Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro

Abstract

IntroductionNeonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production.MethodsWhole 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.ResultsPTX, 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.ConclusionsAge, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.

Pentoxifylline Alone or in Combination with Gentamicin or Vancomycin Inhibits Live Microbe-Induced Proinflammatory Cytokine Production in Human Cord Blood and Cord Blood Monocytes In Vitro

“…We further investigated the immunomodulatory properties of PTX in conjunction with the clinically relevant antimicrobial agents, and we monitored microbial proliferation under these experimental conditions. PTX, as well as all tested antimicrobial agents, was used at clinically relevant concentrations and tested in whole cord blood assays that contain all of the relevant cellular and humoral factors important for antimicrobial host immune responses ( 14 , 46 ).…”

“…Genomic DNA was removed with RNase-free DNase (Qiagen). The concentration and purity of RNA was measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), as previously described ( 14 ), yielding ∼30 ng/µl RNA. Reverse transcription employed high-capacity cDNA reverse transcription kits (Life Technologies), and transcribed cDNA samples were used for real-time PCR-based mRNA expression measurements, which were performed in triplicates with 5 ng of cDNA per 10-μl reaction.…”

“…PTX decreased Toll-like receptor (TLR)-mediated proinflammatory cytokine production with higher efficacy and potency in newborn cord blood compared to adult blood ( 13 ). Furthermore, PTX combined with dexamethasone or azithromycin synergistically inhibited TLR- and inflammasome-mediated proinflammatory cytokine production in human newborn and adult blood in vitro , thus potentially limiting drug exposure and toxicity ( 14 ).…”

Immunomodulation to Prevent or Treat Neonatal Sepsis: Past, Present, and Future

“…This corresponds to the observation of limited clinical benefit in adult sepsis, but promising results from RCTs in neonates. Moreover, PTX's anti-inflammatory effects on PRR signaling are distinct from those of other anti-inflammatory agents such as dexamethasone and azithromycin with which PTX can act in synergy ( 180 ).…”

“…The macrolide azithromycin, specifically inhibits IL-1α and IL-1β secretion and non-canonical inflammasome activation upon TLR agonist, including LPS, stimulation in vitro ( 180 ) and in vivo ( 234 ). A decrease in IL-1β-mediated inflammation has been attributed to destabilizing mRNA levels for NALP3, a key inflammasome component ( 235 ).…”