Ferroptosis contributes to airway epithelial E-cadherin disruption in a mixed granulocytic asthma mouse model.

Aberrant expression of airway epithelial E-cadherin is a key feature of asthma, yet the underlying mechanisms are largely unknown. Ferroptosis is a novel form of regulated cell death involved in asthma pathogenesis. This study was aimed to evaluate the role of ferroptosis and to investigate whether ferroptosis mediates E-cadherin disruption in mixed granulocyte asthma (MGA). Two murine models of MGA were established using toluene diisocyanate (TDI) or ovalbumin with Complete Freund's Adjuvant (OVA/CFA). Specific antagonists of ferroptosis, including Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1) were given to the mice. The allergen-exposed mice displayed markedly shrunk mitochondria in the airway epithelia, with decreased volume and denser staining accompanied by down-regulated GPX4 as well as up-regulated FTH1 and malondialdehyde, which are markers of ferroptosis. Decreased pulmonary expression of E-cadherin was also observed, with profound loss of membrane E-cadherin in the airway epithelia, as well as increased secretion of sE-cadherin. Treatment with Lip-1 not only showed potent protective effects against the allergen-induced airway hyperresponsiveness and inflammatory responses, but also rescued airway epithelial E-cadherin expression and inhibited the release of sE-cadherin. Taken together, our data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in MGA.

New insights into the treatment of polycystic ovary syndrome: HKDC1 promotes the growth of ovarian granulocyte cells by regulating mitochondrial function and glycolysis.

Polycystic ovary syndrome (PCOS) is an endocrine disease, and its pathogenesis and treatment are still unclear. Hexokinase domain component 1 (HKDC1) participates in regulating mitochondrial function and glycolysis. However, its role in PCOS development remains unrevealed. Here, female C57BL/6 mice were intraperitoneally injected with dehydroepiandrosterone (DHEA; 60 mg/kg body weight) to establish an in vivo model of PCOS. In vitro, KGN cells, a human ovarian granular cell line, were used to explore the potential mechanisms. DHEA-treated mice exhibited a disrupted estrus cycle, abnormal hormone levels, and insulin resistance. Dysfunction in mitochondria and glycolysis is the main reason for PCOS-related growth inhibition of ovarian granular cells. Here, we found that the structure of mitochondria was impaired, less ATP was generated and more mitochondrial Reactive Oxygen Species were produced in HKDC1-silenced KGN cells. Moreover, HKDC1 knockdown inhibited glucose consumption and decreased the production of glucose-6-phosphate and lactic acid. Conclusively, HKDC1 protects ovarian granulocyte cells from DHEA-related damage at least partly by preserving mitochondrial function and maintaining glycolysis.

The involvement of interferon regulatory factor 8 in regulating the proliferation of haemocytes in oyster Crassostrea gigas.

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (CgIRF8) in regulating haemocyte proliferation of oyster were further investigated. CgIRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of CgIRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of CgIRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of CgIRF8 protein in haemocytes. After the expression of CgIRF8 was inhibited by the injection of CgIRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of CgGATA and CgSCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein CgCDK2, CgCDC6, CgCDC45 and CgPCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that CgIRF8 was able to activate the CgGATA promoter in HEK293T cells after transfection of CgGATA and CgIRF8. These results collectively indicated that CgIRF8 promoted haemocyte proliferation by regulating the expression of CgGATA and other related genes in the immune response of oyster.

Granulocyte macrophage colony-stimulating factor-induced macrophages of individuals with autism spectrum disorder adversely affect neuronal dendrites through the secretion of pro-inflammatory cytokines.

BACKGROUND: A growing body of evidence suggests that immune dysfunction and inflammation in the peripheral tissues as well as the central nervous system are associated with the neurodevelopmental deficits observed in autism spectrum disorder (ASD). Elevated expression of pro-inflammatory cytokines in the plasma, serum, and peripheral blood mononuclear cells of ASD has been reported. These cytokine expression levels are associated with the severity of behavioral impairments and symptoms in ASD. In a prior study, our group reported that tumor necrosis factor-α (TNF-α) expression in granulocyte-macrophage colony-stimulating factor-induced macrophages (GM-CSF MΦ) and the TNF-α expression ratio in GM-CSF MΦ/M-CSF MΦ (macrophage colony-stimulating factor-induced macrophages) was markedly higher in individuals with ASD than in typically developed (TD) individuals. However, the mechanisms of how the macrophages and the highly expressed cytokines affect neurons remain to be addressed. METHODS: To elucidate the effect of macrophages on human neurons, we used a co-culture system of control human-induced pluripotent stem cell-derived neurons and differentiated macrophages obtained from the peripheral blood mononuclear cells of five TD individuals and five individuals with ASD. All participants were male and ethnically Japanese. RESULTS: Our results of co-culture experiments showed that GM-CSF MΦ affect the dendritic outgrowth of neurons through the secretion of pro-inflammatory cytokines, interleukin-1α and TNF-α. Macrophages derived from individuals with ASD exerted more severe effects than those derived from TD individuals. LIMITATIONS: The main limitations of our study were the small sample size with a gender bias toward males, the use of artificially polarized macrophages, and the inability to directly observe the interaction between neurons and macrophages from the same individuals. CONCLUSIONS: Our co-culture system revealed the non-cell autonomous adverse effects of GM-CSF MΦ in individuals with ASD on neurons, mediated by interleukin-1α and TNF-α. These results may support the immune dysfunction hypothesis of ASD, providing new insights into its pathology.

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

INTRODUCTION: Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. METHODS: EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. RESULTS: A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). CONCLUSION: Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

A novel selective leukocyte depletion human whole blood model reveals the specific roles of monocytes and granulocytes in the cytokine response to Escherichia coli.

The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli (108/mL) for 2 hours at 37 °C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P < 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%-92%) and granulocyte (mean = 97%; 95% confidence interval = 96%-98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coli incubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1ß, IL-2, IL-4, IL-5, IL-17A, interferon-γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α, and fibroblast growth factor-basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1ß, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.

Granulocyte-colony stimulating factor producing cervical cancer with elevated levels of parathyroid hormone-related protein: a case report and literature review.

Systemic effects associated with hormones and cytokines secreted by tumor cells can cause paraneoplastic syndrome. Leukemoid reactions and hypercalcemia are relatively common manifestations of paraneoplastic syndrome. Here, we describe the case of a 90-year-old woman who presented with leukocytosis and hypercalcemia and was diagnosed with granulocyte-colony stimulating factor (G-CSF)-producing cervical cancer with elevated levels of parathyroid hormone-related protein (PTHrP). The patient visited our hospital complaining of general fatigue and anorexia. On admission, she presented with marked leukocytosis, hypercalcemia, and an increase in C-reactive protein level. On the basis of abdominal magnetic resonance imaging and histopathological examination, the patient was diagnosed with cervical cancer. Additional tests confirmed elevated plasma levels of G-CSF, PTHrP, and serum interleukin-6. Immunostaining of pathological specimens of the uterine cervix showed expression of G-CSF in tumor cells. The patient was diagnosed with G-CSF-producing cervical cancer accompanied by elevation of PTHrP levels. As a treatment for hypercalcemia, discontinuation of oral vitamin D derivative and administration of saline and elcatonin were ineffective, and therapeutic intervention with zoledronic acid hydrate was required. Considering the patient's advanced age, surgical resection of cervical cancer was not performed. She died from congestive heart failure approximately 3 months after hospitalization. This case was indicated to be a paraneoplastic syndrome in which G-CSF and PTHrP-induced leukocytosis and hypercalcemia. To the best of our knowledge, there have been no reports of G-CSF-producing cervical cancer with elevated PTHrP levels, and our case is the first report.

The Mechanistic Target of Rapamycin Complex 1 Pathway Contributes to the Anti-Tumor Effect of Granulocyte-Macrophage-Colony-Stimulating Factor-Producing T Helper Cells in Mouse Colorectal Cancer.

INTRODUCTION: The role of granulocyte-macrophage-colony-stimulating factor-producing T helper (ThGM) cells in colorectal cancer (CRC) development remains unclear. This study characterizes the function of ThGM cells in mouse CRC. METHODS: Mouse CRC was induced by administrating azoxymethane and dextran sulfate sodium. The presence of ThGM cells in CRC tissues and the mechanistic target of rapamycin complex 1 (mTORC1) signaling in ThGM cells was detected by flow cytometry. The impact of mTORC1 signaling on ThGM cell function was determined by in vitro culture. The effect of ThGM cells on CRC development was evaluated by adoptive transfer assays. RESULTS: ThGM cells, which expressed granulocyte-macrophage-colony-stimulating factor (GM-CSF), accumulated in CRC tissues. mTORC1 signaling is activated in CRC ThGM cells. mTORC1 inhibition by rapamycin suppressed ThGM cell differentiation and proliferation and resulted in the death of differentiating ThGM cells. mTORC1 inhibition in already differentiated ThGM cells did not induce significant cell death but decreased the expression of GM-CSF, interleukin-2, and tumor necrosis factor-alpha while impeding cell proliferation. Furthermore, mTORC1 inhibition diminished the effect of ThGM cells on driving macrophage polarization toward the M1 type, as evidenced by lower expression of pro-inflammatory cytokines, major histocompatibility complex class II molecule, and CD80 in macrophages after co-culture with rapamycin-treated ThGM cells. Lentivirus-mediated knockdown/overexpression of regulatory-associated protein of mTOR (Raptor) confirmed the essential role of mTORC1 in ThGM cell differentiation and function. Adoptively transferred ThGM cells suppressed CRC growth whereas mTORC1 inhibition abolished this effect. CONCLUSION: mTORC1 is essential for the anti-CRC activity of ThGM cells.

C/EBPß-induced lymphoid-to-myeloid transdifferentiation emulates granulocyte-monocyte progenitor biology.

CCAAT/enhancer-binding protein beta (C/EBPß) induces primary v-Abl immortalized mouse B cells to transdifferentiate (BT, B cell transdifferentiation) into granulocyte-macrophage progenitor-like cells (GMPBTs). GMPBTs maintain cytokine-independent self-renewal, lineage choice, and multilineage differentiation. Single-cell transcriptomics demonstrated that GMPBTs comprise a continuum of myelomonopoietic differentiation states that seamlessly fit into state-to-fate maps of normal granulocyte-macrophage progenitors (GMPs). Inactivating v-Abl kinase revealed the dependence on activated CSF2-JAK2-STAT5 signaling. Deleting IRF8 diminished monopoiesis and enhanced granulopoiesis while removing C/EBPß-abrogated self-renewal and granulopoiesis but permitted macrophage differentiation. The GMPBT culture system is easily scalable to explore the basics of GMP biology and lineage commitment and largely reduces ethically and legislatively debatable, labor-intensive, and costly animal experiments.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins. Nuclear proteome is of particular interest because many nuclear proteins, e.g., transcription factors, regulate functions that determine cell fate. Meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated with all-trans-retinoic acid (ATRA) has shown that the functions and localization of a protein product of the SOWAHD gene are poorly understood. Also, there is no comprehensive information on the SOWAHD gene expression at the protein level. In HL-60 cells, the number of mRNA transcripts of the SOWAHD gene was determined as 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of the SOWAHD protein was measured as 0.27 to 1.25 fmol/µg total protein. The half-life for the protein product of the SOWAHD gene determined using stable isotope pulse-chase labeling was ~19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the content of the SOWAHD protein increased during the ATRA-induced granulocytic differentiation, reached the peak value at 9 h after ATRA addition, and then decreased. Nuclear location and involvement of the SOWAHD protein in the ATRA-induced granulocytic differentiation have been demonstrated for the first time.

Differential expression of interleukin-35 receptor distinguishes different subsets of granulocyte-macrophage-colony-stimulating factor-producing T helper cells in a mouse endometriosis model.

The immune system contributes to the pathophysiology of endometriosis. The role of ThGM cells, which produce granulocyte macrophage-colony-stimulating factor (GM-CSF), in the pathogenesis of endometriosis remains unknown. To analyze the features of ThGM cells in endometriosis, a mouse endometriosis model was established. ThGM cells in the spleen, peritoneal fluid (PF), and endometriotic lesions (EL) were measured by flow cytometry, based on the expression of surface markers and intracellular proteins. Live ThGM cells were sorted according to chemokine receptor expression profiles and their effects on other CD4+ T cell subsets were determined by co-culture assays. An adoptive transfer assay was performed to characterize the effect of ThGM cells on endometriosis. We found that ThGM cells were present in endometriotic PF and EL. Live EL ThGM cells were enriched in CD4+CXCR3-CCR8-CCR4+CCR10+ T cells. EL ThGM cells differentially express interleukin-35 receptor (IL-35R), consisting of an IL-35R+ subset and an IL-35R- subset. The IL-35R+ subset expressed less GM-CSF, interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNF-α) and proliferated slower than the IL-35R- subset. Meanwhile, the IL-35R+ subset was weaker than the IL-35R- subset in promoting the functions of Th1 and Th17 cells. ThGM cell transfer did not influence EL development but significantly alleviated pro-inflammatory cytokines in PF and ELs. Interleukin-35 (IL-35), the ligand of IL-35R, suppressed ThGM cell function and proliferation in an IL-35R-dependent manner. In summary, ThGM cells in the PF and ELs might exacerbate endometriotic inflammation. IL-35 might suppress the function of ThGM cells via IL-35R.

Hematopoietic stem cells undergo a lymphoid to myeloid switch in early stages of emergency granulopoiesis.

Emergency granulopoiesis is the enhanced and accelerated production of granulocytes that occurs during acute infection. The contribution of hematopoietic stem cells (HSCs) to this process was reported; however, how HSCs participate in emergency granulopoiesis remains elusive. Here, using a mouse model of emergency granulopoiesis we observe transcriptional changes in HSCs as early as 4 h after lipopolysaccharide (LPS) administration. We observe that the HSC identity is changed towards a myeloid-biased HSC and show that CD201 is enriched in lymphoid-biased HSCs. While CD201 expression under steady-state conditions reveals a lymphoid bias, under emergency granulopoiesis loss of CD201 marks the lymphoid-to-myeloid transcriptional switch. Mechanistically, we determine that lymphoid-biased CD201+ HSCs act as a first response during emergency granulopoiesis due to direct sensing of LPS by TLR4 and downstream activation of NF-κΒ signaling. The myeloid-biased CD201- HSC population responds indirectly during an acute infection by sensing G-CSF, increasing STAT3 phosphorylation, and upregulating LAP/LAP* C/EBPß isoforms. In conclusion, HSC subpopulations support early phases of emergency granulopoiesis due to their transcriptional rewiring from a lymphoid-biased to myeloid-biased population and thus establishing alternative paths to supply elevated numbers of granulocytes.

Adenosquamous carcinoma of the gallbladder simultaneously producing granulocyte-colony-stimulating factor and parathyroid hormone-related protein.

We report a rare case of adenosquamous carcinoma of the gallbladder which simultaneously produces granulocyte-colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP), confirmed serologically and histologically. A 71-year-old man was examined for a gallbladder tumor with multiple lymph nodes and liver metastases. Histopathological evaluation by endoscopic ultrasound fine-needle aspiration revealed adenosquamous carcinoma of the gallbladder. Laboratory data showed markedly elevated white blood cell (WBC) count of 34,700 µL and corrected serum calcium level of 14.9 mg/dL. Serum G-CSF (191 pg/mL) and PTHrP (23.1 pmol/L) levels were high. Zoledronic acid and calcitonin were administered to treat hypercalcemia, which normalized serum calcium levels. Gemcitabine-cisplatin chemotherapy was started for cStage IVB gallbladder cancer. After chemotherapy initiation, WBCs showed a rapid downward trend; however, the patient suddenly developed acute respiratory distress syndrome; thus, chemotherapy was discontinued. Subsequently, WBC count increased again, and the patient's overall condition deteriorated. The patient died on day 27. Immunohistochemistry using autopsy specimens demonstrated patchy staining for G-CSF in the squamous cell carcinoma portion and diffuse and weak positive staining for PTHrP in the squamous cell carcinoma and poorly differentiated adenocarcinoma portions of the tumor, suggesting simultaneous G-CSF and PTHrP production by the tumor. This is the first report of a patient with gallbladder cancer with serological and histological evidence for G-CSF and PTHrP production.

Single-cell detection of DMSO promoted HL-60 differentiation toward granulocyte based on DC-iDEP for medicine screening.

The most common form of leukemia in adults is acute leukemia. Drug differentiation control is an extremely critical treatment for acute leukemia. Unfortunately, current techniques detecting differentiation control experience long time and complex steps of verification hindering the pace of medicine discovery: flow cytometry and RT-PCR are highly accurate and efficient at a cost of inconvenient fluorescent labeling or a high risk of contamination; conventional staining leads to cell death unavailable for further pharmacological tests. There is a great interest in developing simple, fast, and non-invasive techniques to screen medicine. DC-iDEP is an emerging label-free identification technique taking advantage of the whole cell native biophysical property for sorting cell populations. Here, HL-60 cell line has been used as a model to study the differentiation process toward granulocytes and medicine efficacy. The results showed that DEP succeeded in detecting the DMSO promoted HL-60 differentiation degree by the weighted average characterization factor. This factor is related to the single cell biophysical property, which accumulates to generate differences in each population with distinct constitutions. Furthermore, cichoric acid was investigated to be capable of promoting DMSO-induced differentiation efficiently. Using the change induced by cichoric acid, the HL-60 medicine screening application has been first attempted based on DEP. A rapid, label-free medicine screening method has been established to monitor HL-60 differentiation toward granulocyte and has great potential for medicine screening.

Sepsis induces non-classic innate immune memory in granulocytes.

Secondary infection in patients with sepsis triggers a new wave of inflammatory response, which aggravates organ injury and increases mortality. Trained immunity boosts a potent and nonspecific response to the secondary challenge and has been considered beneficial for the host. Here, using a murine model of polymicrobial infection, we find that the primary infection reprograms granulocytes to boost enhanced inflammatory responses to the secondary infection, including the excessive production of inflammatory cytokines, respiratory burst, and augmented phagocytosis capacity. However, these reprogramed granulocytes exhibit "non-classic" characteristics of innate immune memory. Two mechanisms are independently involved in the innate immune memory of granulocytes: a metabolic shift in favor of glycolysis and fatty acid synthesis and chromatin remodeling leading to the transcriptional inactivity of genes encoding inhibitors of TLR4-initiated signaling pathways. Counteracting the deleterious effects of stressed granulocytes on anti-infection immunity might provide a strategy to fight secondary infections during sepsis.

Interaction of Interleukin-17A with a Th2 Response in a Mouse Model of Allergic Airway Inflammation.

BACKGROUND: A total of 262 million people worldwide suffer from asthma and 461000 people died from it in 2019. Asthma is a disease with different endotypes defined by the granulocytes found in the asthmatic lung. In allergic asthma, the eosinophilic endotype is present, driven by a TH2 response. A TH17 immune response leads to the neutrophil endotype. This often causes uncontrolled asthma and is triggered by pollutants, microbes, and oxidative stress. It has been described that a significant number of patients with eosinophilic asthma develop mixed granulocytic asthma over time. The severity of asthma in the mixed endotype is related to the proportion of neutrophils in the lungs. PURPOSE: In this report, we address the question of how a TH2 response interacts with IL-17A in allergic asthma. METHODS: To this end, we used a mouse model to induce allergic asthma followed by an aerosol challenge with ovalbumin. To investigate the role of IL-17A, we administered IL-17A intranasally during the challenge phase. RESULTS: IL-17A alone did not elicit an immune response, whereas in combination with allergic asthma, it resulted in a shift of the asthmatic endotype from eosinophilic to neutrophilic. TGFß1 was increased in these lungs compared to asthmatic lungs without IL-17A, as was the expression of the IL-17A receptor subunits IL-17RA and IL-17RC. In cultures with human cells, we also found that IL-17A increased the expression of its receptors only in combination with IL-13. We also found this effect for IL-8, which attracts neutrophils in humans. CONCLUSIONS: The TH2 response increased the sensitivity to IL-17A in a mouse asthma model as well as in human cell lines.

Molecular and functional characterization of two granulocyte colony stimulating factors in goldfish (Carassius auratus L.).

Granulocyte colony-stimulating factor (GCSF) is a member of the hematopoietic growth factor family that acts primarily on neutrophils and neutrophilic precursors to promote cell proliferation and differentiation. Although multiple GCSF genes have been found in teleosts, knowledge of their functions during fish hematopoietic development is still limited. Here, we report for the first time the molecular and functional characterization of two goldfish GCSFs (gfGCSF-a and gfGCSF-b). The open reading frame (ORF) of the gfGCSF-a and gfGCSF-b cDNA transcript consisted respectively of 624 bp and 678 bp with its ORF encoding 207 and 225 amino acids (aa), with a 17 aa signal peptide for each gene and a conserved domain of the IL-6 superfamily. Treatment of goldfish head kidney leukocytes (HKLs) with LPS increased gfGCSF-a and gfGCSF-b mRNA expression levels, also exposure of HKLs to either heat-killed or live A. hydrophila, induced transcriptional upregulation of gfGCSF-a and gfGCSF-b levels. Recombinant gfGCSF-a and gfGCSF-b protein (rgGCSF-a and rgGCSF-b) induced a dose-dependent production of TNFα and IL-1ß from goldfish neutrophils. In vitro experiments showed rgGCSF-a and rgGCSF-b differentially promoted the proliferation and differentiation of leukocytes in goldfish. Furthermore, treatment of HKLs with rgGCSF-a showed significant upregulation of mRNA levels of the hematopoietic transcription factor GATA2, Runx1, MafB, and cMyb, while gfGCSF-b induces not only all four transcriptional factors mentioned above but also CEBPα. Our results indicate that goldfish GCSF-a and GCSF-b are important regulators of neutrophil proliferation and differentiation, which could stimulate different stages and lineages of hematopoiesis.

Altered granulocyte count and erythrocyte measures in middle-aged, healthy carriers of APOE and PICALM risk genes for Alzheimer's disease.

APOE­Îµ4 genotype (apolipoprotein E, epsilon 4) is the strongest genetic risk factor for Alzheimer's disease (AD). Despite years of research, it is still not known how it contributes to dementia development. APOE has been implicated in many AD pathology mechanisms, like Aß clearance, brain metabolism, changes within microglia and other glial functions and inflammatory processes. In fact, immunological/inflammatory processes are recently discussed as an important factor in Alzheimer's development and granulocyte profiles changes are reported in patients. However, the exact link between the immune system and risk­genes is unknown. In particular, it is not known whether and how they interact throughout the lifetime, before the disease onset. The aim of the study was to investigate the relationship between granulocyte count and the APOE/PICALM genes in healthy individuals with an increased genetic risk of AD. An exploratory analysis regarding other blood cells was also conducted. Blood samples were collected from 77 healthy middle­aged (50-63 years old) participants, who were also asked to complete a health and life­style questionnaires. Groups with different AD risk­genes were compared. Differences in granulocyte profiles were found in healthy carriers of AD risk­genes who had slightly elevated eosinophil levels as compared to non-risk carriers. An exploratory analysis showed some alteration in mean corpuscular hemoglobin content and concentration (MCH/MCHC) levels between risk­carriers subgroups and non-risk carriers. No other differences in blood count or lipoprotein profile were found between healthy APOE/PICALM risk­carriers and non-risk carriers. Longitudinal studies will reveal if and how those changes contribute to the development of AD pathology.

Relationship of paroxysmal nocturnal hemoglobinuria (PNH) granulocyte clone size to disease burden and risk of major vascular events in untreated patients: results from the International PNH Registry.

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by acquired gene mutations resulting in deficiency of glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins on the surface of blood cells, leading to terminal complement-mediated intravascular hemolysis and increased risk of major adverse vascular events (MAVEs). Using data from the International PNH Registry, this study investigated the relationship between the proportion of GPI-deficient granulocytes at PNH onset and (1) the risk for MAVEs (including thrombotic events [TEs]) and (2) the following parameters at last follow-up: high disease activity (HDA); lactate dehydrogenase (LDH) ratio; fatigue; abdominal pain; and rates of overall MAVEs and TEs. A total of 2813 patients untreated at enrollment were included and stratified by clone size at PNH disease onset (baseline). At last follow-up, higher proportion of GPI-deficient granulocytes (≤ 5% vs. > 30% clone size) at baseline was associated with significantly increased HDA incidence (14% vs. 77%), mean LDH ratio (1.3 vs. 4.7 × upper limit of normal), and rates of MAVEs 1.5 vs. 2.9 per 100 person-years) and TEs (0.9 vs. 2.0 per 100 person-years). Fatigue was evident in 71 to 76% of patients regardless of clone size. Abdominal pain was more frequently reported with clone size > 30%. A larger clone size at baseline appears to indicate a greater disease burden and risk of TEs and MAVEs and may inform decision making among physicians managing PNH patients at risk of experiencing TEs or other MAVEs. ClinicalTrials.gov ID: NCT01374360.

A case of granulocyte-colony-stimulating factor-producing gallbladder cancer with lymph node metastasis together with a literature review.

The granulocyte-colony-stimulating factor (G-CSF) glycoprotein stimulates precursor cell proliferation and differentiation in the bone marrow. Various G-CSF-producing tumors have been reported; they showed early progression and an extremely poor prognosis. Here, we report a case of G-CSF-producing gallbladder cancer with lymph node metastasis. In addition, we reviewed 30 previous case reports of G-CSF-producing gallbladder cancers to elucidate the characteristics and most appropriate treatment. During a routine visit to her local doctor for monitoring of diabetes and hypertension, a 68-year-old female was found to have an elevated white-blood-cell (WBC) count and C-reactive protein (CRP) level, and a gallbladder mass. Laboratory tests revealed a high serum G-CSF level, and imaging revealed a tumor of the gallbladder with regional lymphadenopathy. We diagnosed a G-CSF-producing gallbladder cancer and performed liver resection of segment IVa/V: regional lymph node dissection with extrahepatic bile duct resection. Pathologically, the tumor was a poorly differentiated squamous cell carcinoma. G-CSF immunostaining for tumor cells was positive. She is alive without recurrence at 16 months after surgery. If a patient exhibits a gallbladder tumor, with an elevated WBC count and CRP level but no symptoms of infection, a G-CSF-producing gallbladder cancer should be suspected; radical resection should be performed immediately after diagnosis.