Impact of the inflammasome on hematopoiesis and pyroptosis : pharmacological target discovery and drug validation for the treatment of anemia

Abstract

Chronic diseases and hematopoietic disorders are closely linked to inflammasome dysregulation. In the present PhD thesis, we have used the zebrafish models to elucidate the impact of inflammation, and in particular of the inflammasome and pyroptotic cell death, in hematopoiesis. In the first chapter, we demonstrated that genetic inhibition of the macrophage inflammasome increased macrophage numbers in zebrafish larvae with skin inflammation without affecting erythrocyte or neutrophil counts. Similarly, inhibiting the neutrophil inflammasome resulted in increased neutrophil numbers under inflammatory conditions, without impacting other cell types. Conversely, hyperactivation of the neutrophil inflammasome promoted neutrophil death, which was mitigated by pharmacological inhibition of Gasdermin E (Gsdme), highlighting the myeloid inflammasome's role in pyroptotic cell death via a Gsdme-dependent pathway in zebrafish. In the second chapter, we dilucidated the mechanism of activation of the NLRP1 inflammasome in hematopoietic stem and progenitor cells (HSPCs). Erythroid differentiation of HSPCs activated the ZAKα/P38 kinase axis, promoting NLRP1 phosphorylation, assembly and activation. Inhibition of ZAKα with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in human K562 cells. These findings suggest novel therapeutic avenues for treating hematopoietic disorders associated with chronic inflammation. In the third chapter, the therapeutic potential of ZAKα inhibitors, specifically FDA/EMA- approved tyrosine kinase inhibitors (TKIs), was explored for treating Diamond-Blackfan anemia (DBA). It as found that nilotinib promoted erythroid differentiation in K562 cells by enhancing GATA1 accumulation and inhibiting the ZAKα/P38/NLRP1/CASP1 axis. These effects were confirmed in human CD34+ HSPCs and zebrafish models, where Nilotinib and other TKIs increased erythropoiesis at the expense of myelopoiesis and reduced caspase-1 activity. Furthermore, TKIs also alleviated defective erythroid differentiation in a human RPS19-edited model of DBA and HSPCs from DBA patients. Therefore, the repurposing of TKIs presents a promising therapeutic strategy for DBA and potentially other congenital anemias and myeloid lineage disorders. In the fourth chapter, the non-canonical role of Ninjurin-1 (Ninj1) in HSPC formation was investigated in zebrafish larvae. Ninj1-deficient larvae exhibited reduced neutrophils, erythrocytes, and HSPCs, along with decreased caspase-1 activity, suggesting a broader role beyond its known function in plasma membrane rupture. Ninj1 deficiency alleviated neutrophilia and skin inflammation. Conserved residues critical for zebrafish Ninj1 plasma membrane rupture (PMR) activity were identified through mutant analysis. Ninj1 deficiency also impacted the response to bacterial infection, reducing immune cell recruitment and survival, while overexpression of Ninj1A56P, which is devoid of PMR activity, increased larval susceptibility and myeloid cell recruitment. Importantly, we found that Ninj1 and Ninj2 redundantly regulated hematopoiesis via Wnt signaling. Combined deficiency of both ninjurins drastically reduced all blood cell lineages, underscoring their essential role in HSPC emergence from the hemogenic endothelium. Gene expression, Wnt reporter analysis and genetic experiments confirmed Ninjurins' involvement in HSPC development. These findings highlight Ninjurins as key regulators of HSPC emergence, with potential applications in enhancing HSPC production for regenerative medicine. Our studies collectively advance our understanding of the inflammasome and other molecular pathways in hematopoiesis and inflammation, revealing potential therapeutic strategies for treating hematopoietic disorders.