Neonates rely on their innate immune system, and neutrophils in particular, to recognize and combat life-threatening bacterial infections. Pretreatment with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 agonist, improves survival to polymicrobial sepsis in neonatal mice by enhancing neutrophil recruitment. To understand the response of human neonatal neutrophils to TLR4 stimulation, ex vivo spontaneous neutrophil migration, neutrophil transcriptomics, and cytokine production in the presence and absence of LPS were measured directly from whole blood of adults, term neonates, and preterm neonates. Spontaneous neutrophil migration was measured on novel microfluidic devices with time-lapse imaging for 10 h. Genome-wide neutrophil transcriptomics and plasma cytokine concentrations were also determined. Preterm neonates had significantly fewer spontaneously migrating neutrophils at baseline, and both term and preterm neonates had decreased neutrophil velocity, compared to adults. In the presence of LPS stimulation, the number of spontaneously migrating neutrophils was reduced in preterm neonates compared to term neonates and adults. Neutrophil velocity was not significantly different among groups with LPS stimulation. Preterm neonates upregulated expression of genes associated with the recruitment and response of neutrophils following LPS stimulation, but failed to upregulate the expression of genes associated with antimicrobial and antiviral responses. Plasma levels of IL-1β, IL-6, IL-8, MIP-1α, and TNF-α increased in response to LPS stimulation in all groups, but IL-10 was increased only in term and preterm neonates. In conclusion, age-specific changes in spontaneous neutrophil migration counts are not affected by LPS despite changes in gene expression and cytokine production.
Preterm neonates have reduced spontaneous neutrophil migration compared to term neonates and adults in the absence and presence of TLR4 stimulation.
Preterm and term neonates have reduced neutrophil velocities compared to adults in the absence of TLR4 stimulation but no difference in the presence of TLR4 stimulation.
Unique transcriptomic response to TLR4 stimulation is observed in neutrophils from preterm neonates, term neonates, and adults.
TLR4 stimulation produces an age-specific cytokine response.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Lawn JE, Kerber K, Enweronu-Laryea C, Cousens S (2010) 3.6 million neonatal deaths—what is progressing and what is not? Semin Perinatol 34:371–386
Shane AL, Stoll BJ (2014) Neonatal sepsis: progress towards improved outcomes. J Infect 68(Suppl 1):S24–S32
Khan AM, Morris SK, Bhutta ZA (2017) Neonatal and perinatal infections. Pediatr Clin N Am 64:785–798
Raymond SL, Stortz JA, Mira JC, Larson SD, Wynn JL, Moldawer LL (2017) Immunological defects in neonatal sepsis and potential therapeutic approaches. Front Pediatr 5:14
Raymond SL, Mathias BJ, Murphy TJ, Rincon JC, Lopez MC, Ungaro R, Ellett F, Jorgensen J, Wynn JL, Baker HV et al (2017) Neutrophil chemotaxis and transcriptomics in term and preterm neonates. Transl Res 190:4–15
Wynn JL, Scumpia PO, Winfield RD, Delano MJ, Kelly-Scumpia K, Barker T, Ungaro R, Levy O, Moldawer LL (2008) Defective innate immunity predisposes murine neonates to poor sepsis outcome but is reversed by TLR agonists. Blood 112:1750–1758
Mathias B, Mira JC, Rehfuss JP, Rincon JC, Ungaro R, Nacionales DC, Lopez MC, Baker HV, Moldawer LL, Larson SD (2017) LPS stimulation of cord blood reveals a newborn-specific neutrophil transcriptomic response and cytokine production. Shock (Augusta, Ga) 47:606–614
Aomatsu K, Kato T, Fujita H, Hato F, Oshitani N, Kamata N, Tamura T, Arakawa T, Kitagawa S (2008) Toll-like receptor agonists stimulate human neutrophil migration via activation of mitogen-activated protein kinases. Immunology 123:171–180
Sisson TR, Whalen LE, Telek A (1959) The blood volume of infants. II. The premature infant during the first year of life. J Pediatr 55:430–446
Ellett F, Jorgensen J, Marand AL, Liu YM, Martinez MM, Sein V, Butler KL, Lee J, Irimia D (2018) Diagnosis of sepsis from a drop of blood by measurement of spontaneous neutrophil motility in a microfluidic assay. Nat Biomed Eng 2:207–214
Warner EA, Kotz KT, Ungaro RF, Abouhamze AS, Lopez MC, Cuenca AG, Kelly-Scumpia KM, Moreno C, O'Malley KA, Lanz JD, Baker HV, Martin LC, Toner M, Tompkins RG, Efron PA, Moldawer LL (2011) Microfluidics-based capture of human neutrophils for expression analysis in blood and bronchoalveolar lavage. Lab Investig 91:1787–1795
Arena ET, Rueden CT, Hiner MC, Wang S, Yuan M, Eliceiri KW (2017) Quantitating the cell: turning images into numbers with ImageJ. Wiley Interdiscip Rev Dev Biol 6. https://doi.org/10.1002/wdev.260
Kotz KT, Xiao W, Miller-Graziano C, Qian WJ, Russom A, Warner EA, Moldawer LL, De A, Bankey PE, Petritis BO et al (2010) Clinical microfluidics for neutrophil genomics and proteomics. Nat Med 16:1042–1047
Raymond SL, Lopez MC, Baker HV, Larson SD, Efron PA, Sweeney TE, Khatri P, Moldawer LL, Wynn JL (2017) Unique transcriptomic response to sepsis is observed among patients of different age groups. PLoS One 12:e0184159
Sasaki T, Irie-Sasaki J, Jones RG, Oliveira-dos-Santos AJ, Stanford WL, Bolon B, Wakeham A, Itie A, Bouchard D, Kozieradzki I, Joza N, Mak TW, Ohashi PS, Suzuki A, Penninger JM (2000) Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science (New York, NY) 287:1040–1046
Li Z, Jiang H, Xie W, Zhang Z, Smrcka AV, Wu D (2000) Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction. Science (New York, NY) 287:1046–1049
Hirsch E, Katanaev VL, Garlanda C, Azzolino O, Pirola L, Silengo L, Sozzani S, Mantovani A, Altruda F, Wymann MP (2000) Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation. Science (New York, NY) 287:1049–1053
Sharma N, Akhade AS, Qadri A (2013) Sphingosine-1-phosphate suppresses TLR-induced CXCL8 secretion from human T cells. J Leukoc Biol 93:521–528
Hammond ME, Lapointe GR, Feucht PH, Hilt S, Gallegos CA, Gordon CA, Giedlin MA, Mullenbach G, Tekamp-Olson P (1995) IL-8 induces neutrophil chemotaxis predominantly via type I IL-8 receptors. J Immunol (Baltimore, Md : 1950) 155:1428–1433
Ellis TN, Beaman BL (2004) Interferon-gamma activation of polymorphonuclear neutrophil function. Immunology 112:2–12
Cuenca AG, Joiner DN, Gentile LF, Cuenca AL, Wynn JL, Kelly-Scumpia KM, Scumpia PO, Behrns KE, Efron PA, Nacionales D, Lui C, Wallet SM, Reeves WH, Mathews CE, Moldawer LL (2015) TRIF-dependent innate immune activation is critical for survival to neonatal gram-negative sepsis. J Immunol (Baltimore, Md : 1950) 194:1169–1177
The authors would like to thank all study participants and nursing staff at UF Health Shands Children’s Hospital who made this study possible.
SLR, JLW, LLM, DI, and SDL developed the hypothesis and designed the experiments. FE and DI provided the microfluidic spontaneous migration devices and technical support for the migration assay. SLR, RBH, TJM, JCR, JAS, MCL, RU, and HVB performed the experiments and analyzed the results. SLR, LLM, and SDL prepared the original manuscript. All authors reviewed and revised the final manuscript. No editorial support was used in the preparation of the manuscript.
This work was supported by grants from the National Institute of General Medical Sciences (NIGMS; K08 GM106143, R01 GM097531, and P50 GM111152). SLR was supported by a clinical research training fellowship awarded by the Surgical Infection Society Foundation. RBH and JAS were supported by a NIGMS post-doctoral training grant (T32 GM008721).
The study was approved by the Institutional Review Board prior to initiation. Written informed consent was obtained from the parents or guardians of neonatal subjects prior to sample or data collection and from all adult subjects prior to sample collection.
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
(XLSX 21.5 kb)
The video demonstrates the spontaneous neutrophil migration device in operation. Hoechst stained neutrophils migrate through the erythrocyte filters along the migration channel and then change direction at the posts before continuing to migrate along the channels. Images are recorded every 30 seconds for 10 hours, and the video has been modified to reduce 10 hours of sampling time to two minutes. (MP4 4108 kb)
About this article
Cite this article
Raymond, S.L., Hawkins, R.B., Murphy, T.J. et al. Impact of toll-like receptor 4 stimulation on human neonatal neutrophil spontaneous migration, transcriptomics, and cytokine production. J Mol Med 96, 673–684 (2018). https://doi.org/10.1007/s00109-018-1646-5
- Innate immunity
- Neutrophil migration