Biallelic deleterious germline SH2B3 variants cause a novel syndrome of myeloproliferation and multi-organ autoimmunity

SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss-of-function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss-of-function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi-organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR-Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin-derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL-3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association for a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations

Detection of nucleophosmin and FMS-like tyrosine kinase-3 gene mutations in acute myeloid leukemia

Background and Objectives : Nucleophosmin gene mutations are frequently reported in acute myeloid leukemia (AML) patients with normal karyotype, which is also frequently associated with internal tandem duplication mutations in the FMS-like tyrosine kinase-3 gene. We sought to detect the nucleophosmin and FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutations among Iranian patients with AML and to assess the relationship between these mutations and the subtypes of the disease. Design and Setting : Cross-sectional study of patients referred during 2007 through 2009. Patients and Methods : Bone marrow and peripheral blood samples of 131 AML patients were randomly collected at the time of diagnosis and prior to treatment and the DNA extracted. After amplifying the nucleophosmin and FLT3 gene regions, positive cases were screened by conformation-sensitive gel electrophoresis and agarose gel electrophoresis techniques. Results : Of 131 patients, 23 (17.5%) (0.95% CI=0.107-0.244) had nucleophosmin gene mutations. The highest frequency of such mutations was found among the subtypes of M4 (30.4%), M3 (21.7%) and M5 (17.4%). There was a high frequency of these mutations in the M3 subtype as well as a high frequency of allele D in all subtypes. Also, 21 (16.0%) samples (0.95% CI=0.092-0.229) had FLT3/ITD mutation, of which 8 samples had mutant nucleophosmin (8 of 23, 35%), and another 13 samples had wild-type nucleophosmin gene (13 of 108, 12%). There was a high degree of association between the occurrence of nucleophosmin and FLT3/ITD mutations (P=.012). Conclusion : Our data showed a high frequency of NPM1 mutations in the monocytic subtypes of AML, as well as a high degree of association between the occurrence of NPM1 and FLT3/ITD mutations

A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life

Macrophages protect <i>Talaromyces marneffei</i> conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment <i>in vivo</i>

<div><p>Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen <i>Talaromyces marneffei</i> and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established <i>Aspergillus fumigatus</i> model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions <i>in vivo</i>. Following parenteral inoculation, <i>T</i>. <i>marneffei</i> conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in <i>T</i>. <i>marneffei</i> conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new <i>in vivo</i> model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting <i>T</i>. <i>marneffei’s</i> opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.</p></div

<i>T</i>. <i>marneffei</i> infection of zebrafish at 28°C.

<p>(A-C) Histological time-course following <i>T</i>. <i>marneffei</i> inoculation of zebrafish, stained by Grocott methanamine silver and Evan’s Blue counterstain (i), with adjacent hematoxylin and eosin-stained sections (ii). mpi, minutes post infection; dpi, days post infection. Red arrowheads indicate fungal conidia. White arrowheads indicate Duct of Cuvier (A) and leukocyte with intracellular fungal elements (C). (D) <i>T</i>. <i>marneffei</i> CFU time-course at 28°C following intravascular inoculation of target dose of approximately 150 fungal conidia (actual dose verified by 0 dpi CFU). Different colors indicate 3 independent experiments, mean±SEM, n≥5 embryos/group/experiment. *p = 0.016, **p = 0.0059, ***p = 0.0003 for statistical comparison between 3 and 4 dpi. (E) Embryo survival following intravascular inoculation in (D). Data are pooled embryos from 3 experiments: n = 474 uninfected, 339 infected. NS: not significant.</p

Neutrophils and macrophages play opposing roles during establishment of <i>T</i>. <i>marneffei</i> infection.

<p>(A) Representative images of 52 hpf <i>Tg(mpeg1</i>:<i>mCherry/mpx</i>:<i>EGFP)</i> embryos injected with antisense morpholino oligonucleotides to perturb the balance of neutrophil and macrophage populations. (B) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi corresponding to the aligned treatment groups in panels (Ai-iv), for wild-type (WT) and myeloperoxidase-deficient (<i>mpx</i>^-/-) genotypes as shown. Data are mean±SEM for n≥3 experiments, n≥5 embryos/group/experiment.</p

Depletion of the macrophage niche increases conidial destruction during establishment of infection.

<p>(A) Ablation of macrophages decreases conidial viability in the first 24 hpi. (i) and (ii) show representative images of nitroreductase-dependent metronidazole-mediated macrophage ablation following treatment of transgenic embryos (ii) versus untreated control embryos (i). Ablation efficiency is quantified in (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s010" target="_blank">S9A Fig</a>). (B) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi in macrophage-replete control (<i>Tg(mpeg1</i>:<i>Gal4FF/UAS-E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> negative, treated with 10 mM metronidazole) compared to macrophage-depleted (<i>Tg(mpeg1</i>:<i>Gal4FF/ UAS- E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> positive, treated with 10 mM metronidazole). (C) Ablation of neutrophils increases conidial viability in the first 24 hpi. (i) and (ii) show representative images of Nitroreductase-dependent metronidazole-mediated neutrophil ablation in treated (ii) versus diluent-treated control embryos (i). Ablation efficiency is quantified in (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007063#ppat.1007063.s010" target="_blank">S9B and S9C Fig</a>). (D) <i>T</i>. <i>marneffei</i> CFU numbers at 24 hpi in neutrophil-replete control (<i>Tg(mpx</i>:<i>KalTA4/UAS-E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> negative, treated with 10 mM metronidazole) compared to neutrophil-depleted (<i>Tg(mpx</i>:<i>KalTA4/ UAS- E1b</i>:<i>Eco</i>.<i>nfsB-mCherry)</i> positive, treated with 10 mM metronidazole) Data are mean±SEM, n≥5 embryos/group/experiment, n≥3 experiments.</p