Chronic inflammatory diseases or long-term infections are associated to hematopoietic disorders. We have recently identified an evolutionarily conserved signaling pathways in hematopoietic stem and progenitor cells (HSPC) that mediates the cleavage of the master erythroid transcription factor GATA1 by the canonical inflammasome, a cytosolic molecular platform which is activated upon inflammatory insults. This pathway promotes myelopoiesis at the expenses of erythropoiesis and, therefore, is critically involved in the hematopoietic lineage bias associated to chronic inflammatory diseases.

Zebrafish model: Genetic inactivation of Nlrx, Inter1 and Inter2 was achieved by CRISPR/Cas9 technology. gRNA/Cas9 complexes for zebrafish injection were prepared with 500 ng/ml EnGen Cas9 NLS from Streptococcus pyogenes and 100 ng/ml tracrRNA with negative control, Nlrx, Interactor 1 or Interactor 2 crRNA in 300 mM KCl buffer, incubated for 5 min at 95º and used directly without further storage. Mobilized CD34+ from healthy donor culture: hNLRX was knockdown in human mobilized CD34+ progenitor cells from healthy donors by nucleoporation of Cas9/gRNA ribonucleoprotein complexes. Cells were then cultured in methylcellulose medium with appropriate recombinant cytokines for two weeks. Cell culture of K562 and HEK293T: Transcript levels of NLRX, INTERACTOR 2 and INTERACTOR 3 were determined by RT-qPCR in hemin-stimulated K562 cells. HEK293T cells were transfected with ASC-GFP, NLRX, Inter1 and Inter2 expression constructs using lipofectamine 2000 to induce NLRX-dependent ASC speck formation.. Protein levels were detected by western blot.

Here we demonstrate that the most studied and promiscuous sensor of the inflammasome, the NLRP3, was not involved in this pathway. A novel sensor, that we called NLRX, was dramatically upregulated in human K562 upon erythroid differentiation. Genetic inhibition of NLRX promoted erythropoiesis in both zebrafish and human CD34+ HSPC. Mass spectrometry assays in K562 cells revealed two interactors that acted as negative regulators of NLRX upon reconstitution of this inflammasome in human HEK293T. Genetic experiments in zebrafish showed that both interactors regulated hematopoiesis through the inhibition of the Nlrx inflammasome in HSPC.

Collectively, our results shed light into the complex regulation of hematopoiesis and identify novel targets for therapeutic interventions in hematopoietic disorders associated to chronic inflammation.