Investigation and Management of Endocrinopathies in Thalassaemia Major

A combination of sub-therapeutic chelation and subsequent iron overload are regarded as the principal drivers of endocrine dysfunction in thalassaemia. The clinical presentation of endocrine complications and their timing of onset can be highly variable, in part due to population heterogeneity but also variation in chelation strategies. Endocrinopathies commonly associated with thalassaemia include: growth delay; pubertal delay; gonadal dysfunction; thyroid disorders; parathyroid and adrenal gland impairment; impaired bone metabolism; and type 2 diabetes mellitus. In this chapter we summarise the main presentations of endocrine disorder in thalassaemia, summarising their epidemiology, clinical presentation and pathophysiologic basis. Furthermore, we review screening, monitoring and treatment strategies, with particular regard to the UK Thalassaemia Society’s 2016 National Standards

Hepcidin is regulated by promoter-associated histone acetylation and HDAC3.

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency

Hepcidin is regulated by promoter-associated histone acetylation and HDAC3

Hepcidin regulates systemic iron homeostasis. Suppression of hepcidin expression occurs physiologically in iron deficiency and increased erythropoiesis but is pathologic in thalassemia and hemochromatosis. Here we show that epigenetic events govern hepcidin expression. Erythropoiesis and iron deficiency suppress hepcidin via erythroferrone-dependent and -independent mechanisms, respectively, in vivo, but both involve reversible loss of H3K9ac and H3K4me3 at the hepcidin locus. In vitro, pan-histone deacetylase inhibition elevates hepcidin expression, and in vivo maintains H3K9ac at hepcidin-associated chromatin and abrogates hepcidin suppression by erythropoietin, iron deficiency, thalassemia, and hemochromatosis. Histone deacetylase 3 and its cofactor NCOR1 regulate hepcidin; histone deacetylase 3 binds chromatin at the hepcidin locus, and histone deacetylase 3 knockdown counteracts hepcidin suppression induced either by erythroferrone or by inhibiting bone morphogenetic protein signaling. In iron deficient mice, the histone deacetylase 3 inhibitor RGFP966 increases hepcidin, and RNA sequencing confirms hepcidin is one of the genes most differentially regulated by this drug in vivo. We conclude that suppression of hepcidin expression involves epigenetic regulation by histone deacetylase 3.Hepcidin controls systemic iron levels by inhibiting intestinal iron absorption and iron recycling. Here, Pasricha et al. demonstrate that the hepcidin-chromatin locus displays HDAC3-mediated reversible epigenetic modifications during both erythropoiesis and iron deficiency

Antiviral activity of bone morphogenetic proteins and activins

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN

Antiviral functions of bone morphogenetic proteins and the activins

Previous work in the Drakesmith lab has revealed a novel anti-HCV function of bone morphogenetic protein 6 (BMP6), a TGF&beta;-superfamily cytokine unrelated to type I IFN. Recombinant BMP6 is antiviral against both replication-competent HCV and a full-length genomic replicon model. Data presented in this thesis demonstrate that an anti-HCV effect extends to multiple BMPs and segregates with ability to ligate the type I BMP receptor. Canonically, the type I BMP receptor signals intracellularly via phosphorylation of SMAD1/5/8 transcription factors. Prior work in the lab shows that BMP6 exerts both type I IFN-dependent and type I IFN-independent antiviral effects. In terms of delineating mechanistic basis for the latter, we have formulated a model whereby BMP6 induces cell cycle arrest in phases characterized by reduced cytosolic nucleotide availability, and which are therefore less permissive to viral replication. A recent report indicates that another TGF&beta;-type cytokine, activin B, is able to signal through a nonclassical type I BMP receptor dependent mechanism. Activin A and B have multiple established roles in innate immunity and inflammatory responses. However, no direct link between activin A and B and the early response to viral infection has been described. Given their "immune precedent" within the literature, and their high level of structural and phylogenetic homology to the BMPs, both activin A and B represented promising candidates to explore for an antiviral effect. Our data indicate that activin A mRNA, encoded by the INHBA gene, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and also in PBMCs. In vitro infection of A549 lung adenocarcinoma-derived cells and Huh7 hepatoma-derived cells with the murine paramyxovirus Sendai Virus also elicits robust INHBA induction. In vitro dengue virus infection also elicits INHBA upregulation by Huh7.5 hepatoma cells. In vivo, infection of mice with influenza A PR8 also elicits induction of activin A message within the lung. Treatment of Huh7 cells with activin A increases transcription of multiple type I IFN transduction elements; moreover, co-incubation of Huh7 cells with IFN&alpha; and either activin A or B augments transcriptional induction of key anti-HCV enzymes. This boosting of type I IFN extends to a functional enhancement: activin A elicits a synergistic, dose-dependent enhancement of both type I and type III IFNâs antiviral effect against a full-length HCV genomic replicon. In a full-length genomic replicon model of HCV, both activin A and B alone exert a potent, dose-dependent antiviral effect that is contingent upon signalling via type I BMP receptor. A component of the activins' antiviral effect does not require intact type I IFN signalling. A small-molecule inhibitor of signalling downstream of type I IFN receptor blocks the anti-HCV effect of IFN&alpha; but does not impair the antiviral effects of activin A. Both BMP6 and activin A exert dose-dependent antiviral effects against Hepatitis B Virus infection in vitro. Of note, SMAD1/5/8-binding sites have been identified in the promoter sequences of multiple antiviral Interferon Stimulated Genes (ISG), providing a possible route for the enhancement of ISG induction by the SMAD1/5/8 axis. Furthermore, strong topological homology exists between of the transactivation domains of the SMADs and Interferon Response Factors (IRF), which postulated to have diverged from a common ancestor in early metazoans. Preliminary bioinformatic analyses reveal striking parallels between the genome-wide binding profiles of activated SMAD1 and IRF1, including proximal to genes encoding antiviral effectors. The observations presented in this study may represent the first characterization of a non-IFN intracellular antiviral response in human cells, with implications for the development of targeted therapies against diverse viral diseases. Moreover, these data reveal a novel facet of activin biology, in addition to in part elucidating the nature of the genomic interactions between BMP-SMAD and IFNIRF signalling.</p

Antiviral functions of bone morphogenetic proteins and the activins

Previous work in the Drakesmith lab has revealed a novel anti-HCV function of bone morphogenetic protein 6 (BMP6), a TGFβ-superfamily cytokine unrelated to type I IFN. Recombinant BMP6 is antiviral against both replication-competent HCV and a full-length genomic replicon model. Data presented in this thesis demonstrate that an anti-HCV effect extends to multiple BMPs and segregates with ability to ligate the type I BMP receptor. Canonically, the type I BMP receptor signals intracellularly via phosphorylation of SMAD1/5/8 transcription factors. Prior work in the lab shows that BMP6 exerts both type I IFN-dependent and type I IFN-independent antiviral effects. In terms of delineating mechanistic basis for the latter, we have formulated a model whereby BMP6 induces cell cycle arrest in phases characterized by reduced cytosolic nucleotide availability, and which are therefore less permissive to viral replication. A recent report indicates that another TGFβ-type cytokine, activin B, is able to signal through a nonclassical type I BMP receptor dependent mechanism. Activin A and B have multiple established roles in innate immunity and inflammatory responses. However, no direct link between activin A and B and the early response to viral infection has been described. Given their "immune precedent" within the literature, and their high level of structural and phylogenetic homology to the BMPs, both activin A and B represented promising candidates to explore for an antiviral effect. Our data indicate that activin A mRNA, encoded by the INHBA gene, is induced upon activation of RIG-I, MDA5 and TLR7/8 viral nucleic acid sensors in vitro, across multiple cell lines and also in PBMCs. In vitro infection of A549 lung adenocarcinoma-derived cells and Huh7 hepatoma-derived cells with the murine paramyxovirus Sendai Virus also elicits robust INHBA induction. In vitro dengue virus infection also elicits INHBA upregulation by Huh7.5 hepatoma cells. In vivo, infection of mice with influenza A PR8 also elicits induction of activin A message within the lung. Treatment of Huh7 cells with activin A increases transcription of multiple type I IFN transduction elements; moreover, co-incubation of Huh7 cells with IFNα and either activin A or B augments transcriptional induction of key anti-HCV enzymes. This boosting of type I IFN extends to a functional enhancement: activin A elicits a synergistic, dose-dependent enhancement of both type I and type III IFN’s antiviral effect against a full-length HCV genomic replicon. In a full-length genomic replicon model of HCV, both activin A and B alone exert a potent, dose-dependent antiviral effect that is contingent upon signalling via type I BMP receptor. A component of the activins' antiviral effect does not require intact type I IFN signalling. A small-molecule inhibitor of signalling downstream of type I IFN receptor blocks the anti-HCV effect of IFNα but does not impair the antiviral effects of activin A. Both BMP6 and activin A exert dose-dependent antiviral effects against Hepatitis B Virus infection in vitro. Of note, SMAD1/5/8-binding sites have been identified in the promoter sequences of multiple antiviral Interferon Stimulated Genes (ISG), providing a possible route for the enhancement of ISG induction by the SMAD1/5/8 axis. Furthermore, strong topological homology exists between of the transactivation domains of the SMADs and Interferon Response Factors (IRF), which postulated to have diverged from a common ancestor in early metazoans. Preliminary bioinformatic analyses reveal striking parallels between the genome-wide binding profiles of activated SMAD1 and IRF1, including proximal to genes encoding antiviral effectors. The observations presented in this study may represent the first characterization of a non-IFN intracellular antiviral response in human cells, with implications for the development of targeted therapies against diverse viral diseases. Moreover, these data reveal a novel facet of activin biology, in addition to in part elucidating the nature of the genomic interactions between BMP-SMAD and IFNIRF signalling.</p