Multiplex analysis of toll-like receptor-stimulated neonatal cytokine response

Abstract

Background: The human neonate's increased susceptibility to bacterial infections is not completely understood. Toll-like receptors (TLRs) have been recognized as pattern-recognition receptors critical to the innate immune response. TLR function in neonates, however, remains incompletely defined.

Objective: To examine regulatory and proinflammatory cytokine responses to TLR-1-6 stimulation of cord blood compared to adult blood.

Methods: We stimulated cord blood with ligands for each of TLRs 1-6 and compared these responses to adult controls. The following TLR ligands were utilized: Pam3CSK4 (TLR-1 and 2), zymosan (TLR-2 and 6), poly I:C (TLR-3), LPS (TLR-4), and flagellin (TLR-5). Cytokine production was measured with an assay developed in-house utilizing multi-analyte technology.

Results: TLR-1-6 stimulation produced higher concentrations of proinflammatory cytokines (IL-1beta, IL-6, and IL-8) in cord blood compared to adult blood, with the exception of TLR-4-stimulated TNF-alpha production, which was significantly lower in cord blood (319 pg/ml) compared to adult blood (645 pg/ml; p = 0.027). In contrast, TLR-1-6 stimulation resulted in decreased concentrations of Th1 and Th2 cytokines in cord blood compared to adult blood, with significantly diminished production of IL-12 (TLRs 1/2, 2/6, 3 and 4), IL-13 (TLR-1-6), and IL-10 (TLR-4).

Conclusion: Cord blood production of regulatory Th1 and Th2 cytokines following TLR stimulation is decreased compared to that of adult blood. In contrast, TLR-stimulated proinflammatory cytokine production was markedly higher in neonates than in adults, with the exception of TLR-4-induced TNF-alpha production. The human neonate's increased susceptibility to bacterial infections may be related to abnormal TLR responsiveness, with enhanced proinflammatory and decreased regulatory cytokine production.