mon1-CcZ1 complex

What happens to Rab4 and Rab11 as the phagosome matures?

Receptors are taken off the phagosome to be recycled.

What does Rab7 do?

Recruits RILP and ORP1L, adaptor proteins for dyein.

What does dyein do in phagosome maturation?

Tethers to phagosomes and walks down towards the negative end of microtubules so they have move towards the nucleus, where ribosomes are located.

How is acidification linked to phagosome maturation?

Acidification drives enzyme activation, membrane fusion, and pathogen destruction during phagosome maturation. pH drops from ~7 to ~4 upon the formation of the phagolysosome.

• Proton Pump (V-ATPase): Actively pumps H⁺ ions into the phagosome to lower pH.

• Activation of Enzymes: Acidic pH activates hydrolytic enzymes (proteases, lipases) for degradation.

• Membrane Fusion: Acidification promotes phagosome fusion with lysosomes to form phagolysosomes.

• Reactive Oxygen Species (ROS): Supports optimal NADPH oxidase activity for pathogen killing.

• Antigen Processing: Enhances degradation of pathogens for MHC class II presentation.

Describe the respiratory (ROS) burst.

The release of ROS into the phagosome. This involves the assembly of a huge complex.

• Driven by NADPH oxidase, which assembles on the phagosome membrane after uptake.

• Varies depending on the cell type.

How is there variation in phagosome maturation?

The activation of phagocytic receptors triggers a range of receptor-dependent pathways.

• FcR induces strong inflammation and killing than CR.

• Phagocytic uptake of apoptotic bodies actively suppresses inflammation.

The immune environment also can influence variation in phagosome maturation.

• Macrophage polarisation can alter the speed of maturation and levels of ROS.

• Ageing has an impact on monocyte and macrophage function.

Describe the waste disposal process following degradation.

• Ingestible compounds are exocytosed.

• Possible manipulated by intracellular pathogens.

Describe encapsulation as an evasion strategy.

Hiding pathogen epitopes (PAMPs) e.g. peptidoglycan- capsule not well-recognised. Method of evading direct recognition.

Describe layering as an evasion strategy.

The outer layer of the Histoplasma capsulatum cell wall is alpha(1,3)-glucan, masking beta-glucans from Dectin-1. Method of evading direct recognition.

Describe GAP mimicry as a evasion strategy.

GAP mimicry turns GTPases off, disrupting phagosome maturation.

What are evasion strategies pathogens use to avoid destruction?

• Direct recognition evasion via encapsulation and layering

• Phagocytosis evasion by disabling GTPases.

• Phagosome maturation evasion via the blocking and inhibiting key processes of phagosome maturation and phagocytosis e.g. Leishmania using LPG to block fusion of late endosomes and lysosomes; Mycobacterium uses lots of VFs and lipids to pause maturation in the early stages.

• ROS evasion via the production of detoxifying enzymes such as superoxide dismutase and catalase

• Acid evasion via the production of Listeria LLO toxin, causing the lysis of the phagosome.

• Complement opsonisation evasion via the production of proteins scavenging C3b

• Antibody antibody evasion via the production of proteins that bind Fc and IgG portions; production of proteases e.g. IgA