A novel role for the inflammasome in the regulation of hematopoiesis

Abstract

The inflammasomes are molecular structures localized in the cytosol which activate the immune response. Although several key components of the inflammasome have already been characterized, the mechanism that establishes a relationship between inflammasome and hematopoiesis is still unknown. We have previously demonstrated that the inflammasome plays an important role in erythropoiesis through the direct cleavage of the master erythroid transcription factor GATA1 by its main effector caspase-1 (CASP1). Fine tuning of GATA1 protein level by the inflammasome determines both erythroid-myeloid decision of hematopietic stem and progenitor cells (HSPCs) and terminal erythroid differentiation. This mechanism is conserved in zebrafish, mouse, and human. Using the unique advantages of the zebrafish model for genetic and pharmacological screening in combination with biochemical and functional studies in human erythroleukemic K562 cells, we report here that pharmacological inhibition of caspase-1 rescued the hematopoietic alterations of zebrafish models of neutrophilic inflammation and Diamond-Blackfan anemia (DBA). In addition, we have identified that the NLRP1 inflammasome is the one responsible for the regulation hematopoiesis via the regulation of erythroid-myeloid lineage decision of HSPCs. Thus, inhibition of Nlrp1 in zebrafish resulted in a reduced number of neutrophils and macrophages, and a concomitant increased erythrocyte count, while the emergency of HSPCs was unaffected. Furthermore, inhibition of the Nlrp1 inflammasome also alleviated neutrophilia in a zebrafish model of neutrophilic inflammation. The crosstalk between inflammasome and hematopoiesis has clinical implications, since chronic inflammatory diseases are usually associated to hematopoietic lineage bias, including neutrophilia and anemia. For this reason, we have performed an FDA/EMA-approved compound screening to identify NLRP1 inhibitors. We have found that spironolactone specifically inhibited NLRP1 inflammasome in HEK293T cells and, more importantly, it facilitated erythroid differentiation of K562 cells and human primary HSPCs, and phenocopied the effects of genetic inhibition of Caspa (caspase a, the functional analogue of mammalian caspase-1) and Nlrp1 in zebrafish. Furthermore, it is important to highlight that spironolactone robustly alleviated the impaired erythropoiesis of HSPCs from a DBA patient with the common mutation in RSP19 and, strikingly, without affecting myelopoiesis. Since spironolactone is an FDA/EMA-approved drug, it is attractive for repurposing in the treatment of DBA. Another important issue that needs to be clarified is the mechanism of action of spironolactone, since its analogue eplerenone failed to inhibit the NLRP1 inflammasome and to regulate hematopoiesis. Consequently, it is crucial to know if the structural differences between these two compounds determine their different activity on NLRP1 inflammasome. Finally, we report an additional level of regulation within this crosstalk, involving a GATA switch (GATA1 displaces GATA2) that occurs at a primate conserved sequence located at the intron 7 of CASP1 gene upon induction of erythroid differentiation. Deletion of the binding sites of GATA present in this sequence by CRISPR-Cas9 technology in human erythroleukemic K562 cells facilitates terminal erythroid differentiation. Luciferase reporter assays revealed that this sequence showed promoter activity, which was negatively regulated by GATA1, and drove transcription of a short transcript in both K562 and CD34+ HSPCs encoding a 6 kDa polypeptide, which was stabilized upon erythroid differentiation. Therefore, additional experiments must be performed to illuminate the mechanism of action of the transcript and whether it encodes a protein or rather acts as a non-coding RNA.