A deep dive into future therapies for microcytic anemias and clinical considerations.

INTRODUCTION: Microcytic anemias (MA) have frequent or rare etiologies. New discoveries in understanding and treatment of microcytic anemias need to be reviewed. AREAS COVERED: Microcytic anemias with a focus on the most frequent causes and on monogenic diseases that are relevant for understanding biocellular mechanisms of MA. All treatments except gene therapy, with a focus on recent advances. PubMed search with references selected by expert opinion. EXPERT OPINION: As the genetic and cellular backgrounds of dyserythropoiesis will continue to be clarified, collaboration with bioengineering of treatments acting specifically at the protein domain level will continue to provide new therapies in hematology as well as oncology and neurology.

Targeting Serotonin With Common Antidepressants Induces Rapid Recovery From Cytopenia.

The hematopoietic system uses several, yet undiscovered, factors to adapt to stresses such as chemotherapy, infections, or bleeding. Serotonin is commonly known as a neurotransmitter but is also produced and used in peripheral organs. In particular, we have shown that serotonin synthesized in the bone marrow is necessary for erythroid progenitors' survival and proliferation. Serotonin levels can be increased by FDA approved antidepressants called selective serotonin reuptake inhibitors (SSRI). In this work, we report a previously unknown role of SSRI in the recovery of cytopenia, after autologous hematopoietic stem cell transplantation in patients and after sub-lethal irradiation in mice. We also observed an unexpected cooperation between SSRI and G-CSF on the improvement of the 3 hematopoietic lineages. Of note, SSRI do not seem to affect blood cells production in the absence of stress-induced hematopoiesis. We propose that the serotonergic system could be a valuable therapeutic target in stress-induced cytopenia, especially as a rescue after radiation or chemotherapy.

Neuropilin-1 cooperates with PD-1 in CD8+ T cells predicting outcomes in melanoma patients treated with anti-PD1.

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8+ T cells activity in the context of the antitumor immune response. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1+CD8+ T cells, cooperates and enhances PD-1 activity. In mice, CD8+ T cells specific deletion of Nrp1 improves anti-PD1 antibody antitumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8+ T cells predicts poor outcome of patients treated with anti-PD1. NRP1 is a promising target to overcome resistance to anti-PD1 therapies.

Enhanced Renewal of Erythroid Progenitors in Myelodysplastic Anemia by Peripheral Serotonin.

Tryptophan as the precursor of several active compounds, including kynurenine and serotonin, is critical for numerous important metabolic functions. Enhanced tryptophan metabolism toward the kynurenine pathway has been associated with myelodysplastic syndromes (MDSs), which are preleukemic clonal diseases characterized by dysplastic bone marrow and cytopenias. Here, we reveal a fundamental role for tryptophan metabolized along the serotonin pathway in normal erythropoiesis and in the physiopathology of MDS-related anemia. We identify, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. In vivo studies demonstrate that pharmacological increase of serotonin levels using fluoxetine, a common antidepressant, has the potential to become an important therapeutic strategy in low-risk MDS anemia refractory to erythropoietin.

Serotonin, hematopoiesis and stem cells.

A large number of studies have focused on the role of serotonin as a neurotransmitter in the central nervous system, although only a small percentage of the body's serotonin (∼5%) can be found in the mature brain of mammals. In the gut, the enterochromaffin cells are scattered in the enteric epithelium from the stomach through the colon and produce over 95% of the body's serotonin. Since the generation of tryptophan hydroxylase (Tph1 and Tph2) knockout mice, unsuspected roles have been identified for serotonin synthesized outside the brain. Moreover, the murine model deficient in peripheral serotonin (Tph1-/-) is a unique experimental tool for exploring the molecular and cellular mechanisms involving serotonin's local effects through microserotonergic systems. In this review, we focus on peripheral serotonin and its role on progenitor or stem cells as well as on hematopoietic progenitors. We discuss the possible role of serotonin in hematopoietic diseases, and whether targeting the serotonergic system could be of therapeutic value for the regulation of normal and pathological hematopoiesis.

Human CD4- invariant NKT lymphocytes regulate graft versus host disease.

Despite increasing evidence for a protective role of invariant (i) NKT cells in the control of graft-versus-host disease (GVHD), the mechanisms underpinning regulation of the allogeneic immune response in humans are not known. In this study, we evaluated the distinct effects of human in vitro expanded and flow-sorted human CD4+ and CD4- iNKT subsets on human T cell activation in a pre-clinical humanized NSG mouse model of xenogeneic GVHD. We demonstrate that human CD4- but not CD4+ iNKT cells could control xenogeneic GVHD, allowing significantly prolonged overall survival and reduced pathological GVHD scores without impairing human T cell engraftment. Human CD4- iNKT cells reduced the activation of human T cells and their Th1 and Th17 differentiation in vivo. CD4- and CD4+ iNKT cells had distinct effects upon DC maturation and survival. Compared to their CD4+ counterparts, in co-culture experiments in vitro, human CD4- iNKT cells had a higher ability to make contacts and degranulate in the presence of mouse bone marrow-derived DCs, inducing their apoptosis. In vivo we observed that infusion of PBMC and CD4- iNKT cells was associated with decreased numbers of splenic mouse CD11c+ DCs. Similar differential effects of the iNKT cell subsets were observed on the maturation and in the induction of apoptosis of human monocyte-derived dendritic cells in vitro. These results highlight the increased immunosuppressive functions of CD4-versus CD4+ human iNKT cells in the context of alloreactivity, and provide a rationale for CD4- iNKT selective expansion or transfer to prevent GVHD in clinical trials.

Disrupting IGF Signaling in Adult Mice Conditions Leanness, Resilient Energy Metabolism, and High Growth Hormone Pulses.

Growth hormone (GH) and insulinlike growth factor (IGF) promote aging and age-related pathologies. Inhibiting this pathway by targeting IGF receptor (IGF-1R) is a promising strategy to extend life span, alleviate age-related diseases, and reduce tumor growth. Although anti-IGF-1R agents are being developed, long-term effects of IGF-1R blockade remain unknown. In this study, we used ubiquitous inducible IGF-1R knockout (UBIKOR) to suppress signaling in all adult tissues and screened health extensively. Surprisingly, UBIKOR mice showed no overt defects and presented with rather inconspicuous health, including normal cognition. Endocrine GH and IGF-1 were strongly upregulated without causing acromegaly. UBIKOR mice were strikingly lean with coordinate changes in body composition and organ size. They were insulin resistant but preserved physiological energy expenditure and displayed enhanced fasting metabolic flexibility. Thus, long-term IGF-1R blockade generated beneficial effects on aging-relevant metabolism, but exposed to high GH. This needs to be considered when targeting IGF-1R to protect from neurodegeneration, retard aging, or fight cancer.

Mutations in ACTRT1 and its enhancer RNA elements lead to aberrant activation of Hedgehog signaling in inherited and sporadic basal cell carcinomas.

Basal cell carcinoma (BCC), the most common human cancer, results from aberrant activation of the Hedgehog signaling pathway. Although most cases of BCC are sporadic, some forms are inherited, such as Bazex-Dupré-Christol syndrome (BDCS)-a cancer-prone genodermatosis with an X-linked, dominant inheritance pattern. We have identified mutations in the ACTRT1 gene, which encodes actin-related protein T1 (ARP-T1), in two of the six families with BDCS that were examined in this study. High-throughput sequencing in the four remaining families identified germline mutations in noncoding sequences surrounding ACTRT1. These mutations were located in transcribed sequences encoding enhancer RNAs (eRNAs) and were shown to impair enhancer activity and ACTRT1 expression. ARP-T1 was found to directly bind to the GLI1 promoter, thus inhibiting GLI1 expression, and loss of ARP-T1 led to activation of the Hedgehog pathway in individuals with BDCS. Moreover, exogenous expression of ACTRT1 reduced the in vitro and in vivo proliferation rates of cell lines with aberrant activation of the Hedgehog signaling pathway. In summary, our study identifies a disease mechanism in BCC involving mutations in regulatory noncoding elements and uncovers the tumor-suppressor properties of ACTRT1.

The role of 5-HT2B receptors in mitral valvulopathy: bone marrow mobilization of endothelial progenitors.

BACKGROUND AND PURPOSE: Valvular heart disease (VHD) is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the 5-HT system is associated with VHD. Here, we investigated the contribution of 5-HT receptors in a model of valve degeneration induced by nordexfenfluramine, the main metabolite of the anorexigens, dexfenfluramine and benfluorex. EXPERIMENTAL APPROACH: Nordexfenfluramine was infused chronically (28 days) in mice ((WT and transgenic Htr2B -/- , Htr2A -/- , and Htr2B/2A -/- ) to induce mitral valve lesions. Bone marrow transplantation was also carried out. Haemodynamics were measured with echocardiography; tissues and cells were analysed by histology, immunocytochemistry, flow cytometry and RT -qPCR. Samples of human prolapsed mitral valves were also analysed. KEY RESULTS: Chronic treatment of mice with nordexfenfluramine activated 5-HT2B receptors and increased valve thickness and cell density in a thick extracellular matrix, mimicking early steps of mitral valve remodelling. Lesions were prevented by 5-HT2A or 5-HT2B receptor antagonists and in transgenic Htr2B -/- or Htr2A/2B -/- mice. Surprisingly, valve lesions were mainly formed by numerous non-proliferative CD34+ endothelial progenitors. These progenitors originated from bone marrow (BM) as revealed by BM transplantation. The initial steps of mitral valve remodelling involved mobilization of BM-derived CD34+ CD31+ cells by 5-HT2B receptor stimulation. Analysis of human prolapsed mitral valves showing spontaneous degenerative lesions, demonstrated the presence of non-proliferating CD34+ /CD309+ /NOS3+ endothelial progenitors expressing 5-HT2B receptors. CONCLUSIONS AND IMPLICATIONS: BM-derived endothelial progenitor cells make a crucial contribution to the remodelling of mitral valve tissue. Our data describe a new and important mechanism underlying human VHD.

An activin receptor IIA ligand trap corrects ineffective erythropoiesis in ß-thalassemia.

The pathophysiology of ineffective erythropoiesis in ß-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of ß-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with ß-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in ß-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in ß-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT(2B), is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT(2B) receptors by circulating c-kit(+) precursor cells, whereas mice lacking 5-HT(2B) receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT(2B) receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT(2B) receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34(+) or mice c-kit(+) progenitor cells in the presence of a 5-HT(2B) receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT(2B) receptors on bone marrow lineage progenitors is critical for the development of PAH.

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice.

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.

Downregulation of basophil-derived IL-4 and in vivo T(H)2 IgE responses by serotonin and other organic cation transporter 3 ligands.

BACKGROUND: Murine basophils can contribute to the T(H)2 polarization of the immune response by providing rapidly large amounts of IL-4, which suggests that pharmacologic downregulation of this cytokine might provide a strategy to attenuate pathologies associated with excessive production. OBJECTIVE: We examined a number of physiological and pharmacologic ligands of the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines, for their inhibitory effect on IL-4 production by basophils, selecting the most efficient compounds for in vivo evaluation in basophil-dependent experimental models. METHODS: IL-4 production by basophils isolated ex vivo or from bone marrow cultures was assessed in response to various stimuli with or without biogenic monoamines or pharmacologic analogs. Selected compounds were administered in vivo to examine their effect on levels of circulating IgE generated during a basophil-dependent T(H)2 response and on basophil activation in mice receiving IL-33. RESULTS: We found a drastic decrease in IL-4 production by stimulated basophils on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by basophils through the specific high-affinity transporters serotonin transporter and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or Slc22a3) but inhibits their function exclusively through the latter. This downregulation is likewise observed in vivo in response to 5-HT and other OCT3 ligands, as well as in human basophils sorted from PMBCs of nonatopic donors. CONCLUSIONS: We provide evidence for a new means of downregulating IL-4 production by basophils, both in vitro and in vivo, through OCT3 targeted by 5-HT and pharmacologic ligands.

Essential roles of enteric neuronal serotonin in gastrointestinal motility and the development/survival of enteric dopaminergic neurons.

The gut contains a large 5-HT pool in enterochromaffin (EC) cells and a smaller 5-HT pool in the enteric nervous system (ENS). During development, enteric neurons are generated asynchronously. We tested hypotheses that serotonergic neurons, which arise early, affect development/survival of later-born dopaminergic, GABAergic, nitrergic, and calcitonin gene-related peptide-expressing neurons and are essential for gastrointestinal motility. 5-HT biosynthesis depends on tryptophan hydroxylase 1 (TPH1) in EC cells and on TPH2 in neurons; therefore, mice lacking TPH1 and/or TPH2 distinguish EC-derived from neuronal 5-HT. Deletion of TPH2, but not TPH1, decreased myenteric neuronal density and proportions of dopaminergic and GABAergic neurons but did not affect the extrinsic sympathetic innervation of the gut; intestinal transit slowed in mice lacking TPH2 mice, but gastric emptying accelerated. Isolated enteric crest-derived cells (ENCDCs) expressed the serotonin reuptake transporter (SERT) and 15 subtypes of 5-HT receptor. Addition of 5-HT to cultures of isolated ENCDCs promoted total and dopaminergic neuronal development. Rings of SERT-immunoreactive terminal axons surrounded myenteric dopaminergic neurons and SERT knock-out increased intestinal levels of dopamine metabolites, implying that enteric dopaminergic neurons receive a serotonergic innervation. Observations suggest that constitutive gastrointestinal motility depends more on neuronal than EC cell serotonin; moreover, serotonergic neurons promote development/survival of some classes of late-born enteric neurons, including dopaminergic neurons, which appear to innervate and activate in the adult ENS.

Serotonin activates dendritic cell function in the context of gut inflammation.

Mucosal inflammation in the gut is characterized by infiltration of innate and adaptive immune cells and by an alteration in serotonin-producing enterochromaffin cells. We investigated the role of serotonin in the function of dendritic cells (DCs) and sequential T-cell activation in relation to generation of gut inflammation. DCs isolated from tryptophan hydroxylase-1-deficient (TPH1(-/-)) mice, which have reduced serotonin in the gut, and wild-type (TPH1(+/+)) mice with or without dextran sulfate sodium (DSS)-induced colitis were stimulated with lipopolysaccharide to assess interleukin-12 (IL-12) production. Isolated DCs from TPH1(+/+) and TPH1(-/-) mice were also cocultured with CD4(+) T cells of naive TPH1(+/+) mice to assess the role of serotonin in priming T cells. In addition, serotonin-pulsed DCs were transferred to TPH1(-/-) mice to assess the effect on DSS-induced colitis. Consistent with a reduced severity of colitis, DCs from DSS-induced TPH1(-/-) mice produced less IL-12 compared with the TPH1(+/+) mice. In vitro serotonin stimulation restored the cytokine production from TPH1(-/-) DCs and adoptive transfer of serotonin-pulsed DCs into TPH1(-/-) up-regulated colitis. Furthermore, CD4(+) T cells primed by TPH1(-/-) DCs produce reduced the levels of IL-17 and interferon-γ. This study provides novel information on serotonin-mediated immune signaling and promotion of interactions between innate and adaptive immune responses in the context of gut inflammation, which may ultimately lead to improved strategies to combat gut inflammatory disorders.

Serotonin has a key role in pathogenesis of experimental colitis.

BACKGROUND & AIMS: Mucosal changes in inflammatory bowel disease are characterized by ulcerative lesions accompanied by a prominent infiltrate of immune cells as well as alteration in serotonin (5-hydroxytryptamine [5-HT])-producing enterochromaffin cells. We investigated the role of 5-HT in colonic inflammation in mice. METHODS: Colitis was induced with dextran sulfate sodium or dinitrobenzene sulfonic acid in tryptophan hydroxylase 1-deficient (TPH1(-/-)) mice, which have markedly reduced 5-HT in the gastrointestinal tract, and in mice given the 5-HT synthesis inhibitor parachlorophenylalanine. RESULTS: Delayed onset, decreased severity of clinical disease, and significantly lower macroscopic and histologic damage scores were observed in TPH1(-/-) mice, compared with wild-type mice, and in mice given parachlorophenylalanine after induction of colitis by dextran sulfate sodium. This was associated with down-regulation of macrophage infiltration and production of proinflammatory cytokines. 5-HT stimulated production of proinflammatory cytokines from macrophages collected from the peritoneal cavity of wild-type mice; this process was inhibited by a nuclear factor kappaB inhibitor, indicating a critical role for nuclear factor kappaB signaling in 5-HT-mediated activation of immune cells. Restoration of 5-HT levels in TPH1(-/-) mice by the 5-HT precursor 5-hydroxytryptophan increased the severity of DSS-induced colitis. We also observed significant reduction in severity of colitis in TPH1(-/-) mice after induction of dinitrobenzene sulfonic acid-induced colitis. CONCLUSIONS: 5-HT is involved in the pathogenesis of inflammation in experimental colitis. These findings provide insight into the mechanisms of gastrointestinal inflammation and could lead to new therapeutic strategies for inflammatory disorders.

Targeting conditional gene modification into the serotonin neurons of the dorsal raphe nucleus by viral delivery of the Cre recombinase.

Delivery of viral vectors encoding the Cre recombinase is showing promise to target gene modification in specific brain regions. Here we describe the targeting of the dorsal raphe nucleus (DRN), which contains the majority of the serotonin (5-HT) neurons projecting to the forebrain. First, we demonstrate successful transgene expression in the mouse DRN by stereotaxic delivery of the AdnlslacZ adenoviral vector. Second, we show that expression of the Cre recombinase can be achieved in the 5-HT neurons by optimized injection of the Adcre vector. Using reporter mice in which Cre activity induces beta-galactosidase (beta-gal) expression, we demonstrate efficient Cre-mediated recombination and persistence of beta-gal positive 5-HT neurons at least 1 month postinjection. Together, these results demonstrate that viral delivery provides a valuable method to target Cre recombination throughout the murine DRN and thus to study 5-HT neurotransmission by conditional gene modification.