Neutrophil infiltration of the chorioamnion-decidua tissue at the maternal-fetal interface (chorioamnionitis) is a leading cause of prematurity, fetal inflammation, and perinatal mortality. We induced chorioamnionitis in preterm rhesus macaques by intraamniotic injection of LPS. Here, we show that, during chorioamnionitis, the amnion upregulated phospho-IRAK1-expressed neutrophil chemoattractants CXCL8 and CSF3 in an IL-1-dependent manner. IL-1R blockade decreased chorio-decidua neutrophil accumulation, neutrophil activation, and IL-6 and prostaglandin E2 concentrations in the amniotic fluid. Neutrophils accumulating in the chorio-decidua had increased survival mediated by BCL2A1, and IL-1R blockade also decreased BCL2A1+ chorio-decidua neutrophils. Readouts for inflammation in a cohort of women with preterm delivery and chorioamnionitis were similar to findings in the rhesus macaques. IL-1 is a potential therapeutic target for chorioamnionitis and associated morbidities.

Chorioamnionitis is associated with preterm labor and fetal inflammatory response syndrome (FIRS), causing fetal organ injury and morbidity particularly in extremely premature infants. However, the effects of inflammation on the fetal immune system remain poorly understood, due to the difficulty of studying immune development in infants. Therefore, we used the model of intra-amniotic (IA) LPS administered at ~80% gestation in rhesus monkeys to cause chorioamnionitis and FIRS that is similar in human pathology. Importantly, the frequency of IL-17+ and IL-22+ CD4+ T-cells increased in the spleen of LPS-exposed fetuses, while Treg frequency decreased. These changes persisted for at least 48h. Notably, Th17 cytokines were predominantly expressed by FoxP3+CD4+ T-cells and not by their FoxP3− counterparts. Bi-functional IL17+FoxP3+ exhibited a phenotype of inflammatory Treg (RORcHigh/+, HeliosLow/−, IL-2+, IFNγ+ and IL-8+) compared to typical FoxP3+ cells. Diminished splenic Treg frequency in LPS-exposed fetuses was associated with inadequate Treg generation in the thymus. Mechanistically, the emergence of inflammatory Treg was largely dependent on IL-1 signaling. However, blockage of IL-1R signaling did not abolish the deleterious effects of LPS on Treg frequency in the thymus or spleen. Collectively, we demonstrate that a prenatal inflammatory environment leads to inadequate Treg generation in the thymus with a switch of splenic Treg towards an inflammatory phenotype. Both processes likely contribute to the pathogenesis of chorioamnionitis. Approaches to manipulate Treg numbers and function could thus be useful therapeutically to alleviate FIRS in preterm infants.

Accumulation of activated neutrophils at the feto-maternal interface is a defining hallmark of intrauterine inflammation (IUI) that might trigger an excessive immune response during pregnancy. Mechanisms responsible of this massive neutrophil recruitment are poorly investigated. We have previously showed that intraamniotic injection of LPS in rhesus macaques induced a neutrophil predominant inflammatory response similar to that seen in human IUI. Here, we demonstrate that anti-TNF antibody (Adalimumab) inhibited ∼80% of genes induced by LPS involved in inflammatory signaling and innate immunity in chorio-decidua neutrophils. Consistent with the gene expression data, TNF-blockade decreased LPS-induced neutrophil accumulation and activation at the feto-maternal interface. We also observed a reduction in IL-6 and other pro-inflammatory cytokines but not prostaglandins concentrations in the amniotic fluid. Moreover, TNF-blockade decreased mRNA expression of inflammatory cytokines in the chorio-decidua but not in the uterus, suggesting that inhibition of TNF-signaling decreased the inflammation in a tissue-specific manner within the uterine compartment. Taken together, our results demonstrate a predominant role for TNF-signaling in modulating the neutrophilic infiltration at the feto-maternal interface during IUI and suggest that blockade of TNF-signaling could be considered as a therapeutic approach for IUI, the major leading cause of preterm birth.

Regulatory T-cells (Tregs) mediate their suppressive action by acting directly on conventional T-cells (Tcons) or dendritic cells (DCs). One mechanism of Treg suppression is the increase of cyclic adenosine 3′,5′-monophosphate (cAMP) levels in target cells. Tregs utilize cAMP to control Tcon responses, such as proliferation and cytokine production. Tregs also exert their suppression on DCs, diminishing DC immunogenicity by downmodulating the expression of costimulatory molecules and actin polymerization at the immunological synapse. The Treg-mediated usage of cAMP occurs through two major mechanisms. The first involves the Treg-mediated influx of cAMP in target cells through gap junctions. The second is the conversion of adenosine triphosphate into adenosine by the ectonucleases CD39 and CD73 present on the surface of Tregs. Adenosine then binds to receptors on the surface of target cells, leading to increased intracellular cAMP levels in these targets. Downstream, cAMP can activate the canonical protein kinase A (PKA) pathway and the exchange protein activated by cyclic AMP (EPAC) non-canonical pathway. In this review, we discuss the most recent findings related to cAMP activation of PKA and EPAC, which are implicated in Treg homeostasis as well as the functional alterations induced by cAMP in cellular targets of Treg suppression.

HDLs appear to affect regulatory T cell (Treg) homeostasis, as suggested by the increased Treg counts in HDL-treated mice and by the positive correlation between Treg frequency and HDL-cholesterol levels in statin-treated healthy adults. However, the underlying mechanisms remain unclear. Herein, we show that HDLs, not LDLs, significantly decreased the apoptosis of human Tregs in vitro, whereas they did not alter naïve or memory CD4 T cell survival. Similarly, oleic acid bound to serum albumin increased Treg survival. Tregs bound and internalized high amounts of HDL compared with other subsets, which might arise from the higher expression of the scavenger receptor class B type I by Tregs; accordingly, blocking this receptor hindered HDL-mediated Treg survival. Mechanistically, we showed that HDL increased Treg ATP concentration and mitochondrial activity, enhancing basal respiration, maximal respiration, and spare respiratory capacity. Blockade of FA oxidation by etoxomir abolished the HDL-mediated enhanced survival and mitochondrial activity. Our findings thus suggest that Tregs can specifically internalize HDLs from their microenvironment and use them as an energy source. Furthermore, a novel implication of our data is that enhanced Treg survival may contribute to HDLs' anti-inflammatory properties.

Differentiation of regulatory Treg (Treg) in the periphery is critical to control inflammatory processes. Although polarization of inducible Treg (iTreg) often occurs in an inflammatory environment, the effects exerted by inflammation on human iTreg differentiation have not been extensively studied. We observed that IL-1β significantly reduced the frequency of FOXP3+ T cells under iTreg-polarizing conditions. Mechanistically, we show that IL-1β activated mTORC1 and downstream upregulated hypoxia inducible factor-1 (HIF-1α) expression. Using specific inhibitors, we demonstrated that both steps were critical in the deleterious effect of IL-1β on Treg differentiation. Chemical stabilization of HIF-1α by Dimethyloxalylglycine (DMOG) also significantly impaired iTreg differentiation. Interestingly, while IL-1β-treated cells exhibited only minor changes in metabolism, DMOG treatment decreased iTreg mitochondrial respiration and increased their glycolytic capacity. In conclusion, exposure to inflammatory stimuli profoundly inhibits human Treg differentiation HIF-1α dependent, suggesting that targeting HIF-1α could be a strategy to foster iTreg differentiation in an inflammatory milieu. However, IL-1β deleterious effect does not appear to be completely driven by metabolic changes. These data thus suggest that several mechanisms contribute to the regulation of iTreg differentiation, but the timing and respective requirement for each pathway vary depending on the milieu in which iTreg differentiate.

Regulatory Tcells (Tregs) limit contact between Dendritic cells (DCs) and conventional T cells (Tcons), decreasing the formation of aggregates as well as down-modulating the expression of co-stimulatory molecules by DCs, thus decreasing DC immunogenicity and abrogating T-cell activation. Despite the importance of this Treg-cell function, the capacity of Tregs from term and preterm neonates to suppress DCs, and the suppressive mechanisms they use, are still undefined. We found that, relative to adult Tregs, activated Tregs from human neonates expressed lower FOXP3 and CTLA-4, but contained higher levels of cAMP. We developed an in vitro model in which Treg function was measured at a physiological ratio of 1 Treg for 10 Tcon and 1 monocyte-derived DC, as Treg target. Term and preterm Tregs failed to suppress the formation of DC-Tcon aggregates, in contrast to naïve and memory Tregs from adults. However, neonatal Tregs diminished DC and Tcon activation as well as actin polymerization at the immunological synapses. In addition, CTLA-4 and cAMP were the main suppressive molecules used by neonatal Treg. Altogether, both preterm and term neonatal Tregs appear less functional than adult Tregs, and this defect is consistent with the general impairment of CD4 cell function in newborns.

Chorioamnionitis, a potentially serious inflammatory complication of pregnancy, is associated with the development of an inflammatory milieu within the amniotic fluid surrounding the developing fetus. When chorioamnionitis occurs, the fetal lung finds itself in the unique position of being constantly exposed to the consequent inflammatory meditators and/or microbial products found in the amniotic fluid. This exposure results in significant changes to the fetal lung, such as increased leukocyte infiltration, altered cytokine, and surfactant production, and diminished alveolarization. These alterations can have potentially lasting impacts on lung development and function. However, studies to date have only begun to elucidate the association between such inflammatory exposures and lifelong consequences such as lung dysfunction. In this review, we discuss the pathogenesis of and fetal immune response to chorioamnionitis, detail the consequences of chorioamnionitis exposure on the developing fetal lung, highlighting the various animal models that have contributed to our current understanding and discuss the importance of fetal exposures in regard to the development of chronic respiratory disease. Finally, we focus on the clinical, basic, and therapeutic challenges in fetal inflammatory injury to the lung, and propose next steps and future directions to improve our therapeutic understanding of this important perinatal stress.