Stat3 Regulates Developmental Hematopoiesis and Impacts Myeloid Cell Function via Canonical and Non-Canonical Modalities.

INTRODUCTION: Signal transducer and activator of transcription (STAT) 3 is extensively involved in the development, homeostasis, and function of immune cells, with STAT3 disruption associated with human immune-related disorders. The roles ascribed to STAT3 have been assumed to be due to its canonical mode of action as an inducible transcription factor downstream of multiple cytokines, although alternative noncanonical functional modalities have also been identified. The relative involvement of each mode was further explored in relevant zebrafish models. METHODS: Genome editing with CRISPR/Cas9 was used to generate mutants of the conserved zebrafish Stat3 protein: a loss of function knockout (KO) mutant and a mutant lacking C-terminal sequences including the transactivation domain (ΔTAD). Lines harboring these mutations were analyzed with respect to blood and immune cell development and function in comparison to wild-type zebrafish. RESULTS: The Stat3 KO mutant showed perturbation of hematopoietic lineages throughout primitive and early definitive hematopoiesis. Neutrophil numbers did not increase in response to lipopolysaccharide (LPS) or granulocyte colony-stimulating factor (G-CSF) and their migration was significantly diminished, the latter correlating with abrogation of the Cxcl8b/Cxcr2 pathway, with macrophage responses perturbed. Intriguingly, many of these phenotypes were not shared by the Stat3 ΔTAD mutant. Indeed, only neutrophil and macrophage development were disrupted in these mutants with responsiveness to LPS and G-CSF maintained, and neutrophil migration actually increased. CONCLUSION: This study has identified roles for zebrafish Stat3 within hematopoietic stem cells impacting multiple lineages throughout primitive and early definitive hematopoiesis, myeloid cell responses to G-CSF and LPS and neutrophil migration. Many of these roles showed conservation, but notably several involved noncanonical modalities, providing additional insights for relevant diseases.

Janus Kinase 3 (JAK3): A Critical Conserved Node in Immunity Disrupted in Immune Cell Cancer and Immunodeficiency.

The Janus kinase (JAK) family is a small group of protein tyrosine kinases that represent a central component of intracellular signaling downstream from a myriad of cytokine receptors. The JAK3 family member performs a particularly important role in facilitating signal transduction for a key set of cytokine receptors that are essential for immune cell development and function. Mutations that impact JAK3 activity have been identified in a number of human diseases, including somatic gain-of-function (GOF) mutations associated with immune cell malignancies and germline loss-of-function (LOF) mutations associated with immunodeficiency. The structure, function and impacts of both GOF and LOF mutations of JAK3 are highly conserved, making animal models highly informative. This review details the biology of JAK3 and the impact of its perturbation in immune cell-related diseases, including relevant animal studies.

Immune Factors, Immune Cells and Inflammatory Diseases.

The immune system comprises distinct innate and adaptive arms, each of which contains many layers to provide a coordinated, sequential immune response to insults [...].

Myeloproliferative Neoplasms: Diseases Mediated by Chronic Activation of Signal Transducer and Activator of Transcription (STAT) Proteins.

Myeloproliferative neoplasms (MPNs) are hematopoietic diseases characterized by the clonal expansion of single or multiple lineages of differentiated myeloid cells that accumulate in the blood and bone marrow. MPNs are grouped into distinct categories based on key clinical presentations and distinctive mutational hallmarks. These include chronic myeloid leukemia (CML), which is strongly associated with the signature BCR::ABL1 gene translocation, polycythemia vera (PV), essential thrombocythemia (ET), and primary (idiopathic) myelofibrosis (PMF), typically accompanied by molecular alterations in the JAK2, MPL, or CALR genes. There are also rarer forms such as chronic neutrophilic leukemia (CNL), which involves mutations in the CSF3R gene. However, rather than focusing on the differences between these alternate disease categories, this review aims to present a unifying molecular etiology in which these overlapping diseases are best understood as disruptions of normal hematopoietic signaling: specifically, the chronic activation of signaling pathways, particularly involving signal transducer and activator of transcription (STAT) transcription factors, most notably STAT5B, leading to the sustained stimulation of myelopoiesis, which underpins the various disease sequalae.

Cish knockout mice exhibit similar outcomes to malaria infection despite altered hematopoietic responses.

The Cytokine-inducible Src homology 2 domain-containing (CISH) protein is a negative feedback regulator induced by cytokines that play key roles in immunity and erythropoiesis. Single nucleotide polymorphisms (SNPs) in the human CISH gene have been associated with increased susceptibility to severe malaria disease. To directly assess how CISH might influence outcomes in the BALB/c model of malaria anemia, CISH knockout (Cish-/-) mice on this background were infected with Plasmodium berghei and their hematopoietic responses, cytokine production and ability to succumb to severe malaria disease evaluated. Despite basal erythrocytic disruption, upon P. berghei infection, the Cish -/- mice were better able to maintain peripheral blood cell counts, hemoglobin levels and a steady-state pattern of erythroid differentiation compared to wild-type (Cish+/+) mice. Ablation of CISH, however, did not influence the outcome of acute malaria infections in either the BALB/c model or the alternative C57BL/6 model of experimental cerebral malaria, with the kinetics of infection, parasite load, weight loss and cytokine responses being similar between Cish+/+ and Cish-/- mice, and both genotypes succumbed to experimental cerebral malaria within a comparable timeframe.

Role of Cytokine-Inducible SH2 Domain-Containing (CISH) Protein in the Regulation of Erythropoiesis.

The cytokine-inducible SH2 domain-containing (CISH) protein was the first member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators discovered, being identified in vitro as an inducible inhibitor of erythropoietin (EPO) signaling. However, understanding of the physiological role played by CISH in erythropoiesis has remained limited. To directly assess the function of CISH in this context, mice deficient in CISH were characterized with respect to developmental, steady-state, and EPO-induced erythropoiesis. CISH was strongly expressed in the fetal liver, but CISH knockout (KO) mice showed only minor disruption of primitive erythropoiesis. However, adults exhibited mild macrocytic anemia coincident with subtle perturbation particularly of bone marrow erythropoiesis, with EPO-induced erythropoiesis blunted in the bone marrow of KO mice but enhanced in the spleen. Cish was expressed basally in the bone marrow with induction following EPO stimulation in bone marrow and spleen. Overall, this study indicates that CISH participates in the control of both basal and EPO-induced erythropoiesis in vivo.

Analysis of Potential Non-Canonical or Alternate STAT5 Functions in Immune Development and Growth.

BACKGROUND: Signal transducer and activator of transcription (STAT) proteins play key roles in development, growth, and homeostasis. These roles have principally been assigned to their "canonical" function as inducible transcriptional activators acting downstream of cytokines and other factors. However, variant "non-canonical" functions have also been identified. The potential in vivo role for non-canonical STAT functions was investigated in the zebrafish model. METHODS: Two zebrafish Stat5.1 mutants were generated using CRISPR/Cas9 that should impact canonical functionality: one with a deleted transactivation domain (ΔTAD) and another with a disrupted tyrosine motif (ΔTM). Immune cell development, growth, and adiposity of these Stat5.1 mutants were assessed in comparison to a Stat5.1 knockout (KO) mutant in which both canonical and non-canonical functions were ablated. RESULTS: Both the ΔTAD and ΔTM mutants showed significantly reduced embryonic T lymphopoiesis, similar to the KO mutant. Additionally, adult ΔTAD and ΔTM mutants displayed a decrease in T cell markers in the kidney, but not as severe as the KO, which also showed T cell disruption in the spleen. Severe growth deficiency and increased adiposity were observed in all mutants, but ΔTAD showed a more modest growth defect whereas ΔTM exhibited more profound impacts on both growth and adiposity, suggesting additional gain-of-function activity. CONCLUSIONS: These results indicate that canonical Stat5.1 plays a major role in T cell development and growth throughout the lifespan and non-canonical Stat5.1 functions also contribute to aspects of adult T lymphocyte development and growth, with alternate functions impacting growth and adiposity.

The Role of Cytokine-Inducible SH2 Domain-Containing Protein (CISH) in the Regulation of Basal and Cytokine-Mediated Myelopoiesis.

Cytokine-inducible SH2 domain-containing protein (CISH) is a member of the suppressor of cytokine signaling (SOCS) family of negative feedback regulators shown to play crucial roles in lymphoid cell development and function as well as appetite regulation. It has also been implicated in the control of signaling downstream of the receptors for the cytokines granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in myeloid cells. To investigate the physiological role of CISH in myelopoiesis, mice deficient in CISH were analyzed basally and in response to administration of these cytokines. CISH knockout (KO) mice possessed basally elevated neutrophils in the blood, bone marrow, and spleen compared to wild-type (WT) mice. During GM-CSF-induced myelopoiesis, the frequency of neutrophils, myeloid dendritic cells (DCs), and CFU-M in the bone marrow was higher in the KO, as were the neutrophils and CFU-G in the spleen. In contrast, no differences were observed between KO and WT mice during G-CSF-induced myelopoiesis apart from an elevated frequency of CFU-G and CFU-M in the spleen. This work has identified a role for CISH in the negative regulation of granulopoiesis, including that mediated by GM-CSF.

Cytokine Receptors in Development, Homeostasis and Disease.

This Special Issue represents a collective celebration of the cytokine receptor superfamily and the myriad of functions mediated by these important molecules in development and homeostasis, as well as their disruption in disease [...].

Development of the national priority assistive product list in Malawi.

PURPOSE: In 2016 WHO launched the priority assistive products list (APL) consisting of 50 products and recommended that using this as a reference, countries should develop their own contextually relevant national APLs. This paper describes the development of Malawi's APL. METHODS: Two hundred and ninety-six persons with disabilities participated in a rapid Assistive Technology Assessment (rATA) survey. Six focus group discussions (FGDs) with people with various types of disabilities were conducted. The rATA questionnaire and FGDs collected data on assistive products (APs) participants used, APs they needed and the challenges they experienced. Data collection was done in six districts spread across the three regions in Malawi. All age groups were included in the survey. Persons with disabilities aged less than 18 participated but went with their guardians. All persons who participated in this study provided consent. Survey and FGDs results were presented at an APL consensus meeting with policymakers, service providers, disabled peoples' organizations and development partners in the disability sector. Based on the results and further discussions, a consensus was reached on the priority APs for Malawi. RESULTS: More than a third of respondents used wheelchairs (32%), followed by auxiliary crutches (25%), walking sticks (13%), reading glasses (11%), prosthesis (10%), elbow crutches (9%) and orthosis (8%). There is also a high demand for products such as pull-up underwear (incontinence products) (79%), hearing aids (70%), reading glasses (59%) and diapers (63%). After intensive discussions during a consensus meeting, an agreement was reached on the 22 priority APs for Malawi. CONCLUSION: There is a wide range of APs being used by people with different functional limitations in Malawi. There is also a demand for APs that are not readily available. When developing an APL, the list should include products in use, those in demand, and those recommended by service providers.Implications for rehabilitationFollowing the development of the priority assistive products list (APL) by WHO, member states should develop their own contextually based APL.The development of the APL should be based on research evidence.All key stakeholders including persons with disabilities and other functional limitations, government, and development partners should participate in this process.The APL should be part of the national health system or community services.The Department of Disability and Elderly Affairs in the Ministry of Gender, Community Development, being the Government of Malawi line ministry coordinating disability issues participated actively in this study including inviting participants in the stakeholders' validation workshop.

Correlates of intimate partner violence among pregnant and parenting adolescents: a cross-sectional household survey in Blantyre District, Malawi.

BACKGROUND: Despite efforts from the government and developmental partners to eliminate gender-based violence, intimate partner violence (IPV) remains a pervasive global health and human rights problem, affecting up to 753 million women and girls globally. Few studies on IPV have focused on pregnant and parenting adolescent (PPA) girls in Africa, although the region has the highest rates of adolescent childbearing. This limited attention results in the neglect of pregnant and parenting adolescents in policies and interventions addressing IPV in the region. Our study examined IPV prevalence and its individual, household, and community-level correlates among pregnant and parenting adolescent girls (10-19 years) in Blantyre District, Malawi. METHODS: We collected data from a cross-section of pregnant and parenting adolescent girls (n = 669) between March and May 2021. The girls responded to questions on socio-demographic and household characteristics, lifetime experience of IPV (i.e., sexual, physical, and emotional violence), and community-level safety nets. We used multilevel mixed-effect logistic regression models to examine the individual, household, and community-level factors associated with IPV. RESULTS: The lifetime prevalence of IPV was 39.7% (n = 266), with more girls reporting emotional (28.8%) than physical (22.2%) and sexual (17.4%) violence. At the individual level, girls with secondary education (AOR: 1.72; 95% CI: 1.16-2.54), who engaged in transactional sex (AOR: 2.29; 95% CI: 1.35-3.89), and accepted wife-beating (AOR: 1.97; 95% CI: 1.27-3.08) were significantly more likely to experience IPV compared to those with no education/primary education, who never engaged in transactional sex and rejected wife beating. Girls aged 19 (AOR: 0.49; 95% CI: 0.27-0.87) were less likely to report IPV than those aged 13-16. At the household level, girls with fair and poor partner support had higher odds of experiencing IPV, but the effect size did not reach a significant level in the parsimonious model. A high perception of neighborhood safety was associated with a lower likelihood of experiencing IPV (AOR: 0.81; 95% CI: 0.69-0.95). CONCLUSION: Intimate partner violence is rife among pregnant and parenting adolescent girls in Malawi, underscoring the need for appropriate interventions to curb the scourge. Interventions addressing IPV need to target younger adolescents, those engaging in transactional sex, and those having weaker community-level safety nets. Interventions to change social norms that drive the acceptance of gender-based violence are also warranted.

Zebrafish: A Relevant Genetic Model for Human Primary Immunodeficiency (PID) Disorders?

Primary immunodeficiency (PID) disorders, also commonly referred to as inborn errors of immunity, are a heterogenous group of human genetic diseases characterized by defects in immune cell development and/or function. Since these disorders are generally uncommon and occur on a variable background profile of potential genetic and environmental modifiers, animal models are critical to provide mechanistic insights as well as to create platforms to underpin therapeutic development. This review aims to review the relevance of zebrafish as an alternative genetic model for PIDs. It provides an overview of the conservation of the zebrafish immune system and details specific examples of zebrafish models for a multitude of specific human PIDs across a range of distinct categories, including severe combined immunodeficiency (SCID), combined immunodeficiency (CID), multi-system immunodeficiency, autoinflammatory disorders, neutropenia and defects in leucocyte mobility and respiratory burst. It also describes some of the diverse applications of these models, particularly in the fields of microbiology, immunology, regenerative biology and oncology.

Socs3b regulates the development and function of innate immune cells in zebrafish.

Introduction: Suppressor of cytokine signaling 3 (SOCS3) is a critical component of the negative feedback regulation that controls signaling by cytokines and other factors thereby ensuring that important processes such as hematopoiesis and inflammation occur at appropriate levels. Methods: To gain further insights into SOCS3 function, the zebrafish socs3b gene was investigated through analysis of a knockout line generated using CRISPR/Cas9-mediated genome editing. Results: Zebrafish socs3b knockout embryos displayed elevated numbers of neutrophils during primitive and definitive hematopoiesis but macrophage numbers were not altered. However, the absence of socs3b reduced neutrophil functionality but enhanced macrophage responses. Adult socs3b knockout zebrafish displayed reduced survival that correlated with an eye pathology involving extensive infiltration of neutrophils and macrophages along with immune cell dysregulation in other tissues. Discussion: These findings identify a conserved role for Socs3b in the regulation of neutrophil production and macrophage activation.

A zebrafish model of growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1).

Signal transducer and activator of transcription (STAT) proteins act downstream of cytokine receptors to facilitate changes in gene expression that impact a range of developmental and homeostatic processes. Patients harbouring loss-of-function (LOF) STAT5B mutations exhibit postnatal growth failure due to lack of responsiveness to growth hormone as well as immune perturbation, a disorder called growth hormone insensitivity syndrome with immune dysregulation 1 (GHISID1). This study aimed to generate a zebrafish model of this disease by targeting the stat5.1 gene using CRISPR/Cas9 and characterising the effects on growth and immunity. The zebrafish Stat5.1 mutants were smaller, but exhibited increased adiposity, with concomitant dysregulation of growth and lipid metabolism genes. The mutants also displayed impaired lymphopoiesis with reduced T cells throughout the lifespan, along with broader disruption of the lymphoid compartment in adulthood, including evidence of T cell activation. Collectively, these findings confirm that zebrafish Stat5.1 mutants mimic the clinical impacts of human STAT5B LOF mutations, establishing them as a model of GHISID1.

Signal Transducer and Activator of Transcription Proteins at the Nexus of Immunodeficiency, Autoimmunity and Cancer.

The signal transducer and activator of transcription (STAT) family of proteins has been demonstrated to perform pivotal roles downstream of a myriad of cytokines, particularly those that control immune cell production and function. This is highlighted by both gain-of-function (GOF) and loss-of-function (LOF) mutations being implicated in various diseases impacting cells of the immune system. These mutations are typically inherited, although somatic GOF mutations are commonly observed in certain immune cell malignancies. This review details the growing appreciation of STAT proteins as a key node linking immunodeficiency, autoimmunity and cancer.

Contribution of Signal Transducer and Activator of Transcription 3 (STAT3) to Bone Development and Repair.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated canonically by numerous cytokines and other factors, with significant roles in immunity, immune diseases, and cancer. It has also been implicated in several human skeletal disorders, with loss-of-function (LOF) mutations associated with aberrant skeletal development. To gain further insights, two zebrafish STAT3 lines were investigated: a complete LOF knockout (KO) mutant and a partial LOF mutant with the transactivation domain truncated (ΔTAD). Consistent with other studies, the KO mutants were smaller, with reduced length in early embryos exacerbated by a decreased growth rate from 5 days postfertilization (dpf). They displayed skeletal deformities that approached 80% incidence by 30 dpf, with a significant reduction in early bone but not cartilage formation. Further analysis additionally identified considerable abrogation of caudal fin regeneration, concomitant with a paucity of infiltrating macrophages and neutrophils, which may be responsible for this. Most of these phenotypes were also observed in the ΔTAD mutants, indicating that loss of canonical STAT3 signaling was the likely cause. However, the impacts on early bone formation and regeneration were muted in the ΔTAD mutant, suggesting the potential involvement of noncanonical functions in these processes.

B cell lymphoma 6A regulates immune development and function in zebrafish.

BCL6A is a transcriptional repressor implicated in the development and survival of B and T lymphoctyes, which is also highly expressed in many non-Hodgkin's lymphomas, such as diffuse large B cell lymphoma and follicular lymphoma. Roles in other cell types, including macrophages and non-hematopoietic cells, have also been suggested but require further investigation. This study sought to identify and characterize zebrafish BCL6A and investigate its role in immune cell development and function, with a focus on early macrophages. Bioinformatics analysis identified a homologue for BCL6A (bcl6aa), as well as an additional fish-specific duplicate (bcl6ab) and a homologue for the closely-related BCL6B (bcl6b). The human BCL6A and zebrafish Bcl6aa proteins were highly conserved across the constituent BTB/POZ, PEST and zinc finger domains. Expression of bcl6aa during early zebrafish embryogenesis was observed in the lateral plate mesoderm, a site of early myeloid cell development, with later expression seen in the brain, eye and thymus. Homozygous bcl6aa mutants developed normally until around 14 days post fertilization (dpf), after which their subsequent growth and maturation was severely impacted along with their relative survival, with heterozygous bcl6aa mutants showing an intermediate phenotype. Analysis of immune cell development revealed significantly decreased lymphoid and macrophage cells in both homozygous and heterozygous bcl6aa mutants, being exacerbated in homozygous mutants. In contrast, the number of neutrophils was unaffected. Only the homozygous bcl6aa mutants showed decreased macrophage mobility in response to wounding and reduced ability to contain bacterial infection. Collectively, this suggests strong conservation of BCL6A across evolution, including a role in macrophage biology.

Zebrafish Model of Severe Combined Immunodeficiency (SCID) Due to JAK3 Mutation.

JAK3 is principally activated by members of the interleukin-2 receptor family and plays an essential role in lymphoid development, with inactivating JAK3 mutations causing autosomal-recessive severe combined immunodeficiency (SCID). This study aimed to generate an equivalent zebrafish model of SCID and to characterize the model across the life-course. Genome editing of zebrafish jak3 created mutants similar to those observed in human SCID. Homozygous jak3 mutants showed reduced embryonic T lymphopoiesis that continued through the larval stage and into adulthood, with B cell maturation and adult NK cells also reduced and neutrophils impacted. Mutant fish were susceptible to lymphoid leukemia. This model has many of the hallmarks of human SCID resulting from inactivating JAK3 mutations and will be useful for a variety of pre-clinical applications.

Phylogenetic and Expression Analysis of Fos Transcription Factors in Zebrafish.

Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer.

Cytokine Receptor-Like Factor 3 (CRLF3) Contributes to Early Zebrafish Hematopoiesis.

Cytokine receptor-like factor 3 (CRLF3) is an ancient protein conserved across metazoans that contains an archetypal cytokine receptor homology domain (CHD). This domain is found in cytokine receptors present in bilateria, including higher vertebrates, that play key roles in a variety of developmental and homeostatic processes, particularly relating to blood and immune cells. However, understanding of CRLF3 itself remains very limited. This study aimed to investigate this evolutionarily significant protein by studying its embryonic expression and function in early development, particularly of blood and immune cells, using zebrafish as a model. Expression of crlf3 was identified in mesoderm-derived tissues in early zebrafish embryos, including the somitic mesoderm and both anterior and posterior lateral plate mesoderm. Later expression was observed in the thymus, brain, retina and exocrine pancreas. Zebrafish crlf3 mutants generated by genome editing technology exhibited a significant reduction in primitive hematopoiesis and early definitive hematopoiesis, with decreased early progenitors impacting on multiple lineages. No other obvious phenotypes were observed in the crlf3 mutants.