No abstract

Human newborns are susceptible to microbial infection related to incompletely defined aspects of the neonatal immune system. To characterize neonatal innate immunity, we studied production of two early response cytokines in response to Toll-like receptor (TLR)-activating microbial stimuli in vitro: the proinflammatory cytokine tumor necrosis factor (TNF)-␣ and IL-6, a multifunctional cytokine with antiinflammatory and Th2-polarizing properties. Neonatal cord blood responses to multiple TLR agonists, including poly dI:dC (TLR3), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), and CpG DNA (TLR9), are characterized by a higher IL-6/TNF-␣ ratio than in adult peripheral blood. Robust LPS-induced IL-6 production is due to both neonatal cellular (monocyte-) and humoral (serum-) factors. Remarkably, serum collected from newborns during the first week of life demonstrates higher IL-6/TNF-␣ ratios than does cord blood, associated with elevations of the IL-6-inducible acute phase reactants CRP and LPS-binding protein in the first days of life. A high ratio of stimulus-induced IL-6/TNF-␣ production is likely to profoundly modulate both innate and adaptive immune responses in the human newborn.

Background Newborns suffer frequent infection and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant, but may confer a Th2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes (Mos) demonstrate impaired Th1-polarizing responses to many TLR agonists due to plasma adenosine acting via cAMP. TLR8 agonists, including imidazoquinolines (IMQs) such as the small synthetic 3M-002, induce adult-level TNF from neonatal Mos, but the scope and mechanisms of IMQ-induced activation of neonatal Mos and Mo-derived dendritic cells (MoDCs) have not been reported. Objectives To characterize IMQ-induced activation of neonatal Mos and MoDCs. Methods Neonatal cord and adult peripheral blood Mos and MoDCs were cultured in autologous plasma; Alum- and TLR agonist-induced cytokines and co-stimulatory molecules were measured. TLR8 and inflammasome function were assayed using siRNA and western blotting/caspase-1 inhibitory peptide, respectively. The ontogeny of TLR8 agonist–induced cytokine responses was defined in Rhesus macaque whole blood ex vivo. Results IMQs were more potent and effective than Alum at inducing TNF and IL-1β from Mos. 3M-002 induced robust TLR pathway transcriptome activation and Th1-polarizing cytokine production in neonatal and adult Mos and MoDCs, signaling via TLR8 in an adenosine/cAMP- refractory manner. Newborn MoDCs displayed impaired LPS/ATP-induced caspase-1-mediated IL-1β production, but robust 3M-002-induced caspase-1-mediated inflammasome activation independent of exogenous ATP. TLR8-IMQs induced robust TNF and IL-1β in whole blood of Rhesus macaques at birth and infancy. Conclusions IMQ TLR8 agonists engage adenosine-refractory TLR8 and inflammasome pathways to induce robust Mo and MoDC activation and represent promising neonatal adjuvants.

Identification of safe, effective treatment strategies to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the Federal Drug Administration has yet to approve any agent for a mass-casualty radiation disaster indication. As preparative treatments for hematopoietic stem cell transplantation (HSCT) produce toxicities similar to such radiation exposures, we studied patients early after myeloablative HSCT to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity. We tested this idea in mice. A single 7 Gy radiation dose, which was 95% lethal by 30 days, was followed 24 hours later by twice daily subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum anti-bacterial activity including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle/fluoroquinolone, combined rBPI21/fluoroquinolone treatment improved survival, accelerated hematopoietic recovery and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 and fluoroquinolones initiated 24 hours after lethal irradiation, combined with their favorable bioactivity and safety profiles in critically-ill humans, suggest the potential clinical utility of this radiation mitigation strategy and support its further evaluation.