Whole newborn and adult blood was treated with PTX (50–200 μM), DEX (10−10–10−7 M), or AZI (2.5–20 μM), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity.
Results
PTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation.
PTX preferentially inhibited pro-inflammatory cytokines especially TNF.
DEX inhibited IL-10 in newborn, and TNF, IL-1β, IL-6 and interferon-α in newborn and adult blood.
AZI inhibited R848-induced TNF, IL-1β, IL-6 and IL-10, and LPS-induced IL-1β and IL-10.
(PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1β, and IL-6, and R848-induced IL-1β and interferon-α,
while (PTX+AZI) synergistically decreased induction of TNF, IL-1β, and IL-6.
Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA.
