Models for in vivo and in vitro studies on basophils

1. Models for in vivo and in vitro studies on basophils Trainer: Franco Falcone

2. 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

3. 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!

4. Transgenic models

5. 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)

6. 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

7. 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

8. 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

9. 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

10. Goal: 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

11. Cpa3 is highly expressed in mast cells • Efficient mast cell depletion in all tested tissues, except for spleen

12. 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

13. PCA: Passive cutaneous anaphylaxis, Mast cell-dependent reaction • CAI: Chronic allergic inflammation, basophil-dependent reaction • (contribution of mast cells ignored • here?) PCA (mast cells) CAI (basophils)

14. 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

15. 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

16. Acquired resistance to ticks • Resistance measured as the time of feeding • Resistance is mast cell-dependent • FceRI and antibody independent

17. Recruitment of basophils to tick-feeding sites during the second infestation

18. 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

19. 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?)

20. 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

21. 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