ALBERT

Description: Langmuir DOI: 10.1021/la301347t

Description: Author Correction: Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma Author Correction: Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma, Published online: 20 September 2018; doi:10.1038/s41467-018-06428-2 Author Correction: Commensal microflora-induced T cell responses mediate progressive neurodegeneration in glaucoma

Description: Thrombopoietin (TPO) is essential for normal megakaryopoiesis, and mice and humans lacking the TPO receptor c-Mpl have significantly impaired platelet production. However, in the c-Mpl-null mouse model platelet counts, while reduced to ~10% of normal, are not zero, suggesting that another cytokine is able to support some degree of residual thrombopoiesis. We and others have reported that elimination or severe reduction of stem cell factor, G-CSF, IL-3, IL-6 or IL-11 does not eliminate residual thrombopoiesis. Because megakaryocytes (MKs) and erythrocytes are derived from a common progenitor, we asked if erythropoietin (EPO) can stimulate thrombopoiesis in c-Mpl-null mice. We administered 90 u recombinant EPO or vehicle by subcutaneous injection every 3 days to c-Mpl-null or WT control mice and measured baseline and weekly platelet counts. In three independent experiments, at 2 weeks platelet counts in c-Mpl-null mice receiving EPO were significantly higher that at baseline (5–7 mice per group, average of mean platelet counts 425,000/mm3 vs. 285,000/mm3, p=0.0015). There was a trend towards higher platelet counts in WT mice receiving EPO but this did not reach statistical significance. No difference in platelet counts was observed in mice injected with vehicle. In one experiment c-Mpl-null or WT mice were injected with EPO for 4 weeks and the platelet response in the c-Mpl-null animals was sustained for the duration of the experiment. Western blotting showed that murine MKs express the EPO receptor. To determine if EPO stimulates MK production directly we stimulated WT murine MKs in vitro with either 6 u/ml EPO, 100 ng/ml rhTPO or both and monitored activation of ERK and STAT5 signaling by immunoblotting. Stimulation of MKs with EPO resulted in phosphorylation of ERK and STAT5 (15- and 14-fold above baseline, respectively), compared to TPO (97- and 75-fold above baseline). Stimulation with EPO and TPO together had an additive effect (phospho-ERK increased 121-fold and phospho-STAT5 increased 100-fold). To determine if EPO acts primarily on early or late MKs, we harvested bone marrow from c-Mpl-null mice after 2 weeks of treatment with EPO or vehicle and measured CFU-MK frequency and MK ploidy. Although there was a small increase in the frequency of CFU-MK in mice treated with EPO compared to vehicle, these differences were not significant (n=3, p=0.7), possibly due to the difficulty in assaying CFU-MK in vitro without TPO. In addition, EPO did not significantly enhance MK ploidy in c-Mpl-null mice, although MKs in the 32N and greater peaks were slightly more numerous. Therefore, we conclude that EPO can augment platelet production in the absence of c-Mpl signaling, although it is not yet clear if EPO primarily acts on early or late cells. Additional experiments are underway to determine if ablating EPO receptors in a TPO-null mouse model will eliminate residual thrombopoiesis. These findings may have clinical relevance for treating patients with congenital amegakaryocytic thrombocytopenia and other causes of thrombocytopenia in which c-Mpl signaling is impaired.

Description: Abstract 1737 Myeloproliferative disorders (MPDs) are a heterogeneous group of bone marrow disorders characterized by increases in one or more blood cell lineages. A single, somatic mutation in JAK2 (V617F) is responsible for many of the features of Philadelphia chromosome-negative MPDs (Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis). Clinically, the most common cause of death in these patients is arterial thrombosis; however some patients display a bleeding diathesis. Although the incidence and potential causes of dysfunctional hemostasis in patients with MPDs has been studied extensively, the critical regulating factors are unclear and therefore it has been difficult to develop an effective therapeutic regimen for these complications. As the formation of stable thrombi requires interactions between endothelial cells, platelets and leukocytes, we have recently generated mice that express human JAK2V617F in each of these cell lineages by crossing a JAK2V617F/Flip-Flop (FF1) mouse with mice expressing lineage-specific Cre recombinases. These crosses have generated the following mice; 1) Tie2-Cre/FF1, where JAK2V617F is expressed in all hematopoietic and endothelial cells, 2) Pf4-Cre/FF1, where JAK2V617F expression is limited to platelets, and 3) LysM-Cre/FF1, where JAK2V617F expression is limited to leukocytes. Expression of human JAK2V617F was confirmed in megakaryocytes, platelets, leukocytes and endothelial cells (Tie2-Cre/FF1), megakaryocytes and platelets (Pf4-Cre/FF1) and leukocytes (LysM-Cre/FF1) by conventional and real-time PCR. Of the 3 mouse strains, only Tie2-Cre/FF1 exhibited a MPD phenotype. Platelet counts were significantly increased compared to Tie2-Cre controls (at 3 months, Tie2-Cre: 779 (±61)/ml;Tie2-Cre/FF1: 2943 (± 217)/ml) without significant increases in any other cells types. Tie2-Cre/FF1 mice also exhibit greatly increased number of CFU-MKs and bone marrow derived megakaryocytes. Therefore, Tie2-Cre/FF1 mouse exhibits an ET-like phenotype. Although circulating platelet counts did not increase in Pf4-Cre/FF1 mice, we did observe an increase in the number of CFU-MKs in colony assays. Next we determined the roles of the lineage-restricted JAK2V617F expression on hemostasis in vitro and in vivo. Aggregometry on washed platelets showed no significant difference between any group and their controls in response to PAR4 (100–400mM), ADP (2–20mM) or collagen (1–10mg/ml). Additionally, we were unable to show a significant difference in GPIIbIIIa activation or surface expression of P-selectin in response to the same agonists. Despite no clear platelet abnormalities in any of the 3 mouse lineages, we identified significant hemostatic abnormalities in vivo in Tie2-Cre/FF1 mice. Tail bleeding time was significantly increased in Tie2-Cre/FF1 mice compared to Tie2-Cre controls (Tie2-Cre average, 2min 47secs; Tie2-Cre/FF1, 6mins 37secs) while Tie2-Cre/FF1 mice also exhibited an increased occurrence of re-bleeding compared to Tie2-Cre controls. Additionally, we performed FeCl3 carotid artery occlusion assays to better determine in vivo thrombosis. We found that at 10% FeCl3, Tie2-Cre control mice exhibited complete artery occlusion in approximate 6 min. In contrast, Tie2-Cre/FF1 mice failed to show any sign of arterial occlusion throughout the duration of the experiment (30 min). Given the significant increase in platelet numbers in Tie2-Cre/FF1 mice, we next determined if acquired von Willibrand Disease (VWD) could account for prolonged bleeding and reduced clotting; plasma vW Factor levels by ELISA were normal. In contrast to Tie2-Cre/FF1 mice, neither the PF4-Cre/FF1 or LysM-Cre/FF1 mice exhibit dysfunctional thrombosis. These data provide compelling evidence that expression of JAK2V617F in cells other than just platelets or just leukocytes is necessary to generate the hemostatic abnormalities seen in patients with MPDs. Recent findings show that some patients express endothelial JAK2V617F and patients with ET exhibit increased numbers of circulating endothelial progenitors. Thus, our data is consistent with the hypothesis that expression of JAK2V617F in endothelial cells, in addition to hematopoietic cells results in the bleeding diathesis seen in patients with MPDs. Disclosures: No relevant conflicts of interest to declare.