Characterising the transcriptome of hypersegmented human neutrophils

Background: Mature human neutrophils are characterised by their multilobed nuclear morphology. Neutrophil hypersegmentation, a pathologic nuclear phenotype, has been described in the alveolar compartment of patients with acute respiratory distress syndrome and in several other contexts. This study aimed to characterise the transcriptional changes associated with neutrophil hypersegmentation. Methods: A model of hypersegmentation was established by exposing healthy peripheral blood neutrophils to the angiotensin converting enzyme inhibitor (ACEi) captopril. Laser capture microdissection (LCM) was then adapted to isolate a population of hypersegmented neutrophils. Transcriptomic analysis of microdissected hypersegmented neutrophils was undertaken using ribonucleic acid (RNA) sequencing. Differential gene expression (DEG) and enrichment pathway analysis were conducted to investigate the mechanisms underlying hypersegmentation. Results: RNA-Seq analysis revealed the transcriptomic signature of hypersegmented neutrophils, with five genes differentially expressed. VCAN, PADI4 and DUSP4 were downregulated, while LTF and PSMC4 were upregulated. Modulated pathways included histone modification, protein-DNA complex assembly and antimicrobial humoral response. The role of PADI4 was further validated using the small molecule inhibitor, Cl-amidine. Conclusions: Hypersegmented neutrophils display a marked transcriptomic signature, characterised by the differential expression of five genes. This study provides insights into the mechanisms underlying neutrophil hypersegmentation and describes a novel method to isolate and sequence neutrophils based on their morphologic subtype.

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Transcriptional landscape of PTEN loss in primary prostate cancer

BMC Cancer ◽

10.1186/s12885-021-08593-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Eddie Luidy Imada ◽

Diego Fernando Sanchez ◽

Wikum Dinalankara ◽

Thiago Vidotto ◽

Ericka M. Ebot ◽

...

Keyword(s):

Prostate Cancer ◽

Meta Analysis ◽

Pten Loss ◽

Primary Prostate Cancer ◽

Expression Atlas ◽

Transcriptomic Signature ◽

Transcriptional Changes ◽

New Biomarkers ◽

Adaptive Immune Systems ◽

Transcriptional Landscape

Abstract Background PTEN is the most frequently lost tumor suppressor in primary prostate cancer (PCa) and its loss is associated with aggressive disease. However, the transcriptional changes associated with PTEN loss in PCa have not been described in detail. In this study, we highlight the transcriptional changes associated with PTEN loss in PCa. Methods Using a meta-analysis approach, we leveraged two large PCa cohorts with experimentally validated PTEN and ERG status by Immunohistochemistry (IHC), to derive a transcriptomic signature of PTEN loss, while also accounting for potential confounders due to ERG rearrangements. This signature was expanded to lncRNAs using the TCGA quantifications from the FC-R2 expression atlas. Results The signatures indicate a strong activation of both innate and adaptive immune systems upon PTEN loss, as well as an expected activation of cell-cycle genes. Moreover, we made use of our recently developed FC-R2 expression atlas to expand this signature to include many non-coding RNAs recently annotated by the FANTOM consortium. Highlighting potential novel lncRNAs associated with PTEN loss and PCa progression. Conclusion We created a PCa specific signature of the transcriptional landscape of PTEN loss that comprises both the coding and an extensive non-coding counterpart, highlighting potential new players in PCa progression. We also show that contrary to what is observed in other cancers, PTEN loss in PCa leads to increased activation of the immune system. These findings can help the development of new biomarkers and help guide therapy choices.

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Microarray analysis reveals an inflammatory transcriptomic signature in peripheral blood for sciatica

BMC Neurology ◽

10.1186/s12883-021-02078-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Yi Wang ◽

Guogang Dai ◽

Ling Jiang ◽

Shichuan Liao ◽

Jiao Xia

Keyword(s):

Functional Analysis ◽

Network Analysis ◽

Peripheral Blood ◽

Differentially Expressed ◽

Healthy Controls ◽

Toll Like Receptor ◽

Qrt Pcr ◽

Transcriptomic Signature ◽

Transcriptional Changes ◽

Key Genes

Abstract Background Although the pathology of sciatica has been studied extensively, the transcriptional changes in the peripheral blood caused by sciatica have not been characterized. This study aimed to characterize the peripheral blood transcriptomic signature for sciatica. Methods We used a microarray to identify differentially expressed genes in the peripheral blood of patients with sciatica compared with that of healthy controls, performed a functional analysis to reveal the peripheral blood transcriptomic signature for sciatica, and conducted a network analysis to identify key genes that contribute to the observed transcriptional changes. The expression levels of these key genes were assessed by qRT-PCR. Results We found that 153 genes were differentially expressed in the peripheral blood of patients with sciatica compared with that of healthy controls, and 131 and 22 of these were upregulated and downregulated, respectively. A functional analysis revealed that these differentially expressed genes (DEGs) were strongly enriched for the inflammatory response or immunity. The network analysis revealed that a group of genes, most of which are related to the inflammatory response, played a key role in the dysregulation of these DEGs. These key genes are Toll-like receptor 4, matrix metallopeptidase 9, myeloperoxidase, cathelicidin antimicrobial peptide, resistin and Toll-like receptor 5, and a qRT-PCR analysis validated the higher transcript levels of these key genes in the peripheral blood of patients with sciatica than in that of healthy controls. Conclusion We revealed inflammatory characteristics that serve as a peripheral blood transcriptomic signature for sciatica and identified genes that are essential for mRNA dysregulation in the peripheral blood of patients with sciatica.

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Electrospun Polydioxanone Loaded With Chloroquine Modulates Template-Induced NET Release and Inflammatory Responses From Human Neutrophils

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.652055 ◽

2021 ◽

Vol 9 ◽

Author(s):

Allison E. Fetz ◽

Shannon E. Wallace ◽

Gary L. Bowlin

Keyword(s):

Growth Factor ◽

Inflammatory Responses ◽

Human Peripheral Blood ◽

Early Time ◽

Human Neutrophils ◽

Late Time ◽

Dependent Manner ◽

Blood Neutrophils ◽

Net Release

The implantation of a biomaterial quickly initiates a tissue repair program initially characterized by a neutrophil influx. During the acute inflammatory response, neutrophils release neutrophil extracellular traps (NETs) and secrete soluble signals to modulate the tissue environment. In this work, we evaluated chloroquine diphosphate, an antimalarial with immunomodulatory and antithrombotic effects, as an electrospun biomaterial additive to regulate neutrophil-mediated inflammation. Electrospinning of polydioxanone was optimized for rapid chloroquine elution within 1 h, and acute neutrophil-biomaterial interactions were evaluated in vitro with fresh human peripheral blood neutrophils at 3 and 6 h before quantifying the release of NETs and secretion of inflammatory and regenerative factors. Our results indicate that chloroquine suppresses NET release in a biomaterial surface area–dependent manner at the early time point, whereas it modulates signal secretion at both early and late time points. More specifically, chloroquine elution down-regulates interleukin 8 (IL-8) and matrix metalloproteinase nine secretion while up-regulating hepatocyte growth factor, vascular endothelial growth factor A, and IL-22 secretion, suggesting a potential shift toward a resolving neutrophil phenotype. Our novel repurposing of chloroquine as a biomaterial additive may therefore have synergistic, immunomodulatory effects that are advantageous for biomaterial-guided in situ tissue regeneration applications.

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Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases

mBio ◽

10.1128/mbio.02031-20 ◽

2021 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Tatiane S. Lima ◽

Sharmila Mallya ◽

Allen Jankeel ◽

Ilhem Messaoudi ◽

Melissa B. Lodoen

Keyword(s):

Cell Death ◽

Toxoplasma Gondii ◽

Life Span ◽

Peripheral Blood ◽

Productive Infection ◽

Human Neutrophils ◽

Serum Starvation ◽

Rna Seq ◽

Content Type ◽

Blood Neutrophils

ABSTRACT Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

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Identification and characterization of a unique subpopulation (CALLA/CD10/negative) of human neutrophils manifesting a heightened chemotactic response to activated complement

Blood ◽

10.1182/blood.v70.5.1624.bloodjournal7051624 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1624-1629

Author(s):

RT McCormack ◽

RD Nelson ◽

DE Chenoweth ◽

TW LeBien

Keyword(s):

Lymphoblastic Leukemia ◽

Low Frequency ◽

Chemotactic Response ◽

Human Neutrophils ◽

Normal Peripheral Blood ◽

Cd10 Expression ◽

Blood Neutrophils

We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10- negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.

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R213G polymorphism in SOD3 protects against bleomycin-induced inflammation and attenuates induction of proinflammatory pathways

Physiological Genomics ◽

10.1152/physiolgenomics.00053.2018 ◽

2018 ◽

Vol 50 (9) ◽

pp. 807-816 ◽

Cited By ~ 6

Author(s):

Anastacia M. Garcia ◽

Ayed Allawzi ◽

Philip Tatman ◽

Laura Hernandez-Lagunas ◽

Kalin Swain ◽

...

Keyword(s):

Inflammatory Responses ◽

Rna Seq ◽

Single Nucleotide ◽

Enhanced Resolution ◽

The Matrix ◽

Pathways Analysis ◽

Transcriptional Changes ◽

Differential Gene ◽

Glycine Substitution ◽

Downstream Processes

Extracellular superoxide dismutase (EC-SOD), one of three mammalian SOD isoforms, is the sole extracellular enzymatic defense against superoxide. A known human single nucleotide polymorphism (SNP) in the matrix-binding domain of EC-SOD characterized by an arginine-to-glycine substitution at position 213 (R213G) redistributes EC-SOD from the matrix into extracellular fluids. We previously reported that knock-in mice harboring the human R213G SNP (R213G mice) exhibited enhanced resolution of inflammation with subsequent protection against fibrosis following bleomycin treatment compared with wild-type (WT) littermates. Herein we set out to determine the underlying pathways with RNA-Seq analysis of WT and R213G lungs 7 days post-PBS and bleomycin. RNA-Seq analysis uncovered significant differential gene expression changes induced in WT and R213G strains in response to bleomycin. Ingenuity Pathways Analysis was used to predict differentially regulated up- and downstream processes based on transcriptional changes. Most prominent was the induction of inflammatory and immune responses in WT mice, which were suppressed in the R213G mice. Specifically, PKC signaling in T lymphocytes, IL-6, and NFΚB signaling were opposed in WT mice when compared with R213G. Several upstream regulators such as IFNγ, IRF3, and IKBKG were implicated in the divergent responses between WT and R213G mice. Our data suggest that the redistributed EC-SOD due to the R213G SNP attenuates the dysregulated inflammatory responses observed in WT mice. We speculate that redistributed EC-SOD protects against dysregulated alveolar inflammation via reprogramming of recruited immune cells toward a proresolving state.

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The chemokinetic response of human neutrophils

Blood ◽

10.1182/blood.v67.4.1036.1036 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1036-1042 ◽

Cited By ~ 7

Author(s):

TH Howard

Keyword(s):

Dose Dependence ◽

Human Neutrophils ◽

Maximal Response ◽

Computer Assisted ◽

Cellular Inflammatory Response ◽

Blood Neutrophils ◽

Response Increase ◽

Flow Through ◽

The Mean ◽

Serial Addition

Abstract A computer-assisted single cell assay that allows quantification of the locomotive behavior of individual cells and a flow-through system that allows study of response of individual cells to stimulation were utilized to study the chemokinetic response of neutrophils. The range of basal mean rate of locomotion (mROL) and chemokinetic response to 10(-9) mol/L formylmethionyl leucyl phenylalanine (FMLP) was determined for neutrophils of eight normal adults. The basal mROL was 8.2 +/- 1.5 um/min and 6.2 +/- 1.0 um/min; the rate after 10(-9) mol/L fMLP was 12.1 +/- 2.1 and 9.5 +/- 1.8 um/min in 2.0 g% and 0.05 g% HSA, respectively. The mean increase in ROL for neutrophils was 50%. Assay with the flow-through system shows that the chemokinetic response-- increase in mROL of a population of neutrophils in response to 10(-9) mol/L--is due to an increase in ROL when cells are actively moving and not due to a decrease in the amount of time the cell spends inactive. Studies of individual cells within the populations show that chemokinetic response to 10(-9) mol/L fMLP is highly variable. The majority of cells (77%) respond with an increase in ROL; the minority (23%) are nonresponders that characteristically move at ROL greater than or equal to 14 um/min prior to stimulation and do not change ROL or exhibit a net decline in ROL in response to 10(-9) mol/L fMLP. The dose response of a population of neutrophils and of individual neutrophils to serial addition of 10(-10) to 10(-6) mol/L fMLP shows that the fMLP dose dependence for maximal chemokinetic response is highly variable among individual cells. Seventeen percent of cells do not respond to any fMLP concentration; 25% of neutrophils exhibit maximal response to 10(-10) mol/L fMLP, while 50% and 25% of cells showed peak chemokinetic response to 10(-9) mol/L and greater than or equal to 10(-8) mol/L fMLP, respectively. These studies document the variability in the locomotive responses of peripheral blood neutrophils. Understanding the causes of variability in the chemokinetic responsiveness of individual neutrophils may improve our understanding of how the cellular inflammatory response in man can be modulated.

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Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold

Blood ◽

10.1182/blood.v65.2.423.423 ◽

1985 ◽

Vol 65 (2) ◽

pp. 423-432 ◽

Cited By ~ 83

Author(s):

E Cramer ◽

KB Pryzwansky ◽

JL Villeval ◽

U Testa ◽

J Breton-Gorius

Keyword(s):

Human Peripheral Blood ◽

Ultrastructural Localization ◽

Human Neutrophils ◽

Cell Fractionation ◽

Thin Sections ◽

The Matrix ◽

Blood Neutrophils ◽

Microscopic Studies ◽

Specific Localization ◽

Monospecific Antibodies

Abstract Colloidal gold was used as a marker for immunoelectron microscopy to localize lactoferrin (LF) and myeloperoxidase (MPO) in human peripheral blood neutrophils. Cells were reacted with monospecific antibodies against LF or MPO and then with gold-labeled antiglobulin. MPO cytochemistry was also associated with immunologic detection of LF. Immunologic labeling of thin sections after embedding in glycol methacrylate gave good ultrastructural morphology and specific localization of both proteins. MPO was detected in the large azurophil granules, whereas LF was consistently localized in the matrix of another population of morphologically distinct granules, smaller and more numerous than azurophil granules. When cytochemical detection of MPO was coupled with immunologic detection of LF, LF was observed in the population of MPO-negative granules, which were identified as specific. This was confirmed on cells that were permeabilized with saponin and stained for LF and MPO before embedding. No other neutrophil organelles displayed labeling for LF; other blood cells also were unreactive for LF. In the bone marrow, myeloblast and promyelocyte granulations were not stained and LF-containing granules appeared at the myelocyte stage. In conclusion, we confirm previous biochemical and light microscopic studies by ultrastructural demonstration of LF and MPO in two categories of granules, the specific and azurophil granules, respectively. The method described in this article avoids disruption caused by cell fractionation procedures. In the future, other intragranular proteins can be localized by a similar approach.

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Characterization of BMS-911543, a Functionally Selective Small Molecule Inhibitor of JAK2

Blood ◽

10.1182/blood.v116.21.4112.4112 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4112-4112 ◽

Cited By ~ 2

Author(s):

Ashok V Purandare ◽

Animesh Pardanani ◽

Theresa McDevitt ◽

Marco Gottardis ◽

Terra Lasho ◽

...

Keyword(s):

Small Molecule ◽

Cell Types ◽

Small Molecule Inhibitor ◽

Janus Kinase ◽

Jak Inhibitors ◽

In Vivo Models ◽

Molecule Inhibitor ◽

Transcriptional Changes ◽

Dose Levels

Abstract Abstract 4112 We report the characterization of BMS-911543, a potent and functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a reversible inhibitor of JAK2 with a biochemical IC50 of 0.001 μ M and Ki of 0.48 nM. It has over 74- and 350-fold selectivity against the other JAK family members, JAK3 and JAK1, respectively. Further, examination of > 450 other kinases did not reveal significant inhibitory activity for this JAK2 inhibitor. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased anti-proliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species (mouse, rat, dog and monkey) with sustained pathway suppression being observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of <2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. BMS-911543 was also compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other disorders characterized by constitutively active JAK2 signaling. Disclosures: Purandare: Bristol-Myers Squibb: Employment. McDevitt:Bristol-Myers Squibb: Employment. Gottardis:Bristol-Myers Squibb: Employment. You:Bristol-Myers Squibb: Employment. Lombardo:Bristol_Myers Squibb: Employment. Penhallow:Bristol-Myers Squibb: Employment. Vuppugalla:Bristol-Myers Squibb: Employment. Trainor:Bristol-Myers Squibb: Employment. Lorenzi:Bristol-Myers Squibb: Employment.

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Children with Invasive Staphylococcus aureus Disease Exhibit a Potently Neutralizing Antibody Response to the Cytotoxin LukAB

Infection and Immunity ◽

10.1128/iai.01558-13 ◽

2013 ◽

Vol 82 (3) ◽

pp. 1234-1242 ◽

Cited By ~ 33

Author(s):

Isaac P. Thomsen ◽

Ashley L. DuMont ◽

David B. A James ◽

Pauline Yoong ◽

Benjamin R. Saville ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antibody Response ◽

High Titer ◽

Humoral Response ◽

Neutralizing Antibody ◽

Human Neutrophils ◽

Serum Samples ◽

Marked Rise ◽

Content Type

ABSTRACTDespite the importance ofStaphylococcus aureusas a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity ofS. aureustoward human neutrophils. Children with culture-provenS. aureusinfection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers againstS. aureusexotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently describedS. aureusbicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicityin vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies toS. aureusantigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is producedin vivoand that it elicits a functional humoral response.

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