Myeloid cell differentiation Flashcards
How do HSCs choose their lineage?
The chromatin is regulating which genes are turned on and off and which transcription factors are to be expressed which is giving the identity to the cell.
If cells go into a particular direction, can they still change their fate?
In healthy, in vivo setting if a cell commits towards certain lineage then it usually does not change their lineage anymore. However, in the lab it can be manipulated by turning on different transcription factors.
What guides the cell fate decisions?
- Intrinsic factors (transcription factors which regulate genes)
- Extrinsic factors (molecules, cytokines and cells around them which give signals)
What do colony-stimulating-factors (CSF) do?
They can make progenitor cells to differentiate into a particular colony, e.g. macrophage-colony-stimulating- factor will make them to differentiate into macrophages.
What is a semi-solid medium?
It is a very thick medium and you can vortex your cells there and each cell will be in one place and it will not diffuse, then these cells if are progenitors will differentiate into certain cells and for colonies which can be observed under a microscope.
What are examples of extrinsic factors?
- IL-3 is required for proliferation, it is required in differentiation into many different lineages and is not lineage specific (megakaryocyte, erythrocyte, granulocyte, monocyte etc.)
- TPO - specific for megakaryocytes
- EPO - specific for erythrocytes
- M-CSF and GM-CSF - specific for monocyte/macrophage
- GM-CSF - specific for granulocytes
What signalling pathway is typical for type I cytokines (IL-3, IL-5, IL-6, GM-CSF, G-CSF)
JAK/STAT signalling pathway
How does JAK/STAT pathway work?
- When cytokine engages with its receptor, that leads to the phosphorylation of tyrosines by JAK kinase. This gives a signal to recruit other proteins, here STAT which gets activated by phosphorylation. Once activates it travels into the nucleus, finds specific DNA motifs and promotes expression of target genes. Then STAT becomes inactivated and travels back to the cytosol.
- Genes which are expressed as a result of JAK/STAT pathway are essential for cell survival, cell proliferation and functional activation
What are transcription factors?
They are proteins found in the nucleus which bind to specific sequences in the DNA and induce changes in the transcriptional program. They can have activator or repressor function.
What is the importance of chromatin with transcription factors?
As transcription factors recognise a short sequence, which we will find a lot within the genome, the chromatin, which is tightly wrapped around nucleosomes it does not give access to transcription factors to bind.
Do transcription factors work alone or in complexes?
They are usually in complexes to get the enhancer-promoter to be open and be active.
What happens after transcription factor binds?
It recruits co-activators and chromatin remodelling complexes, which allows other transcription factors to bind as well
What are examples of intrinsic factors?
- PU-1 (granulocyte, monocyte)
- GATA-1 (megakaryocyte, erythrocyte)
- C/EBP - neutrophil/eosinophil
-Gfi-1 - neutrophil, known for its inhibitory function
How is the balance kept?
- You need a balance as if you will have a lot erythrocytes, you will have good transport of oxygen but if you dont have myeloid cells then you will have problem when you get an infection. So the balance need to be kept and then for example if you lose red blood cells or require more oxygen for example mountain climbing then there will be more EPO produced etc.
- If you need CMP to differentiate into an erythroid cell then you need signals for erythroid genes and transcription factors but at the same time you need to reduce the signals for the myeloid genes and other way around.
How do we get a myeloid lineage?
Myeloid transcription factors will give positive feedback to themselves and other myeloid transcription factors and will inhibit the expression of erythroid transcription factors. E.g. PU-1 will bind to GATA-1 and repress GATA-1 bound genes.
Where is PU-1 expressed?
It is expressed in both myeloid and lymphoid cells, however, it is quickly turned off in the lymphoid lineage, only B cells retain the expression but at lower levels than myeloid cells. It is required for granulocyte/monocyte lineage and is turned off in megakaryocyte/erythrocyte lineage
What happens to PU-1 KO mice?
They die soon after birth, they have a lack of myeloid cells, B and T cells. Even PU-1 expression is turned off in T cells to allow them to mature, they still need PU-1 to get to that stage (Ka Sin Mak et al. 2011).
How can we track the PU-1 expression in different progenitor cells?
- KI GFP into PU-1 gene
- Flow cytometry for Lineage-negative cells
- Separate GFP positive and GFP negative cells from each other
- By flow cytometry, you can look at different cell populations and their GFP expression (Back et al. 2005)
How does PU-1 act negatively on erythropoiesis?
- it’s KO causes premature differentiation of erythrocytes which leads to their apoptosis.
- it’s overexpression leads to anaemia due to the block in erythrocyte differentiation.
How do we know that PU-1 is an important regulator of myeloid differentiation?
Because when Iwasaki et al. 2005 make conditional KO mouse they saw decrease in. HSCs, loss of repopulation ability and absence of CMPs and GMPs. When excised from CMPs and GMPs cells did not mature properly, hence PU-1 is important in all stages of myeloid differentiation (Iwasaki et al. 2005)
How do GMPs become macrophages or neutrophils?
- GMP has expression of PU-1 and C-EBPalpha
- To become neutrophil the expression of C/EBPalpha increases and PU-1 expression stays the same. This recruits Gfi-1
- To become macrophage the expression of PU-1 increases and C/EBPalpha expression stays the same. This recruits Egr-1.2 and Nab-2
- Gfi-1 and Egr-1.2/Nab-2 inhibit each other
How can cytokines modulate cell fate decisions?
They do that by regulating lineage transcription factor.
What Dahl et al. 2003 did is they had PUER cells and they gave tamoxifen which binds to PU-1 ER complex and leads to PU-1 transport into the nucleus.
- If cells got low Pu-1 concentration they became neutrophils
- If cells got high PU-1 concentration they became macrophages
- If cells got high PU-1 concentration and IL-3 it supported their differentiation into macrophages
- But if cells got high PU-1 concentration and then G-CSF they differentiated into neutrophils
What is GATA-1
A transcription factor, regulator of erythroid/megakaryocyte lineage. GATA-1 can bind on its own but usually, it binds as a complex which gives more specificity.
What does master regulator mean?
- It is a transcription factor which is essential for specific lineage differentiation
- It represses other potential lineage programs
- They can reprogram certain specific cell lineage when introduced into cells
What can happen when the balance is lost?
Due to the loss of certain transcription factors, there may be mutations which will lead to cells gaining proliferative abilities. Usually, this happens in the progenitor stages, and you develop leukaemia.
what is AML-ETO?
-It is a subtype of acute myeloid leukaemia where ETO binds to RUNX1 which interferes with generation of granulocytes. This leads to high myeloblast counts and leads to decrease in C/EBPalpha and PU-1 levels.
- Mutations in C/EBPalpha are very common in acute myeloid leukaemia, however, the KO does not lead to leukaemia is the improper function which leads to it.
What is the connection between PU-1 and AML?
The knockdown of PU-1 leads to the accumulation of pre-differentiated cells and they are more likely to develop into AML.
What is a myelodysplastic syndrome?
It is a type of blood cancer where bone marrow produces immature blood cells rather than healthy ones, it is characterised by the decreased level of PU-1.
What is innate immunity?
Innate immunity is already there and does not need further development to start an immune response. The innate immune response is very quick, within hours but it has low specificity and no memory. It is the same response every time it encounters a pathogen.
What are the steps of immune response?
- Pathogen enters the body
- It is recognised by macrophages/neutrophils
- Production of pro-inflammatory cytokines
- Entrance of macrophages and neutrophils into the tissue from the bloodstream
- Engulfment of microbes by innate immune cells
- Up-take of microbial antigens by dendritic cells and presentation of antigens to T cells in lymphoid tissues
- Support of complement
- Tissue repair by macrophages
What are characteristics of neutrophils?
- Most numerous types of leukocytes
- Short-lived (6h)
- Multi-segmented nucleus
- Contain different types of granules for microbial killing (lysozyme, collagenase etc)
- Phagocytes
- NETosis
What are characteristics of mononuclear phagocytes (monocytes/macrophages)
- granular cyoplasm with lysosomes and phagocytic vacuoles
- circulate in the blood as immature monocyte or is based in the tissue as tissue resident macrophage
- when it enters the tissue from blood, monocytes differentiate into mature macrophages
- main function is phagocytosis of pathogens and dead host cells
- They produce cytokines to instruct immune response
- They can present antigens to T cells
- They promote tissue repair by stimulating angiogenesis and matrix synthesis
- Long-lived (several days) and can proliferate at the site of infection
- They can have different sub-types (mostly M1 and M2)
What are characteristics of mast cells?
- They reside in the skin and mucosal epithelium
- They have receptors for IgE and IgG antibodies and bind them
- They release granules (cytokines and histamine) upon binding to the antigen
- Defense against worms
What are characteristics of basophils?
- There is a low number of them in the blood
- Functional very similar to mast cells
- contain granules that bind basic dyes (that’s where their name comes from)
What are the characteristics of eosinophils?
- There is a low number of them, and they are present in peripheral tissues like mucosa of the respiratory and gastrointestinal tracts.
- Number increases in inflammation
- They contain granules which damage parasites but can also damage host tissue
- Take part in allergy
What are examples of APCs?
Antigen-presenting cells:
- Dendritic cells - lymphoid tissues
- Follicular dendritic cells - germinal centres
- Macrophages - at the site of infection
- B cells - lymphoid tissues
What are characteristics of dendritic cells?
- They are the main APCs
- They are present in skin, lymphoid tissues, mucosal epithelium and organ parenchyma
- They differentiate from monocyte precursor
- They produce cytokines like type 1 interferons
- They travel to secondary lymphoid tissues to present antigens to T cells
How does the migration of leukocyte happen from blood to tissue?
- Leukocyte is slowed down and captured by selectins (P and E-selectin on endothelium -> glycoproteins on leukocyte or L-selectin on leukocyte -> ligand on endothelium)
- Rolling
- Activation of chemokine gradient
- Slow rolling
- Stronger adhesion by Integrins (LFA-1 or VLA-4 -> ICAM-1) or (Mac-1 on monocytes -> ICAM-1)
- Paracellular or transcellular migration into the tissue
What are chemokines doing?
- They instruct movement of leukocytes
- They are produced by leukocytes, endothelial and epithelial cells or fibroblasts
- Their secretion is induced by pathogen recognition and inflammatory cytokines
- Chemokine receptors are G-protein coupled receptors expressed on leukocytes
- They increase integrin affinity
- They regulate traffic through the lymphoid tissues
- They guide DCs from the site of infection into the draining lymph node
What can innate immune cells recognise using PRRs?
PAMPs and DAMPs
What are PAMPs?
Pathogen-associated molecular patterns, they are specific molecular structures of pathogens for example:
- Nucleic acid (ssRNA, dsRNA) of viruses or bacteria
- Proteins (Pilin, Flagellin) of bacteria
- cell wall lipids (LPS, lipoteichoic acid) of gram-negative or gram-positive bacteria
- Carbohydrates (Mannan, Dectin glucans) of fungi and bacteria
What are DAMPs?
Damage-associated molecular patterns, are produced by damaged cells due to infection or injury, for example:
- Stress-induced proteins (HSPs)
- Nuclear proteins (HMGB1)
Which cells express pattern recognition receptors (PRRs)?
- Macrophages
- Neutrophils
- DCs
- Epithelial and endothelial cells
What are examples of PRRs?
- Toll-like-receptors (TLR) - extracellular and intracellular
- Lectin
- Scavenger receptor
- nod-like-receptor (NLR)
- RIG-like-receptor (RLR)
TLR
-TLR1- LPS - extracellular
-TLR5- Flagellin - extracellular
-TLR3 - dsRNA - intracellular
-TLR7 and TLR8 - ssRNA - intracellular
-TLR9 - CpG DNA - intracellular
-TLR2 and TLR4 recognise DAMPs - extracellular
What TLRs need to do to become active?
They need to dimerise either homodimerise (e.g. TLR4 with another TLR4) or they can dimerise with another TLR (e.g. TLR2 with TLR1). They also require additional proteins to form a dimer.
What are main adaptor molecules of TLRs?
- MyD88
- TRIF (TIR domain containing adaptor inducing IFN-beta)
What is the outcome of TLR signalling?
NF-kB and AP-1:
- regulate expression of pro-inflammatory genes (cytokines TNF, IL-1, IL-6, chemokines, endothelial adhesion molecules, co-stimulatory molecules) and lead to the state of acute inflammation
IRFs (interferon response factor):
- regulate expression of type 1 interferon (IFNalpha/beta genes)
- secretion of IFNalpha/beta and lead to the antiviral state
What are cytosolic receptors?
They are PRRs in the cytoplasm. They detect infection in the cytoplasm (bacteria and viruses). For example:
- NOD-like receptors (NLRs)
- RIG-like receptors (RLRs)
NLRs
- NLRs can recognise different pathogen components and toxins. They are signalling through NF-kB activation through adapter molecule RIP2, which leads to the inflammation state
- They can be activated by microbial products and cytoplasmic changes and form an inflammasome complex, which secretes IL-1beta
RLRs
- They recognise ss and dsRNA
- They can distinguish between viral and cellular RNA
- Binding of RNA results in activation of IRF3 and IRF7 or NF-kB depending on adapter molecules
- They lead to antiviral state by production of type 1 interferons
What can the complement system do ?
The complement can recognise microbes outside the host cell and mark them (opsonisation) for phagocytosis by macrophages, neutrophils or DCs or the complement can as well directly kill infected cells.
What is the complement?
It is a group of proteins which are synthesised by the liver, and they are present in the bloodstream
Which proteins of the complement are the most important and why?
C2, C3 and C4 because they get cleaved into two pieces, and their cleavage leads to the activation of the pathway
What are three pathways by which the complement can activate?
- Classical pathway (C3a and C5a which attract phagocytes)
- MB-lectin pathway (C3b leads to opsonisation of pathogen for phagocytes)
- Alternative pathway (C5b forms a membrane attack complex with C6, C7, C8 and C9 and kills the infected cell)
In all of these pathways, C3 needs to be cleaved
How proteins cleave in the complement?
They cleave into two pieces:
Ca: is the smaller piece which is soluble and is a chemoattractant for neutrophils
Cb: which is the bigger piece which binds to microbe membrane
What cytokines are in acute inflammation?
- TNF
- IL-1
- IL-6
TNF
- Produced by macrophages, DCs etc
- Binds as a trimer to a trimeric receptor
- leads to apoptosis and cell death or activation of NF-kB and AP1
IL-1
- produced by macrophages, neutrophils, epithelial and endothelial cells
- binds to IL-1 receptor (TIR domain)
- leads to activation of NF-kB and AP1
IL-6
- produced by PAMP-activated macrophages, fibroblasts, endothelial cells
- binds IL-6 receptor (signals through JAK/STAT pathway)
- it stimulates neutrophil generation in the bone marrow
IL-12
- produced by macrophages and DCs
- Stimulates IFN-y production by NK cells and T cells
- enhances NK and CD8+ T cells activation
- promotes Th1 cell differentiation
What are two different types of macrophages?
-M1
-M2
How to regulate innate immunity?
- IL-10 inhibits production of IL-1, IL-12 and TNF
- TLR and cytokine signalling inhibitors (SHP-1, SOCS)
- IL-1RA (IL-1 receptor agonist, binds to IL-1 receptor but doesn’t stimulate downstream signalling)
- Decoy receptors (cytokine receptors with similar affinity to their ligand, but without signalling function)
- Autophagy genes (destruction of IL-1 processing inflammasomes)
What can happen if acute inflammation gets out of control?
Septic shock. This is when cytokines produced due to severe infection enter the bloodstream and act systemic. They cause organ damage and, ultimately, failure.
