Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation

Jansen, Kirstin; Blimkie, Darren; Furlong, Jeff; Hajjar, Adeline M.; Rein‐Weston, Annie; Crabtree, Juliet; Reikie, Brian A.; Wilson, Christopher; Kollmann, Tobias R.

doi:10.1016/j.jim.2008.04.013

Cited by 45 publications

(92 citation statements)

References 29 publications

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“…We used a polychromatic flow cytometric assay and gating strategy, as described elsewhere (23,24) and Abs to HLA-DR (clone TU36; BD Biosciences), CD14 (clone M5E2; BD Biosciences), CD123 (clone 6H6; eBioscience), CD11c (clone 3.9; eBioscience), IFN-a (clone A11 from Antigenix America or 7N4-1 from BD Biosciences), and IL-6 (clone MQ2-13A5; eBioscience). The integrated mean fluorescence intensity (iMFI) (25) was used as readout for IFN-a and IL-6 production.…”

Section: Polychromatic Flow Cytometry Analysismentioning

confidence: 99%

“…As controls, cells either were stimulated with sucrose in HBSS/HEPES buffer (stabilizing agent for virus), 1 mg/ml dsRNA lacking a 59 triphosphate (ctl-dsRNA) in complex with LyoVec (InvivoGen), or 1 mg/ml R848 (InvivoGen), or were left unstimulated. For intracellular cytokine staining, a protein transport inhibitor mixture containing brefeldin A and monensin (eBioscience) was added for the final 3 h (isolated pDCs) or 6 h (MNCs) (23).…”

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confidence: 99%

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Attenuation of Respiratory Syncytial Virus–Induced and RIG-I–Dependent Type I IFN Responses in Human Neonates and Very Young Children

Marr

Wang

Kam

et al. 2014

The Journal of Immunology

101

105

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Newborn infants, including those born at term without congenital disorders, are at high risk of severe disease from respiratory syncytial virus (RSV) infection. Indeed, our current local surveillance data demonstrate that approximately half of children hospitalized with RSV were ≤3 mo old, and 74% were born at term. Informed by this clinical epidemiology, we investigated antiviral innate immune responses in early life, with the goal of identifying immunological factors underlying the susceptibility of infants and young children to severe viral lower respiratory tract infections. We compared RSV-induced innate cytokine production in blood mononuclear cells from neonates, young children aged 12–59 mo, and healthy adults. RSV-induced IFN-α production was primarily mediated by plasmacytoid dendritic cells (pDCs), and was significantly lower in term infants and young children < 5 y of age than in adults (p < 0.01). RSV-induced IFN-α production in human pDCs proceeded independently of endosomal TLRs, and human pDCs from healthy adult donors produced IFN-α in a retinoic acid–inducible gene I protein (RIG-I)–dependent manner. Of interest, young age and premature birth were independently associated with attenuated RIG-I–dependent IFN-α responses (p < 0.01). In contrast to IFN-α production, proinflammatory IL-6 responses to RSV were mediated by monocytes, appeared less dependent on RIG-I, and were significantly impaired only among preterm infants, not in term infants and young children. Our results suggest that human pDCs are less functional in early life, which may contribute to the increased susceptibility of infants and young children to severe RSV disease.

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Section: Polychromatic Flow Cytometry Analysismentioning

confidence: 99%

mentioning

confidence: 99%

Attenuation of Respiratory Syncytial Virus–Induced and RIG-I–Dependent Type I IFN Responses in Human Neonates and Very Young Children

Marr

Wang

Kam

et al. 2014

The Journal of Immunology

101

105

View full text Add to dashboard Cite

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“…Whole blood was diluted 1:2 with RPMI 1640 medium with L-glutamine (Gibco, Grand Island, NY) and stimulated with saturating doses of the TLR4 agonist lipopolysaccharide (LPS, 100 ng/ml; InvivoGen, San Diego, CA), the TLR7/8 agonist R848 (1 μg/ml; InvivoGen), or unstimulated, for 24 h at 37 °C in 5% CO 2 atmosphere in prediluted aliquots round-bottom 96-well microplates. The concentration of TLR agonists used in this study corresponded to peak stimulating concentrations, as determined in our previous studies (7,25). Following stimulation, cell supernatants were stored at −80 °C for batches analyses.…”

Section: Immune Stimulation and Cytokine Measuresmentioning

confidence: 99%

Attenuated innate immune defenses in very premature neonates during the neonatal period

et al. 2015

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Background: Antimicrobial responses have been shown to be profoundly attenuated in very preterm neonates when examined on cord blood. However, we lack data on these responses at the time these neonates are most vulnerable to infections. Methods: Multiple cytokine responses to two prototypic Toll-like receptor (TLR) agonists: lipopolysaccharide (LPS) (TLR4) and R848 (TLR7/8) were prospectively measured in preterm neonates born ≤30 wk of gestation (n = 50) during the first 28 d of age using whole blood and single-cell multiparameter flow cytometry assays. Results were compared to term neonates (n = 30) and adult controls (n = 25). results: In preterm neonates, LPS and R848 responses remained attenuated in both cord blood and in the first 28 d of age. These responses showed significant maturation over time after adjusting for gestational age and were confirmed in monocytes and dendritic cells on a per-cell basis. We detected no major contribution of chorioamnionitis, maternal antenatal corticosteroids or magnesium sulfate treatment, labor, or mode of delivery to the maturation of cytokine responses. conclusion: Innate immune antimicrobial defenses are profoundly attenuated developmentally in very preterm neonates during the neonatal period, suggesting that exogenous factors drive the sustained systemic inflammation that has been linked to increased morbidities in these infants.d espite improvements in neonatal health care, mortality from infections has continued to increase in very preterm neonates over the last two decades, owing to the improved survival of smaller and more vulnerable infants (1). However, the underlying immunological basis of their high morbidities and high vulnerability to sepsis remains poorly understood (2). To defend themselves against infection, neonates rely on first-line, innate immune defense mechanisms. These mechanisms include the detection of specific microbial molecular structures called pathogenassociated molecular patterns through specialized receptors termed pattern recognition receptors (PRR), but also other defense mechanisms such as phagocytosis and antigen presentation to the adaptive immune system.It is widely stated that the high risk of neonatal sepsis observed in these infants is due to immature immune defense mechanisms. However, data are lacking on the development of immune functions beyond measures performed on umbilical cord blood. Recent studies have reported reduced antigen-presenting receptor expression during the first week of age in infants born below 33 wk of gestation (3,4). In contrast, phagocytic functions in these infants are more comparable to term infants (5). Toll-like receptors (TLRs) play a predominant role as a major family of PRR that act as essential "detectors" in the recognition of microbial pathogens by the innate immune system. We and others have shown that TLR-induced cytokine immune reactivity are profoundly attenuated in cord blood of very preterm neonates and develops asynchronously over the last trimester of gestation (6-10). These infants als...

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“…Innate immune responsiveness was quantified using published techniques (18)(19)(20). Umbilical cord blood was obtained from healthy, full-term infants delivered by elective Caesarian section before the onset of labor.…”

Section: Quantifying Innate Immune Responsivenessmentioning

confidence: 99%

Functional Genetic Variation inNFKBIAand Susceptibility to Childhood Asthma, Bronchiolitis, and Bronchopulmonary Dysplasia

Ali

Hirschfeld

Mayer

et al. 2013

The Journal of Immunology

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Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.

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Polychromatic flow cytometric high-throughput assay to analyze the innate immune response to Toll-like receptor stimulation

Cited by 45 publications

References 29 publications

Attenuation of Respiratory Syncytial Virus–Induced and RIG-I–Dependent Type I IFN Responses in Human Neonates and Very Young Children

Attenuation of Respiratory Syncytial Virus–Induced and RIG-I–Dependent Type I IFN Responses in Human Neonates and Very Young Children

Attenuated innate immune defenses in very premature neonates during the neonatal period

Functional Genetic Variation inNFKBIAand Susceptibility to Childhood Asthma, Bronchiolitis, and Bronchopulmonary Dysplasia

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