Repercussion of megakaryocyte-specific Gata1 Loss on megakaryopoiesis and the hematopoietic precursor compartment

During hematopoiesis, transcriptional programs are essential for the commitment and differentiation of progenitors into the different blood lineages. GATA1 is a transcription factor expressed in several hematopoietic lineages and essential for proper erythropoiesis and megakaryopoiesis. Megakaryocyte-specific genes, such as GP1BA, are known to be directly regulated by GATA1. Mutations in GATA1 can lead to dyserythropoietic anemia and pseudo gray-platelet syndrome. Selective loss of Gata1 expression in adult mice results in macrothrombocytopenia with platelet dysfunction, characterized by an excess of immature megakaryocytes. To specifically analyze the impact of Gata1 loss in mature committed megakaryocytes, we generated Gata1-Lox|Pf4-Cre mice (Gata1cKOMK). Consistent with previous findings, Gata1cKOMK mice are macrothrombocytopenic with platelet dysfunction. Supporting this notion we demonstrate that Gata1 regulates directly the transcription of Syk, a tyrosine kinase that functions downstream of Clec2 and GPVI receptors in megakaryocytes and platelets. Furthermore, we show that Gata1cKOMK mice display an additional aberrant megakaryocyte differentiation stage. Interestingly, these mice present a misbalance of the multipotent progenitor compartment and the erythroid lineage, which translates into compensatory stress erythropoiesis and splenomegaly. Despite the severe thrombocytopenia, Gata1cKOMK mice display a mild reduction of TPO plasma levels, and Gata1cK-OMK megakaryocytes show a mild increase in Pf4 mRNA levels; such a misbalance might be behind the general hematopoietic defects observed, affecting locally normal TPO and Pf4 levels at hematopoietic stem cell niches. © 2016 Meinders et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Mild dyserythropoiesis and beta-like globin gene expression imbalance due to the loss of histone chaperone ASF1B

The expression of the human β-like globin genes follows a well-orchestrated developmental pattern, undergoing two essential switches, the first one during the first weeks of gestation (ε to γ), and the second one during the perinatal period (γ to β). The γ- to β-globin gene switching mechanism includes suppression of fetal (γ-globin, HbF) and activation of adult (β-globin, HbA) globin gene transcription. In hereditary persistence of fetal hemoglobin (HPFH), the γ-globin suppression mechanism is impaired leaving these individuals with unusual elevated levels of fetal hemoglobin (HbF) in adulthood. Recently, the transcription factors KLF1 and BCL11A have been established as master regulators of the γ- to β-globin switch. Previously, a genomic variant in the KLF1 gene, identified by linkage analysis performed on twenty-seven members of a Maltese family, was found to be associated with HPFH. However, variation in the levels of HbF among family members, and those from other reported families carrying genetic variants in KLF1, suggests additional contributors to globin switching. ASF1B was downregulated in the family members with HPFH. Here, we investigate the role of ASF1B in γ- to β-globin switching and erythropoiesis in vivo. Mouse-human interspecies ASF1B protein identity is 91.6%. By means of knockdown functional assays in human primary erythroid cultures and analysis of the erythroid lineage in Asf1b knockout mice, we provide evidence that ASF1B is a novel contributor to steady-state erythroid differentiation, and while its loss affects the balance of globin expression, it has no major role in hemoglobin switching

Abundance of Early Functional HIV-Specific CD8+ T Cells Does Not Predict AIDS-Free Survival Time

Background T-cell immunity is thought to play an important role in controlling HIV infection, and is a main target for HIV vaccine development. HIV-specific central memory CD8+ and CD4+ T cells producing IFNγ and IL-2 have been associated with control of viremia and are therefore hypothesized to be truly protective and determine subsequent clinical outcome. However, the cause-effect relationship between HIV-specific cellular immunity and disease progression is unknown. We investigated in a large prospective cohort study involving 96 individuals of the Amsterdam Cohort Studies with a known date of seroconversion whether the presence of cytokine-producing HIV-specific CD8+ T cells early in infection was associated with AIDS-free survival time. Methods and Findings The number and percentage of IFNγ and IL-2 producing CD8+ T cells was measured after in vitro stimulation with an overlapping Gag-peptide pool in T cells sampled approximately one year after seroconversion. Kaplan-Meier survival analysis and Cox proportional hazard models showed that frequencies of cytokine-producing Gag-specific CD8+ T cells (IFNγ, IL-2 or both) shortly after seroconversion were neither associated with time to AIDS nor with the rate of CD4+ T-cell decline. Conclusions These data show that high numbers of functional HIV-specific CD8+ T cells can be found early in HIV infection, irrespective of subsequent clinical outcome. The fact that both progressors and long-term non-progressors have abundant T cell immunity of the specificity associated with low viral load shortly after seroconversion suggests that the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression

Dissecting platelet proteomics to understand the pathophysiology of immune thrombocytopenia : studies in mouse models

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by enhanced platelet clearance and defective platelet production. Diagnosis by exclusion and trial-and-error treatment strategies is common practice, and despite the advancement in treatment options, many patients remain refractory. Although the existence of different pathophysiological entities is acknowledged, we are still far from stratifying and understanding ITP. To investigate, we sought to dissect the platelet proteome dynamics in so-called passive and active preclinical ITP mouse models, with which we propose to phenocopy respectively acute/newly diagnosed and persistent/chronic stages of ITP in humans. We obtained the platelet proteome at the thrombocytopenic stage and after platelet count recovery (reached naturally or by IVIg-treatment, depending on the model). Although most of the proteomic alterations were common to both ITP models, there were model-specific protein dynamics that accompanied and explained alterations in platelet aggregation responses, as measured in the passive ITP model. The expression dynamics observed in Syk may explain, extrapolated to humans and pending validation, the increased bleeding tendency of patients with ITP when treated with fostamatinib as third or later- as opposed to second line of treatment. We propose that the platelet proteome may give diagnostic and prognostic insights into ITP and that such studies should be pursued in humans

Dissecting platelet proteomics to understand the pathophysiology of immune thrombocytopenia: studies in mouse models

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by enhanced platelet clearance and defective platelet production. Diagnosis by exclusion and trial-and-error treatment strategies is common practice, and despite the advancement in treatment options, many patients remain refractory. Although the existence of different pathophysiological entities is acknowledged, we are still far from stratifying and understanding ITP. To investigate, we sought to dissect the platelet proteome dynamics in so-called passive and active preclinical ITP mouse models, with which we propose to phenocopy respectively acute/newly diagnosed and persistent/chronic stages of ITP in humans. We obtained the platelet proteome at the thrombocytopenic stage and after platelet count recovery (reached naturally or by IVIg-treatment, depending on the model). Although most of the proteomic alterations were common to both ITP models, there were model-specific protein dynamics that accompanied and explained alterations in platelet aggregation responses, as measured in the passive ITP model. The expression dynamics observed in Syk may explain, extrapolated to humans and pending validation, the increased bleeding tendency of patients with ITP when treated with fostamatinib as third or later- as opposed to second line of treatment. We propose that the platelet proteome may give diagnostic and prognostic insights into ITP and that such studies should be pursued in humans

Long-term highly active antiretroviral therapy in chronic HIV-1 infection: evidence for reconstitution of antiviral immunity

In this study we investigated the long-term effect of highly active antiretroviral therapy (HAART) on HIV-specific CD4+ T-cell responses in comparison with virus-specific CD4+ T-cell responses against the persistent herpes viruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV). To this end, HIV- and herpes virus-specific cellular immune responses were measured longitudinally in 10 seroconverters with long-term follow-up including 55 months of successful suppression of viral load by HAART. HIV- and CMV-specific CD4+ T cells producing interferon-gamma (IFNgamma) or interleukin-2 (IL-2) were analysed as well as proliferative capacity. EBV-specific CD4+ T cells were determined using a 12-day ex vivo assay. Initiation of HAART resulted in a transient increase of HIV-specific IL-2(+)IFNgamma(+)CD4(+) T cells and, to a lesser extent, IL-2(+)CD4(+) T cells. Long-term HAART resulted in an increase in HIV-, CMV- and EBV-specific CD4+ T-cell proliferative capacity. The increase in HIV- and herpes-virus-specific CD4+ T-cell proliferative capacity after 55 months of HAART suggests that the improved proliferative response is not specific for HIV, but reflects a more general improvement of antiviral immune responses, which is induced by HAAR

Shift of CMV-specific CD4+ T-cells to the highly differentiated CD45RO-CD27- phenotype parallels loss of proliferative capacity and precedes progression to HIV-related CMV end-organ disease

To identify factors related to progression to CMV end-organ disease, cytokine production, proliferative capacity and phenotype of CMV-specific CD4(+) T-cells were analysed longitudinally. Numbers of IFNgamma(+)CD4(+) and IFNgamma(+)IL-2(+)CD4(+) T-cells tended to decrease in individuals progressing to AIDS with CMV end-organ disease (AIDS-CMV), whereas they remained detectable in long-term asymptomatics (LTAs) and progressors to AIDS with opportunistic infections (AIDS-OI). In parallel, CMV-specific proliferative capacity was lost in AIDS-CMV. Initially, the majority of the CMV-specific IFNgamma(+)CD4(+) T-cells were of the CD45RO(+)CD27(-) subset, but during progression to AIDS-CMV a shift in phenotype to the CD45RO(-)CD27(-) subset was observed. Our data indicate that a decrease in CMV-specific cytokine production and proliferative capacity precedes progression to AIDS-CMV. Accumulation of CD4(+) T-cells with a CD45RO(-)CD27(-) phenotype suggests that persistent antigen exposure drives differentiation of CMV-specific CD4(+) T-cells towards a poorly proliferating, and highly differentiated "effector" subset, which eventually fails to produce IFNgamma in patients developing AIDS-CM

Pathogen reduction treatment using riboflavin and ultraviolet light impairs platelet reactivity toward specific agonists in vitro

Recent studies showed that Mirasol pathogen reduction treatment (PRT) leads to increased P-selectin expression and increased oxygen and glucose consumption in resting platelets (PLTs). This study investigates the effect of PRT on PLT activation. Untreated or Mirasol-treated PLTs were analyzed at different time points during storage. Microaggregation upon stimulation with phorbol myristate acetate (PMA), convulxin, and ristocetin was measured. Alpha granule contents and release upon thrombin stimulation were assessed by flow cytometry and Western blotting. PLT spreading was determined on collagen-coated glass slides. Mirasol PRT led to spontaneous aggregation (hyperreactivity), as measured by flow cytometry in the absence of agonist throughout storage time. PMA-induced aggregation was significantly higher in Mirasol PRT PLTs compared to controls. Aggregation in response to convulxin and ristocetin was significantly lower and directly influenced by storage time after Mirasol PRT, compared to untreated stored PLT concentrates. Despite the reported hyperreactivity of resting PLTs, PLT activation with thrombin on Day 8 after Mirasol PRT resulted in less P-selectin-positive PLTs. Furthermore, platelet factor 4 (PF4) secretion was reduced upon thrombin stimulation on Day 8 after PRT compared to controls. Significantly decreased spreading of Mirasol PRT PLTs over collagen-coated slides was observed directly after PRT and persisted throughout storage. Mirasol PRT leads to hyperreactive PLTs, probably caused by continuous basal degranulation through storage time. This results in a reduction in the degranulation capacity upon acute stimulation, which influences PLT spreading, but not overtly microaggregation. The clinical relevance needs to be investigate