Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse ...human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.
NADPH oxidases (NOX) have many biological roles, but their regulation to control production of potentially toxic ROS molecules remains unclear. A previously identified insertion sequence of 21 ...residues (called NIS) influences NOX activity, and its predicted flexibility makes it a good candidate for providing a dynamic switch controlling the NOX active site. We constructed NOX2 chimeras in which NIS had been deleted or exchanged with those from other NOXs (NIS1, 3 and 4). All contained functional heme and were expressed normally at the plasma membrane of differentiated PLB-985 cells. However, NOX2-ΔNIS and NOX2-NIS1 had neither NADPH-oxidase nor reductase activity and exhibited abnormal translocation of p47phox and p67phox to the phagosomal membrane. This suggested a functional role of NIS. Interestingly after activation, NOX2-NIS3 cells exhibited superoxide overproduction compared with wild-type cells. Paradoxically, the Vmax of purified unstimulated NOX2-NIS3 was only one-third of that of WT-NOX2. We therefore hypothesized that post-translational events regulate NOX2 activity and differ between NOX2-NIS3 and WT-NOX2. We demonstrated that Ser486, a phosphorylation target of ataxia telangiectasia mutated kinase (ATM kinase) located in the NIS of NOX2 (NOX2-NIS), was phosphorylated in purified cytochrome b558 after stimulation with phorbol 12-myristate-13-acetate (PMA). Moreover, ATM kinase inhibition and a NOX2 Ser486Ala mutation enhanced NOX activity whereas a Ser486Glu mutation inhibited it. Thus, the absence of Ser486 in NIS3 could explain the superoxide overproduction in the NOX2-NIS3 mutant. These results suggest that PMA-stimulated NOX2-NIS phosphorylation by ATM kinase causes a dynamic switch that deactivates NOX2 activity. We hypothesize that this downregulation is defective in NOX2-NIS3 mutant because of the absence of Ser486.
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•NIS sequence is a flexible structure supporting a functional role in NOX enzymes.•NIS sequence of NOX2 regulates its NADPH oxidase activity in phagocytes.•ATM kinase is activated by ROS production in phagocytes after PMA stimulation.•Ser486 phosphorylation in NIS-NOX2 by ATM inhibits the NADPH oxidase activity.
The molecular mechanisms that underlie T-cell quiescence are poorly understood. In the present study, we report a primary immunodeficiency phenotype associated with MST1 deficiency and primarily ...characterized by a progressive loss of naive T cells. The in vivo consequences include recurrent bacterial and viral infections and autoimmune manifestations. MST1-deficient T cells poorly expressed the transcription factor FOXO1, the IL-7 receptor, and BCL2. Conversely, FAS expression and the FAS-mediating apoptotic pathway were up-regulated. These abnormalities suggest that increased cell death of naive and proliferating T cells is the main mechanism underlying this novel immunodeficiency. Our results characterize a new mechanism in primary T-cell immunodeficiencies and highlight a role of the MST1/FOXO1 pathway in controlling the death of human naive T cells.
Inherited defects of granule-dependent cytotoxicity led to the life-threatening immune disorder hemophagocytic lymphohistiocytosis (HLH), characterized by uncontrolled CD8 T-cell and macrophage ...activation. In a cohort of HLH patients with genetic abnormalities expected to result in the complete absence of perforin, Rab27a, or syntaxin-11, we found that disease severity as determined by age at HLH onset differed significantly, with a severity gradient from perforin (early onset) > Rab27a > syntaxin-11 (late onset). In parallel, we have generated a syntaxin-11–deficient (Stx11−/−) murine model that faithfully reproduced the manifestations of HLH after lymphocytic choriomeningitis virus (LCMV) infection. Stx11−/− murine lymphocytes exhibited a degranulation defect that could be rescued by expression of human syntaxin-11 but not expression of a C-terminal–truncated mutant. Comparison of the characteristics of LCMV infection-induced HLH in the murine counterparts of the 3 human conditions revealed a similar gradient in the phenotypic severity of HLH manifestations. Strikingly, the severity of HLH was not correlated with the LCMV load and not fully with differences in the intensity of cytotoxic activity. The capacity of antigen presentation differed in vivo between Rab27a- and Syntaxin-11–deficient mutants. Our data indicate that cytotoxic effectors may have other immune-regulatory roles in addition to their role in controlling viral replication.
•Syntaxin-11–deficient (Stx11−/−) murine model faithfully reproduced the manifestations of HLH after LCMV infection.•HLH severity differed significantly with a severity gradient from perforin (early onset) Rab27a syntaxin-11 (late onset).
DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also ...involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature ("FILS syndrome") in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients' T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder.
JAK2 inhibition therapy is used to treat patients suffering from myeloproliferative neoplasms (MPN). Conflicting data on this therapy are reported possibly linked to the types of inhibitors or ...disease type. Therefore, we decided to compare in mice the effect of a JAK2 inhibitor, Fedratinib, in MPN models of increasing severity: polycythemia vera (PV), post‐PV myelofibrosis (PPMF) and rapid post‐essential thrombocythemia MF (PTMF). The models were generated through JAK2 activation by the JAK2V617F mutation or MPL constant stimulation. JAK2 inhibition induced a correction of splenomegaly, leucocytosis and microcytosis in all three MPN models. However, the effects on fibrosis, osteosclerosis, granulocytosis, erythropoiesis or platelet counts varied according to the disease severity stage. Strikingly, complete blockade of fibrosis and osteosclerosis was observed in the PPMF model, linked to correction of MK hyper/dysplasia, but not in the PTMF model, suggesting that MF development may also become JAK2‐independent. Interestingly, we originally found a decreased in the JAK2V617F allele burden in progenitor cells from the spleen but not in other cell types. Overall, this study shows that JAK2 inhibition has different effects according to disease phenotypes and can (i) normalize platelet counts, (ii) prevent the development of marrow fibrosis/osteosclerosis at an early stage and (iii) reduce splenomegaly through blockage of stem cell mobilization in the spleen.
GS-0387 (momelotinib, formerly CYT387) is a type I JAK1/2 inhibitor. Phase 1/2 trial results in primary myelofibrosis (PMF) or post-polycythemia vera/essential thrombocythemia MF patients showed that ...GS-0387 was well tolerated and reduced splenomegaly and general symptoms. More interestingly, GS-0387 relieved anemia in 59% of patients, with 70% of patients requiring red blood cell transfusion becoming transfusion independent for a minimum of 12 weeks.1 Therefore, the purpose of this study was to verify if this benefit on anemia response could be transposed to a mouse model of human primary myelofibrosis (PMF).
For this purpose we used the TPOHIGH retroviral (RV) model that mimics the development of human PMF including its progressing anemia. In this model, hyperactivation of MPL, as observed in human PMF through gain-of-function mutations, is caused by high levels of circulating thrombopoietin (TPO) through an adoptive transfer of TPO retrovirally transduced bone marrow (BM) cells in C57/Bl6 recipient mice. After transplantation, mice developed a 2 stepped disease with a myeloproliferative phase characterized by high thrombocythemia, leukocytosis and anemia (hemoglobin ≈ 12 g/dL, hematocrit ≈35 %; normal levels: 16 g/dL and 45 %, respectively) and a marrow insufficiency phase characterized by thrombocytopenia, leukopenia and severe anemia (hemoglobin ≈ 8 g/dL, hematocrit level ≈22%) caused by high grade fibrosis and osteosclerosis. GS-0387 was administered as a single daily oral dose (SID) during the myeloproliferative phase in two studies with 47 and 68 days of treatment and during the marrow insufficiency phase with 101 days of treatment.
GS-0387 was well tolerated at ca 80mpk with an MTD of approximately 100mpk. GS-0387 was first tested during the myeloproliferative phase, 5 weeks post transplantation of the TPO transduced BM cells. A significant improvement in anemia was observed. Starting 20 weeks after treatment, hemoglobin concentration in GS-0387-treated mice remained higher (8.5±0.4 g/dL, n=7) than in vehicle-treated mice (6.8 ±0.3 g/dL, n=7) for at least 35 days of treatment (p<0.01). Treatment with GS-0387 commencing during the marrow insufficiency phase (10 weeks post transplantation), did not significantly improve anemia, possibly due to the dramatic picture of the disease in terminal stage.
Improvement of anemia during the myeloproliferative phase was associated with an increase over 35 days of treatment in:
- Hematocrit levels (23±1% vs 27±1%, vehicle vs GS-0387)
- Red blood cell numbers (5.5±0.2 vs 5.7±0.3 x109/mL, vehicle vs GS-0387)
- Mean corpuscular volume (MCV), from microcytosis to normal levels (44.2±0.2 to 47.75 fL, vehicle vs GS-0387)
An increase in erythroblast and early erythroid progenitor cell numbers was also observed in the spleen of GS-0387-treated mice compared to vehicle-treated mice. None of these increases, except for MCV, were observed during the 90 day treatment in the marrow insufficiency phase, in which no anemia response was observed.
Besides improving anemia, GS-0387 prevented the development of leukocytosis and thrombocytosis and reduced splenomegaly associated with the disease without deleterious effects on hematopoiesis at efficacious doses.
GS-0387 was tested in a murine model of human PMF. It reduced the anemia associated with the development of myelofibrosis, as reported during clinical trials of patients suffering from MF. Furthermore, GS-0387, as expected from a JAK1/2 inhibitor reduced splenomegaly, thrombocytosis and leukocytosis associated with the disease. Improvement of anemia was associated with an increase in RBC, erythroid precursor and progenitor cells suggesting that GS-0387 stimulates the early stages of erythropoiesis. Further experiments are in progress to investigate whether changes in levels of cytokines known to positively or negatively effect erythropoiesis are seen. Regulation of iron metabolism, which could account for the improvement of anemia induced by GS-0387 is also being investigated.
1. Pardanani A, Laborde RR, Lasho TL, et al. Safety and efficacy of CYT387, a JAK1 and JAK2 inhibitor, in myelofibrosis. Leukemia. 2013;27(6):1322-1327.
Villeval:YMBioSciences: Research Funding. Off Label Use: JAK1/2 Inhibitor GS-0387 (momelotinib) / Human Primary Myelofibrosis. Burns:YMBioSciences: Employment; Gilead: Consultancy. Smith:YMBioSciences: Employment; Gilead: Consultancy.
Inherited defects of granule-dependent cytotoxicity led to the life-threatening immune disorder hemophagocytic lymphohistiocytosis (HLH), characterized by uncontrolled CD8 T-cell and macrophage ...activation. In a cohort of HLH patients with genetic abnormalities expected to result in the complete absence of perforin, Rab27a, or syntaxin-11, we found that disease severity as determined by age at HLH onset differed significantly, with a severity gradient from perforin (early onset) > Rab27a > syntaxin-11 (late onset). In parallel, we have generated a syntaxin-11–deficient (
Stx11
−/−
) murine model that faithfully reproduced the manifestations of HLH after lymphocytic choriomeningitis virus (LCMV) infection.
Stx11
−/−
murine lymphocytes exhibited a degranulation defect that could be rescued by expression of human syntaxin-11 but not expression of a C-terminal–truncated mutant. Comparison of the characteristics of LCMV infection-induced HLH in the murine counterparts of the 3 human conditions revealed a similar gradient in the phenotypic severity of HLH manifestations. Strikingly, the severity of HLH was not correlated with the LCMV load and not fully with differences in the intensity of cytotoxic activity. The capacity of antigen presentation differed in vivo between Rab27a- and Syntaxin-11–deficient mutants. Our data indicate that cytotoxic effectors may have other immune-regulatory roles in addition to their role in controlling viral replication.
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