Aerobic exercise training mitigates tumor growth and cancer-induced splenomegaly through modulation of non-platelet platelet factor 4 expression.

Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.

Akkermansia muciniphila induces slow extramedullary hematopoiesis via cooperative IL-1R/TLR signals.

Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the spleen, a process termed extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria regulate not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbes on hematopoietic pathology remains unclear. Here, we find that systemic single injections of Akkermansia muciniphila (A. m.), a mucin-degrading bacterium, rapidly activate BM myelopoiesis and slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we term delayed EMH. Mechanistically, delayed EMH triggered by A. m. is mediated entirely by the MYD88/TRIF innate immune signaling pathway, which persistently stimulates splenic myeloid cells to secrete interleukin (IL)-1α, and in turn, activates IL-1 receptor (IL-1R)-expressing splenic HSPCs. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) or IL-1α partially diminishes A. m.-induced delayed EMH, while inhibition of both pathways alleviates splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Notch1/Hes1­PTEN/AKT/IL­17A feedback loop regulates Th17 cell differentiation in mouse psoriasis­like skin inflammation.

IL­17A, the effector cytokine of T helper (Th) 17 cells, plays a crucial role in the pathogenesis of psoriasis. The Notch1 and PI3K/AKT signaling pathways are implicated in Th17 cell differentiation and IL­17A production. The present study aimed to evaluate the regulatory effect of the Notch1/hairy and enhancer of split 1 (Hes1)­PTEN/AKT/IL­17A feedback loop on Th17 cell differentiation via the PI3K/AKT inhibitor LY294002 in a mouse model of psoriasis. Mice were randomly divided into 3 groups: a control group, a model group [5% imiquimod (IMQ)­induced group] and an intervention group (5% IMQ­induced plus LY294002­treated group). Skin structural characteristics were recorded and evaluated by hematoxylin and eosin staining. The weights of the spleens and inguinal lymph nodes were measured. Th17 cell percentage, as well as the mRNA and protein expression levels of Notch1, Notch1 intracellular domain (NICD1), Hes1, PTEN, AKT, phosphorylated (p)­AKT, mTOR complex 1 (mTORC1), p­mTORC1, S6 kinase (S6K)1, S6K2 and IL­17A were detected in skin samples of the three experimental groups. Additionally, splenic mononuclear cells from model mice were treated by 10 and 50 µM LY294002 to further evaluate its regulatory effect on Notch1/Hes1­PTEN/AKT/IL­17A feedback loop. Increased Th17 cell percentage, increased expression of Notch1, NICD1, Hes1, AKT, p­AKT, mTORC1, p­mTORC1, S6K1, S6K2 and IL­17A, and decreased PTEN levels were observed in model mice alongside marked psoriasis­like skin inflammation, splenomegaly and lymphadenopathy. LY294002 treatment significantly alleviated the severity of psoriasis­like skin inflammation in the intervention mice, attenuated the degree of epidermal hyperplasia and dermal inflammatory cell infiltration, and mitigated splenomegaly and lymphadenopathy. In addition, LY294002 treatment reversed the increased Th17 cell percentage, as well as the increased expression of Notch1, NICD1, Hes1, AKT, p­AKT, mTORC1, p­mTORC1, S6K1, S6K2 and IL­17A, and the decreased expression of PTEN. In vitro study from 5% IMQ­induced mouse splenic mononuclear cells presented that high dose of LY294002 exerted more obviously regulatory effect on Notch1/Hes1­PTEN/AKT/IL­17A feedback loop. The current findings suggested that the Notch1/Hes1­PTEN/AKT/IL­17A feedback loop regulates Th17 cell differentiation within the disease environment of psoriasis. Blocking the Notch1/Hes1­PTEN/AKT/IL­17A feedback loop may thus be a potential therapeutic method for management of psoriatic inflammation.

Oxaliplatin-induced haematological toxicity and splenomegaly in mice.

PURPOSE: Haematological toxicities occur in patients receiving oxaliplatin. Mild anaemia (grade 1-2) is a common side effect and approximately 90% of recipients develop measurable spleen enlargement. Although generally asymptomatic, oxaliplatin-induced splenomegaly is independently associated with complications following liver resection for colorectal liver metastasis and separately with poorer patient outcomes. Here, we investigated oxaliplatin-induced haematological toxicities and splenomegaly in mice treated with escalating dosages comparable to those prescribed to colorectal cancer patients. METHODS: Blood was analysed, and smears assessed using Wright-Giemsa staining. Paw coloration was quantified as a marker of anaemia. Spleen weight and morphology were assessed for abnormalities relating to splenomegaly and a flow cytometry and multiplex cytokine array assessment was performed on splenocytes. The liver was assessed for sinusoidal obstructive syndrome. RESULTS: Blood analysis showed dose dependent decreases in white and red blood cell counts, and significant changes in haematological indices. Front and hind paws exhibited dose dependent and dramatic discoloration indicative of anaemia. Spleen weight was significantly increased indicating splenomegaly, and red pulp tissue exhibited substantial dysplasia. Cytokines and chemokines within the spleen were significantly affected with temporal upregulation of IL-6, IL-1α and G-CSF and downregulation of IL-1ß, IL-12p40, MIP-1ß, IL-2 and RANTES. Flow cytometric analysis demonstrated alterations in splenocyte populations, including a significant reduction in CD45+ cells. Histological staining of the liver showed no evidence of sinusoidal obstructive syndrome but there were signs suggestive of extramedullary haematopoiesis. CONCLUSION: Chronic oxaliplatin treatment dose dependently induced haematological toxicity and splenomegaly characterised by numerous physiological and morphological changes, which occurred independently of sinusoidal obstructive syndrome.

PKN1 kinase-negative knock-in mice develop splenomegaly and leukopenia at advanced age without obvious autoimmune-like phenotypes.

Protein kinase N1 (PKN1) knockout (KO) mice spontaneously form germinal centers (GCs) and develop an autoimmune-like disease with age. Here, we investigated the function of PKN1 kinase activity in vivo using aged mice deficient in kinase activity resulting from the introduction of a point mutation (T778A) in the activation loop of the enzyme. PKN1[T778A] mice reached adulthood without external abnormalities; however, the average spleen size and weight of aged PKN1[T778A] mice increased significantly compared to aged wild type (WT) mice. Histologic examination and Southern blot analyses of spleens showed extramedullary hematopoiesis and/or lymphomagenesis in some cases, although without significantly different incidences between PKN1[T778A] and WT mice. Additionally, flow cytometry revealed increased numbers in B220+, CD3+, Gr1+ and CD193+ leukocytes in the spleen of aged PKN1[T778A] mice, whereas the number of lymphocytes, neutrophils, eosinophils, and monocytes was reduced in the peripheral blood, suggesting an advanced impairment of leukocyte trafficking with age. Moreover, aged PKN1[T778A] mice showed no obvious GC formation nor autoimmune-like phenotypes, such as glomerulonephritis or increased anti-dsDNA antibody titer, in peripheral blood. Our results showing phenotypic differences between aged Pkn1-KO and PKN1[T778A] mice may provide insight into the importance of PKN1-specific kinase-independent functions in vivo.

Noncirrhotic Portal Hypertension: Current and Emerging Perspectives.

Idiopathic portal hypertension (IPH) and extrahepatic portal venous obstruction (EHPVO) are prototype noncirrhotic causes of portal hypertension (PHT), characterized by normal hepatic venous pressure gradient, variceal bleeds, and moderate to massive splenomegaly with preserved liver synthetic functions. Infections, toxins, and immunologic, prothrombotic and genetic disorders are possible causes in IPH, whereas prothrombotic and local factors around the portal vein lead to EHPVO. Growth failure, portal biliopathy, and minimal hepatic encephalopathy are long-term concerns in EHPVO. Surgical shunts and transjugular intrahepatic portosystemic shunt resolve the complications secondary to PHT. Meso-Rex shunt is now the standard-of-care surgery in children with EHPVO.

MCM8- and MCM9 Deficiencies Cause Lifelong Increased Hematopoietic DNA Damage Driving p53-Dependent Myeloid Tumors.

Hematopoiesis is particularly sensitive to DNA damage. Myeloid tumor incidence increases in patients with DNA repair defects and after chemotherapy. It is not known why hematopoietic cells are highly vulnerable to DNA damage. Addressing this question is complicated by the paucity of mouse models of hematopoietic malignancies due to defective DNA repair. We show that DNA repair-deficient Mcm8- and Mcm9-knockout mice develop myeloid tumors, phenocopying prevalent myelodysplastic syndromes. We demonstrate that these tumors are preceded by a lifelong DNA damage burden in bone marrow and that they acquire proliferative capacity by suppressing signaling of the tumor suppressor and cell cycle controller RB, as often seen in patients. Finally, we found that absence of MCM9 and the tumor suppressor Tp53 switches tumorigenesis to lymphoid tumors without precedent myeloid malignancy. Our results demonstrate that MCM8/9 deficiency drives myeloid tumor development and establishes a DNA damage burdened mouse model for hematopoietic malignancies.

Notch Signaling in Nestin-Expressing Cells in the Bone Marrow Maintains Erythropoiesis via Macrophage Integrity.

Notch signaling plays pivotal roles in both hematopoietic stem/progenitor and their niche cells. Myeloproliferative phenotypes are induced by disruption of Notch signaling in nonhematopoietic bone marrow (BM) cells. Nestin-expressing cells in the BM reportedly represent a component of the hematopoietic stem cell niche. We established mice in which rare Nestin-expressing cells in the BM were marked by green fluorescent protein, and Notch signaling was conditionally disrupted in these cells specifically. We observed impairment of erythropoiesis in the BM accompanying splenomegaly with BM hematopoietic programs in other lineages undisturbed. Transplantation experiments revealed that the microenvironmental rather than the hematopoietic cells were attributable to these phenotypes. We further found that the erythroid-island-forming ability of BM central macrophages was compromised along with the transcriptional upregulation of interleukin-6. Various Inflammatory conditions hamper BM erythropoiesis, which often accompanies extramedullary hematopoiesis. The mouse model demonstrated here may be of relevance to this common pathophysiologic condition. Stem Cells 2019;37:924-936.

TLR7 Agonism Accelerates Disease and Causes a Fatal Myeloproliferative Disorder in NZM 2410 Lupus Mice.

Murine models of lupus, both spontaneous and inducible, are valuable instruments to study SLE pathogenesis. Accelerants such as Type I IFN are often used to trigger earlier disease onset. We used a topical TLR7 agonist, previously reported to induce lupus-like disease in WT mice within weeks, to validate this data in C57BL/6j mice, and to test TLR7 agonism as an accelerant in lupus-prone NZM2410 mice. We found that TLR7-stimulated B6 and NZM2410 mice had significantly reduced survival and exhibited profound splenomegaly with significantly reduced B cells (4 vs. 40%), and T cells (8 vs. 31%). Spleen pathology and IHC revealed massive expansion of F4/80+ cells in TLR7-treated mice consistent with histiocytosis. While resiqimod treatment caused mild autoimmunity in B6 mice and accelerated autoimmunity in NZM2410 mice, it did not cause significant nephritis or proteinuria in either strain (renal function intact at death). Given the macrophage expansion, cytopenias, and disruption of normal splenic lymphoid follicle architecture, histiocytic sarcoma is favored as the cause of death. An alternative etiology is a macrophage activation syndrome (MAS)-like syndrome, since the mice also had a transaminitis and histologic hemophagocytosis in the setting of their rapid mortality. For investigators who are focused on murine models of lupus nephritis, this model is not ideal when utilizing B6 mice, however topical resiqimod may prove useful to accelerate autoimmunity and nephritis in NZM2410 mice, or potentially to investigate secondary complications of lupus such as histiocytic diseases or macrophage activation like syndromes.

CML with Megakaryocytic Blast Crisis: Report of 3 Cases.

Chronic myelogenous leukemia (CML) is a chronic myeloproliferative neoplasm consistently associated with the BCR-ABL1 fusion gene located in the Philadelphia chromosome. The Blast Phase is diagnosed when blasts are ≥20% of the peripheral blood white cell count or of bone marrow nucleated cells or when there is an extramedullary blast proliferation. Megakaryocytic blast crisis as the presenting manifestation of CML is extremely rare and only 7 reported cases were found in the literature. Out of 34 cases of CML-Blast Phase between April 2015 and June 2016, 3 cases showed megakaryocytic differentiation. 2 of these presented in Blast phase as the first manifestation of CML and the third case was a known case of CML-Chronic phase. Flow cytometric immunophenotyping was performed on peripheral blood/bone marrow using 6- color flow cytometer Navios. On CD45 vs SSC two distinct populations of blasts were seen in two cases and single population in the third case. All the 3 cases were positive for CD61, cCD41, cCD61 confirming the megakaryocytic lineage. The clinical features, morphologic and cytogenetic findings help in the identification and distinction of megakaryocytic blast phase of CML from Acute Megakayoblastic Leukemia. The diagnosis of such rare presentation of CML is essential for determining the choice of treatment. Therefore including a megakaryocytic marker in the primary flow cytometry panel is important so that these cases are not under-diagnosed as Acute myeloid leukemia because of expression of CD13 and CD33 only.

Captopril mitigates splenomegaly and myelofibrosis in the Gata1low murine model of myelofibrosis.

Allogeneic stem cell transplantation is currently the only curative therapy for primary myelofibrosis (MF), while the JAK2 inhibitor, ruxolitinib. Has been approved only for palliation. Other therapies are desperately needed to reverse life-threatening MF. However, the cell(s) and cytokine(s) that promote MF remain unclear. Several reports have demonstrated that captopril, an inhibitor of angiotensin-converting enzyme that blocks the production of angiotensin II (Ang II), mitigates fibrosis in heart, lung, skin and kidney. Here, we show that captopril can mitigate the development of MF in the Gata1low mouse model of primary MF. Gata1low mice were treated with 79 mg/kg/d captopril in the drinking water from 10 to 12 months of age. At 13 months of age, bone marrows were examined for fibrosis, megakaryocytosis and collagen expression; spleens were examined for megakaryocytosis, splenomegaly and collagen expression. Treatment of Gata1low mice with captopril in the drinking water was associated with normalization of the bone marrow cellularity; reduced reticulin fibres, splenomegaly and megakaryocytosis; and decreased collagen expression. Our findings suggest that treating with the ACE inhibitors captopril has a significant benefit in overcoming pathological changes associated with MF.

Caveolin-1 is dispensable for early lymphoid development, but plays a role in the maintenance of the mature splenic microenvironment.

OBJECTIVE: Caveolin-1 (CAV1) is known for its role as both a tumor suppressor and an oncogene, harboring a highly context-dependent role within a myriad of malignancies and cell types. In an immunological context, dysregulation of CAV1 expression has been shown to alter immunological signaling functions and suggests a pivotal role for CAV1 in the facilitation of proper immune responses. Nonetheless, it is still unknown how Cav1-deficiency and heterozygosity would impact the development and composition of lymphoid organs in mice. Herein, we investigated the impacts of Cav1-dysregulation on the lymphoid organs in young (12 weeks) and aged (36 weeks) Cav1+/+, Cav1+/-, and Cav1-/- mice. RESULTS: We observed that only Cav1-deficiency is associated with persistent splenomegaly at all timepoints. Furthermore, no differences in overall body weight were detected (and without sexual dimorphisms). Both aged Cav1+/- and Cav1-/- mice present with decreased CD19+CD22+ B cells and secondary-follicle atrophy, specifically in the spleen, compared with wild-type controls and irrespective of splenomegaly status. Consequently, the demonstrated effects on B cell homeostasis and secondary follicle characteristics prompted our investigation into follicle-derived human B-cell lymphomas. Our investigation points toward CAV1 as a dysregulated protein in follicle-derived B-cell malignancies without harboring a differential expression between more aggressive and indolent hematological malignancies.

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

RATIONALE: Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE: Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS: We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS: Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

Should rituximab replace splenectomy in the management of splenic marginal zone lymphoma?

BACKGROUND: SMZL is a relatively rare low grade B-cell lymphoma, characterized usually by an indolent clinical behavior. Since there is no prospective randomized trials to establish the best treatment approach, decision on therapeutic management should be based on the available retrospective series. Based on these data, rituximab and splenectomy appear to be the most effective. Splenectomy represented the standard treatment modality until early 2000s. More than 90% of the patients present quick amelioration of splenomegaly related symptoms along with improvement of cytopenias related to hypersplenism. The median progression free survival was 8.25 years in the largest series of patients published so far, while the median 5- and 10- year OS were 84% and 67%, respectively. Responses to splenectomy are not complete since extrasplenic disease persists. Patients with heavy bone marrow infiltration, lymphadenopathy or other disease localization besides the spleen are not good candidates for splenectomy. Furthermore splenectomy is a major surgical procedure accompanied by acute perioperative complications as well as late toxicities mainly due to infections. For that reasons splenectomy is not appropriate for elderly patients or patients with comorbidities with a high surgical risk. On the other hand rituximab monotherapy displays high efficacy with minimal toxicity. Several published series have shown an ORR more than 90%, with high CR rates (∼50%). The 10-year PFS and OS were 63% and 85%, respectively in a series of 104 SMZL patients. The role of rituximab maintenance has been investigated by only one group. Based on these data, maintenance with rituximab further improved the quality of responses by increasing significantly the CR rates (from 42% at the end of induction to 71% at the end of maintenance treatment), as well as the duration of responses: 7-year PFS was 75% for those patients who received maintenance vs 39% for those who did not (p < 0.0004). However no difference in OS has been noticed between the two groups, so far. Summarizing the above data, it is obvious that Rituximab monotherapy is associated with high response rates, long response duration and favorable safety profile, rendering it as the treatment of choice in SMZL.

The Schistosomiasis SpleenOME: Unveiling the Proteomic Landscape of Splenomegaly Using Label-Free Mass Spectrometry.

Schistosomiasis is a neglected parasitic disease that affects millions of people worldwide and is caused by helminth parasites from the genus Schistosoma. When caused by S. mansoni, it is associated with the development of a hepatosplenic disease caused by an intense immune response to the important antigenic contribution of adult worms and to the presence of eggs trapped in liver tissue. Although the importance of the spleen for the establishment of immune pathology is widely accepted, it has received little attention in terms of the molecular mechanisms operating in response to the infection. Here, we interrogated the spleen proteome using a label-free shotgun approach for the potential discovery of molecular mechanisms associated to the peak of the acute phase of inflammation and the development of splenomegaly in the murine model. Over fifteen hundred proteins were identified in both infected and control individuals and 325 of those proteins were differentially expressed. Two hundred and forty-two proteins were found upregulated in infected individuals while 83 were downregulated. Functional enrichment analyses for differentially expressed proteins showed that most of them were categorized within pathways of innate and adaptive immunity, DNA replication, vesicle transport and catabolic metabolism. There was an important contribution of granulocyte proteins and antigen processing and presentation pathways were augmented, with the increased expression of MHC class II molecules but the negative regulation of cysteine and serine proteases. Several proteins related to RNA processing were upregulated, including splicing factors. We also found indications of metabolic reprogramming in spleen cells with downregulation of proteins related to mitochondrial metabolism. Ex-vivo imunophenotyping of spleen cells allowed us to attribute the higher abundance of MHC II detected by mass spectrometry to increased number of macrophages (F4/80+/MHC II+ cells) in the infected condition. We believe these findings add novel insights for the understanding of the immune mechanisms associated with the establishment of schistosomiasis and the processes of immune modulation implied in the host-parasite interactions.

Clinical and Laboratory Features of CD5-Negative Chronic Lymphocytic Leukemia.

BACKGROUND Chronic lymphocytic leukemia (CLL) usually expresses CD5 antigen. However, 7-20% of patients are CD5 negative. We report here a series of 19 CD5-negative B-CLL cases. MATERIAL AND METHODS We reviewed 19 consecutive CD5-negative B-CLL cases seen in our medical center from 2009 to 2015 and compared them with 105 CD5-positive B-CLL cases. The two groups were compared in terms of clinical parameters, laboratory parameters, and survival characteristics. RESULTS Lymphadenopathy was present in 31.5% of the CD5-negative group and 51.4% of the CD5-positive group (p=0.029). Splenomegaly was present in 42.1% of the CD5-negative group and 16.1% of the CD5-positive group (p=0.029). There was no difference between the groups in terms of Binet A, B, and C stages (p=0.118, p=0.051, and p=0.882, respectively). The median thrombocyte count was 144×109/L and 160×109/L in the CD5-negative and CD5-positive groups, respectively (p=0.044). There was no difference between the two groups in terms of median neutrophil count (p=0.169). The mean lymphocyte count was 43.2±4.0×10^9/L and 36.7±3.2×10^9/L in the CD5-negative and CD5-positive groups, respectively (p=0.001). There was no difference between the groups in terms of autoimmune hemolytic anemia and autoimmune thrombocytopenia. In five-year follow-up, 84.2% of CD5-negative patients and 90.5% of CD5-positive patients were alive (p=0.393). CONCLUSIONS We found more isolated splenomegaly, less lymphadenopathy, a higher lymphocyte count, and a lower thrombocyte count in the CD5-negative group. There was no difference between the groups in terms of clinical stage, autoimmune phenomena, hemoglobin and neutrophil count, and survival.

Non-invasive MRI biomarkers for the early assessment of iron overload in a humanized mouse model of ß-thalassemia.

ß-thalassemia (ßT) is a genetic blood disorder causing profound and life threatening anemia. Current clinical management of ßT is a lifelong dependence on regular blood transfusions, a consequence of which is systemic iron overload leading to acute heart failure. Recent developments in gene and chelation therapy give hope of better prognosis for patients, but successful translation to clinical practice is hindered by the lack of thorough preclinical testing using representative animal models and clinically relevant quantitative biomarkers. Here we demonstrate a quantitative and non-invasive preclinical Magnetic Resonance Imaging (MRI) platform for the assessment of ßT in the γß0/γßA humanized mouse model of ßT. Changes in the quantitative MRI relaxation times as well as severe splenomegaly were observed in the heart, liver and spleen in ßT. These data showed high sensitivity to iron overload and a strong relationship between quantitative MRI relaxation times and hepatic iron content. Importantly these changes preceded the onset of iron overload cardiomyopathy, providing an early biomarker of disease progression. This work demonstrates that multiparametric MRI is a powerful tool for the assessment of preclinical ßT, providing sensitive and quantitative monitoring of tissue iron sequestration and cardiac dysfunction- parameters essential for the preclinical development of new therapeutics.

The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.

Epithelial-to-mesenchymal-transition (EMT) is critical for normal embryogenesis and effective postnatal wound healing, but is also associated with cancer metastasis. SNAIL, ZEB, and TWIST families of transcription factors are key modulators of the EMT process, but their precise roles in adult hematopoietic development and homeostasis remain unclear. Here we report that genetic inactivation of Zeb2 results in increased frequency of stem and progenitor subpopulations within the bone marrow (BM) and spleen and that these changes accompany differentiation defects in multiple hematopoietic cell lineages. We found no evidence that Zeb2 is critical for hematopoietic stem cell self-renewal capacity. However, knocking out Zeb2 in the BM promoted a phenotype with several features that resemble human myeloproliferative disorders, such as BM fibrosis, splenomegaly, and extramedullary hematopoiesis. Global gene expression and intracellular signal transduction analysis revealed perturbations in specific cytokine and cytokine receptor-related signaling pathways following Zeb2 loss, especially the JAK-STAT and extracellular signal-regulated kinase pathways. Moreover, we detected some previously unknown mutations within the human Zeb2 gene (ZFX1B locus) from patients with myeloid disease. Collectively, our results demonstrate that Zeb2 controls adult hematopoietic differentiation and lineage fidelity through widespread modulation of dominant signaling pathways that may contribute to blood disorders.

Tumor necrosis factor drives increased splenic monopoiesis in old mice.

Aging is accompanied by changes in hematopoiesis and consequently in leukocyte phenotype and function. Although age-related changes in bone marrow hematopoiesis are fairly well documented, changes in extramedullary hematopoiesis are less well described. We observed that 18-22-mo-old mice had larger spleens than young controls and found that the enlargement was caused by increased monopoiesis. Because extramedullary hematopoiesis is often driven by inflammation, we hypothesized that the chronic, low-level inflammation that occurs with age is a causal agent in splenomegaly. To test this theory, we compared the number of monocytes in 18-mo-old tumor necrosis factor-knockout mice, which are protected from age-associated inflammation, and found that they did not have increased extramedullary monopoiesis. To determine whether increased splenic monopoiesis is caused by intrinsic changes in the myeloid precursors that occur with age or by the aging microenvironment, we created heterochronic bone marrow chimeras. Increased splenic monopoiesis occurred in old recipient mice, regardless of the age of the donor mouse, but not in young recipient mice, demonstrating that these cells respond to signals from the microenvironment. These data suggest that decreasing the inflammatory microenvironment with age would be an effective strategy for reducing inflammatory diseases propagated by cells of myeloid lineage, which increase in number with age.