Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

ETV6::ACSL6 translocation-driven super-enhancer activation leads to eosinophilia in acute lymphoblastic leukemia through IL-3 overexpression.

ETV6::ACSL6 represents a rare genetic aberration in hematopoietic neoplasms and is often associated with severe eosinophilia, which confers an unfavorable prognosis requiring additional anti-inflammatory treatment. However, since the translocation is unlikely to produce a fusion protein, the mechanism of ETV6::ACSL6 action remains unclear. Here, we performed multi-omics analyses of primary leukemia cells and patient-derived xenografts from an acute lymphoblastic leukemia (ALL) patient with ETV6::ACSL6 translocation. We identified a super-enhancer located within the ETV6 gene locus, and revealed translocation and activation of the super-enhancer associated with the ETV6::ACSL6 fusion. The translocated super-enhancer exhibited intense interactions with genomic regions adjacent to and distal from the breakpoint at chromosomes 5 and 12, including genes coding inflammatory factors such as IL-3. This led to modulations in DNA methylation, histone modifications, and chromatin structures, triggering transcription of inflammatory factors leading to eosinophilia. Furthermore, the bromodomain and extraterminal domain (BET) inhibitor synergized with standard-of-care drugs for ALL, effectively reducing IL-3 expression and inhibiting ETV6::ACSL6 ALL growth in vitro and in vivo. Overall, our study revealed for the first time a cis-regulatory mechanism of super-enhancer translocation in ETV6::ACSL6ALL, leading to an ALL-accompanying clinical syndrome. These findings may stimulate novel treatment approaches for this challenging ALL subtype.

RUNX1 loss renders hematopoietic and leukemic cells dependent on IL-3 and sensitive to JAK inhibition.

Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC).

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells.

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

Nuclear factor interleukin 3 (NFIL3) participates in regulation of the NF-κB-mediated inflammation and antioxidant system in Litopenaeus vannamei under ammonia-N stress.

Nuclear factor interleukin 3 (NFIL3) is a critical upstream regulator of the NF-κB pathway. Nevertheless, the detailed molecular mechanism of NFIL3 and its function in shrimp have not been well characterized. In the present study, NFIL3 was identified and characterized from Litopenaeus vannamei. Molecular feature analysis revealed that the open reading frame (ORF) of LvNFIL3 was 2963 bp, which codes for a polypeptide of 516 amino acids with a conserved basic region leucine zipper (bZIP) domain. Sequence alignments and phylogenetic tree analysis showed that the amino acid sequence of LvNFIL3 shared 18.82%-98.07% identity with that of NFIL3 in other species, and was closely related to Penaeus monodon NFIL3. A core promoter in the 5' flanking region of LvNFIL3 was essential for regulation of transcription. LvNFIL3 mRNA was highly expressed in gills and hepatopancreas. Subcellular localization of the protein was observed almost exclusively in the nucleus. Amplification of mRNA by RT-qPCR showed that LvNFIL3 was induced in shrimp gills, hepatopancreas, and muscle after ammonia-N stress. Moreover, silencing of LvNFIL3 increased the mortality of shrimp exposed to ammonia-N. Furthermore, dual-luciferase reporter assay data suggested that LvNFIL3 was capable of activating the NF-κB pathway. Conversely, knockdown of LvNFIL3 decreased NF-κB homolog (Dorsal and Relish) and IkB homolog (Cactus) expression, as well as expression of anti-inflammatory cytokine (IL-16) and five antioxidant-related genes (HO-1, Mn-SOD, CAT, GPx, and GST), whereas NF-κB repressing factor (NKRF) and inflammation-related genes (TNFα and Spz) were upregulated. More importantly, LvNFIL3 knockdown exacerbated the pathology in hepatopancreas exposed to ammonia-N, and the total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) were significantly decreased, resulting in a significant increased lipid peroxidation and protein carbonization. Taken together, these data suggest that LvNFIL3 was involved in ammonia-N tolerance in L. vannamei by regulating the inflammation and antioxidant system through the NF-κB pathway.

Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation.

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

A STUB1 ubiquitin ligase/CHIC2 protein complex negatively regulates the IL-3, IL-5, and GM-CSF cytokine receptor common ß chain (CSF2RB) protein stability.

The IL-3, IL-5, and GM-CSF family of cytokines play an essential role in the growth, differentiation, and effector functions of multiple hematopoietic cell types. Receptors in this family are composed of cytokine-specific α chains and a common ß chain (CSF2RB), responsible for the majority of downstream signaling. CSF2RB abundance and stability influence the magnitude of the cellular response to cytokine stimulation, but the exact mechanisms of regulation are not well understood. Here, we use genetic screens in multiple cellular contexts and cytokine conditions to identify STUB1, an E3 ubiquitin ligase, and CHIC2 as regulators of CSF2RB ubiquitination and protein stability. We demonstrate that Stub1 and Chic2 form a complex that binds Csf2rb and that genetic inactivation of either Stub1 or Chic2 leads to reduced ubiquitination of Csf2rb. The effects of Stub1 and Chic2 on Csf2rb were greatest at reduced cytokine concentrations, suggesting that Stub1/Chic2-mediated regulation of Csf2rb is a mechanism of reducing cell surface accumulation when cytokine levels are low. Our study uncovers a mechanism of CSF2RB regulation through ubiquitination and lysosomal degradation and describes a role for CHIC2 in the regulation of a cytokine receptor.

MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα.

Interleukin-3 (IL-3) is a hematopoietic growth factor and critical regulator of inflammatory response such as sepsis. IL-3 binds to IL-3 receptor α (IL-3Rα), which is then associated with IL-3Rß to initiate signaling. How IL-3-triggered physiological and pathological effects are regulated at the receptor level is unclear. Here, we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates IL-3-triggered signaling. MARCH3 is associated with IL-3Rα, mediates its K48-linked polyubiquitination at K377 and promotes its proteasomal degradation. MARCH3-deficiency promotes IL-3-triggered transcription of downstream effector genes and IL-3-induced expansion of myeloid cells. In the cecal ligation and puncture (CLP) model of sepsis, MARCH3-deficiency aggravates IL-3-ampified expression of inflammatory cytokines, organ damage and inflammatory death. Our findings suggest that regulation of IL-3Rα by MARCH3 plays an important role in IL-3-triggered physiological functions and inflammatory diseases.

Clinical utility of next generation sequencing to detect IGH/IL3 rearrangements [t(5;14)(q31.1;q32.1)] in B-lymphoblastic leukemia/lymphoma.

The t(5;14)(q31.1;q32.1) associated with B-lymphoblastic leukemia/lymphoma (B-ALL/LBL) is a rare, recurrent genetic abnormality recognized as a distinct entity by the 2017 World Health Organization (WHO) classification. In these cases, the IGH enhancer region (14q32.1) is juxtaposed to the vicinity of the IL3 gene (5q31.1), resulting in increased production of interleukin-3 (IL3) and subsequently a characteristic reactive eosinophilia. B-ALL with t(5;14)(q31.1;q32.1) may have a low lymphoblast count that can complicate detection of t(5;14)(q31.1;q32.1) by conventional chromosome studies. We have identified four patients with IGH/IL3 rearrangements despite normal conventional chromosome studies in each case [one patient had a non-clonal t(5;14)(q31;q32) finding]. Fluorescence in situ hybridization utilizing a laboratory-developed IGH break-apart probe set identified IGH rearrangements in three of four cases, and a next generation sequencing (NGS) based assay, mate-pair sequencing (MPseq), was required to characterize the IGH/IL3 rearrangements in each case. Three patients demonstrated a balanced t(5;14)(q31.1;q32.1) while one patient had a cryptic insertion of the IL3 gene into the IGH region. These results demonstrate that NGS-based assays, such as MPseq, confer an advantage in the detection of IGH/IL3 rearrangements that are otherwise challenging to characterize by traditional cytogenetic methodologies.

Bovine ß-lactoglobulin-induced passive systemic anaphylaxis model using humanized NOG hIL-3/hGM-CSF transgenic mice.

Food allergy is a common disease caused by intake of allergen-containing foods, such as milk, eggs, peanuts and wheat. Systemic anaphylaxis is a severe hypersensitive allergic reaction resulting from degranulation of mast cells or basophils after cross-linking of surface high-affinity IgE receptors (Fcε-RI) with allergen-specific IgE and allergens. In this study, we developed a novel human mast cell/basophil-engrafted mouse model that recapitulates systemic anaphylaxis triggered by ß-lactoglobulin (BLG), a major allergen found in cow's milk. Human CD34+ hematopoietic stem cells were transferred into NOG (non-Tg) or NOG hIL-3/hGM-CSF transgenic (Tg) mice. After 14-16 weeks, bovine BLG-specific human IgE was intravenously injected into humanized mice, followed by intravenous or oral bovine BLG exposure 1 day later. Body temperature in Tg, but not in non-Tg, mice gradually decreased within 10 min, and 80% of Tg mice died within 1 h by intravenous BLG exposure. Serum histamine levels and anaphylaxis scores in Tg mice were markedly increased compared to non-Tg mice. Furthermore, these allergic symptoms were significantly inhibited by epinephrine treatment of the Tg mice. Therefore, the current NOG hIL-3/hGM-CSF Tg mouse model may be useful for development of novel anaphylaxis drugs for treatment of food allergies and for safety assessment of low-allergenicity extensively hydrolyzed cow's milk whey protein-based infant formulas.

The Tumor Milieu Promotes Functional Human Tumor-Resident Plasmacytoid Dendritic Cells in Humanized Mouse Models.

Particular interest to harness the innate immune system for cancer immunotherapy is fueled by limitations of immune checkpoint blockade. Plasmacytoid dendritic cells (pDC) are detected in a variety of solid tumors and correlate with poor clinical outcome. Release of type I interferons in response to toll-like-receptor (TLR)7 and TLR9 activation is the pDC hallmark. Mouse and human pDC differ substantially in their biology concerning surface marker expression and cytokine production. Here, we employed humanized mouse models (HIS) to study pDC function. We performed a comprehensive characterization of transgenic, myeloid-enhanced mouse strains (NOG-EXL and NSG-SGM3) expressing human interleukin-3 (hIL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) using identical humanization protocols. Only in HIS-NOG-EXL mice sufficient pDC infiltration was detectable. Therefore, we selected this strain for subsequent tumor studies. We analyzed pDC frequency in peripheral blood and tumors by comparing HIS-NOG-EXL with HIS-NOG mice bearing three different ovarian and breast tumors. Despite the substantially increased pDC numbers in peripheral blood of HIS-NOG-EXL mice, we detected TLR7/8 agonist responsive and thus functional pDCs only in certain tumor models independent of the mouse strain employed. However, HIS-NOG-EXL mice showed in general a superior humanization phenotype characterized by reconstitution of different myeloid subsets, NK cells and B cells producing physiologic IgG levels. Hence, we provide first evidence that the tumor milieu but not genetically introduced cytokines defines intratumoral (i.t.) frequencies of the rare pDC subset. This study provides model systems to investigate in vivo pro- and anti-tumoral human pDC functions.

CRISPR-Cas9-Mediated ELANE Mutation Correction in Hematopoietic Stem and Progenitor Cells to Treat Severe Congenital Neutropenia.

Severe congenital neutropenia (SCN) is a monogenic disorder. SCN patients are prone to recurrent life-threatening infections. The main causes of SCN are autosomal dominant mutations in the ELANE gene that lead to a block in neutrophil differentiation. In this study, we use CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)6 as a donor template delivery system to repair the ELANEL172P mutation in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs). We used a single guide RNA (sgRNA) specifically targeting the mutant allele, and an sgRNA targeting exon 4 of ELANE. Using the latter sgRNA, ∼34% of the known ELANE mutations can in principle be repaired. We achieved gene correction efficiencies of up to 40% (with sgELANE-ex4) and 56% (with sgELANE-L172P) in the SCN patient-derived HSPCs. Gene repair restored neutrophil differentiation in vitro and in vivo upon HSPC transplantation into humanized mice. Mature edited neutrophils expressed normal elastase levels and behaved normally in functional assays. Thus, we provide a proof of principle for using CRISPR-Cas9 to correct ELANE mutations in patient-derived HSPCs, which may translate into gene therapy for SCN.

Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33 % less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.

Expression of eosinophil ß chain-signaling cytokines receptors, outer-membrane integrins, and type 2 inflammation biomarkers in severe non-allergic eosinophilic asthma.

BACKGROUND: Severe non-allergic eosinophilic asthma (SNEA) is a rare asthma phenotype associated with severe clinical course, frequent exacerbations, and resistance to therapy, including high steroid doses. The key feature is type 2 inflammation with predominant airway eosinophilia. Eosinophil maturation, activation, survivability, and recruitment are mainly induced by interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) through their receptors on eosinophil surface and related with integrins activation states. The aim of the study was to estimate the expression of eosinophil ß chain-signaling cytokines receptors, outer-membrane integrins, and serum-derived type 2 inflammation biomarkers in SNEA. METHODS: We examined 8 stable SNEA patients with high inhaled steroid doses, 12 steroid-free patients with non-severe allergic asthma (AA), 12 healthy subjects (HS). Blood eosinophils were isolated using Ficol gradient centrifugation and magnetic separation. Eosinophils were lysed, and mRNA was isolated. Gene expressions of IL-5Rα, IL-3Rα, GM-CSFRα, and α4ß1, αMß2 integrins were analyzed using quantitative real-time reverse transcription polymerase chain reaction. Type 2 inflammation activity was evaluated measuring exhaled nitric oxide concentration (FeNO) collected with the electrochemical sensing device. Serum IL-5, IL-3, GM-CSF, periostin, chemokine ligand (CCL) 17 and eotaxin concentrations were assessed by enzyme-linked immunosorbent assay. RESULTS: Eosinophils from SNEA patients demonstrated significantly increased gene expression of IL-3Rα, IL-5Rα and GM-CSFRα as well as α4, ß1 and αM integrin subunits compared with the AA group. The highest IL-5 serum concentration was in the SNEA group; it significantly differed compared with AA and HS. GM-CSF serum levels were similar in the SNEA and AA groups and were significantly lower in the HS group. No differences in serum IL-3 concentration were found among all groups. Furthermore, serum levels of eotaxin, CCL17 and FeNO, but not periostin, differed in all groups, with the highest levels in SNEA patients. CONCLUSIONS: Eosinophil demonstrated higher expression of IL-3, IL-5, GM-CSF α-chain receptors and α4, ß1, αM integrins subunits in SNEA compared with the AA group. Additionally, SNEA patients had increased serum levels of IL-5, eotaxin and CCL-17. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03388359.