Purified Neonatal Plasmacytoid Dendritic Cells Overcome Intrinsic Maturation Defect with TLR Agonist Stimulation

Abstract

Neonates are more susceptible than adults to viral and bacterial diseases. We hypothesized that plasmacytoid dendritic cells, the cells that provide large amounts of IFN-α in response to Toll-like receptor 9 (TLR9) agonists, are defective in neonates. To assess the intrinsic functionality of plasmacytoid dendritic cells from neonates we compared IFN-α production by plasmacytoid dendritic cells derived from neonates versus adults in both whole blood and in purified plasmacytoid dendritic cells. TLR9-stimulation of whole blood from adults and neonates resulted in comparable amounts of IFN-α production. However, we observed small but significant differences in IFN-α production from purified CD123+ plasmacytoid dendritic cells from neonates after stimulation with the TLR9 ligand CpG-DNA. Furthermore, we assessed surface expression of co-stimulatory molecules on plasmacytoid dendritic cells after stimulation. While purified CD123+ plasmacytoid dendritic cells from adults up-regulated co-stimulatory molecules CD80 and CD86 with IL-3 alone those from neonates required the addition of CpG-DNA to reach adult levels. Therefore, the intrinsic deficiencies of neonatal plasmacytoid dendritic cells can be mitigated by TLR9 agonists. These results are consistent with the observation that vaccines that effect strong adjuvant activity on dendritic cells can induce protective responses in neonates.Neonates are more susceptible than adults to severe disease following exposure to viruses and bacteria (1). A lack of pre-existing memory T-and B-cells as well as diminished T-cell responses (2), reduced numbers of dendritic cells (DC), and a bias toward Th2 immunity (3) are contributors, but likely don't entirely explain this observation. One hypothesis is that neonatal DC are functionally immature. One manifestation of such immaturity may be that neonatal DC respond suboptimally to pathogens via Toll-like receptor (TLR) agonist stimulation and therefore are lacking the ability to provide a link between innate and adaptive immune responses (4). Unlike myeloid DC, plasmacytoid DC (pDC) express high levels of the pathogen-recognizing receptor TLR9 (4). Once stimulated with TLR9 agonists such as viral (5,6) as well as bacterial DNA (7,8) pDC produce large amounts of IFN-α. In addition, pDC can stimulate allogeneic T-cells and pDC exposed to virus are capable of directly priming T-cells in mice (9). Furthermore, influenza-exposed pDC can stimulate antigen-experienced influenza-specific CD4 and CD8 T-cell clones to produce IFN-γ suggesting a role for pDC in stimulation of memory T-cell responses (10).We hypothesized that DC from the plasmacytoid subset in human neonates were functionally defective. Previous studies of DC either in vivo or using mixed cell populations that contained nonDC (11,12) showed that neonatal DC may be defective while more recent studies using purified populations (13,14) defects. Therefore, to distinguish intrinsic deficiencies from extrinsic factors we studied responses in whole blood and in highly purifie...

Toll-Like Receptor 9-Mediated Protection of Enterovirus 71 Infection in Mice Is Due to the Release of Danger-Associated Molecular Patterns

“…Thus, the induction of neurological manifestations may not be due to different expression levels of TLR9 in neonatal and adult mice. However, neonatal pDCs are impaired in their ability to mature and produce the levels of IFN-␣ common in adult mice (44,45). Furthermore, we confirmed that pDCs are involved in protective mechanisms by using anti-PDCA1 antibody to deplete pDCs before EV71 infection.…”

Variables to be controlled in the assessment of blood innate immune responses to Toll-like receptor stimulation

“…Ida et al (2006) who compared IFN-α and TNF-α production by ICS only, described a complete dropout of IFN-α production in previously cryopreserved pDC, while TNF-α production in cDC and pDC appeared unaffected. Both Ida et al and our data are in complete agreement, but stand in direct conflict with that of Gold et al (2006) who described that the IFN-α production of pDC in response to TLR9 stimulation produced ‘equivalent results’ for fresh and cryopreserved cells; however, they did not provide the detailed data to support this. Yet another group categorically states that there are no differences between fresh and cryopreserved cells (Upham et al, 2002); however, they did not provide data to support this conclusion, and reference reports as support that only compared T cell responses between fresh and cryopreserved samples, not innate immune responses to TLR stimulation (Upham et al, 1995).…”

TLR9 agonist, but not TLR7/8, functions as an adjuvant to diminish FI-RSV vaccine-enhanced disease, while either agonist used as therapy during primary RSV infection increases disease severity

“…The ability of TLR agonists to modulate immunity provides new strategies that may improve vaccine design. In particular, TLR agonists have been shown to stimulate mature immune responses in neonates [38]. We therefore tested the ability of TLR7/8 and TLR9 agonists (1) to serve as adjuvants and modulate FI-RSV-enhanced disease immunization and (2) to serve as a potential therapeutic agent and alter pathogenesis during live RSV infection of naïve or immunized mice.…”