Accumulation of immune-suppressive CD4 + T cells in aging - tempering inflammaging at the expense of immunity.

The 'immune risk profile' has been shown to predict mortality in the elderly, highlighting the need to better understand age-related immune dysfunction. While aging leads to many defects affecting all arms of the immune system, this review is focused on the accrual of immuno-suppressive CD4 + T cell populations, including FoxP3 + regulatory T cells, and subsets of IL-10-producing T follicular helper cells. New data suggest that such accumulations constitute feedback mechanisms to temper the ongoing progressive low-grade inflammation that develops with age, the so-called "inflammaging", and by doing so, how they have the potential to promote healthier aging. However, they also impair effector immune responses, notably to infections, or vaccines. These studies also reinforce the idea that the aged immune system should not be considered as a poorly functional version of the young one, but more as a dynamic system in which CD4 + T cells, and other immune/non-immune subsets, differentiate, interact with their milieu and function differently than in young hosts. A better understanding of these unique interactions is thus needed to improve effector immune responses in the elderly, while keeping inflammaging under control.

Interactions Between HDL and CD4+ T Cells: A Novel Understanding of HDL Anti-Inflammatory Properties.

Several studies in animal models and human cohorts have recently suggested that HDLs (high-density lipoproteins) not only modulate innate immune responses but also adaptative immune responses, particularly CD4+ T cells. CD4+ T cells are central effectors and regulators of the adaptive immune system, and any alterations in their homeostasis contribute to the pathogenesis of cardiovascular diseases, autoimmunity, and inflammatory diseases. In this review, we focus on how HDLs and their components affect CD4+ T-cell homeostasis by modulating cholesterol efflux, immune synapsis, proliferation, differentiation, oxidative stress, and apoptosis. While the effects of apoB-containing lipoproteins on T cells have been relatively well established, this review focuses specifically on new connections between HDL and CD4+ T cells. We present a model where HDL may modulate T cells through both direct and indirect mechanisms.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

The induction of preterm labor in rhesus macaques is determined by the strength of immune response to intrauterine infection.

Intrauterine infection/inflammation (IUI) is a major contributor to preterm labor (PTL). However, IUI does not invariably cause PTL. We hypothesized that quantitative and qualitative differences in immune response exist in subjects with or without PTL. To define the triggers for PTL, we developed rhesus macaque models of IUI driven by lipopolysaccharide (LPS) or live Escherichia coli. PTL did not occur in LPS challenged rhesus macaques, while E. coli-infected animals frequently delivered preterm. Although LPS and live E. coli both caused immune cell infiltration, E. coli-infected animals showed higher levels of inflammatory mediators, particularly interleukin 6 (IL-6) and prostaglandins, in the chorioamnion-decidua and amniotic fluid (AF). Neutrophil infiltration in the chorio-decidua was a common feature to both LPS and E. coli. However, neutrophilic infiltration and IL6 and PTGS2 expression in the amnion was specifically induced by live E. coli. RNA sequencing (RNA-seq) analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon (IFN) response, chemotaxis, sumoylation, and iron homeostasis were up-regulated in the E. coli group compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.

Delayed Microbial Maturation Durably Exacerbates Th17-driven Asthma in Mice.

Microbial maturation disrupted by early-life dysbiosis has been linked with increased asthma risk and severity; however, the immunological mechanisms underpinning this connection are poorly understood. We sought to understand how delaying microbial maturation drives worsened asthma outcomes later in life and its long-term durability. Drinking water was supplemented with antibiotics on Postnatal Days 10-20. To assess the immediate and long-term effects of delaying microbial maturation on experimental asthma, we initiated house dust mite exposure when bacterial diversity was either at a minimum or had recovered. Airway hyperresponsiveness, histology, pulmonary leukocyte recruitment, flow cytometric analysis of cytokine-producing lymphocytes, and assessment of serum IgG1 (Immunoglobulin G1) and IgE (Immunoglobulin E) concentrations were performed. RT-PCR was used to measure IL-13 (Interleukin 13)-induced gene expression in sequentially sorted mesenchymal, epithelial, endothelial, and leukocyte cell populations from the lung. Delayed microbial maturation increased allergen-driven airway hyperresponsiveness and Th17 frequency compared with allergen-exposed control mice, even when allergen exposure began after bacterial diversity recovered. Blockade of IL-17A (Interleukin 17A) reversed the airway hyperresponsiveness phenotype. In addition, allergen exposure in animals that experienced delayed microbial maturation showed signs of synergistic signaling between IL-13 and IL-17A in the pulmonary mesenchymal compartment. Delaying microbial maturation in neonates promotes the development of more severe asthma by increasing Th17 frequency, even if allergen exposure is initiated weeks after microbial diversity is normalized. In addition, IL-17A-aggravated asthma is associated with increased expression of IL-13-induced genes in mesenchymal, but not epithelial cells.

Apolipoprotein E-containing HDL decreases caspase-dependent apoptosis of memory regulatory T lymphocytes.

Plasma levels of HDL cholesterol are inversely associated with CVD progression. It is becoming increasingly clear that HDL plays important roles in immunity that go beyond its traditionally understood roles in lipid transport. We previously reported that HDL interaction with regulatory T cells (Treg) protected them from apoptosis, which could be a mechanism underlying the broad anti-inflammatory effect of HDL. Herein, we extend our work to show that HDL interacts mainly with memory Treg, particularly with the highly suppressive effector memory Treg, by limiting caspase-dependent apoptosis in an Akt-dependent manner. Reconstitution experiments identified the protein component of HDL as the primary driver of the effect, though the most abundant HDL protein, apolipoprotein A-I (APOA1), was inactive. In contrast, APOE-depleted HDL failed to rescue effector memory Treg, suggesting the critical role of APOE proteins. HDL particles reconstituted with APOE, and synthetic phospholipids blunted Treg apoptosis at physiological concentrations. The APOE3 and APOE4 isoforms were the most efficient. Similar results were obtained when lipid-free recombinant APOEs were tested. Binding experiments showed that lipid-free APOE3 bound to memory Treg but not to naive Treg. Overall, our results show that APOE interaction with Treg results in blunted caspase-dependent apoptosis and increased survival. As dysregulation of HDL-APOE levels has been reported in CVD and obesity, our data bring new insight on how this defect may contribute to these diseases.

IRAK1 Is a Critical Mediator of Inflammation-Induced Preterm Birth.

Preterm birth (PTB) is a major cause of neonatal mortality and morbidity, often triggered by chorioamnionitis or intrauterine inflammation (IUI) with or without infection. Recently, there has been a strong association of IL-1 with PTB. We hypothesized that IL-1R-associated kinase 1 (IRAK1), a key signaling mediator in the TLR/IL-1 pathway, plays a critical role in PTB. In human fetal membranes (FM) collected immediately after birth from women delivering preterm, p-IRAK1 was significantly increased in all the layers of FM with chorioamnionitis, compared with no-chorioamnionitis subjects. In a preterm rhesus macaque model of IUI given intra-amniotic LPS, induction of p-IRAK1 and downstream proinflammatory signaling mediators were seen in the FM. In a C57BL/6J wild-type PTB mouse model of IUI given intrauterine LPS, an IRAK1 inhibitor significantly decreased PTB and increased live birth in a dose-dependent manner. Furthermore, IRAK1 knockout mice were protected from LPS-induced PTB, which was seen in wild-type controls. Activation of IRAK1 was maintained by K63-mediated ubiquitination in preterm FM of humans with chorioamnionitis and rhesus and mouse IUI models. Mechanistically, IRAK1 induced PTB in the mouse model of IUI by upregulating expression of COX-2. Thus, our data from human, rhesus, and mouse demonstrates a critical role IRAK1 in IUI and inflammation-associated PTB and suggest it as potential therapeutic target in IUI-induced PTB.

Regulatory T cells in retroviral infections.

Tight regulation of immune responses is not only critical for preventing autoimmune diseases but also for preventing immunopathological damage during infections in which overactive immune responses may be more harmful for the host than the pathogen itself. Regulatory T cells (Tregs) play a critical role in this regulation, which was discovered using the Friend retrovirus (FV) mouse model. Subsequent FV studies revealed basic biological information about Tregs, including their suppressive activity on effector cells as well as the molecular mechanisms of virus-induced Treg expansion. Treg suppression not only limits immunopathology but also prevents complete elimination of pathogens contributing to chronic infections. Therefore, Tregs play a complex role in the pathogenesis of persistent retroviral infections. New therapeutic concepts to reactivate effector T-cell responses in chronic viral infections by manipulating Tregs also came from work with the FV model. This knowledge initiated many studies to characterize the role of Tregs in HIV pathogenesis in humans, where a complex picture is emerging. On one hand, Tregs suppress HIV-specific effector T-cell responses and are themselves targets of infection, but on the other hand, Tregs suppress HIV-induced immune hyperactivation and thus slow the infection of conventional CD4+ T cells and limit immunopathology. In this review, the basic findings from the FV mouse model are put into perspective with clinical and basic research from HIV studies. In addition, the few Treg studies performed in the simian immunodeficiency virus (SIV) monkey model will also be discussed. The review provides a comprehensive picture of the diverse role of Tregs in different retroviral infections and possible therapeutic approaches to treat retroviral chronicity and pathogenesis by manipulating Treg responses.

Interleukin 2 Promotes Hepatic Regulatory T Cell Responses and Protects From Biliary Fibrosis in Murine Sclerosing Cholangitis.

In the multidrug resistance protein 2 (Mdr2)-/- mouse model, low phospholipid bile instigates biliary epithelial injury, sterile inflammation, and fibrosis, thereby recapitulating disease mechanisms implicated in biliary atresia (BA) and primary sclerosing cholangitis. We hypothesize that T lymphocytes contribute to the biliary injury and fibrosis in murine sclerosing cholangitis (SC) and that they are susceptible to suppression by regulatory T cells (Tregs). In juvenile Mdr2-/- mice, intrahepatic CD8+ lymphocytes were expanded, and contraction of intrahepatic Tregs coincided with rising serum alanine transferase and alkaline phosphatase (ALP) levels between days 14-30 of life. Antibody-mediated depletion of intrahepatic CD8+ lymphocytes during that time reduced ALP levels and the expression of osteopontin (Opn), a pro-fibrogenic cytokine. Depletion of intrahepatic Tregs with anti-CD25 antibody between days 7-30 increased intrahepatic CD8+ T cells, Opn expression, and fibrosis. Conversely, expansion of intrahepatic Tregs with interleukin 2/anti-interleukin 2 immune complexes (IL-2c) downregulated hepatic expression of Opn and Tnf, reduced frequency of intrahepatic CD8+ lymphocytes, and diminished biliary injury and fibrosis. Treatment with IL-2c upregulated hepatic Treg expression of CD39, an ectonucleotidase capable of hydrolyzing pro-inflammatory adenosine triphosphate. In vitro, Tregs expressing CD39 suppressed the proliferation of hepatic CD8+ lymphocytes from Mdr2-/- mice more efficiently than those lacking CD39. In infants with BA, infiltration of interlobular bile ducts with CD8+ cells was associated with biliary expression of Opn and its transcription was negatively correlated with mRNA expression of Treg-associated genes. Conclusion: Hepatic CD8+ T lymphocytes drive biliary injury and fibrosis in murine SC. Their proliferation is controlled by hepatic Tregs through the purinergic pathway, which is responsive to IL-2c, suggesting that Treg-directed low-dose Il-2 treatment may be considered as therapy for SC.

Lipopolysaccharide-Induced Chorioamnionitis Promotes IL-1-Dependent Inflammatory FOXP3+ CD4+ T Cells in the Fetal Rhesus Macaque.

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 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, whereas regulatory T cell (Treg) frequency decreased. These changes persisted for at least 48 h. Notably, Th17 cytokines were predominantly expressed by FOXP3(+)CD4(+) T cells and not by their FOXP3(-) counterparts. Bifunctional IL-17(+)FOXP3(+) exhibited a phenotype of inflammatory Tregs (RORc(High/+), Helios(Low/-), IL-2(+), IFN-γ(+), and IL-8(+)) compared with 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 Tregs 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 Tregs toward 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.

Maternal house dust mite exposure during pregnancy enhances severity of house dust mite-induced asthma in murine offspring.

BACKGROUND: Atopic status of the mother and maternal exposure to environmental factors are associated with increased asthma risk. Moreover, animal models demonstrate that exposure to allergens in strongly sensitized mothers influences offspring asthma development, suggesting that in utero exposures can influence offspring asthma. However, it is unclear whether maternal exposure to common human allergens such as house dust mite (HDM), in the absence of additional adjuvants, influences offspring asthma development. OBJECTIVE: We sought to determine whether maternal HDM exposure influences asthma development in offspring. METHODS: Pregnant female mice were exposed to PBS or HDM during pregnancy. Using offspring of PBS- or HDM-exposed mothers, the magnitude of HDM or Aspergillus fumigatus (AF) extract-induced airway hyperresponsiveness (AHR), airway inflammation, immunoglobulin production, TH2-associated cytokine synthesis, and pulmonary dendritic cell activity was assessed. RESULTS: Compared with offspring of PBS-exposed mothers, offspring of HDM-exposed mothers demonstrate increased AHR, airway inflammation, TH2 cytokine production, and immunoglobulin levels and a modest decrease in the phagocytic capacity of pulmonary macrophage populations following HDM exposure. Increased sensitivity to AF-induced airway disease was not observed. Offspring of HDM-exposed B-cell-deficient mothers also demonstrated increased HDM-induced AHR, suggesting that transfer of maternal immunoglobulins is not required. CONCLUSIONS: Our data demonstrate that maternal exposure to HDM during pregnancy increases asthma sensitivity in offspring in an HDM-specific manner, suggesting that vertical transmission of maternal immune responses may be involved. These findings have important implications for regulation of asthma risk, and suggest that exposure to HDM in the developed world may have underappreciated influences on the overall prevalence of allergic asthma.

High density lipoproteins selectively promote the survival of human regulatory T cells.

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.

Pro-inflammatory immune responses in leukocytes of premature infants exposed to maternal chorioamnionitis or funisitis.

BACKGROUND: Acute chorioamnionitis contributes to premature birth, and is associated with postbirth complications. How chorioamnionitis impacts neonate's developing immune system has not been well defined. METHODS: Blood from extremely preterm infants (≤28 wk gestation) was drawn at the first, second, and fourth week of life. Blood was either left unstimulated or stimulated for 4 h with PMA/ionomycin. mRNA expression of transcription factors in unstimulated cells (RORC, TBET, GATA3, and Forkhead box protein 3 (FOXP3)) and inflammatory cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-6) in unstimulated and stimulated cells were analyzed. Data were analyzed based on the diagnosis of chorioamnionitis, funisitis and bronchopulmonary dysplasia (BPD). RESULTS: At 1 wk of life, exposure to funisitis, but not maternal chorioamnionitis was associated with an increased expression of RORC and RORC/FOXP3 ratio. These increases in RORC and RORC/FOXP3 ratio were sustained over the 4 wk of follow-up. Leukocytes from infants who developed BPD had increased stimulated and unstimulated IL-4 at the first week of life, but these increases were not sustained over time. In contrast, infants with mild BPD had a sustained decrease in stimulated IL-2. CONCLUSION: Chorioamnionitis exposure, in particular to funisitis, lead to enhanced Th17-like responses that persist for 4 wk after birth. Infants who later developed BPD did not exhibit a strikingly distinct immune profile.

IL-6 and ICOS Antagonize Bim and Promote Regulatory T Cell Accrual with Age.

Regulatory T cells (Tregs), a subset of CD4(+) T cells, dramatically accumulate with age in humans and mice and contribute to age-related immune suppression. Recently, we showed that a majority of accumulating Tregs in aged mice expressed low levels of CD25, and their accrual is associated with declining levels of IL-2 in aged mice. In this study, we further investigated the origin of CD25(lo) Tregs in aged mice. First, aged Tregs had high expression of neuropilin-1 and Helios, and had a broad Vß repertoire. Next, we analyzed the gene expression profile of Tregs, naive T cells, and memory T cells in aged mice. We found that the gene expression profile of aged CD25(lo) Tregs were more related to young CD25(lo) Tregs than to either naive or memory T cells. Further, the gene expression profile of aged Tregs was consistent with recently described "effector" Tregs (eTregs). Additional analysis revealed that nearly all Tregs in aged mice were of an effector phenotype (CD44(hi)CD62L(lo)) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, because in vivo Ab blockade of ICOSL led to a loss of eTregs, and this loss was rescued in Bim-deficient mice. Further, serum levels of IL-6 increased with age and contributed to elevated expression of ICOS on aged Tregs. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting eTreg accrual with age likely through maintenance of ICOS expression.

Loss of Phagocytic and Antigen Cross-Presenting Capacity in Aging Dendritic Cells Is Associated with Mitochondrial Dysfunction.

Impaired functionality of dendritic cells (DCs) significantly contributes to decreased adaptive immune responses in aged hosts. The expression of MHC-peptide on the DC surface is the critical first step in T cell priming, but few studies have addressed the effect of aging on Ag acquisition, processing, and presentation by DCs. In this study, we show that aged murine DCs were less efficient in the cross-presentation of cell-associated Ag and subsequently in the cross-priming of CD8(+) T cells than were their young counterparts. The decreased cross-presentation was associated with a reduction in the frequency of CD8α DCs and merocytic (CD8α(-)CD11b(-))DCs that could endocytose cell-associated Ag, as well as the number and the size of the endocytosed particles in the DC that did internalize cell-associated materials. Mechanistically, phagocytic capacity has been associated with mitochondrial activity and membrane potential (Δψm). Aged DCs exhibited profound signs of mitochondrial dysfunction, illustrated by lower Δψm, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation, and greater proton leak and reactive oxygen species (ROS) production. Mimicking the aged metabolic phenotype in young DCs by pharmacologic manipulation indicated that the reductions in Δψm and ATP impeded the phagocytic capacity whereas ROS interfered with a later step in the cross-presentation process. Conversely, in vitro scavenging of ROS partially restored cross-presentation by aged DCs. Taken together, these data suggest that improvement of aged DC functionality might be feasible in the elderly by targeting metabolic dysfunction or its downstream sequelae, thereby opening new avenues for enhancing vaccine efficiency in this population.

Placental Infection With Ureaplasma species Is Associated With Histologic Chorioamnionitis and Adverse Outcomes in Moderately Preterm and Late-Preterm Infants.

OBJECTIVE: The human Ureaplasma species are the microbes most frequently isolated from placentae of women who deliver preterm. The role of Ureaplasma species has been investigated in pregnancies at <32 weeks of gestation, but currently no studies have determined the prevalence of ureaplasmas in moderately preterm and late-preterm (hereafter, "moderate/late preterm") infants, the largest cohort of preterm infants. METHODS: Women delivering moderate/late preterm infants (n = 477) and their infants/placentae (n = 535) were recruited, and swab specimens of chorioamnion tissue, chorioamnion tissue specimens, and cord blood specimens were obtained at delivery. Swab and tissue specimens were cultured and analyzed by 16S ribosomal RNA polymerase chain reaction (PCR) for the presence of microorganisms, while cord blood specimens were analyzed for the presence of cytokines, chemokines, and growth factors. RESULTS: We detected microorganisms in 10.6% of 535 placentae (443 were delivered late preterm and 92 were delivered at term). Significantly, Ureaplasma species were the most prevalent microorganisms, and their presence alone was associated with histologically confirmed chorioamnionitis in moderate/late preterm and term placentae (P < .001). The presence of ureaplasmas in the chorioamnion was also associated with elevated levels of granulocyte colony-stimulating factor (P = .02). CONCLUSIONS: These findings have important implications for infection and adverse pregnancy outcomes throughout gestation and should be of major consideration for obstetricians and neonatologists.

Intra-amniotic Ureaplasma parvum-Induced Maternal and Fetal Inflammation and Immune Responses in Rhesus Macaques.

BACKGROUND: Although Ureaplasma species are the most common organisms associated with prematurity, their effects on the maternal and fetal immune system remain poorly characterized. METHODS: Rhesus macaque dams at approximately 80% gestation were injected intra-amniotically with 107 colony-forming units of Ureaplasma parvum or saline (control). Fetuses were delivered surgically 3 or 7 days later. We performed comprehensive assessments of inflammation and immune effects in multiple fetal and maternal tissues. RESULTS: Although U. parvum grew well in amniotic fluid, there was minimal chorioamnionitis. U. parvum colonized the fetal lung, but fetal systemic microbial invasion was limited. Fetal lung inflammation was mild, with elevations in CXCL8, tumor necrosis factor (TNF) α, and CCL2 levels in alveolar washes at day 7. Inflammation was not detected in the fetal brain. Significantly, U. parvum decreased regulatory T cells (Tregs) and activated interferon γ production in these Tregs in the fetus. It was detected in uterine tissue by day 7 and induced mild inflammation and increased expression of connexin 43, a gap junction protein involved with labor. CONCLUSIONS: U. parvum colonized the amniotic fluid and caused uterine inflammation, but without overt chorioamnionitis. It caused mild fetal lung inflammation but had a more profound effect on the fetal immune system, decreasing Tregs and polarizing them toward a T-helper 1 phenotype.

Neonatal regulatory T cells have reduced capacity to suppress dendritic cell function.

Regulatory T cells (Treg cells) 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 Treg cells from term and preterm neonates to suppress DCs, and the suppressive mechanisms they use, are still undefined. We found that, relative to adult Treg cells, activated Treg cells 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 Treg cells failed to suppress the formation of DC-Tcon aggregates, in contrast to naïve and memory Treg cells from adults. However, neonatal Treg cells 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 Treg cells appear less functional than adult Treg cells, and this defect is consistent with the general impairment of CD4 cell function in newborns.

Intra-amniotic LPS causes acute neuroinflammation in preterm rhesus macaques.

BACKGROUND: Chorioamnionitis is associated with an increased risk of brain injury in preterm neonates. Inflammatory changes in brain could underlie this injury. Here, we evaluated whether neuroinflammation is induced by chorioamnionitis in a clinically relevant model. METHODS: Rhesus macaque fetuses were exposed to either intra-amniotic (IA) saline, or IA lipopolysaccharide (LPS) (1 mg) 16 or 48 h prior to delivery at 130 days (85 % of gestation) (n = 4-5 animals/group). We measured cytokines in the cerebrospinal fluid (CSF), froze samples from the left brain for molecular analysis, and immersion fixed the right brain hemisphere for immunohistology. We analyzed the messenger RNA (mRNA) levels of the pro-inflammatory cytokines IL-1ß, CCL2, TNF-α, IL-6, IL-8, IL-10, and COX-2 in the periventricular white matter (PVWM), cortex, thalamus, hippocampus, and cerebellum by RT-qPCR. Brain injury was assessed by immunohistology for myelin basic protein (MBP), IBA1 (microglial marker), GFAP (astrocyte marker), OLIG2 (oligodendrocyte marker), NeuN (neuronal marker), CD3 (T cells), and CD14 (monocytes). Microglial proliferation was assessed by co-immunostaining for IBA1 and Ki67. Data were analyzed by ANOVA with Tukey's post-test. RESULTS: IA LPS increased mRNA expression of pro-inflammatory cytokines in the PVWM, thalamus, and cerebellum, increased IL-6 concentration in the CSF, and increased apoptosis in the periventricular area after 16 h. Microglial proliferation in the white matter was increased 48 h after IA LPS. CONCLUSIONS: LPS-induced chorioamnionitis caused neuroinflammation, microglial proliferation, and periventricular apoptosis in a clinically relevant model of chorioamnionitis in fetal rhesus macaques. These findings identify specific responses in the fetal brain and support the hypothesis that neuroinflammatory changes may mediate the adverse neurodevelopmental outcomes associated with chorioamnionitis.

The placental membrane microbiome is altered among subjects with spontaneous preterm birth with and without chorioamnionitis.

BACKGROUND: Preterm birth (PTB) is a leading cause of neonatal morbidity and mortality and is not uncommonly associated with chorioamnionitis. We recently have demonstrated that the placenta harbors a unique microbiome with similar flora to the oral community. We also have shown an association of these placental microbiota with PTB, history of antenatal infection, and excess maternal weight gain. On the basis of these previous observations, we hypothesized that the placental membranes would retain a microbiome community that would vary in association with preterm birth and chorioamnionitis. OBJECTIVE: In the current study, we aimed to examine the differences in the placental membrane microbiome in association with PTB in both the presence and absence of chorioamnionitis and/or funisitis using state-of-the-science whole-genome shotgun metagenomics. STUDY DESIGN: This was a cross-sectional analysis with 6 nested spontaneous birth cohorts (n = 9-15 subjects/cohort): Term gestations without chorioamnionitis, term with chorioamnionitis, preterm without chorioamnionitis, preterm with mild chorioamnionitis, preterm with severe chorioamnionitis, and preterm with chorioamnionitis and funisitis. Histologic analysis was performed with Redline's criteria, and inflammatory cytokines were analyzed in the cord blood. DNA from placental membranes was extracted from sterile swabs collected at delivery, and whole-genome shotgun sequencing was performed on the Illumina HiSeq platform. Filtered microbial DNA sequences were annotated and analyzed with MG-RAST (ie, Metagenomic Rapid Annotations using Subsystems Technology) and R. RESULTS: Subjects were assigned to cohorts on the basis of gestational age at delivery and independent scoring of histologic chorioamnionitis. We found that preterm subjects with severe chorioamnionitis and funisitis had increases in cord blood inflammatory cytokines. Of interest, although the placental membrane microbiome was altered in association with severity of histologic chorioamnionitis (permutational multivariate analysis of variance P = .005), there was no observable impact with either betamethasone or antibiotic treatment. In preterm subjects with chorioamnionitis, we found a high abundance of both urogenital and oral commensal bacteria. These alterations in the microbiome were accompanied by significant variation (P < .05) in microbial metabolic pathways important in the glucose-fed pentose phosphate pathway (term subjects), or glycerophopholipid metabolism, and the biosynthesis of the siderophore group nonribosomal peptides (preterm subjects). CONCLUSION: Consistent with ours and others previous findings, women who experienced spontaneous PTB harbor placental microbiota that further differed by severity of chorioamnionitis. Integrative metagenomic analysis revealed significant variation in distinct bacterial metabolic pathways, which we speculate may contribute to risk of preterm birth with and without severe chorioamnionitis.