Imidazoquinoline Toll-like receptor 8 agonists activate human newborn monocytes and dendritic cells through adenosine-refractory and caspase-1-dependent pathways

Abstract

Background: Newborns have frequent infections and manifest impaired vaccine responses, motivating a search for neonatal vaccine adjuvants. Alum is a neonatal adjuvant but might confer a T(H)2 bias. Toll-like receptor (TLR) agonists are candidate adjuvants, but human neonatal cord blood monocytes demonstrate impaired T(H)1-polarizing responses to many TLR agonists caused by plasma adenosine acting through cyclic AMP. TLR8 agonists, including imidazoquinolines (IMQs), such as the small synthetic 3M-002, induce adult-level TNF from neonatal monocytes, but the scope and mechanisms of IMQ-induced activation of neonatal monocytes and monocyte-derived dendritic cells (MoDCs) have not been reported.

Objective: We sought to characterize IMQ-induced activation of neonatal monocytes and MoDCs.

Methods: Neonatal cord and adult peripheral blood monocytes and MoDCs were cultured in autologous plasma; levels of alum- and TLR agonist-induced cytokines and costimulatory molecules were measured. TLR8 and inflammasome function were assayed by using small interfering RNA 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 monocytes. 3M-002 induced robust TLR pathway transcriptome activation and T(H)1-polarizing cytokine production in neonatal and adult monocytes and MoDCs, signaling through TLR8 in an adenosine/cyclic AMP-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 monocyte and MoDC activation and represent promising neonatal adjuvants.

FIG 1. TLR7/8 and TLR8 agonists are more potent and effective than Alum in inducing TNF and IL-1β from human newborn and adult monocytes

A, and B, newborn or C and D, adult stimulated monocytes for 4 hrs (TNF) or 18 hrs (IL-1β) and supernatants were assayed by ELISA. n = 3–4; * P ≤0.05 (Mann-Whitney test comparing TLR agonist to Alum).

FIG 2. The TLR8 agonist 3M-002 induces greater TLR pathway mRNA up-regulation and Th1-polarizing cytokines/chemokines from neonatal Mos than a TLR7 agonist

Monocytes (5 × 10⁷ cells/mL) stimulated with indicated agonist (50 μM), 4 h. A, Agonist-induced mRNA fold change relative to control (colored lines indicate 2-fold change). B-D; Th1, Th2 (±Th1), Th17 and anti-inflammatory proteins. n = 3–6; * P < 0.05, ** P < 0.01.

FIG 3. TLR8 agonists are refractory to inhibitory adenosine/cAMP and associated with lesser cAMP accumulation in newborn Mos

A, Monocytes, to which adenosine (10 μM) or saline (Sal) added prior to stimulation with agonists (50 μM) for 2 hrs (representative of 3-6 experiments). B, % cytokine. C, cAMP concentrations in agonist treated monocytes. D, 30 min db-cAMP (10 μM) treated monocytes, 4 hr stimulation. n =3–6; * P < 0.05, ** P < 0.01.

FIG 4. Newborn MoDCs display diminished LPS/ATP-induced but robust IMQ-induced caspase-1 activation and IL-1β production

Newborn and adult MoDCs stimulated with LPS (2 hrs) (A, B, C) or 3M-002 (24 hrs) (D, E, F), plus 5 mM ATP as indicated. Supernatant assayed IL-1β (ELISA) (A, D), cell lysates caspase-1 by western blotting/densitometry (B, C, E, F). C, F representative of n = 3; A, D n = 4–6, Asterisk denotes comparison between treatment conditions. Line-bar indicates comparison between groups (newborn and adult). G, Caspase-1 inhibitor Z-WEHD-FMK pretreatment of neonatal MoDCs treated as above. H, IL-1β data from G shown as %. n = 4–6; * P < 0.05, ** P < 0.01, *** P < 0.001.

FIG 5. Proposed mechanisms of Imidazoquinoline TLR8 agonist activation of human newborn monocytes and DCs via adenosine-refractory and caspase-1-dependent pathways

3M-002 agonists are refractory to inhibitory effects of the adenosine/cAMP pathway, which skews neonatal immunity against Th1-polarizition (A), can overcome impaired IL-1β responses of neonatal DCs to LPS/ATP (B) independently of exogenous ATP (C).