Neutrophil Survival Research Articles

Salmonella Typhimurium induces genome-wide expression and phosphorylation changes that modulate immune response, intracellular survival and vesicle transport in infected neutrophils

Salmonella Typhimurium is a food-borne pathogen that causes salmonellosis. When in contact with the host, neutrophils are rapidly recruited to act as first line of defense. To better understand the pathogenesis of this infection, we used an in vitro model of neutrophil infection to perform dual RNA-sequencing (both host and pathogen). In addition, and given that many pathogens interfere with kinase‐mediated phosphorylation in host signaling, we performed a phosphoproteomic analysis. The immune response was overall diminished in infected neutrophils, mainly JAK/STAT and toll-like receptor signaling pathways. We found decreased expression of proinflammatory cytokine receptor genes and predicted downregulation of the mitogen-activated protein (MAPK) signaling pathway. Also, Salmonella infection inhibited interferons I and II signaling pathways by upregulation of SOCS3 and subsequent downregulation of STAT1 and STAT2. Additionally, phosphorylation of PSMC2 and PSMC4, proteasome regulatory proteins, was decreased in infected neutrophils. Cell viability and survival was increased by p53 signaling, cell cycle arrest and NFkB-proteasome pathways activation. Combined analysis of RNA-seq and phosphoproteomics also revealed inhibited vesicle transport mechanisms mediated by dynein/dynactin and exocyst complexes, involved in ER-to-Golgi transport and centripetal movement of lysosomes and endosomes. Among the overexpressed virulence genes from Salmonella we found potential effectors responsible of these dysregulations, such as spiC, sopD2, sifA or pipB2, all of them involved in intracellular replication. Our results suggest that Salmonella induces (through overexpression of virulence factors) transcriptional and phosphorylation changes that increases neutrophil survival and shuts down immune response to minimize host response, and impairing intracellular vesicle transport likely to keep nutrients for replication and Salmonella-containing vacuole formation and maintenance.

Microenvironment Immune Cells in Bone Marrow and Lymph Node Measured By Flow Cytometry Have Prognostic Impact on Survival in Nodal Non-Hodgkin T-Cell Lymphoma

INTRODUCTION Outcomes in nodal peripheral T-cell lymphomas (PTCL) remain poor. Baseline clinical risk indexes have shown to be prognostic but do not imply a change in therapeutic strategies. Many publications have addressed the role of tumor microenvironment (TME) in lymphomagenesis and disease progression. Particularly in PTCL, TME was shown to be profoundly immunosuppressive. Therefore, TME variables that may aid to improve risk stratification and treatment management are urgently needed. AIMS To determine the prognostic impact on survival in PTCL of: -Conventional clinical risk parameters. -The percentage (%) of immune cells (IC) in the bone marrow (BM) and lymph node (LN) measured by flow cytometry (FC). METHODS We retrospectively collected information of patients (pts) with PTCL and available BM aspiration and/or LN FC data at diagnosis, treated at our center between 2012-2021. FC analysis was performed with 8-color panels according to Euroflow protocols. % of BM and LN IC by FC were compared to normal values determined by Matarraz et al. (Cytometry part B 2010) and Battaglia et al. (Immunology 2003), and analyzed in 3 categories: low, normal and high. Survival analysis was performed with Kaplan-Meier (variables compared with log-rank) and multivariate analysis with Cox regression. RESULTS We included 34 pts, with a median age of 59 years. LN and BM FC data were available in 26 and 28 pts, respectively. Table 1 shows baseline patient characteristics. 85.4% received induction with CHOP or CHOP-like regimens (with etoposide or brentuximab). Complete remission (CR) rate: 52.9%. 14 pts received treatment with new drugs (brentuximab, belinostat, romidepsin or lenalidomide) in the relapsed/refractory setting. Only 10/34 pts underwent consolidation with autologous stem cell transplant (ASCT). Chemorefractory disease was the reason to forgo ASCT in 50% (12/24) of the non- transplanted pts and 3/12 underwent consolidation with allogeneic stem cell transplant in 1st CR. Median progression-free (PFS) and overall survival (OS) for the whole cohort were 15 and 72 months (m) respectively, with a median follow up time of 41.2 m. Pts with ALK+ anaplastic T-cell lymphoma had improved PFS (median of 67 vs 8, 9 and 5.6 m for ALK- anaplastic, PTCL NOS and angioimmunoblastic lymphoma, respectively). Regarding prognostic indexes, PIT score was able to discriminate subgroups with different PFS (median of 15.3 m for pts with at least 1 risk factor vs not reached and PFS rate of 100% in those without, p: 0.012). Other clinical parameters such as BM and CNS compromise were associated with inferior PFS (median of 49.7 in BM not involved vs 6.7 m, p: 0.006; and median of 19.2 in pts without CNS compromise vs 3.45 m, p<0.001, respectively). The only TME variable with prognostic impact on PFS was LN CD4/CD8 ratio (median of 30.8, 5.7 and 2.7 m for the low, normal and high subgroups, respectively, p: 0.045). On multivariate analysis, absence of CNS involvement was the only independent predictor of PFS (HR: 0.009, CI95%: 0-0.235, p: 0.005). The only clinical parameter with prognostic impact on OS was performance status (PS) (median of 72.2 vs 0.2 m in pts with PS≤2 and ≥3, respectively). Regarding TME variables, low BM neutrophil % was associated with improved OS with median not reached vs 27.8 m in pts with normal and high values, p: 0.039). BM neutrophil % did not statistically differ in involved vs not involved BM. Interestingly, only 1/9 pts with normal-high BM neutrophil % with relapse disease could achieve CR with 2nd line treatment vs 6/10 in the low % subgroup (likelihood ratio 5.26, p: 0.022). In contrast, no association was found between peripheral blood neutrophil counts and survival in our cohort. On multivariate analysis, only low BM neutrophil % remained as an independent predictor of OS with a HR of 0.12 (CI95%: 0.015-0.96), p: 0.046. CONCLUSIONS Both clinical risk factors and TME variables showed prognostic impact in our cohort of PTCL pts. A high LN CD4/CD8 ratio and a normal-high BM neutrophil % were associated with decreased PFS and OS, respectively. These TME variables are widely accessible and could help refine prediction of prognosis in pts with nodal PTCL. Manipulating TME with therapeutic strategies that promote effector T-cell function and/or suppress the pro-malignant cell effects of myeloid derived cells may improve patient outcomes in this poor risk disease. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Granulocyte colony-stimulating factor therapy as a bridging treatment for pediatric decompensated liver cirrhosis prior to liver transplantation: an open-label randomized clinical trial

Background: Decompensated cirrhosis (DC) in children is the main indication of liver transplantation (LT). The lack of access to liver transplantation and impending clinical complications while awaiting transplantation affect the morbidity and mortality in pretransplant patients. Granulocyte colony-stimulating factor (G-CSF) therapy has shown promising results in adult decompensated cirrhosis as a potential bridging treatment. Our study aimed to identify the effect of G-CSF on pediatric end-stage liver disease (PELD) score, liver function, CD34+ cell mobilization, nutritional status, survival, and short-term side effects in children awaiting LT. Methods: This study was an open-label, randomized controlled trial that included patients with decompensated liver cirrhosis between 3 months and 12 years of age. The intervention group received 12 courses of G-CSF subcutaneous injection (5 μg/kg/day) plus standard medical treatment (SMT) for liver cirrhosis. Results were obtained regarding PELD scores, liver function, CD34+ cell mobilization, changes in leukocyte and neutrophil counts, nutritional status, survival, and side effects within three months. Results: Thirty-five pediatric patients were randomized into the intervention (17 patients) and control (18 patients) groups. During the trial, 14 (82%) in the intervention group completed the treatment. The median ages of the patients in the intervention and control groups were 18 and 14.5 months, respectively. The study's primary outcome identified no statistically significant difference in PELD scores between the intervention and control groups after G-CSF treatment. Liver function tests that showed significant changes in the intervention group compared to the control group were from improvements in alanine aminotransferase (ALT) levels. Other liver function tests, nutritional status, and survival did not. CD34+ cell mobilization was increased in the intervention group compared with the control group, but there was no significant difference. Minor side effects of G-CSF were observed in the intervention group. Conclusion: Multiple doses of G-CSF did not improve the PELD score, nutritional status, and survival after three months but significantly showed temporary improvement in ALT level.

Prospective Trial of Neutrophil/Lymphocyte Ratio and Other Blood Counts as Biomarkers of Survival among Patients with High-Grade Soft Tissue Sarcomas Treated with Pegylated Liposomal Doxorubicin and Ifosfamide.

Simple SummaryPrevious studies have reported an association between the ratio of neutrophils to lymphocytes circulating in blood and outcomes in patients with cancer. This study examined the association between lymphocyte and neutrophil counts and survival in a prospective trial of preoperative chemotherapy for high-grade soft-tissue sarcomas. A statistically significant association between overall survival was observed with the neutrophil/lymphocyte ratio. Our results suggest that a balance between the lymphocyte count and the number of circulating myeloid cells that might suppress lymphocyte function may be predictive of survival in patients with soft-tissue sarcomas. Future research should examine the role of lymphocyte-myeloid cell balance in sarcoma biology.Several studies have reported an association between levels of circulating blood cells, in particular the neutrophil to lymphocyte ratio (absolute neutrophil count (ANC)/absolute lymphocyte count (ALC)) and outcomes in patients with cancer. In the current study, the association between lymphocyte, neutrophil, monocyte, and platelet counts and survival was examined in a prospective trial of preoperative pegylated-liposomal doxorubicin and ifosfamide for high-grade soft-tissue sarcomas. A statistically significant association between overall survival, but not progression free-survival, was observed with the ANC/ALC ratio at a cutoff value of ≥2 and a statistically significant trend using a cutoff of ≥5. Our results suggest that a balance between the lymphocyte count and the number of circulating myeloid cells that can suppress lymphocyte function may be predictive of survival in patients with soft-tissue sarcomas. Future research should therefore examine the role of lymphocyte-myeloid cell balance in sarcoma biology.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

A culture model to analyze the acute biomaterial-dependent reaction of human primary neutrophils in vitro.

Neutrophils play a pivotal role in orchestrating the immune system response to biomaterials, the onset and resolution of chronic inflammation, and macrophage polarization. However, the neutrophil response to biomaterials and the consequent impact on tissue engineering approaches is still scarcely understood. Here, we report an in vitro culture model that comprehensively describes the most important neutrophil functions in the light of tissue repair. We isolated human primary neutrophils from peripheral blood and exposed them to a panel of hard, soft, naturally- and synthetically-derived materials. The overall trend showed increased neutrophil survival on naturally derived constructs, together with higher oxidative burst, decreased myeloperoxidase and neutrophil elastase and decreased cytokine secretion compared to neutrophils on synthetic materials. The culture model is a step to better understand the immune modulation elicited by biomaterials. Further studies are needed to correlate the neutrophil response to tissue healing and to elucidate the mechanism triggering the cell response and their consequences in determining inflammation onset and resolution.

Heat-killed probiotic Lactobacillus plantarum affects the function of neutrophils but does not improve survival in murine burn injury

Probiotics have become of interest as therapeutics in trauma or sepsis-induced inflammation due to their ability to affects the immune response. However, their use is still under debate due to the potential risk of septicemia. Therefore, heat-killed probiotics offer a potential alternative, with recent research suggesting a comparable immunomodulating potential and increased safety. In a previous study, we demonstrated decreased mortality by administration of live Lactobacillus plantarum in a mouse burn-sepsis model. Neutrophils are an essential innate defense against pathogens. Therefore, our present study aims to understand the impact of heat-killed probiotic L. plantarum (HKLP) on neutrophil function. Utilizing an in vitro stimulation with HKLP and a burn-infection in vivo model, we determined that administration of HKLP induced significant release of granulocyte-colony stimulating factor (G-CSF) and stimulated the release of pro-and anti-inflammatory cytokines. HKLP had no impact on neutrophil function, such as phagocytosis, oxidative burst, and NETosis, but increased apoptosis and activated neutrophils. HKLP did not improve survival. Together, contrary to our hypothesis, heat-killed probiotics did not improve neutrophil function and survival outcome in a murine severe burn injury model.

Suppression of Aspergillus fumigatus Germination by Neutrophils Is Enhanced by Endothelial-Derived CSF3 Production.

Infection with Aspergillus fumigatus can cause life-threatening diseases in immunocompromised patients with an unacceptable mortality rate. Angioinvasion is one of the features of severe invasive aspergillosis. Neutrophils are short-lived immune cells regulated by colony-stimulating factor 3 (CSF3) that play a key role in anti-fungal immune responses. To investigate the interactions between A. fumigatus and the host immune cells, such as neutrophils, we stimulated human umbilical vein endothelial cells (HUVECs) with the conidia of A. fumigatus, and co-cultured them with human neutrophils. Apoptosis and functions of neutrophils were analyzed. Our results showed that HUVECs upregulate the expression of CSF3, which could reduce the apoptosis of neutrophils while enhancing their functions. Lack of CSF3 was associated with enhanced apoptosis of neutrophils with impaired function. This work indicated that the CSF3 is required for neutrophil survival and function, at least in the early stages of A. fumigatus infection.

The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001—is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide—a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

Leishmania infantum Defective in Lipophosphoglycan Biosynthesis Interferes With Activation of Human Neutrophils

Visceral leishmaniasis (VL) is often associated with hematologic manifestations that may interfere with neutrophil response. Lipophosphoglycan (LPG) is a major molecule on the surface of Leishmania promastigotes, which has been associated with several aspects of the parasite–vector–host interplay. Here, we investigated how LPG from Leishmania (L.) infantum, the principal etiological agent of VL in the New World, influences the initial establishment of infection during interaction with human neutrophils in an experimental setting in vitro. Human neutrophils obtained from peripheral blood samples were infected with either the wild-type L. infantum (WT) strain or LPG-deficient mutant (∆lpg1). In this setting, ∆lpg1 parasites displayed reduced viability compared to WT L. infantum; such finding was reverted in the complemented ∆lpg1+LPG1 parasites at 3- and 6-h post-infection. Confocal microscopy experiments indicated that this decreased survival was related to enhanced lysosomal fusion. In fact, LPG-deficient L. infantum parasites more frequently died inside neutrophil acidic compartments, a phenomenon that was reverted when host cells were treated with Wortmannin. We also observed an increase in the secretion of the neutrophil collagenase matrix metalloproteinase-8 (MMP-8) by cells infected with ∆lpg1 L. infantum compared to those that were infected with WT parasites. Furthermore, collagen I matrix degradation was found to be significantly increased in ∆lpg1 parasite-infected cells but not in WT-infected controls. Flow cytometry analysis revealed a substantial boost in production of reactive oxygen species (ROS) during infection with either WT or ∆lpg1 L. infantum. In addition, killing of ∆lpg1 parasites was shown to be more dependent on the ROS production than that of WT L. infantum. Notably, inhibition of the oxidative stress with Apocynin potentially fueled ∆lpg1 L. infantum fitness as it increased the intracellular parasite viability. Thus, our observations demonstrate that LPG may be a critical molecule fostering parasite survival in human neutrophils through a mechanism that involves cellular activation and generation of free radicals.

Comparative Genomic Reveals Clonal Heterogeneity in Persistent Staphylococcus aureus Infection.

Persistent infections caused by Staphylococcus aureus remain a clinical challenge. Adaptational mechanisms of the pathogen influencing infection persistence, treatment success, and clinical outcome in these types of infections by S. aureus have not been fully elucidated so far. We applied a whole-genome sequencing approach on fifteen isolates retrieved from a persistent S. aureus infection to determine their genetic relatedness, virulome, and resistome. The analysis of the genomic data indicates that all isolates shared a common clonal origin but displayed a heterogenous composition of virulence factors and antimicrobial resistance. This heterogeneity was reflected by different mutations in the rpoB gene that were related to the phenotypic antimicrobial resistance towards rifampicin and different minimal inhibitory concentrations of oxacillin. In addition, one group of isolates had acquired the genes encoding for staphylokinase (sak) and staphylococcal complement inhibitor (scn), leading to the truncation of the hemolysin b (hlb) gene. These features are characteristic for temperate phages of S. aureus that carry genes of the immune evasion cluster and confer triple conversion by integration into the hlb gene. Modulation of immune evasion mechanisms was demonstrated by significant differences in biofilm formation capacity, while invasion and intracellular survival in neutrophils were not uniformly altered by the presence of the immune evasion cluster. Virulence factors carried by temperate phages of S. aureus may contribute to the course of infection at different stages and affect immune evasion and pathogen persistence. In conclusion, the application of comparative genomic demonstrated clonal heterogeneity in persistent S. aureus infection.

NET Release of Long-Term Surviving Neutrophils.

BackgroundNeutrophil extracellular traps (NETs)—as double-edged swords of innate immunity—are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.MethodsWe analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).ResultsUntreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.ConclusionsG-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.

Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction.

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro. We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.

MicroRNA-Enriched Exosomes from Different Sources of Mesenchymal Stem Cells Can Differentially Modulate Functions of Immune Cells and Neurogenesis.

Adult Mesenchymal stem cells-derived exosomes carry several biologically active molecules that play prominent roles in controlling disease manifestations. The content of these exosomes, their functions, and effect on the immune cells may differ depending on their tissue sources. Therefore, in this study, we purified the exosomes from three different sources and, using the RNA-Seq approach, highly abundant microRNAs were identified and compared between exosomes and parental cells. The effects of exosomes on different immune cells were studied in vitro by incubating exosomes with PBMC and neutrophils and assessing their functions. The expression levels of several miRNAs varied within the different MSCs and exosomes. Additionally, the expression profile of most of the miRNAs was not similar to that of their respective sources. Exosomes isolated from different sources had different abilities to induce the process of neurogenesis and angiogenesis. Moreover, these exosomes demonstrated their varying effect on PBMC proliferation, neutrophil survival, and NET formation, highlighting their versatility and broad interaction with immune cells. The knowledge gained from this study will improve our understanding of the miRNA landscape of exosomes from hMSCs and provide a resource for further improving our understanding of exosome cargo and their interaction with immune cells.

Candida glabrata Hst1-Rfm1-Sum1 complex evolved to control virulence-related genes

C. glabrata is an opportunistic fungal pathogen and the second most common cause of opportunistic fungal infections in humans, that has evolved virulence factors to become a successful pathogen: strong resistance to oxidative stress, capable to adhere and form biofilms in human epithelial cells as well as to abiotic surfaces and high resistance to xenobiotics. Hst1 (a NAD+-dependent histone deacetylase), Sum1 (putative DNA binding protein) and Rfm1 (connector protein) form a complex (HRS-C) and control the resistance to oxidative stress, to xenobiotics (the antifungal fluconazole), and adherence to epithelial cells. Hst1 is functionally conserved within the Saccharomycetaceae family, Rfm1 shows a close phylogenetic relation within the Saccharomycetaceae family while Sum1 displays a distant phylogenetic relation with members of the family and is not conserved functionally. CDR1 encodes for an ABC transporter (resistance to fluconazole) negatively controlled by HRS-C, for which its binding site is located within 223 bp upstream from the ATG of CDR1. The absence of Hst1 and Sum1 renders the cells hyper-adherent, possibly due to the overexpression of AED1, EPA1, EPA22 and EPA6, all encoding for adhesins. Finally, in a neutrophil survival assay, HST1 and SUM1, are not required for survival. We propose that Sum1 in the HRS-C diverged functionally to control a set of genes implicated in virulence: adherence, resistance to xenobiotics and oxidative stress.

Hereditary Chronic Neutrophilic Leukemia in a Four Generation Family without Transformation to Acute Leukemia

Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN) characterized by peripheral blood leukocytosis consisting primarily of segmented neutrophils and band forms, hypercellular bone marrow with granulocytosis, hepatosplenomegaly, and the presence of activating colony-stimulating factor 3 receptor (CSF3R) mutations. Blast transformation occurs frequently in patients with acquired CNL, with a median overall survival of 21 months from diagnosis. Typically, CSF3R mutations in CNL are thought to be somatic; however, we and others have reported rare cases of germline activating CSF3R mutations producing a familial CNL. Here we report the clinical course of a patient with CNL along with definitive evidence of inherited germline transmission of the CSF3R T618I mutation. Spanning four generations, with affected family members of ages 1.6 - 51 years, this is the largest reported pedigree of a family with familial CNL (Figure 1A).The proband is a 49-year-old female referred to our center with a history of lifelong leukocytosis and leukocyte count of 115.1 x 10 9/L with 75% granulocytes and 11% bands, platelet count of 341 x 10 9/L, and hemoglobin of 12.5 g/dL with hematocrit of 38%. The family history was also remarkable for leukocytosis. Prior therapies for the proband included imatinib, splenectomy, and hydroxyurea. Additional testing by our center revealed a T618I CSFR3 mutation, and the absence of mutations in ASXL1 and SETBP1 or a BCR-ABL translocation. Treatment with ruxolitinib resulted in improvement of her leukocyte count to 43.0 x 10 9/L with 73% granulocytes, and reduction in her alkaline phosphatase from 732 IU/L to 296 IU/L. There has been no evidence of gain of any known deleterious somatic mutations that frequently co-occur with somatic T618I CSF3R mutations in CNL in the patient to date.Germline analysis of genomic DNA extracted from cultured mesenchymal stromal cells from the proband and Sanger sequencing demonstrated a heterozygous T618I mutation. Mutational analysis of the proband's family members confirmed a heterozygous CSF3R T618I mutation in all living affected family members, while all unaffected family members tested were homozygous wild type. There has been no evidence of leukemic transformation in any affected family members to date. Mutational analysis was not feasible on the proband's deceased mother and brother with a putative CNL diagnosis due to lack of DNA samples; however, there was no evidence of transformation to acute leukemia in either of the two deceased family members.Because CSF3R can produce anti-apoptotic signaling, we hypothesized that autoactivating T618I mutations could prolong neutrophil survival. Polymorphonuclear cells (PMNs) isolated from the proband and from normal donors were cultured in vitro and apoptosis assessed at 24-hour intervals. Neutrophils expressing the CSF3R T618I had prolonged survival with a >40% decrease in apoptosis after 48 hours in culture (Figure 1B). RNA-seq followed by pathway analysis demonstrated significant decreases in activation of canonical apoptotic pathways in PMNs, including both the extrinsic and mitochondrial dependent pathways. Immunoblotting for candidate anti- and pro-apoptotic proteins revealed increased expression of the anti-apoptotic BCL2 family member MCL1 in T618I-expressing PMNs. Notably, inhibition of MCL1 using S63845 reversed the anti-apoptotic effect induced by ligand-activation of the CSF3R receptor in PMNs (P < 0.001, Figure 1C).In conclusion, we demonstrate hereditary CNL within a large family tree with no observed transformation to acute leukemia in any affected individuals up to age 51, suggesting a potentially more indolent course. Nonetheless, our observations highlight the need for germline testing of patients with CNL to better understand the natural history of CNL. Moreover, our data provide further insight into the pathobiology of CNL and potential novel targets for therapy. [Display omitted] DisclosuresVoorhees: Bristol-Myers Squibb Company.: Other: Data Safety & Monitoring; AbbVie Inc, Bristol-Myers Squibb Company; Consulting Agreement: GlaxoSmithKline, Novartis, Oncopeptides: Other: Advisory Committee.

Hydrogen Peroxide Affects Growth of S. aureus Through Downregulation of Genes Involved in Pyrimidine Biosynthesis.

Reactive oxygen species (ROS) play a crucial role in the cellular defense against S. aureus, as evidenced by the importance of this pathogen in patients lacking the ROS-generating phagocyte NADPH oxidase NOX2. ROS concentrations required to kill S. aureus in vitro are much higher than those found in the phagosome. We therefore hypothesized that sublethal ROS concentrations may play a role in S. aureus gene dysregulation and investigated the in vitro transcriptomic response of S. aureus to sublethal concentrations of hydrogen peroxide (H2O2). A striking observation of these experiments was a coordinated and massive downregulation of genes involved in pyrimidine metabolism. Using transposon insertion mutants, we demonstrated that deletion of carA, a gene involved in pyrimidine synthesis, led to a significant growth defect and to an increased sensitivity of S. aureus to added H2O2. The phenotype of the carA mutant could be reversed through supplementation with the pyrimidine precursor uracil, or with a multicopy vector encoding carA. As opposed to the impact of ROS on extracellular survival, carA deletion did not affect the intracellular survival in neutrophils. Our results raise the possibility that ROS-dependent downregulation of pyrimidine metabolism might be a survival strategy of S. aureus, allowing colonization through intracellular survival, while decreasing the risk of killing the host through dampened extracellular growth.

Involvement of G-CSF, IL-6, and cortisol in transient neutrophilia after marathon races.

The aim of this study was to clarify the mechanisms of the transient increase in neutrophils after running standard marathon races by measurement of cytokines involved in the production and survival of neutrophils, and cortisol. Fourteen male runners who participated in the Hokkaido Marathon, which is the sole marathon race held in summer in Japan, and finished the standard marathon were analyzed sequentially from the start until a maximum of 8days after the finish. Neutrophilia was observed in all runners just after they reached the goal (mean neutrophils: 13226/μL). IL-6, G-CSF, and cortisol, but not GM-CSF, increased at the same time. Time-course studies with complete blood counts, biochemical markers, cytokines, and cortisol showed transient increases in neutrophils, monocytes, myoglobin, high-sensitivity C-reactive protein (hsCRP), G-CSF, IL-6, and cortisol. The increase in hsCRP was delayed 6hours from the first increase in neutrophils. Correlations were observed between the neutrophil count and G-CSF, IL-6, and cortisol (G-CSF; r=.667, IL-6; r=.667, cortisol; r=.623). These results suggest that G-CSF is directly involved, and IL-6 is involved via cortisol in the transient neutrophilia that occurs after marathon races.

Perioperative change in neutrophil count predicts worse survival in esophageal squamous cell carcinoma.

Aims: This study aimed to investigate the relationship between perioperative change in neutrophil count and survival of patients with esophageal squamous cell carcinoma. Method: Neutrophil change (Nc) (whereNc=post-surgeryneutrophil count-pre-surgeryneutrophil count) was counted according to data within 1week before surgery and 2weeks after surgery. Patients were divided into two groups, Nc ≥2.60 and Nc <2.60, according to the median of Nc. Results: Multivariate analysis revealed that Nc≥2.60 was an independent prognostic marker for overall survival. Subgroup analysis suggested that the overall survivalof male patients, patientsaged ≤60 years, patients without vessel invasion and patients without nerve infiltration wasdramatically worse for those with Nc<2.60. Conclusion: Perioperative change in neutrophil count predicts worse survival in esophageal squamous cell carcinoma after surgery.

Targeting multiple cell death pathways extends the shelf life and preserves the function of human and mouse neutrophils for transfusion.

Clinical outcomes from granulocyte transfusion (GTX) are disadvantaged by the short shelf life and compromised function of donor neutrophils. Spontaneous neutrophil death is heterogeneous and mediated by multiple pathways. Leveraging mechanistic knowledge and pharmacological screening, we identified a combined treatment, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which altered neutrophil fate by simultaneously targeting multiple cell death pathways. CLON-G prolonged human and mouse neutrophil half-life in vitro from less than 1 day to greater than 5 days. CLON-G-treated aged neutrophils had equivalent morphology and function to fresh neutrophils, with no impairment to critical effector functions including phagocytosis, bacterial killing, chemotaxis, and reactive oxygen species production. Transfusion with stored CLON-G-treated 3-day-old neutrophils enhanced host defenses, alleviated infection-induced tissue damage, and prolonged survival as effectively as transfusion with fresh neutrophils in a clinically relevant murine GTX model of neutropenia-related bacterial pneumonia and systemic candidiasis. Last, CLON-G treatment prolonged the shelf life and preserved the function of apheresis-collected human GTX products both ex vivo and in vivo in immunodeficient mice. Thus, CLON-G treatment represents an effective and applicable clinical procedure for the storage and application of neutrophils in transfusion medicine, providing a therapeutic strategy for improving GTX efficacy.