Group_1_Presentation - Mast Cell

Report
Models for in vivo
and in vitro studies
on basophils
Trainer: Franco Falcone
Introduction
• Basophils were discovered more than century ago (1879)
• Only recently they have been identify to play a role in
allergy (1972)
• Compared to human, mice have less basophils which were
initially more difficult to identify. First discovery of mouse
basophils by EM in 1981
How to deplete basophils in mice
• No naturaly occuring basophil defficiency in mice
• Antibody induces defficiency
Mar-1
Anti-FcseRI mAb
Ba103
anti-CD200R3 mAb
• Not selective only for basophils!
• Posible activation and/or depletion of mast cells
• Depletion of DC subpopulations (antigen presentation properties?)
• Not optimal depletion efficiency (80 – 90% for Ba103)
• Problematic timing in long term experiments
Other models are needed!
Transgenic models
BasoDTR
DTR transgene
(expression
under CD203c
promotor
Diphteria toxin
injection
Cells express DTR
allowing inducible
deletion
Depletion of
basophils 84%
NA
Aims:
• To reveal contribution of basophils and eosinophils in IgE-CAI in vivo.
• To generate a new mouse models lacking basophils and eosinophils.
•
BasoDTR mice - hDTR espression driven by CD203c promotor
(basophil marker)
•
EoDTR mice - hDTR espression driven by EPO promotor (eosinophil
peroxidase, granule cationic protein)
Basophil-speciphic ablation after DT administration in BasoDTR
mice
• Decrease in basophils in
peripheral blood (84%) and
spleen
• No decrease in basophil
population in bone-marrow!
• qPCR and WB analysis failed to
detect hDTR in bone-marrow
• No effect on peritoneal mast
cells
IgE-mediated chronic allergic (CAI)
reaction in BasoDTR mice
•
•
TNP-spec. IgE i.v. 24 hrs
TNP-OVA i.d.
DT treatment in BasoDTR mice almost completely abolished developement of IgE-CAI
IgE-CAI in EoDTR
•
•
DT added -2days before Ag
DT treatment abolished the IgE-CAI
in EoDTR mice
•
Eosinophils are involved in ear
swelling exacerbation rather than
induction of inflammation
Pros and cons
+ efficient systemic depletion
+ first inducible depletion of eosinophils
- No convincing evidence about CD203c expression in mice basophils and not in other
cell types
- What happens to other cells is not discused (Table 1)
- Only one time poind measured
- Significance for chronic models (IgE-CAI?)
- High concentrations of DT affects also other cells
Aims:
• Generate mast cell and basophil deficient mouse model
Background:
– Based on finding that Myeloid cell leukemia sequence-1 (Mcl-1) is
shown to be an intracellular anti-apoptotic factor
–
Increased apoptosis of cells that do express Cpa3
• Cpa3 is highly expressed in mast
cells
• Efficient mast cell depletion in all
tested tissues, except for spleen
• Cpa3 is expressed in
subpopulation of
basophils
• Efficient basophil
depletion in tested
tissues (56-78%)
• Cpa3 is expressed in
neutrophils, eosinophils,
T cells,b cells
• Other cells type
numbers not affected,
except for RBC
• PCA: Passive cutaneous anaphylaxis,
Mast cell-dependent reaction
PCA (mast cells)
• CAI: Chronic allergic inflammation,
basophil-dependent reaction
(contribution of mast cells ignored
here?)
CAI (basophils)
Pros and cons
+ efficient depletion of mast cells
+ constitutive depletion (e.g. chronic models)
- Quite inefficient depletion of basophils; 22-42% of basophils are still present;
what happens in a more subtle model in which basophils are involved?
- Not a good model for investigating basophils; mast cells are depleted
- No depletion of mast cells in spleen
Aims:
• To asses the role of basophils in acquired tick resistance
Background:
– Basophil depleting antibodies used (Ba103, anti-CD200R3)
– Selective inducible ablation of basophils – Mctp8DTR mice
– hDTR expressed under the control of basophil speciphic promoter of
Mcpt8
Acquired resistance to ticks
• Resistance measured
as the time of feeding
• Resistance is mast celldependent
• FceRI and antibody
independent
Recruitment of basophils to tickfeeding sites during the second
infestation
DT-mediated basophil ablation in
Mctp8DTR mice
• A: DT abolished acquired tick resistance in Mctp8DTR mice
• B: Basophils and mast cell numbers in skin 4 days after the initiation of second
infestation
Pros and cons
+ efficient basophil depletion
+ no other cell types appear to be affected
- High concentrations of DT affects also other cells
- Significance for chronic models (IgE-CAI?)
Conclusions
• Selective ablation of basophils showed to be powerful tool in studying their
role in physiological conditions
• Limitations in basophil specific markers
• Limitations in tools for inducing depletion (DT)
• Constitutive depletion might affect also mast cells numbers (Cpa3-Cre)
• Mcpt8-Cre might be a good alternative
Suggestions
• Search for more specific basophil markers?
• Mctp8 is currently the best option
• GFP expression only in basophils is important control (mctp8 promotor
control, different cells from several tissues were checked)
• Best model (Mctp8) is good only for mice, not for human
• Toxic effects of DT, dose-dependent
• Fast recovery of basophils – should be DT administration repeated?
• Hello kitty – only part of the promotor, mediates moderate Cre expression
• Cpa3 KO has different phenotype than Cpa3-Cre
• Side effects of Cre expression in other tissues?
• Overexpression of basophil population is missing in the literature

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