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Impact of toll-like receptor 4 stimulation on human neonatal neutrophil spontaneous migration, transcriptomics, and cytokine production


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.

Key messages

  • 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.

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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).

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Corresponding author

Correspondence to Shawn D. Larson.

Ethics declarations

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

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)


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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).

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  • Immunology
  • Innate immunity
  • Inflammation
  • Microfluidics
  • Neutrophil migration
  • Lipopolysaccharide