A graded neonatal mouse model of necrotizing enterocolitis demonstrates that mild enterocolitis is sufficient to activate microglia and increase cerebral cytokine expression.

Background: Necrotizing enterocolitis (NEC) is an inflammatory gastrointestinal process that afflicts approximately 10% of preterm infants born in the United States each year, with a mortality rate of 30%. NEC severity is graded using Bell's classification system, from stage I mild NEC to stage III severe NEC. Over half of NEC survivors present with neurodevelopmental impairment during adolescence, a long-term complication that is poorly understood but can occur even after mild NEC. Although multiple animal models exist, none allow the experimenter to control nor represent the gradient of symptom severities seen in NEC patients. We bridge this knowledge gap by developing a graded murine model of NEC and studying its relationship with neuroinflammation across a range of NEC severities. Methods: Postnatal day 3 (P3) C57BL/6 mice were fed a formula containing different concentrations (0% control, 0.25%, 1%, 2%, and 3%) of dextran sodium sulfate (DSS). P3 mice were fed every 3 hours for 72-hours. We collected data on weight gain and behavior (activity, response, body color) during feeding. At the end of the experiment, we collected tissues (intestine, liver, plasma, brain) for immunohistochemistry, immunofluorescence, and cytokine and chemokine analysis. Results: Throughout NEC induction, mice fed higher concentrations of DSS died sooner, lost weight faster, and became sick or lethargic earlier. Intestinal characteristics (dilation, color, friability) were worse in mice fed with higher DSS concentrations. Histology revealed small intestinal disarray among mice fed all DSS concentrations, while higher DSS concentrations resulted in reduced small intestinal cellular proliferation and increased hepatic and systemic inflammation. In the brain, IL-2, G-CSF, and CXCL1 concentrations increased with higher DSS concentrations. Although the number of neurons and microglia in the CA1 hippocampal region did not differ, microglial branching was significantly reduced in DSS-fed mice. Conclusion: We characterize a novel graded model of NEC that recapitulates the full range of NEC severities. We show that mild NEC is sufficient to initiate neuroinflammation and microglia activation. This model will facilitate studies on the neurodevelopmental effects of NEC.

The state and future of pediatric research-an introductory overview : The state and future of pediatric research series.

IMPACT: This is an introduction to an article series devoted to the current state and future of pediatric research. The role of public-private partnerships, influencing factors, challenges, and recent trends in pediatric research are described, with emphasis on funding, drug and device development, physician-scientist training, and diversity. Potential solutions and advocacy opportunities are discussed.

A Neonatal Murine Escherichia coli Sepsis Model Demonstrates That Adjunctive Pentoxifylline Enhances the Ratio of Anti- vs. Pro-inflammatory Cytokines in Blood and Organ Tissues.

Introduction: Neonatal sepsis triggers an inflammatory response that contributes to mortality and multiple organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses pro-inflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that administration of PTX in addition to antibiotics decreases live bacteria-induced pro-inflammatory and/or enhances anti-inflammatory cytokine production in septic neonatal mice without augmenting bacterial growth. Methods: Newborn C57BL/6J mice (< 24 h old) were injected intravenously with 105 colony forming units (CFUs)/g weight of a bioluminescent derivative of the encapsulated clinical isolate Escherichia coli O18:K1. Adequacy of intravenous injections was validated using in vivo bioluminescence imaging and Evans blue. Pups were treated with gentamicin (GENT), PTX, (GENT + PTX) or saline at 0, 1.5, or 4 h after sepsis initiation, and euthanized after an additional 4 h. CFUs and cytokines were measured from blood and homogenized organ tissues. Results: GENT alone inhibited bacterial growth, IL-1ß, and IL-6 production in blood and organs. Addition of PTX to GENT profoundly inhibited E. coli-induced TNF and enhanced IL-10 in blood of newborn mice at all timepoints, whereas it primarily upregulated IL-10 production in peripheral organs (lung, spleen, brain). PTX, whether alone or adjunctive to GENT, did not increase microbial colony counts in blood and organs. Conclusion: Addition of PTX to antibiotics in murine neonatal E. coli sepsis promoted an anti-inflammatory milieu through inhibition of plasma TNF and enhancement of IL-10 production in plasma and organs without increasing bacterial growth, supporting its utility as a potential adjunctive agent for newborn sepsis.

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.

Neonatal sepsis and its accompanying inflammatory response contribute to substantial morbidity and mortality. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses transcription and production of proinflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that PTX decreases live microbe-induced inflammatory cytokine production in newborn blood. Cord blood was stimulated with live microorganisms commonly encountered in newborn sepsis (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, or Candida albicans) and simultaneously treated with antimicrobial agents (gentamicin, vancomycin, or amphotericin B) and/or clinically relevant concentrations of PTX. Microbial colony counts were enumerated by plating, supernatant cytokines were measured by multiplex assay, intracellular cytokines and signaling molecules were measured by flow cytometry, and mRNA levels were measured by quantitative reverse transcription-PCR. PTX inhibited concentration-dependent E. coli-, S. aureus-, S. epidermidis-, and C. albicans-induced tumor necrosis factor (TNF) and E. coli-induced interleukin-1ß (IL-1ß) production in whole blood, with greater suppression of proinflammatory cytokines in combination with antimicrobial agents. Likewise, PTX suppressed E. coli-induced monocytic TNF and IL-1ß, whereby combined PTX and gentamicin led to significantly greater reduction of TNF and IL-1ß. The anti-inflammatory effect of PTX on microbe-induced proinflammatory cytokine production was accompanied by inhibition of TNF mRNA expression and was achieved without suppressing the production of the anti-inflammatory IL-10. Of note, microbial colony counts in newborn blood were not increased by PTX. Our findings demonstrated that PTX inhibited microbe-induced proinflammatory cytokine production, especially when combined with antimicrobial agents, without enhancing microbial proliferation in human cord blood in vitro, thus supporting its utility as candidate adjunctive agent for newborn sepsis.

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.

INTRODUCTION: Neonatal 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. METHODS: 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-1ß, IL-6 and interferon-α in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1ß, IL-6 and IL-10, and LPS-induced IL-1ß and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1ß, and IL-6, and R848-induced IL-1ß and interferon-α, while (PTX+AZI) synergistically decreased induction of TNF, IL-1ß, 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. CONCLUSIONS: Age, 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 inhibits lipopolysaccharide-induced inflammatory mediators in human second trimester placenta explants.

BACKGROUND: Intrauterine infection and inflammation during pregnancy, which leads to up-regulation of inflammatory cytokines and prostaglandin synthesis, has been implicated in the pathogenesis of preterm delivery and other pregnancy complications. Effective preventive and therapeutic strategies to reduce these outcomes are lacking to date. Pentoxifylline (PTX) is a non-specific phosphodiesterase inhibitor which raises intracellular cyclic adenosine monophosphate and decreases production of pro-inflammatory mediators while enhancing anti-inflammatory cytokines. We hypothesized that pentoxifylline will decrease lipopolysaccharide (LPS)-induced pro-inflammatory cytokines production in human placental explants. METHODS: Placental explants derived from normal second trimester human placentas were treated with PTX, stimulated with LPS and cultured at 37 °C in 5% CO2. Conditioned media were assayed for pro- and anti-inflammatory mediators with multiplex immunoassays or ELISA, and explant tissues for mRNA with real time PCR. Means of PTX-treated and untreated samples were compared using paired t tests and Wilcoxon-signed rank tests. RESULTS: PTX preferentially inhibited placental expression and production of LPS-induced pro-inflammatory cytokines including TNF-α (25461 vs. 1908 pg/ml, p < 0.001), IL-1ß (2921 vs. 1067 pg/ml, p < 0.001) and IFN-γ (2190 vs 427 pg/ml, p < 0.001) with relative preservation of anti-inflammatory mediators. The suppressive effects on LPS-induced placental inflammation were independent of the timing of PTX administration in relation to LPS-induced stimulation. CONCLUSION: Our study suggests that PTX attenuates the LPS-induced pro-inflammatory milieu in human placental explants. We speculate that PTX may have utility as a candidate anti-inflammatory agent for prophylaxis and/or treatment of human placental inflammation.

Pentoxifylline inhibits TLR- and inflammasome-mediated in vitro inflammatory cytokine production in human blood with greater efficacy and potency in newborns.

BACKGROUND: Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1ß) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood. METHODS: Newborn cord and adult blood were treated with PTX (50-400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR. RESULTS: Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1ß with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation. CONCLUSION: PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Role of single nucleotide polymorphisms of cytokine genes in spontaneous preterm delivery.

Intra-amniotic infections are implicated in spontaneous preterm delivery (PTD). Certain genetic polymorphisms are associated with increased production of proinflammatory and/or decreased production of anti-inflammatory cytokines, thereby possibly promoting PTD. We determined the relationship between maternal and fetal cytokine gene polymorphisms with occurrence and severity of spontaneous PTD (PTD after spontaneous-onset preterm labor and/or preterm prelabor rupture of membranes) and their association with intrauterine inflammation and infection. DNA from buccal brushings of 80 preterm (gestation < 35 weeks) and 80 matched term mother-infant pairs was assayed for tumor necrosis factor alpha (TNF-alpha [-308G/A]), interferon-gamma (IFN-gamma [+874A/T]), interleukin-6 (IL-6 [-174C/G]), interleukin-10 (IL-10 [-1082G/A, -819C/T, -592C/A]), and transforming growth factor beta1 (TGF-beta1 [T/Ccodon10,G/Ccodon25]) by using polymerase chain reaction (PCR) with sequence-specific primers. The presence of histologic chorioamnionitis was determined for PTDs. Conditioned on maternal IFN-gamma genotypes, fetal high IFN-gamma producing allele (IFN-gamma[+874T]) was associated with spontaneous PTD (odds ratio = 2.3 [1.2-4.4]). Among preterm deliveries, maternal low TGF-beta1 (TGF-beta1 [codon10C]) producing genotypes correlated negatively with gestation. Fetal TNF-alpha (-308G) was significantly associated with histologic chorioamnionitis. Underlying genitourinary infections and/or inflammation were significantly associated with maternal and fetal IL-6 (-174G), fetal TNF-alpha (-308GG), and fetal IL-10 (-1082A). We conclude that certain fetal and maternal cytokine gene polymorphisms may be associated with occurrence and/or severity of spontaneous PTD and with intrauterine inflammation and infection.