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MYSM1 is a chromatin-binding transcriptional cofactor that deubiquitinates histone H2A. Studies of Mysm1-deficient mice have shown that it is essential for hematopoietic stem cell (HSC) function and lymphopoiesis. Human carriers of a rare MYSM1-inactivating mutation display similar lymphopoietic deficiencies. However, the mechanism by which MYSM1 regulates hematopoietic homeostasis remains unclear. Here, we show that Mysm1-deficiency results in p53 protein elevation in many hematopoietic cell types. p53 is a central regulator of cellular stress responses and HSC homeostasis. We thus generated double-knockout mice to assess a potential genetic interaction between Mysm1 and p53 in hematopoiesis. Mysm1(-/-)p53(-/-) mouse characterization showed a full rescue of Mysm1(-/-) developmental and hematopoietic defects. This included restoration of lymphopoiesis, and HSC numbers and functions. These results establish p53 activation as the driving mechanism for hematopoietic abnormalities in Mysm1 deficiency. Our findings may advance the understanding of p53 regulation in hematopoiesis and implicate MYSM1 as a potential p53 cofactor.
Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.
Mice with a constitutive or platelet-specific deletion of the C-type-lectin-like receptor (CLEC-2) exhibit hemorrhaging in the brain at mid-gestation. We sought to investigate the basis of this defect, hypothesizing that it is mediated by the loss of CLEC-2 activation by its endogenous ligand, podoplanin, which is expressed on the developing neural tube. To induce deletion of podoplanin at the 2-cell stage, we generated a podoplanin(fl/fl) mouse crossed to a PGK-Cre mouse. Using 3-dimensional light-sheet microscopy, we observed cerebral vessels were tortuous and aberrantly patterned at embryonic (E) day 10.5 in podoplanin- and CLEC-2-deficient mice, preceding the formation of large hemorrhages throughout the fore-, mid-, and hindbrain by E11.5. Immunofluorescence and electron microscopy revealed defective pericyte recruitment and misconnections between the endothelium of developing blood vessels and surrounding pericytes and neuro-epithelial cells. Nestin-Cre-driven deletion of podoplanin on neural progenitors also caused widespread cerebral hemorrhaging. Hemorrhaging was also seen in the ventricles of embryos deficient in the platelet integrin subunit glycoprotein IIb or in embryos in which platelet α-granule and dense granule secretion is abolished. We propose a novel role for podoplanin on the neuro-epithelium, which interacts with CLEC-2 on platelets, mediating platelet adhesion, aggregation, and secretion to guide the maturation and integrity of the developing vasculature and prevent hemorrhage.
S100A8 and S100A9 are calcium-binding proteins predominantly expressed by neutrophils and monocytes and play key roles in both normal and pathological inflammation. Recently, both proteins were found to promote tumor progression through the establishment of premetastatic niches and inhibit antitumor immune responses. Although S100A8 and S100A9 have been studied in solid cancers, their functions in hematological malignancies remain poorly understood. However, S100A8 and S100A9 are highly expressed in acute myeloid leukemia (AML), and S100A8 expression has been linked to poor prognosis in AML. We identified a small subpopulation of cells expressing S100A8 and S100A9 in AML mouse models and primary human AML samples. In vitro and in vivo analyses revealed that S100A9 induces AML cell differentiation, whereas S100A8 prevents differentiation induced by S100A9 activity and maintains AML immature phenotype. Treatment with recombinant S100A9 proteins increased AML cell maturation, induced growth arrest, and prolonged survival in an AML mouse model. Interestingly, anti-S100A8 antibody treatment had effects similar to those of S100A9 therapy in vivo, suggesting that high ratios of S100A9 over S100A8 are required to induce differentiation. Our in vitro studies on the mechanisms/pathways involved in leukemic cell differentiation revealed that binding of S100A9 to Toll-like receptor 4 (TLR4) promotes activation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2, and Jun N-terminal kinase signaling pathways, leading to myelomonocytic and monocytic AML cell differentiation. These findings indicate that S100A8 and S100A9 are regulators of myeloid differentiation in leukemia and have therapeutic potential in myelomonocytic and monocytic AMLs.
Ibrutinib, a potent and irreversible small-molecule inhibitor of both Bruton's tyrosine kinase and interleukin-2 inducible kinase (ITK), has been used to treat relapsed/refractory chronic lymphocytic leukemia (CLL) with prolongation of progression-free and overall survival. Here, we present 27 patients with relapsed CLL following allogeneic hematopoietic cell transplant (HCT) who subsequently received ibrutinib salvage therapy. Sixteen of these patients were part of multi-institutional clinical trials and achieved an overall response rate of 87.5%. An additional 11 patients were treated at Stanford University following US Food and Drug Administration approval of ibrutinib; 7 (64%) achieved a complete response, and 3 (27%) achieved a partial response. Of the 9 patients treated at Stanford who had mixed chimerism-associated CLL relapse, 4 (44%) converted to full donor chimerism following ibrutinib initiation, in association with disease response. Four of 11 (36%) patients evaluated by ClonoSeq achieved minimal residual disease negativity with CLL <1/10 000 white blood cells, which persisted even after ibrutinib was discontinued, in 1 case even after 26 months. None of the 27 patients developed graft-versus-host-disease (GVHD) following ibrutinib initiation. We postulate that ibrutinib augments the graft-versus-leukemia (GVL) benefit through a T-cell-mediated effect, most likely due to ITK inhibition. To investigate the immune modulatory effects of ibrutinib, we completed comprehensive immune phenotype characterization of peripheral B and T cells from treated patients. Our results show that ibrutinib selectively targets pre-germinal B cells and depletes Th2 helper cells. Furthermore, these effects persisted after drug discontinuation. In total, our results provide evidence that ibrutinib effectively augments GVL without causing GVHD.
Hematopoietic stem cells (HSCs) reside at the top of the hematopoietic hierarchy and are the origin of all blood cells produced throughout an individual's life. The balance between HSC self-renewal and differentiation is maintained by various intrinsic and extrinsic mechanisms. Among these, the molecular pathways that restrict cell cycle progression are critical to the maintenance of functional HSCs. Alterations in the regulation of cell cycle progression in HSCs invariably lead to the development of hematologic malignancies or bone marrow failure syndromes. Here we report that hematopoietic-specific genetic inactivation of Sin3B, an essential component of the mammalian Sin3-histone deacetylase corepressor complex, severely impairs the competitive repopulation capacity of HSCs. Sin3B-deleted HSCs accumulate and fail to properly differentiate following transplantation. Moreover, Sin3B inactivation impairs HSC quiescence and sensitizes mice to myelosuppressive therapy. Together, these results identify Sin3B as a novel and critical regulator of HSC functions.
The term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), IL-18BP-/- mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated IL-18BP-/- mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine Cxcl9 or the transcription factor CIIta, were significantly increased in IL-18BP-/- mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in IL-18BP-/- mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.
The identity of T-cell progenitors that seed the thymus has remained controversial, largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings, we compared the myeloid potential of 2 putative thymus seeding populations, common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs), and the earliest intrathymic progenitor (DN1), using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo, as well as in methylcellulose cultures. MPP, on the other hand, displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic, but nonphysiologic, myeloid potentials of lymphoid progenitors, providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials.
A region of the human von Willebrand factor (VWF) gene between -2812 and the end of the first intron (termed vWF2) was previously shown to direct expression in the endothelium of capillaries and a subset of larger blood vessels in the heart and skeletal muscle. Here, our goal was to delineate the DNA sequences responsible for this effect. A series of constructs containing deletions or mutations of vWF2 coupled to LacZ were targeted to the Hprt locus of mice, and the resulting animals were analyzed for reporter gene expression. The findings demonstrate that DNA sequences between -843 and -620 are necessary for expression in capillary but not large vessel endothelium in heart and skeletal muscle. Further, expression of VWF in capillaries and larger vessels of both tissues required the presence of a native or heterologous intron. In vitro assays implicated a role for ERG-binding ETS motif at -56 in mediating basal expression of VWF. In Hprt-targeted mice, mutation of the ETS consensus motif resulted in loss of LacZ expression in the endothelium of the heart and skeletal muscle. Together, these data indicate that distinct DNA modules regulate vascular bed-specific expression of VWF.
CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.
We describe a mutation in human erythrocyte band 3 (anion exchanger 1; SLC4A1) causing both hereditary spherocytosis and distal renal tubular acidosis. The proband developed a transfusion-dependent, hemolytic anemia following birth. Immunoblotting showed band 3 was reduced to approximately 35% of wildtype; other proteins of the band 3/Rh macrocomplex were also reduced. DNA sequence analysis revealed a novel homozygous mutation, c.2000C>T, leading to the amino acid substitution Ser667Phe. The parents were heterozygous for the same mutation. Sulfate influx in the patient's erythrocytes was approximately 40% wild type. The mutant band 3 produced very little chloride influx when expressed in Xenopus oocytes. Influx was partially rescued by coexpression of glycophorin A and also rescued by coexpression of wild-type band 3. At 2 years of age, an ammonium chloride challenge showed the child has incomplete distal renal tubular acidosis (dRTA). Stable expression of mutant kidney band 3 in both nonpolarized and polarized Madin-Darby canine kidney cells showed that most of the mutant protein was retained in the endoplasmic reticulum. Overall our results suggest that the Ser667Phe does not affect the anion transport function of band 3, but causes a trafficking defect in both erythrocytes and kidney cells.
In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1alpha-mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 x 10(6) and 5.4 x 10(6) neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 x 10(6) neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.
Chemokines and adhesion molecules up-regulated in lymphatic endothelial cells (LECs) during tissue inflammation are thought to enhance dendritic cell (DC) migration to draining lymph nodes, but the in vivo control of this process is not well understood. We performed a transcriptional profiling analysis of LECs isolated from murine skin and found that inflammation induced by a contact hypersensitivity (CHS) response up-regulated the adhesion molecules ICAM-1 and VCAM-1 and inflammatory chemokines. Importantly, the lymphatic markers Prox-1, VEGFR3, and LYVE-1 were significantly down-regulated during CHS. By contrast, skin inflammation induced by complete Freund adjuvant induced a different pattern of chemokine and lymphatic marker gene expression and almost no ICAM-1 up-regulation in LECs. Fluorescein isothiocyanate painting experiments revealed that DC migration to draining lymph nodes was more strongly increased in complete Freund adjuvant-induced than in CHS-induced inflammation. Surprisingly, DC migration did not correlate with the induction of CCL21 and ICAM-1 protein in LECs. Although the requirement for CCR7 signaling became further pronounced during inflammation, CCR7-independent signals had an additional, albeit moderate, impact on enhancing DC migration. Collectively, these findings indicate that DC migration in response to inflammation is stimulus-specific, mainly CCR7-dependent, and overall only moderately enhanced by LEC-induced genes other than CCL21.
Abnormal interaction of sickle red blood cells (SS RBC) with the vascular endothelium has been implicated as a factor in the initiation of vasoocclusion in sickle cell anemia. Both von Willebrand factor (vWf) and thrombospondin (TSP) play important roles in mediating SS RBC-endothelium interaction and can bind to the endothelium via alphaVbeta3 receptors. We have used monoclonal antibodies (MoAb) directed against alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa) integrins to dissect the role of these integrins in SS RBC adhesion. The murine MoAb 7E3 inhibits both alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa), whereas MoAb LM609 selectively inhibits alphaVbeta3, and MoAb 10E5 binds only to alphaIIbbeta3. In this study, we have tested the capacity of these MoAbs to block platelet-activating factor (PAF)-induced SS RBC adhesion in the ex vivo mesocecum vasculature of the rat. Infusion of washed SS RBC in preparations treated with PAF (200 pg/mL), with or without a control antibody, resulted in extensive adhesion of these cells in venules, accompanied by frequent postcapillary blockage and increased peripheral resistance units (PRU). PAF also caused increased endothelial surface and interendothelial expression of endothelial vWf. Importantly, pretreatment ofthe vasculature with either MoAb 7E3 F(ab')(2) or LM609, but not 10E5 F(ab')(2), after PAF almost completely inhibited SS RBC adhesion in postcapillary venules, the sites of maximal adhesion and frequent blockage. The inhibition of adhesion with 7E3 or LM609 was accompanied by smaller increases in PRU and shorter pressure-flow recovery times. Thus, blockade of alphaVbeta3 may constitute a potential therapeutic approach to prevent SS RBC-endothelium interactions under flow conditions. (Blood. 2000;95:368-374)
This study demonstrates a unique and crucial role of plasmacytoid dendritic cells (pDCs) and pDC-derived type I interferons (IFNs) in the pathogenesis of mouse coronavirus infection. pDCs controlled the fast replicating mouse hepatitis virus (MHV) through the immediate production of type I IFNs. Recognition of MHV by pDCs was mediated via TLR7 ensuring a swift IFN-alpha production following encounter with this cytopathic RNA virus. Furthermore, the particular type I IFN response pattern was not restricted to the murine coronavirus, but was also found in infection with the highly cytopathic human severe acute respiratory syndrome (SARS) coronavirus. Taken together, our results suggest that rapid production of type I IFNs by pDCs is essential for the control of potentially lethal coronavirus infections.
Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with beta1- and beta3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCgamma2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in alpha-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for alpha-granule secretion and therefore may play a central role in the regulation of platelet function.
To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp(+) murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp(+) human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease.
A hematopoietic cell transplantation regimen was adapted from a preclinical model that used reduced-intensity conditioning (RIC) and protected against graft-versus-host disease (GVHD) by skewing residual host T-cell subsets to favor regulatory natural killer T cells. One hundred eleven patients with lymphoid (64) and myeloid (47) malignancies received RIC using total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) followed by the infusion of granulocyte colony-stimulating factor-mobilized grafts. Included were 34 patients at least 60 years of age, 32 patients at high risk of lymphoma relapse after disease recurrence following prior autologous transplantation, and 51 patients at high risk of developing GVHD due to lack of a fully human leukocyte antigen (HLA)-matched related donor. Durable chimerism was achieved in 97% of patients. Cumulative probabilities of acute GVHD (grades II-IV) were 2 and 10% of patients receiving related and unrelated donor grafts. Nonrelapse mortality (NRM) at 1 year was less than 4%. Cumulative incidence of chronic GVHD was 27%. The 36-month probability of overall and event-free survival was 60% and 40%, respectively. Disease status at start of conditioning and the level of chimerism achieved after transplantation significantly impacted clinical outcome. The high incidence of sustained remission among patients with active disease at time of transplantation suggests retained graft-versus-tumor reactions. Active trial registration currently at clinicaltrials.gov under IDs of NCT00185640 and NCT00186615.
Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies.
The histone acetyltransferase MOZ (MYST3, KAT6A) is the target of recurrent chromosomal translocations fusing the MOZ gene to CBP, p300, NCOA3, or TIF2 in particularly aggressive cases of acute myeloid leukemia. In this study, we report the role of wild-type MOZ in regulating B-cell progenitor proliferation and hematopoietic malignancy. In the Eμ-Myc model of aggressive pre-B/B-cell lymphoma, the loss of just one allele of Moz increased the median survival of mice by 3.9-fold. MOZ was required to maintain the proliferative capacity of B-cell progenitors, even in the presence of c-MYC overexpression, by directly maintaining the transcriptional activity of genes required for normal B-cell development. Hence, B-cell progenitor numbers were significantly reduced in Moz haploinsufficient animals. Interestingly, we find a significant overlap in genes regulated by MOZ, mixed lineage leukemia 1, and mixed lineage leukemia 1 cofactor menin. This includes Meis1, a TALE class homeobox transcription factor required for B-cell development, characteristically upregulated as a result of MLL1 translocations in leukemia. We demonstrate that MOZ localizes to the Meis1 locus in pre-B-cells and maintains Meis1 expression. Our results suggest that even partial inhibition of MOZ may reduce the proliferative capacity of MEIS1, and HOX-driven lymphoma and leukemia cells.
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