S. L. Raymond1, R. B. Hawkins1, T. J. Murphy1, J. C. Rincon1, J. A. Stortz1, M. Lopez2, R. Ungaro1, H. V. Baker2, J. L. Wynn3, L. L. Moldawer1, S. D. Larson1 1University Of Florida College Of Medicine,Department Of Surgery,Gainesville, FL, USA 2University Of Florida College Of Medicine,Department Of Molecular Genetics And Microbiology,Gainesville, FL, USA 3University Of Florida College Of Medicine,Department Of Pediatrics,Gainesville, FL, USA
Introduction: Neonates rely predominantly on the innate immune system to recognize and combat life-threatening bacterial infections. Neutrophils, a key component of the innate immune system, therefore play a crucial role in neonatal survival. In neonatal murine models, toll-like receptor (TLR) agonists have shown promise in improving survival to polymicrobial sepsis by enhancing neutrophil recruitment. To better understand the transcriptomic response in human neonates to TLR 4 stimulation, we measured neutrophil transcriptomics using microfluidic approaches that require less than 0.5 ml of blood.
Methods: Whole blood samples from six young adults (21-45 years old), six term neonates (gestational age >37 weeks), and six preterm neonates (gestational age <37 weeks) were collected and incubated for 2 hours with and without TLR 4 stimulation by lipopolysaccharide (final concentration of 100 ng/ml). CD66b+ cells were subsequently captured by positive selection on antibody coated microfluidic devices, and genome-wide expression analyses were performed. Ingenuity Pathway Analysis software was utilized to predict biological functions of significant genes. Significant pathways were determined using a –log p-value greater than 1.3 and a z-score greater than 2 or less than -2.
Results: Supervised analysis identified 1517 unique genes whose expression significantly differed between age groups in response to TLR 4 stimulation (p<0.001, FC>1.5). Biological function predictions suggest that both adults and neonates had a significant overexpression of genes involved in immune response of cells, cell-mediated response, and inflammatory response following TLR 4 stimulation. Adults and term neonates also had overexpression of genes associated with antimicrobial and antiviral response. Preterm neonates failed to upregulate these same pathways but had a significant overexpression of genes involved in the recruitment and response of neutrophils which was not observed in adults.
Conclusion: Microfluidic techniques were utilized on whole blood to identify unique neutrophil transcriptomic profiles among different age groups in response to TLR 4 stimulation. These novel data provide important insights into the neonatal neutrophil response.