A JAGN1-associated severe congenital neutropenia zebrafish model revealed an altered G-CSFR signaling and UPR activation.

ABSTRACT: A variety of autosomal recessive mutations in the JAGN1 gene cause severe congenital neutropenia (CN). However, the underlying pathomechanism remains poorly understood, mainly because of the limited availability of primary hematopoietic stem cells from JAGN1-CN patients and the absence of animal models. In this study, we aimed to address these limitations by establishing a zebrafish model of JAGN1-CN. We found 2 paralogs of the human JAGN1 gene, namely jagn1a and jagn1b, which play distinct roles during zebrafish hematopoiesis. Using various approaches such as morpholino-based knockdown, CRISPR/Cas9-based gene editing, and misexpression of a jagn1b harboring a specific human mutation, we successfully developed neutropenia while leaving other hematopoietic lineages unaffected. Further analysis of our model revealed significant upregulation of apoptosis and genes involved in the unfolded protein response (UPR). However, neither UPR nor apoptosis is the primary mechanism that leads to neutropenia in zebrafish. Instead, Jagn1b has a critical role in granulocyte colony-stimulating factor receptor signaling and steady-state granulopoiesis, shedding light on the pathogenesis of neutropenia associated with JAGN1 mutations. The establishment of a zebrafish model for JAGN1-CN represents a significant advancement in understanding the specific pathologic pathways underlying the disease. This model provides a valuable in vivo tool for further investigation and exploration of potential therapeutic strategies.

TH1 cytokines induce senescence in AML.

Cancer testis antigen PRAME is over-expressed in a variety of malignant cells but is not or minimally expressed in normal non-germ line cells. Adoptive transfer of PRAME-specific T cells is thus under investigation in clinical trials as an innovative therapeutic option for acute myeloid leukemia (AML). However, their senescence-inducing activity has not been studied. This study therefore examines senescence induction in AML cells by PRAME-specific TH1 cells. Analysis of cell cycle and marker expression demonstrate that the supernatants of antigen-stimulated PRAME-specific TH1 cells induce senescence in AML cell lines Kasumi and Nomo-1 through combinative IFN-γ and TNF-α. Additionally IFN-γ and TNF-α secreted by TCR-activated Vδ2+ or CMV-specific T cells can also drive these AML cell lines into terminal growth arrest. G1/0 arrest is also suggested in patient-derived AML by TH1 cytokines or supernatants from Zoledronate-stimulated or aCD3/aCD28-stimulated PBMCs. Thus, we show for the first time that senescence is induced in AML cells by combined IFN-γ and TNF-α, and that these cytokines can be derived either from TCR-engineered CD4+ T cells, or intriguingly from Virus-specific as well as innate Vδ2+ T cells responding to their cognate antigens, namely T-cell responses targeting an antigen that is NOT expressed by the leukemic cells.

A zebrafish model for HAX1-associated congenital neutropenia.

Severe congenital neutropenia (CN) is a rare heterogeneous group of diseases, characterized by a granulocytic maturation arrest. Autosomal recessive mutations in the HAX1 gene are frequently detected in affected individuals. However, the precise role of HAX1 during neutrophil differentiation is poorly understood. To date, no reliable animal model has been established to study HAX1-associated CN. Here we show that knockdown of zebrafish hax1 impairs neutrophil development without affecting other myeloid cells and erythrocytes. Furthermore, we have found that interference with the Hax1 function decreases the expression level of key target genes of the granulocyte-colony stimulating factor (G-CSF) signaling pathway. The reduced neutrophil numbers in the morphants could be reversed by G-CSF, which is also the main therapeutic intervention for patients who have CN. Our results demonstrate that zebrafish is a suitable model for HAX1-associated neutropenia. We anticipate that this model will serve as an in vivo platform to identify new avenues for developing tailored therapeutic strategies for CN patients, particularly for those individuals that do not respond to the G-CSF treatment.