Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd -deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9–TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.

The transcriptional repressor Hairy Enhancer of Split 1 (HES1) plays an essential role in the development of many organs by promoting the maintenance of stem/progenitor cells, controlling the reversibility of cellular quiescence, and regulating both cell fate decisions. Deletion of Hes1 in mice results in severe defects in multiple organs and is lethal in late embryogenesis. Here we have investigated the role of HES1 in hematopoiesis using a hematopoietic lineage-specific Hes1 knockout mouse model.We found that while Hes1 is dispensable for steady-state hematopoiesis, Hes1deficient hematopoietic stem cells (HSCs) undergo exhaustion under replicative stress. Loss of Hes1 upregulates the expression of genes involved in PPARγ signaling and fatty acid metabolism pathways, and augments fatty acid oxidation (FAO) in Hes1 f/f Vav1Cre HSCs and progenitors. Functionally, PPARγ targeting or FAO inhibition ameliorates the repopulating defects of Hes1 f/f Vav1Cre HSCs through improving quiescence in HSCs. Lastly, transcriptome analysis reveals that disruption of Hes1 in hematopoietic lineage alters expression of genes critical to HSC function, PPARγ signaling, and fatty acid metabolism. Together, our findings identify a novel role of HES1 in regulating stress hematopoiesis and provide mechanistic insight into the function of HES1 in HSC maintenance. K E Y W O R D S fatty acid metabolism, hairy enhancer of Split 1, hematopoietic reconstitution capacity, hematopoietic stem progenitor cells, PPARγ signaling pathway, replicative stress

Cyclooxygenase (COX)-dependent production of prostaglandins (PGs) is known to play important roles in tumorigenesis. PGD 2 has recently emerged as a key regulator of tumor- and inflammation-associated functions. Here we show that mesenchymal stromal cells (MSCs) from patients with acute myeloid leukemia (AML) or normal MSCs overexpressing COX2 promote proliferation of co-cultured hematopoietic stem and progenitor cells (HSPCs), which can be prevented by treatment with COX2 knockdown or TM30089, a specific antagonist of the PGD 2 receptor CRTH2. Mechanistically, we demonstrate that PGD 2 -CRTH2 signaling acts directly on type 2 innate lymphoid cells (ILC2s), potentiating their expansion and driving them to produce Interleukin-5 (IL-5) and IL-13. Furthermore, IL-5 but not IL-13 expands CD4 + CD25 + IL5Rα + T regulatory cells (Tregs) and promotes HSPC proliferation. Disruption of the PGD 2 -activated ILC2-Treg axis by specifically blocking the PGD 2 receptor CRTH2 or IL-5 impedes proliferation of normal and malignant HSPCs. Conversely, co-transfer of CD4 + CD25 + IL5Rα + Tregs promotes malignant HSPC proliferation and accelerates leukemia development in xenotransplanted mice. Collectively, these results indicate that the mesenchymal source of PGD 2 promotes proliferation of normal and malignant HSPCs through activation of the ILC2-Treg axis. These findings also suggest that this novel PGD 2 -activated ILC2-Treg axis may be a valuable therapeutic target for cancer and inflammation-associated diseases.