Rapid development of myeloproliferative neoplasm in mice with Ptpn11D61Y mutation and haploinsufficient for Dnmt3a

PTPN11 gain-of-function mutation is the most common mutation found in patients with juvenile myelomonocytic leukemia and DNMT3A loss occurs in over 20% of acute myeloid leukemia patients. We studied the combined effect of both Ptpn11 gain-of-function mutation (D61Y) and Dnmt3a haploinsufficiency on mouse hematopoiesis, the presence of which has been described in both juvenile myelomonocytic leukemia and acute myeloid leukemia patients. Double mutant mice rapidly become moribund relative to any of the other genotypes, which is associated with enlargement of the spleen and an increase in white blood cell counts. An increase in the mature myeloid cell compartment as reflected by the presence of Gr1+Mac1+ cells was also observed in double mutant mice relative to any other group. Consistent with these observations, a significant increase in the absolute number of granulocyte macrophage progenitors (GMPs) was seen in double mutant mice. A decrease in the lymphoid compartment including both T and B cells was noted in the double mutant mice. Another significant difference was the presence of extramedullary erythropoiesis with increased erythroid progenitors in the spleens of Dnmt3a+/-;D61Y mice relative to other groups. Taken together, our results suggest that the combined haploinsufficiency of Dnmt3a and presence of an activated Shp2 changes the composition of multiple hematopoietic lineages in mice relative to the individual heterozygosity of these genes

Protein-tyrosine Phosphatase Shp2 Positively Regulates Macrophage Oxidative Burst

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores

Hematological parameters of FTL-Tg mice treated with iron dextran (Fe) (n = 13) compared to age-matched FTL-Tg untreated (Control (no Fe)) (n = 10).

<p>DFP-treated FTL-Tg mice at a low dose (DFP₅₀) (n = 11) or a high dose (DFP₁₀₀) (n = 14) were compared to age-matched FTL-Tg untreated (Control (no DFP)) (<i>n =</i> 11). The following hematological parameters were measured: RBC, red blood cells number (x 10⁶/ml); WBC, white blood cells (x 10³/ml); Hb, hemoglobin (g/dl); HtC, hematocrit (%), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), Platelet (Pt), and mean platelet volume (MPV). Significant differences compared to controls (p < 0.05) are indicated by *. Values are mean ± SEM.</p

Serum iron levels and UIBC levels in iron-treated mice.

<p>Serum iron levels were significantly (p < 0.01) increased in iron treated mice (145.9 ± 10.6 μg/dl) compared to untreated mice (104.3 ± 2.2 μg/dl) (A). A decrease of UIBC levels in iron treated mice was observed but did not reach statistical significance (p = 0.6055) (B). For serum studies, FTL-Tg untreated (control; n = 10, 5 males and 5 females) and treated (n = 12, 6 males and 6 females) mice were analyzed. Samples were analyzed by a two-tailed t-test and results considered significant for p < 0.05.</p

Ferritin and iron deposition in the kidney and spleen of DFP-treated mice.

<p>Histological and immunohistochemical studies of DFP-treated and control FTL-Tg mice (A-F). Analysis of paraffin embedded sections from control (A, D), DFP₅₀ (B, E), and DFP₁₀₀ (C, F) treated FTL-Tg mice. Sections shown are from kidney (A-C) and spleen (D-F). Sections were immunostained with Abs against the mutant L chain (A-C) or stained with the Perls’ Prussian blue method (D-F). Original magnifications x10 (D-F), x40 (A-C). Western blot analysis of protein samples from the kidney using antibodies specific for the L, Lm, and H chains (B). β-actin was used as loading control. Representative blots are shown for five control, three DFP₅₀, and four DFP₁₀₀ male mice. Densitometric analysis from three independent experiments shows statistical significant differences between the controls and DFP-treated mice in the supernatant (G) and the pellet (H). (*p < 0.05). By the colorimetric ferrozine method, a decrease in the levels of non-heme iron in the kidney of DFP-treated FTL-Tg mice compared with non-treated FTL-Tg controls was observed in the supernatant (DFP₅₀, p = 0.0190; DFP₁₀₀, p = 0.0299) (I) and the pellet (DFP₁₀₀, p = 0.0032) (J).</p

Western blot analysis and non-heme iron of liver of iron-loaded and control FTL-Tg mice.

<p>The levels of wild type L and H polypeptides in the liver in the supernatant (A) and the pellet (B) were determined by western blot. β-actin was used as loading control. The vertical lines in the panels denote non-adjacent bands from the same blot. Representative blots are shown for four male mice on each group. Densitometric analysis from three independent experiments shows a statistical significant difference between the controls and iron-loaded mice (*p < 0.05). By the colorimetric ferrozine method, a significant increase in the levels of non-heme iron in the liver of iron-treated FTL-Tg mice compared with non-treated FTL-Tg controls was observed in the supernatant (p < 0.0001) (C) and the pellet (p = 0.0004) (D).</p

Histological and immunohistochemical studies of iron-loaded and control FTL-Tg mice.

<p>Analysis of paraffin embedded sections from FTL-Tg control (A, C, E, G, I, K, M) and iron-loaded (B, D, F, H, J, L, N) mice. Sections shown are from kidney (A, B), adipose tissue (C, D), spleen (E-H), and liver (I-N). Sections were immunostained with Abs against the mutant L chain (A-D, I, J) and against the H chain (E, F, K, L), and stained with the Perls’ Prussian blue method (G, H, M, N). Original magnifications x10 (G, H), x20 (E), x40 (A-D, F, H-N).</p