How abundant are microglia?
Distributed ubiquitously throughout CNS accounting for approx. 10% of brain cells, but varies depending on region (0.5%-16.6%)
What morphology to microglia assume in the normal healthy brain?
They are morphological and phenotypically plastic cells of the CNS parenchyma:
• Highly ramified resting morphology
• When they become activated: they develop shorter processes and enlarged soma
• Amoeboid: rounded, ‘classic phagocytic macrophage’. Source of microglia in adult healthy brain?
What are the types of macrophages found in the brain?
- Ramified microglia: Immune surveillance of the brain parenchyma
- Perivascular macrophage: Surveillance of the perivascular space between parenchyma and blood. On the brain side of the BBB, not in the brain.
- Choroid plexus macrophage: The site of immune cell entry and CSF production in the ventricles
- Meningeal macrophages: associated with meningeal blood vessels; circulate in the CSF and over surface of the brain (antigen presentation for CNS infection occurs in this space). These are very important population, as the primary place for antigen presentation happens in the meningeal spaces.
Where do the CNS macrophages originate from?
- Ramified microglia originate from the common myeloid progenitors from the yolk sac.
- Perivascular macrophages, choroid plexus macrophages and meningeal macrophages originate from the granulocyte-monocyte progenitor from the bone marrow.
What evidence supports the myeloid origin of the microglia?
- Differentiation of myeloid lineage cells, including macrophages, fails in mice with a deletion of the transcription factor PU.1. No microglia are detected in the CNS of PU.1 null mice, therefore they have a myeloid origin.
- Myeloid progenitors of the yolk sac are the source of the embryonic wave of myeloid cells that colonize the CNS
- Occurs before onset of haematopoiesis or the onset of vascularisation
Where do expansion of CNS macrophages occur?
Microglial expansion occurs through in-situ proliferation. Other macrophage populations proliferate in the bone marrow.
What are the functions of microglia during development?
Microglia have important functions during development. Though these are much the same as the adult functions, as there is a lot of cell death during development due to synaptic pruning etc, they have an important role in circuitry modelling. Functions include:
• Crucial scavenger function
• Phagocytose debris from naturally occurring cell death. Their appearance correlates with programmed cell death during development. Remember, neurones are overproduced during development.
• Remove cells in remodelling of foetal brain
• Eliminate axonal projections - as axonal projections and cells are overproduced.
• Immune surveillance
How do microglia modulate synaptogenesis?
Microglia are well positioned to monitor neuronal firing activity and synaptic function. Microglia steer their processes towards active synapses. They passively monitor these synapses, but also actively respond to neuronal activity. They also surround inactive synapses, and drive PCD [programmed cell death] during development and apoptosis and removal of adult neurons. Neuronal apoptosis happens after the microglia has surrounded it! It is a very active process. This is beneficial to avoid inflammatory response to neuronal debris.
What signalling molecules do microglia use to drive synaptic pruning or maturation of synapses?
- Drive the pruning & elimination of synapses via C3-CR3 signalling
- Drive maturation of synapses via CX3CL1/CX3CR1
What is the source of microglia in the adult brain?
- This occurs mainly through proliferation of resident parenchymal microglia.
- However when this is insufficient, there can also be migration of amoeboid perivascular cells and rarely as ramified microglial cells into parenchyma via vasculature and meninges.
- When resident microglia can no longer divide, there can be invasion of microglia derived from bone marrow progenitor cells.
Describe the nature of neuronal signalling to microglia
Neurons provide calming inputs to keep microglial cells in a quiescent state:
• Interaction of the neuronal membrane protein CD200 with the myeloid cell receptor CD200R on microglia
• High levels of fractalkine (CX3C family of chemokines) in normal neurons prevents MG activation - MG express CX3CR1)
• Microglial expression of molecules for antigen presentation is inhibited by electrically active neurons
What are the function of microglia in the adult brain?
Surveillance:
- Occupy own spacial territory
- Monitor the extracellular environment
- Sense homeostatic disturbances or changes in structure
- Interact with other cells to receive signals concerning changes in state.
Immune Response:
- Can undergo a rapid transformation into altered or reactive state (reactive microgliosis)
- Initiate an immune response
Neuroprotection:
- Support endangered neurones or interfere with potential threats to tissue integrity.
Describe the surveillance features of microglia
- Occupy own spatial territory
Microglia occupy their own space - i.e. have their own “territory” where they survey that space. They do not contact each other - each cell has it’s own territory. - Monitor the extracellular environment, and
- Sense homeostatic disturbances or changes in structure
Microglia are highly dynamic in vivo, but are not migratory in their resting state. Their cell bodies remain generally fixed, but their processes are highly motile, undergoing continual cycles of extension and withdrawal. Microglia sample the extracellular environment in a random fashion. - Interact with other cells to receive signals concerning changes in state – their processes directly contact neurons, astrocytes and blood vessels
They interact with basically every other cell type:
○ Blood vessels - important to initiate an immune response.
○ Astrocytes
○ Neurones
○ Dendrites and synapses
But they don’t seem to make permanent contacts via e.g. gap junctions
What are the changes microglia undergo in becoming ‘reactive’?
Change morphologically from a highly ramified resting state, to shorter processes and enlarged soma. They also exhibit:
○ Migration (whereas it did not in the ramified state)
○ Proliferation
○ Upregulation of innate immune cell surface receptors
○ Phagocytosis
○ Upregulation of antigen-presenting capabilities
○ Secretion of pro-inflammatory mediators and cytotoxic factors
○ Secretion of anti-inflammatory compounds and neurotrophic factors
What role to microglia have in initiating an immune response?
In healthy a brain, the BBB restricts access of immune cells from the blood. Microglial cells have end feet on endothelial cells to communicate with inflammatory cells.
Microglia initiate an immune response by phagocytosis of invading pathogens:
○ Microglia recognise micro-organisms through carbohydrate and lipid motifs with PRRs [pattern
recognition receptors], CR3 or Fc receptors.
○ Extend pseudopodia around microbe and microbe is endocytosed into a phagosome - same as any other macrophage
○ Cell lysosomes containing microbiocidal oxygen metabolites and enzymes fuse with the phagosome to digest the microbe
○ Undigested fragments released to outside Pattern Recognition Receptors (PRRs)
They can then initiate a chronic inflammatory response:
○ Regulation of T-cells responses through presentation of antigen
○ Critical event for generation of protective T-cell responses
○ Activated microglia upregulate MHC class II
○ Antigen expressed on cell surface in association with MHC class II
○ Interaction between T-cell receptor and MHC class II
Stimulate CD4 T-helper cells to produce cytokines
Activated microglia in the M1 phenotype, themselves produce pro-inflammatory cytokines, chemokines, and express MHC Class II, iNOS etc.
Describe the neuroprotective role of microglia after axotomy
Axon transection in the periphery results in rapid microgliosis in the facial nerve nucleus in the CNS. In other words, axotomy in the periphery causes reaction of microglia in the cell body of the neurone in the CNS. This leads us to believe that neurons produce rescue signals that mediate microglial activation
Immediately after axotomy:
○ Axotomy triggers immediate microglial response, where cell become activated.
○ Microglial activation is initiated by fractalkine signalling or massive release of ATP from distressed neurones.
○ Microglia proliferate as more cells required to meet increased trophic demand of injured neurons
Days after axotomy:
○ Axotomised perikarya [cell body of a neurone] are surrounded by activated microglia
○ It was thought that microglia then strip away the synapses, but it was found that they do not display phagocytotic activity (depends on extent of injury)
○ Microglia may release factors (e.g. TGFα) to protect neurons and promote regeneration
○ Microglia displace axosomatic terminals (not strip away synapses) so microglial and neuronal membranes become opposed . Deafferentation protects axotomised neurons from afferent excitatory impulses (while the neurone recovers). Also facilitates exchange of signalling or trophic molecules between them.
Several weeks after axotomy:
○ Microglial activation subsides coinciding with successful regeneration. Once target reinnervation has been achieved and animals regain whisker movement, the number of microglia decreases – cells die by apoptosis.
Describe the role of microglia in Alzheimer’s and MS
Microglia accumulate around amyloid plaques in the Alzheimer’s brain. Amoeboid macrophages appear to be absent. Looks as if the microglia think they are able to cope? This is in contrast to MS.
In MS there is wide-scale activation of microglia in the white matter. There is also activation in the grey matter, regardless if there is a lesion? There is also an absence of amoeboid cells.
In an MS lesion, you see lots of macrophages. Within an active region of MS, 45% of macrophage-like cells in active MS lesions are derived form the resident microglial pool, while the rest is from monocytes.
In an aminal model of MS (EAE), it suggests that monocyte derived macrophages associated with Nodes of Ranvier initiate demyelination, while microglial derived macrophages appear to clear debris.
How can activated microglia modulate inflammation?
- Chemokines (attracting immune cells): MIP-1a, MIP1- bmMCP-1, RANTES
- Anti-inflammatory cytokines: IL-10, TGF-b, IL-1ra
- Pro-inflammatory cytokines: IL-1, TNF-a, IL-6, IL- 12, IL15, IL-18
- Death receptor ligands: TNF-a, FasL, TRAIL
- Prostanoids: PGD2, PGE2, Thromboxane B2
- Cytotoxic Factors: NO, ROS, glutamate
What phenotypic states are activated macrophages (not microglia) thought to exist in?
Peripheral macrophages are known to exist in different activation states, primarily M1 and M2;
• M1 = classical activated state. Associated with production of inflammatory cytokines, reactive oxygen species and cytotoxicity.
• M2 = anti-inflammatory phenotype. Associated with production of anti-inflammatory cytokines, wound repair and debris clearance.
There is much debate to whether you can translate this, from ameboid-like cells of the peripheral body to dendritic cells of the CNS (microglia).
Discuss the evidence to support the polarised state hypothesis.
- Peripheral macrophages exist in an M1 or M2 phenotype
- There is much debate to whether you can translate this, from ameboid-like cells of the peripheral body to dendritic cells of the CNS (microglia)
In Vitro studies to polarise microglia (induction of a specific state through cytokines) found that there can be two phenotypes:
• M1 polarised microglia produce: TNF,IL1β,IL6,IL12,CCL8,CXCL9/10,CCR7,CD80
• M1 polarised microglia express: MHC class II, CD68, iNOS
• M2 polarised microglia produce: L10, IL4, CCL1/20, CXCL1/13, TGFβ, BDNF, IGF, CD209, CD163
• M2 polarised microglia express: Arginase 1, Ym1 (heparin binding lectin), mannose receptor
However, remember in biology that there is no binary system - it will all be in a spectrum - there will be all kinds of things in between M1 and M2. It is now suggested there are more phenotypes:
• M2a – suppression of inflammation
• M2b – following exposure to immune complexes
• M2c – tissue remodeling and matrix deposition
…how useful is this classification really?
There is extensive accumulating evidence that microglial activation contributes to neuronal damage. Microglial dysregulation and over-reaction are suggested to be neurotoxic. However, there is no evidence of this from whole animal studies. It is not yet known if sustained or excessive microglial activation is detrimental to neuronal/glial survival.
It may be instead that microglia activate astrocytes to become neurotoxic. Some have labelled astrocytes A1 and A2.
What are the features of polarised activated microglia?
In Vitro studies to polarise microglia (induction of a specific state through cytokines) found that there can be two phenotypes:
• M1 polarised microglia produce: TNF,IL1β,IL6,IL12,CCL8,CXCL9/10,CCR7,CD80
• M1 polarised microglia express: MHC class II, CD68, iNOS
• M2 polarised microglia produce: L10, IL4, CCL1/20, CXCL1/13, TGFβ, BDNF, IGF, CD209, CD163
• M2 polarised microglia express: Arginase 1, Ym1 (heparin binding lectin), mannose receptor
