Peripheral Neutrophils Research Articles

Peripheral neutrophils and naive CD4 T cells predict the development of heart failure following acute myocardial infarction: A bioinformatic study

IntroductionHeart failure (HF) secondary to acute myocardial infarction (AMI) is still a worldwide problem with a high mortality rate. The current study aimed to explore early and reliable predictive biomarkers of HF following AMI. MethodsThe gene expression profile GSE59867 was downloaded from GEO. Array data from peripheral blood mononuclear cells (PBMCs) was used from 46 control patients and 111 patients with AMI at four time points: (i) first day of AMI; (ii) 4-6 days after AMI; (iii) one month after AMI; and (iv) six months after AMI. Among the 111 AMI patients, nine with HF and eight without HF were studied. CIBERSORT was used to analyze the relative proportions of immune cells in PBMCs. The proportions of immune cells in different groups were compared. Differentially expressed genes (DEGs) were analyzed with R language packages. ResultsThe percentages of monocytes and neutrophils increased significantly on the first day of AMI, and then decreased gradually. The percentage of regulatory T cells increased significantly 4-6 days after AMI, while the percentage of resting memory CD4 cells, CD8 T cells, and resting natural killer cells decreased significantly on the first day of AMI, and then increased gradually. Patients who developed HF had a significantly higher proportion of neutrophils in PBMCs on the first day of AMI, but had a significantly lower proportion of naive CD4 T cells. Two shared genes, interleukin-1 receptor 2 (IL1R2) and leucine-rich repeat neuronal protein 3 (LRRN3), were found to have potentially important roles in predicting the development of HF following AMI. ConclusionA higher proportion of neutrophils and a lower proportion of naive CD4 T cells in PBMCs on the first day of AMI may be correlated with the development of HF following AMI. IL1R2 and LRRN3 may exert functions in the development of HF following AMI.

In Gastric Cancer Patients Receiving Neoadjuvant Chemotherapy Systemic Inflammation Response Index is a Useful Prognostic Indicator.

Background: The preoperative systemic inflammation response index (SIRI), based on peripheral neutrophil (N), monocyte (M), and lymphocyte (L) counts, has shown mounting evidence as an effective prognostic indicator in some malignant tumors. The aim of the present study was to evaluate the prognostic significance of pre-treatment SIRI in gastric cancer patients who received neoadjuvant chemotherapy (NACT). Methods: This retrospective study comprised 107 patients with advanced gastric cancer treated with NACT between July 2007 and September 2015 in our hospital. SIRI was calculated from peripheral venous blood samples obtained prior to treatment. The best cutoff value for SIRI by receiver operating characteristic (ROC) curve was 1.2 (low SIRI <1.21, high SIRI ≥1.21). The clinical outcomes of disease-free survival (DFS) and overall survival (OS) were analyzed by Kaplan-Meier survival analysis and compared using the log-rank test. Univariate and multivariate analyses were performed by the Cox proportional hazards regression model. Results: The results demonstrated that the low SIRI group was statistically associated with gender, primary tumor site, white blood cell, neutrophil, and monocyte counts, NLR (neutrophil to lymphocyte ratio), MLR (monocyte to lymphocyte ratio), and PLR (platelet to lymphocyte ratio). The SIRI was predictive for DFS and OS by univariate and multivariate analysis; the low SIRI group had better median DFS and OS than the high SIRI group (median DFS 27.03 vs. 22.33 months, median OS 29.73 vs. 24.43 months). The DFS and OS in the low SIRI group were longer than the high SIRI group. Conclusions: SIRI may qualify as a useful, reliable, and convenient prognostic indicator in patients with advanced gastric cancer to help physicians to provide personalized prognostication for gastric cancer patients treated with NACT.

Soluble epoxide hydrolase inhibitor attenuates BBB disruption and neuroinflammation after intracerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is a devastating disease with high mortality and morbidity. Soluble epoxide hydrolase (sEH) is the key enzyme in the epoxyeicosatrienoic acids (EETs) signaling. sEH inhibition has been demonstrated to have neuroprotective effects against multiple brain injuries. However, its role in the secondary injuries after ICH has not been fully elucidated. Here we tested the hypothesis that 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent and highly selective sEH inhibitor, suppresses inflammation and the secondary injuries after ICH. Adult male C57BL/6 mice were subjected to a collagenase-induced ICH model. TPPU alleviated blood-brain barrier damage, inhibited inflammatory response, increased M2 polarization of microglial cells, reduced the infiltration of peripheral neutrophils. In addition, TPPU attenuated neuronal injury and promoted functional recovery. The results suggest that sEH may represent a potential therapeutic target for the treatment of ICH.

Prognostic Significance of Admission Systemic Inflammation Response Index in Patients With Spontaneous Intracerebral Hemorrhage: A Propensity Score Matching Analysis.

Intracerebral hemorrhage (ICH) accounts for ~15% of all strokes and is associated with high mortality and disability rates. The systemic inflammation response index (SIRI) is a novel systemic inflammatory marker based on peripheral neutrophil, monocyte, and lymphocyte counts. This study aimed to evaluate the prognostic significance of admission SIRI in patients with spontaneous ICH and compare its predictive ability with that of the neutrophil-to-lymphocyte ratio (NLR). This retrospective study was conducted based on a prospectively collected database of patients with ICH between June 2016 and January 2019. Propensity score matching (PSM) was conducted to adjust for potential imbalances in the clinical parameters. A total of 403 patients were included in the original cohort. The optimal SIRI cut-off value was 2.76. After 1:1 PSM based on potential confounding variables, a new cohort containing 262 patients was established for further analysis. In the original cohort, SIRI served as an independent predictor of 3-month functional outcome [odds ratio (OR), 1.302; 95% CI, 1.120–1.512; p = 0.001] and 1-month mortality (OR, 1.072; 95% CI, 1.020–1.126; p = 0.006), while NLR was independently associated with only 3-month functional outcomes (OR, 1.051; 95% CI, 1.004–1.100; p = 0.031) and not 1-month mortality. The same applied to the PSM cohort. Receiver operating characteristic analyses and predictive models indicated that in most instances, SIRI was superior to NLR and their components in predicting the outcomes of patients with ICH. Our study found that SIRI is determined to be an independent predictive indicator for ICH patients in 3-month functional outcomes and 1-month mortality. The prognostic predictive ability of SIRI was stronger than that of NLR.

PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke

There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell–specific PKM2−/− mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe−/−). Controls were littermate PKM2fl/flLysMCre– or PKM2fl/flLysMCre–Apoe−/− mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell–specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

The Functional Roles of RNAs Cargoes Released by Neutrophil-Derived Exosomes in Dermatomyositis.

Dermatomyositis (DM) is an idiopathic inflammatory myopathy characterized by cutaneous manifestations. We first identified the profiles of noncoding RNAs (lncRNAs and miRNAs) in peripheral neutrophil exosomes (EXOs) of DM patients and explored their potential functional roles. Bioinformatics analyses were performed with R packages. Real-time quantitative PCR was used to validate the altered RNAs in DM neutrophil EXO-stimulated human dermal microvascular endothelial cells (HDMECs) and human skeletal muscle myoblasts (HSkMCs). In DM neutrophil EXOs, 124 upregulated lncRNAs (with 1,392 target genes), 255 downregulated lncRNAs (with 1867 target genes), 17 upregulated miRNAs (with 2,908 target genes), and 15 downregulated miRNAs (with 2,176 target genes) were identified. GO analysis showed that the differentially expressed (DE) lncRNAs and DE miRNAs participated in interleukin-6 and interferon-beta production, skeletal muscle cell proliferation and development, and endothelial cell development and differentiation. KEGG analysis suggested that DE lncRNAs and DE miRNAs were enriched in the PI3K–Akt, MAPK, AMPK and FoxO signalling pathways. Many novel and valuable DE lncRNAs and DE miRNAs interacted and cotargeted in the PI3K–Akt, MAPK, AMPK and FoxO signalling pathways. Our study suggests that neutrophil EXOs participate in DM pathogenesis through lncRNAs and miRNAs in the PI3K–Akt, MAPK, AMPK and FoxO signalling pathways.

Abstract 107: PKM2 Promotes Neutrophil Activation And Cerebral Thrombo-inflammation: Therapeutic Implications For Ischemic Stroke

In recent years compelling evidence has emerged that implicates role of metabolic reprogramming in the modulation of thrombosis and inflammation. The dimeric form of the metabolic enzyme pyruvate kinase muscle 2 (PKM2) enters nucleus and exerts protein kinase activity. We found that nuclear PKM2 levels were upregulated in neutrophils after the onset of ischemic stroke both in humans and in mice. Therefore, we evaluated the role of PKM2 in promoting thrombo-inflammation and ischemic brain injury.We generated novel myeloid cell-specific PKM2 -/- mice on wild-type ( PKM2 fl/fl LysMCre + ) and hyperlipidemic background ( PKM2 fl/fl LysMCre + Apoe -/- ). We observed that genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps following cerebral ischemia/reperfusion. In the filament and autologous clot/rtPA stroke models, irrespective of sex, deletion of PKM2 in myeloid cells either in wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrin(ogen), platelet (CD41- positive) deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. Utilizing small molecule inhibitor (ML265) that inhibits PKM2 dimerization, we evaluated ex vivo thrombosis using human whole blood in a microfluidic flow chamber system. We observed a fivefold reduction in the thrombus growth rate in ML265-treated group. Similarly, ML265 treatment in mice resulted into significantly reduced poststroke neutrophil hyperactivation and improved short-term and long-term functional outcomes following stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery following reperfusion.

1305P Peripheral low-density neutrophils in the response to immunotherapy in untreated non-small cell lung cancer

Different groups have recently revealed the usefulness of immune cell quantification in peripheral blood to predict response to immune checkpoint inhibitors (ICI). Our team found an association between CD4+ CD27- CD28- T cells and response to ICI in pretreated NSCLC patients (Zuazo M, 2019), however these findings were not applicable in frontline ICI. We evaluated a cohort of untreated NSCLC patients receiving ICI alone or in combination with chemotherapy (CT+IT).

Dysconnectivity of a brain functional network was associated with blood inflammatory markers in depression

ObjectiveThere is increasing evidence for a subgroup of major depressive disorder (MDD) associated with heightened peripheral blood inflammatory markers. In this study, we aimed to understand the mechanistic brain-immune axis in inflammation-linked depression by investigating associations between functional connectivity (FC) of brain networks and peripheral blood immune markers in depression. MethodsResting-state functional magnetic resonance imaging (fMRI) and peripheral blood inflammatory markers (C-reactive protein; CRP, interleukin-6; IL-6 and immune cells) were collected on N = 46 healthy controls (HC; CRP ≤ 3 mg/L) and N = 83 cases of depression, stratified further into low CRP cases (loCRP cases; ≤ 3 mg/L; N = 50) and high CRP cases (hiCRP cases; > 3 mg/L; N = 33). In a two-part analysis, network-based statistics (NBS) was firstly used to ascertain whole-brain FC differences in HC vs hiCRP cases. Secondly, we investigated the association between this network of interconnected brain regions and continuous measures of peripheral CRP (N = 83), IL-6 (N = 72), neutrophils and CD4+ T-cells (N = 36) in depression cases only. ResultsCase-control NBS testing revealed a single network of abnormally attenuated FC in the high CRP depression cases compared to healthy controls. Connections within this network were mainly between brain regions located in the left insula/frontal operculum and posterior cingulate cortex, which were assigned to ventral attention and default mode canonical fMRI networks respectively. Within-group analysis across all depression cases, secondarily demonstrated that FC within the identified network significantly negatively scaled with CRP, IL-6 and neutrophils. ConclusionsThe findings suggest that inflammation is associated with disruption of functional connectivity within a brain network deemed critical for interoceptive signalling, e.g. accurate communication of peripheral bodily signals such as immune states to the brain, with implications for the pathogenesis of inflammation-linked depression.

Increased Neutrophil Aging Contributes to T Cell Immune Suppression by PD-L1 and Arginase-1 in HIV-1 Treatment Naïve Patients.

Neutrophils are characterized by their heterogeneity. They fight against pathogens and are involved in tissue injury repair and immune system regulation. Neutrophils have an extremely short life span in the peripheral blood and undergo aging after being released from the bone marrow. The over-aggregation of aged neutrophils is associated with phenotypical and functional changes. Here, we aimed to investigate the dynamics of neutrophil aging and its relationship with T cell exhaustion in HIV-1 infection, as they are not well understood. In this study, we enrolled 23 treatment naïve (TN) patients, 23 individuals that had received antiretroviral therapy (ART), and 21 healthy controls (HC). In these cohorts, we measured the degree of neutrophil aging, and its possible correlation with T cell dysfunction. In TN patients, peripheral neutrophils showed a more distinct aging phenotype and were over-activated compared to those in ART-treated patients. The degree of neutrophil aging was positively correlated with HIV-1 RNA viral load and negatively correlated with CD4+ T cell count. Moreover, aged neutrophils had impaired reactive oxygen species (ROS) production after lipopolysaccharide (LPS) stimulation, and were characterized by increased PD-L1 and arginase-1 expression in a time-dependent manner. Aged neutrophils demonstrated an increased inhibition of IFN-γ and TNF-α secretion by CD8+ T cell compared to non-aged neutrophils. The inhibition effect could be partially reversed by blocking PD-L1 and arginase-1 in vitro, and LPS was identified as an important activator of neutrophil aging. These results provide evidence that dampening neutrophil aging may provide a novel approach to recover T cell dysfunction in patients with HIV-1 infection.

Alpha-1 Antitrypsin Augmentation Inhibits Proteolysis of Neutrophil Membrane Voltage-Gated Proton Channel-1 in Alpha-1 Deficient Individuals.

Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.

Changes in the gut microbiota: a possible factor influencing peripheral blood immune indexes in non-obese diabetic mice.

Intestinal flora changes were found in patients and animals with type 1 diabetes (T1D). However, few studies have provided any explicit clues of changes in highly disease related commensal microbiota before disease onset and their relationships with disordered peripheral immune cells. We conducted 16S rRNA microbiota analysis of non-obese diabetic (NOD) mice from weaning to diabetes onset to identify highly disease related microbes and performed Spearman correlation analysis between anomalous flora and peripheral immune cells. We found NOD mice had increased exclusive bacteria and decreased community richness or diversity, besides, with the features of decreased abundance of Bacteroidetes and increased abundance of Firmicutes, Proteobacteria or Deferribacteres and remarkable fluctuations of genus relative abundance. Furthermore, kinds of highly T1D related genus and their strong correlations with peripheral immune cells, especially neutrophils, were discovered. Microbial changes in NOD mice differed from that of ICR mice and highly disease associated microbes have strong correlations with the peripheral neutrophil ratio, which provide evidence that neutrophils are possibly involved in the pathogenesis of T1D.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

Peripheral Neutrophil- Lymphocyte Ratio as a Predictor of Seizure in Preterm Infants with Intra-Ventricular Hemorrhage

Background: Preterm infants with Intra-Ventricular Hemorrhage (IVH) are at risk for developing significant complications, including post hemorrhagic hydrocephalus and seizures. Neonatal seizures are the most common overt manifestation of neurological dysfunction in the newborn, and is associated with short- and long-term adverse effects.&#x0D; Objective: The aim of the study is to evaluate the value of Peripheral Neutrophil- lymphocyte ratio (NLR) as a predictor of seizure in preterm infants with intra-ventricular hemorrhage.&#x0D; Methods: This prospective cohort study that comprised 60 Preterm infants with IVH admitted at NICU pediatric department Tanta university hospital from November 2019 to May 2020, Preterm infants were divided into two groups according to incidence of seizure.&#x0D; Preterm infants in this study subjected to Careful history taking, clinical examination and investigations (laboratory and Trans-cranial ultrasound) as well as analysis of result and follow up clinical status for development of seizure.&#x0D; Results: There was a statistically highly significant difference regarding NLR and development of seizure with p value &lt;0.001 (NLR ≥ 2.3 with sensitivity 96%, specificity 93%, Area under the curve 0.849 and accuracy 84.9%).&#x0D; Conclusion: NLR is a predictor of seizure in preterm infants with intra-ventricular hemorrhage.

Assessment of the In Vivo Release and Biocompatibility of Novel Vesicles Containing Zinc in Rats.

This paper is focused on the in vivo release and biocompatibility evaluation in rats of some novel systems entrapping zinc chloride in lipid vesicles. The particles were prepared by zinc chloride immobilization inside lipid vesicles made using phosphatidylcholine, stabilized with 0.5% chitosan solution, and dialyzed for 10 h to achieve a neutral pH. The submicrometric systems were physico-chemically characterized. White Wistar rats, assigned into four groups of six animals each, were treated orally with a single dose, as follows: Group I (control): deionized water 0.3 mL/100 g body weight; Group II (Zn): 2 mg/kg body weight (kbw) zinc chloride; Group III (LV-Zn): 2 mg/kbw zinc chloride in vesicles; Group IV (LVC-Zn): 2 mg/kbw zinc chloride in vesicles stabilized with chitosan. Haematological, biochemical, and immune parameters were assessed after 24 h and 7 days, and then liver fragments were collected for histopathological examination. The use of zinc submicrometric particles—especially those stabilized with chitosan—showed a delayed zinc release in rats. No substantial changes to blood parameters, plasma biochemical tests, serum complement activity, or peripheral neutrophils phagocytic capacity were noted; moreover, the tested substances did not induce liver architectural disturbances. The obtained systems provided a delayed release of zinc, and showed good biocompatibility in rats.

Activation of T Cells Specific to Endogenous Retroviral Peptides: Possible Association with Clinical Response to Azacitidine in Myeloid Malignancies

DNA methyltransferase inhibitors (DNMTi's), 5-azacytidine (5-aza-CR, azacitidine) and 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine), are drugs of choice for patients with higher-risk myelodysplastic syndromes (MDS). Although DNMTi's have shown clinical benefits, their mode of action is not completely understood. In vitro studies have shown that low-dose DNMTi causes up-regulation of transcripts derived from human endogenous retroviruses (HERVs) in target cells that may give rise to double and single stranded RNA formation that are sensed by the cells as foreign. This in turn induces the viral defense pathway that elicits an interferon response, which may play an important role in the efficacies of DNMTi's. In this study we wanted to explore whether DNMTi-treatment in patients causes up-regulation of HERVs and formation of HERV-derived peptides in vivo that can stimulate an adaptive immune response against the malignant cells.We have developed a novel technology that allows multi-parallel assessment of T-cell reactivity for >1000 different peptide-MHC complexes in a single sample (Bentzen et al., Nat Biotechnol, 2016). To identify recognition of HERV-derived peptides by CD8+ T cells we created a library of 1174 such antigens coupled to the most frequent HLA-I types (HLA-A*01:01, HLA-A*02:01, HLA-B*07:02 and HLA-B*08:01). Peripheral blood mononuclear cells from 26 patients (17 MDS, 5 AML and 4 CMML) with matching HLA-I types and treated with azacitidine were sampled before and during treatment. Cells from onset of treatment and at the starting day of third and fifth treatment cycle were used in our multiplex technology to identify activated CD8+ T cells recognizing HERV-derived peptides embedded in their respective HLA class I molecules. For a comparative analysis we also included known exogenous viral antigens to assess whether the enhancement in CD8+ T-cell responses was a general phenomenon or restricted to the selected antigens. Moreover, CD8+ T cells from a number of healthy donors were investigated with the same peptide library for normalization. Sorted CD14+ and CD3+ cells from four CMML patients were analyzed at transcript-level by RNA sequencing for up-regulation of HERVs and viral defense genes. Besides, DNA from peripheral neutrophils or from mononuclear cells from 18 of the patients was sequenced with a targeted 20 gene panel, including genes frequently mutated in MDS. Clinical characteristics and treatment responses according to the IWG criteria were collected from patient files.Nine of the 26 patients showed a clinical response to the treatment. Of these nine patients four had a significant induction of specific CD8+ T cells targeting HERV-derived peptides. For the non-responding patients the equivalent number was two of 17 patients (p<0.05); one of them with stable disease during treatment until undergoing allogeneic hematopoietic stem cell transplantation. When investigating individual HERV families across all patients we found a widespread induction of specific CD8+ T cells targeting HERV-derived peptides. The preliminary RNA sequencing data revealed that HERVs and downstream viral defense genes are indeed up-regulated in the patients during treatment and that more HERVs are up-regulated in the malignant cells compared with T cells. Conclusively, the up-regulation of HERVs and induction of immune responses, specifically adaptive immunity against HERV-derived peptides, may be essential for the patients' responses to this class of drugs. These findings point towards a rationale for combining DNMTi's with immunotherapy.A.D.Ø, S.K.S. and A-M.B. as well as K.G. and S.R.H. contributed equally. DisclosuresTreppendahl:Novo Nordisk: Employment. Grønbæk:Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Combinatorial CSF3R and RUNX1 Mutations Involved in the Leukemic Progression of Severe Congenital Neutropenia Confer a G-CSF-Dependent Premalignant State in Mice

Background: Severe congenital neutropenia (SCN) is a genetically heterogeneous disorder characterized by a lack of peripheral neutrophils, leading to life-threatening bacterial infections. The disease is alleviated by sustained G-CSF treatment, which restores neutrophil production in the majority of patients. While on G-CSF therapy, SCN patients frequently develop hematopoietic clones with acquired CSF3R mutations. The appearance of CSF3R mutant clones is seen in multiple genetic subtypes of SCN and is strongly associated with progression towards MDS or AML. At this stage, CSF3R mutant clones acquire secondary mutations, most often (in ~75% of cases) in RUNX1 . The majority of these mutations affect the Runt homology domain (RHD) of RUNX1. This combination of CSF3R and RUNX1 mutations is exclusively seen in SCN/MDS/AML, suggesting a unique pattern of leukemic progression of SCN. However, the molecular and cellular consequences of combined CSF3R and RUNX1 mutations and the effects of sustained G-CSF therapy on leukemia development are unknown.Aim: To determine the cooperative role of CSF3R and RUNX1 mutations in sequential steps of malignant transformation in vivo and to investigate how sustained G-CSF therapy affects this process.Methods: We used our previously generated Csf3r -d715 mouse strain, which copies the most frequent G-CSFR truncation mutant in SCN patients. Lethally irradiated wild type mice were transplanted with Csf3r-d715 HSPCs transduced with an IRES-GFP backbone lentiviral vector expressing RHD mutant RUNX1-D171N or empty vector control. Mice were treated 3x per week with G-CSF or PBS (solvent control). Biweekly, peripheral blood (PB) samples were analyzed for GFP-marked engraftment, immunophenotypes and cytology. At regular intervals, in vitro progenitor cell colony assays were performed. Bone marrow (BM) and spleen samples were used for phenotypic analyses and secondary transplantations. RNA-seq experiments were carried out on FACS purified LK populations to identify molecular pathways associated with the observed phenotypes.Results: Mice transplanted with Csf3r -d715/ RUNX1 -D171N (RHD mutant) BM cells started to accumulate GFP-marked Lin negative, c-Kit positive (LK) progenitor cells in the PB, 6 weeks after initiation of G-CSF treatment. The percentages of GFP+ LK cells in the PB ranged from 0.7% to 27.8% (Mean=5.9%; n=11) and fluctuated over time in individual mice, indicating that they were maintained but not vastly expansive at this stage. This phenotype was fully dependent on the combined presence of the Csf3r and RUNX1 mutations and was only seen in the G-CSF treated mice. The PB-LK cells had a blast-like morphology and formed myeloid colonies in semi-solid cultures. Transcriptome analysis of in vitro expanded LK cells by RNA-seq and gene set enrichment analysis (GSEA) showed that the combination of Csf3r -d715 and RUNX1 -D171N mutations and G-CSF stimulation selectively enhanced c-Myc , NF-κB and E2F1-driven transcriptional programs. Of note, fully differentiated GFP-positive neutrophils were also formed, both in vitro and in vivo and mice did not succumb to this pre-leukemic condition. Csf3r -d715/ RUNX1 -D171N BM cells isolated after 30 weeks could successfully engraft secondary recipients (n=6), again resulting in sustained G-CSF dependent accumulation of immature blast (LK) cells in the PB. In these mice, the blast cells in the BM exceeded 85% at week 7 after initiation of G-CSF treatment. At this stage, the blasts had become entirely defective in neutrophil differentiation, indicating that they had now undergone full transformation to AML. RNA-seq and genomic analyses of these AML blasts are currently underway to assess the molecular steps involved in the transition from the premalignant to the fully transformed state.Conclusion: The combination of Csf3r -d715 and RUNX1 -D171N (RHD) mutations frequently seen in SCN/MDS/AML perturbs myeloid development in an in vivo transplant model in a G-CSF dependent manner. A sustained pre-leukemic state characterized by the selective accumulation of LK cells is seen in primary recipients, followed by a fully transformed AML phenotype in secondary recipients. This model is attractive for unraveling the molecular steps of malignant transformation driven by CSF3R and RUNX1 mutations and for preclinical testing of new therapeutic options for SCN/AML. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Significance of Hematopoietic-cell Serglycin for the Survival of Hepatocellular Carcinoma: A Single-center Retrospective Study.

Inflammation-related changes in peripheral blood cells and blood proteins are prognostic factors for survival in hepatocellular carcinoma (HCC), but their usefulness is limited by an active bacterial infection. This study investigated whether infection interfered with the predictive value of serglycin, a proteoglycan found in hematopoietic cells, on survival in HCC. Patients with hepatitis B virus (HBV)-induced HCC, 100 without and 30 with a bacterial infection, and 30 healthy adult controls were enrolled retrospectively. Baseline clinical data collected before treatment with transarterial chemoembolization (TACE) was evaluated, and serglycin expression was assayed by flow cytometry. Receiver operating characteristic (ROC) curve analysis identified serglycin cutoff values for patient stratification. Cox regression and Kaplan-Meier analyses were performed to identify predictors of overall survival (OS). Serglycin levels in peripheral blood cells were higher in both groups of HCC patients than in the control group. Cholinesterase, lung metastasis, average neutrophil serglycin fluorescence intensity, and aspartate aminotransferase levels were associated with survival risk. Barcelona Clinic Liver Cancer stage A was associated with a good prognosis of OS. The intensity of serglycin fluorescence in peripheral neutrophils was independently predictive of survival in HCC, and its value was not limited by a bacterial infection. The method presented here is a simple and feasible way to predict prognosis in HCC patients with TACE.

Oral dimethyl fumarate induces changes within the peripheral neutrophil compartment of patients with psoriasis that are linked with skin improvement.

Dimethyl fumarate (DMF) is a treatment for moderate-to-severe psoriasis and multiple sclerosis. DMF therapy typically improves skin inflammation within the first 3months of treatment. DMF is a prodrug that generates the hydroxycarboxylic acid receptor 2 (HCA2) agonist, monomethyl fumarate (MMF). Despite widespread clinical use, DMF's mechanism of action is not fully understood. We wished to characterize the changes induced by DMF in peripheral neutrophils within the first 3months of treatment to better understand its early antipsoriatic effects. Flow cytometry was used to assess T-cell and neutrophil frequencies, apoptosis and activation phenotype. In vitro culture of neutrophils with DMF and MMF was used to evaluate apoptosis and HCA2 internalization. Serum levels of neutrophil degranulation products were measured by enzyme-linked immunosorbent assay. Patients with psoriasis had significantly higher leucocyte counts at baseline compared with controls, with a large population of pro-inflammatory CD62Llo CD11bbright neutrophils. Analysis revealed that DMF treatment reduced the frequency of CD62Llo CD11bbright neutrophils and serum levels of neutrophil activation markers. This reduction was not linked to increased apoptosis. Our results reveal a novel invivo effect of DMF therapy on pro-inflammatory neutrophils that likely contributes to this treatment's antipsoriatic efficacy.

Systemic Inflammation Response Index is a Prognostic Risk Factor in Patients with Hepatocellular Carcinoma Undergoing TACE.

BackgroundMounting evidence has shown that systemic inflammation response index (SIRI), a novel prognostic biomarker based on peripheral lymphocyte, neutrophil and monocyte counts, is associated with poor prognosis for several tumors. However, the prognostic value of SIRI in patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE) is elusive. Herein, we aimed to evaluate the correlation between SIRI and clinical outcomes in these patients.MethodsA total of 194 consecutive patients who underwent TACE were included in this study. Patients were stratified into high and low SIRI groups based on the cut-off value using receiver operating characteristic (ROC) analysis. Independent risk factors for tumor response were analyzed using forward stepwise logistic regression. A one-to-one propensity score matching (PSM) was conducted to compare progression-free survival (PFS) and overall survival (OS) between low and high SIRI patients. The discriminatory power of the combination of number of tumors and SIRI in predicting initial TACE response was evaluated by ROC analysis.ResultsPatients were divided into high SIRI (> 0.88) and low SIRI (≤ 0.88) groups. High SIRI (p = 0.003) and more than three tumors (p = 0.002) were significantly related to poorer tumor response. Moreover, the low SIRI group had longer PFS and OS than the high SIRI group (both P < 0.05) before and after PSM. Combination of SIRI and number of tumors can improve the predictive ability to predict initial TACE response with an area under the curve (AUC) of 0.678.ConclusionPretreatment peripheral blood SIRI was found to be an independent predictor of tumor response and clinical outcomes in patients with HCC undergoing TACE. Patients with high SIRI may have a poor prognosis.