An in-vivo zebrafish model with cecr1b deficiency recapitulates human DADA2 phenotype

Deficiency of adenosine deaminase 2 (DADA2) is a rare and potentially lethal genetic disease caused by loss-of-function mutations in the ADA2 (CECR1) gene. Patients affected by DADA2 display neutropenia and bone marrow (BM) aplasia, immune dysregulation, systemic inflammation, vasculitis leading to vasculopathy and recurrent hemorrhagic strokes. Current therapies, mainly based on anti-TNF agents or allogeneic hematopoietic stem progenitor cell (HSPCs) transplantation, are not always resolutive. Advances in therapeutical treatments are hampered by the poor knowledge of the pathogenetic mechanisms and the lack of an animal model. Indeed, rodents do not have an ADA2 orthologue. Zebrafish (Danio rerio) harbors two ADA2 orthologues, cecr1a and cecr1b, with the latter sharing a conserved function with ADA2 and a specific expression during HSCs differentiation. A cecr1b morpholino-mediated knock-down (KD) model showing neutropenia and intracranial hemorrhages has been described in the literature. Taking advantage of this model, we have characterized in detail the hematopoietic phenotypes resulting from cecr1b downregulation and investigated the cellular mechanisms implicated in DADA2 pathogenesis. We found that, although neutrophils number was significantly reduced in cecr1b-KD embryos, their sensing and migrating capacity towards an inflammatory stimulus were unaltered. Moreover, neutropenia was rescued by systemic administration of granulocyte-colony stimulating factor (G-CSF), which stimulates granulocyte production. The neutropenic condition of cecr1b-KD embryos was the consequence of an early hematopoietic defect. Indeed, cecr1b-KD embryos showed a low number of HSPCs in the caudal hematopoietic tissue, reminiscent of BM aplasia of severe DADA2 cases. Although HSPC survival was not affected, these cells exhibited a reduced proliferation capacity giving rise to cytopenia. As in DADA2 patients, cecr1b-KD also induced constitutive inflammation sustained by the pro-inflammatory cytokines il-1, il-6, tnf, ifn-3. Notably, a successful correction of the hematopoietic and inflammatory phenotypes was achieved by administrating recombinant human ADA2, demonstrating that these defects are a direct cause of cecr1b loss of function. In conclusion, our data provide, for the first time, the mechanistic link between DADA2 and hematological abnormalities and neutropenia observed in patients and provided a valuable in-vivo model to develop new targeted therapies for this lethal disease.