Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice

There is an unmet need for novel therapies to treat acute myeloid leukemia (AML) and the associated relapse that involves persistent leukemia stem cells ...

The Pennsylvania State University

Prostaglandin D and its cyclopentenone metabolites [cyclopentenone prostaglandins (CyPGs)], Δprostaglandin J and 15-deoxy-Δ-prostaglandin J, act through 2 GPCRs, d-type prostanoid 1 and the chemoattractant receptor homologous molecule expressed on type 2 T-helper cells (Crth2). In addition to its role in allergy and asthma, the role of Crth2 in the resolution of inflammation, to mediate the proresolving functions of endogenous CyPGs, is not well understood. We investigated the regulation of LPS or zymosan-induced inflammatory response by signals from the Crth2 receptor in macrophages that lack Crth2 expression [knockout (KO)]. Increased expression of proinflammatory genes, including -α, was observed in Crth2 KO cells. Targeting the endogenous biosynthetic pathway of CyPGs with indomethacin or HQL79, which inhibit cyclooxygenases or hematopoietic prostaglandin D synthase, respectively, or use of Crth2 antagonists recapitulated the proinflammatory phenotype as in Crth2 KO cells. Ligand-dependent activation of Crth2 by 13,14-dihydro-15-keto-prostaglandin D increased Ca influx through store-operated Ca entry (SOCE) accompanied by the up-regulation of stromal interaction molecule 1 and calcium release-activated calcium modulator 1 expression, suggesting that the proresolution effects of CyPG-dependent activation of SOCE could be mediated by Crth2 during inflammation. Interestingly, Crth2 signaling down-regulated the Ca-regulated heat stable protein 1 that stabilizes Tnf-α mRNA  the increased expression of microRNA 155 to dampen inflammatory responses triggered through the TNF-α-NF-κB axis. In summary, these studies present a novel regulatory role for Crth2 during inflammatory response in macrophages.-Diwakar, B. T., Yoast, R., Nettleford, S., Qian, F., Lee, T.-J., Berry, S., Huffnagle, I., Rossi, R. M., Trebak, M., Paulson, R. F., Prabhu, K. S. Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages  changes in intracellular calcium.

Crth2 receptor signaling down-regulates lipopolysaccharide-induced NF-κB activation in murine macrophages  changes in intracellular calcium.

Selenium (Se) is an essential trace element that functions in the form of the 21st amino acid, selenocysteine (Sec) in a defined set of proteins. Se deficiency is associated with pathological conditions in humans and animals, where incorporation of Sec into selenoproteins is reduced along with their expression and catalytic activity. Supplementation of Se-deficient population with Se has shown health benefits suggesting the importance of Se in physiology. An interesting paradigm to explain, in part, the health benefits of Se stems from the observations that selenoprotein-dependent modulation of inflammation and efficient resolution of inflammation relies on mechanisms involving a group of bioactive lipid mediators, prostanoids, which orchestrate a concerted action toward maintenance and restoration of homeostatic immune responses. Such an effect involves the interaction of various immune cells with these lipid mediators where cellular redox gatekeeper functions of selenoproteins further aid in not only dampening inflammation, but also initiating an effective and active resolution process. Here we have summarized the current literature on the multifaceted roles of Se/selenoproteins in the regulation of these bioactive lipid mediators and their immunomodulatory effects.

Selenium and selenoproteins in prostanoid metabolism and immunity.

Anemic stress induces stress erythropoiesis, which rapidly generates new erythrocytes to restore tissue oxygenation. Stress erythropoiesis is best understood in mice where it is extramedullary and occurs primarily in the spleen. However, both human and mouse stress erythropoiesis use signals and progenitor cells that are distinct from steady-state erythropoiesis. Immature stress erythroid progenitors (SEPs) are derived from short-term hematopoietic stem cells. Although the SEPs are capable of self-renewal, they are erythroid restricted. Inflammation and anemic stress induce the rapid proliferation of SEPs, but they do not differentiate until serum erythropoietin (Epo) levels increase. Here we show that rather than directly regulating SEPs, Epo promotes this transition from proliferation to differentiation by acting on macrophages in the splenic niche. During the proliferative stage, macrophages produce canonical Wnt ligands that promote proliferation and inhibit differentiation. Epo/Stat5-dependent signaling induces the production of bioactive lipid mediators in macrophages. Increased production of prostaglandin J2 (PGJ2) activates peroxisome proliferator-activated receptor γ (PPARγ)-dependent repression of Wnt expression, whereas increased production of prostaglandin E2 (PGE2) promotes the differentiation of SEPs.

Epo receptor signaling in macrophages alters the splenic niche to promote erythroid differentiation.

Enteropathogenic  (EPEC) leads to adverse colonic inflammation associated with poor resolution of inflammation and loss of epithelial integrity. Micronutrient trace element selenium (Se) is incorporated into selenoproteins as the 21st amino acid, selenocysteine (Sec). Previous studies have shown that such an incorporation of Sec into the selenoproteome is key for the anti-inflammatory functions of Se in macrophages and other immune cells. An intriguing mechanism underlying the anti-inflammatory and pro-resolving effects of Se stems from the ability of selenoproteins to skew arachidonic acid metabolism from pro-inflammatory mediators, prostaglandin E (PGE) toward anti-inflammatory mediators derived from PGD, such as 15-deoxy-Δ- prostaglandin J (15d-PGJ), via eicosanoid class switching of bioactive lipids. The impact of Se and such an eicosanoid-class switching mechanism was tested in an enteric infection model of gut inflammation by , a murine equivalent of EPEC. C57BL/6 mice deficient in Se (Se-D) experienced higher mortality when compared to those on Se adequate (0.08 ppm Se) and Se supplemented (0.4 ppm Se) diets following infection. Decreased survival was associated with decreased group 3 innate lymphoid cells (ILC3s) and T helper 17 (Th17) cells in colonic lamina propria of Se-D mice along with deceased expression of epithelial barrier protein Zo-1. Inhibition of metabolic inactivation of PGE by 15-prostaglandin dehydrogenase blocked the Se-dependent increase in ILC3 and Th17 cells in addition to reducing epithelial barrier integrity, as seen by increased systemic levels of FITC-dextran following oral administration; while 15d-PGJ administration in Se-D mice alleviated the effects by increasing ILC3 and Th17 cells. Mice lacking selenoproteins in monocyte/macrophages via the conditional deletion of the tRNA showed increased mortality post infection. Our studies indicate a crucial role for dietary Se in the protection against inflammation following enteric infection via immune mechanisms involving epithelial barrier integrity.

The Essential Role of Selenoproteins in the Resolution of -Induced Intestinal Inflammation.

Interleukin-4 (IL-4) is a signature cytokine pivotal in Type 2 helper T cell (Th2) immune response, particularly in allergy and hypersensitivity. Interestingly, IL-4 increases endogenous levels of prostaglandin D (PGD ) and its metabolites, Δ -prostaglandin J (Δ -PGJ ) and 15-deoxy-Δ -prostaglandin J (15d-PGJ ), collectively called cyclopentenone PGs (CyPGs). However, the therapeutic role of IL-4 in hematologic malignancies remains unclear. Here, we employed a murine model of acute myeloid leukemia (AML), where human MLL-AF9 fusion oncoprotein was expressed in hematopoietic progenitor cells, to test the effect of IL-4 treatment in vivo. Daily intraperitoneal treatment with IL-4 at 60 µg/kg/d significantly alleviated the severity of AML, as seen by decreased leukemia-initiating cells (LICs). The effect of IL-4 was mediated, in part, by the enhanced expression of hematopoietic- PGD  synthase (H-PGDS) to effect endogenous production of CyPGs, through autocrine and paracrine signaling mechanisms. Similar results were seen with patient-derived AML cells cultured ex vivo with IL-4. Use of GW9662, a peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, suggested endogenous CyPGs-PPARγ axis mediated p53-dependent apoptosis of LICs by IL-4. Taken together, our results reveal a beneficial role of IL-4 treatment in AML suggesting a potential therapeutic regimen worthy of clinical trials in patients with AML.

Fenghua Qian

The Pennsylvania State University

Intra-Peritoneal Transplantation for Generating Acute Myeloid Leukemia in Mice