An Integrative Approach for Immune Monitoring of Human Health and Disease by Advanced Flow Cytometry Methods

Tirouvanziam, Rabindra; Diaz, Daisy; Gernez, Yaël; Laval, Julie; Crubezy, Monique; Makam, Megha

doi:10.1002/9783527634286.ch12

Cited by 16 publications

(25 citation statements)

References 47 publications

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“…Samples were also stained with cholera toxin B (Life Sciences); after a wash in PBS-EDTA and permeabilization with Perm I buffer (BD Biosciences), they were stained with the DNA-binding probe DRAQ5 (Life Sciences). The combination of cholera toxin B and DRAQ5 enables robust gating of live neutrophils from blood and airway samples (30), as illustrated in Supplemental Fig. 1A.…”

Section: Flow Cytometric Analysis Of Nutrient Transporter Expressionmentioning

confidence: 99%

“…Control stainings combined anti-mFc or -rFc with cholera toxin B and DRAQ5, in the absence of RBD ligands. After a final wash in Perm I buffer at room temperature, samples were analyzed on a LSR II flow cytometer (BD Biosciences), and expression of nutrient transporters was determined after fluorescence compensation (FlowJo software; TreeStar) (30). Data are reported as differential median fluorescence intensity (delta MFI) between the specific RBD staining and the corresponding background control for each individual sample.…”

Section: Flow Cytometric Analysis Of Nutrient Transporter Expressionmentioning

confidence: 99%

“…1B. After a final wash in Perm I buffer, samples were analyzed on an ImageStreamX system (Amnis), and expression and localization (nuclear versus cytoplasmic) of vigilin and LC3 were quantified (IDEAS software) after fluorescence compensation (30,34).…”

Section: Multispectral Imaging Flow Cytometric Analysis Of Vigilin Anmentioning

confidence: 99%

See 2 more Smart Citations

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Laval

Touhami

Herzenberg

et al. 2013

The Journal of Immunology

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Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

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Section: Flow Cytometric Analysis Of Nutrient Transporter Expressionmentioning

confidence: 99%

Section: Flow Cytometric Analysis Of Nutrient Transporter Expressionmentioning

confidence: 99%

See 1 more Smart Citation

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Laval

Touhami

Herzenberg

et al. 2013

The Journal of Immunology

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“…Pregenomic, genomic and postgenomic investigations for vaccine development, possible from such materials, have not been greatly hindered by the absence of robust in vitro culture systems (Box 3.3) [11,13,19]. Immune response studies are possible with the currently available resources and increasingly so and in greater depth than even a few years ago thanks to the advancement of technologies requiring comparatively small blood volumes [20,21]. Such modern technologies are facilitating the exploration of both innate and adaptive immune responses.…”

Section: Box 3 1 Special Biological Features Of Plasmodium Vivax Inmentioning

confidence: 99%

“…Many such protocols rely on direct staining of freshly collected blood (or can be minimally modified to become so), followed by rapid fixation and preservation until later ana lysis [20], which makes them compelling tools for field-based studies of malaria. With a simple centrifugation step prior to staining, innate immune profiling methods can be combined with plasma isolation and use for proteomics or metabolomics studies, which opens exciting opportunities for field-based systems biology approaches.…”

Section: Box 34 Cells Of the Innate Immune Responsementioning

confidence: 99%

Plasmodium vivaxvaccine surrogate markers of protection: dawning of a new era

Galinski¹,

Tirouvanziam

Moreno³

2014

Malaria Vaccine Development: Over 40 Years of Trials and Tribulations

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Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis

Forrest

Ingersoll

Preininger

et al. 2018

Journal of Leukocyte Biology

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130

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Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

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An Integrative Approach for Immune Monitoring of Human Health and Disease by Advanced Flow Cytometry Methods

Cited by 16 publications

References 47 publications

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Plasmodium vivaxvaccine surrogate markers of protection: dawning of a new era

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis

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