Dataset Information

Extracellular Vesicles-mediated recombinant IL-10 protects against ascending infection-associated preterm birth by reducing fetal inflammatory response.

ABSTRACT:

Background

Fetal inflammatory response mediated by the influx of immune cells and activation of pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is the major determinant of infection-associated preterm birth (PTB, live births < 37 weeks of gestation).

Objective

To reduce the incidence of PTB by minimizing inflammation, extracellular vesicles (EVs) were electroporetically engineered to contain anti-inflammatory cytokine interleukin (IL)-10 (eIL-10), and their efficacy was tested in an ascending model of infection (vaginal administration of E. coli) induced PTB in mouse models.

Study design

EVs (size: 30-170 nm) derived from HEK293T cells were electroporated with recombinant IL-10 at 500 volts and 125 Ω, and 6 pulses to generate eIL-10. eIL-10 structural characters (electron microscopy, nanoparticle tracking analysis, ExoView [size and cargo content] and functional properties (co-treatment of macrophage cells with LPS and eIL-10) were assessed. To test efficacy, CD1 mice were vaginally inoculated with E. coli (10¹⁰CFU) and subsequently treated with either PBS, eIL-10 (500ng) or Gentamicin (10mg/kg) or a combination of eIL-10+gentamicin. Fetal inflammatory response in maternal and fetal tissues after the infection or treatment were conducted by suspension Cytometer Time of Flight (CyTOF) using a transgenic mouse model that express red fluorescent TdTomato (mT+) in fetal cells.

Results

Engineered EVs were structurally and functionally stable and showed reduced proinflammatory cytokine production from LPS challenged macrophage cells in vitro. Maternal administration of eIL-10 (10 µg/kg body weight) crossed feto-maternal barriers to delay E. coli-induced PTB to deliver live pups at term. Delay in PTB was associated with reduced feto-maternal uterine inflammation (immune cell infiltration and histologic chorioamnionitis, NF-κB activation, and proinflammatory cytokine production).

Conclusions

eIL-10 administration was safe, stable, specific, delayed PTB by over 72 hrs and delivered live pups. The delivery of drugs using EVs overcomes the limitations of in-utero fetal interventions. Protecting IL-10 in EVs eliminates the need for the amniotic administration of recombinant IL-10 for its efficacy.

SUBMITTER: Kammala AK

PROVIDER: S-EPMC10437065 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Extracellular Vesicles-mediated recombinant IL-10 protects against ascending infection-associated preterm birth by reducing fetal inflammatory response.

Kammala Ananth Kumar AK Mosebarger Angela A Radnaa Enkhtuya E Rowlinson Emma E Vora Natasha N Fortunato Stephen J SJ Sharma Surendra S Safarzadeh Melody M Menon Ramkumar R

Frontiers in immunology 20230804

<h4>Background</h4>Fetal inflammatory response mediated by the influx of immune cells and activation of pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is the major determinant of infection-associated preterm birth (PTB, live births < 37 weeks of gestation).<h4>Objective</h4>To reduce the incidence of PTB by minimizing inflammation, extracellular vesicles (EVs) were electroporetically engineered to contain anti-inflammatory cytokine interleukin (IL)-10 (eIL-10), and ...[more]

PMID: 37600782

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Project description:Preterm birth increases the risk of adverse birth outcomes and is the leading cause of neonatal mortality. A significant cause of preterm birth is in utero infection with vaginal microorganisms. These vaginal microorganisms are often recovered from the amniotic fluid of preterm birth cases. A vaginal microorganism frequently associated with preterm birth is group B streptococcus (GBS), or Streptococcus agalactiae However, the molecular mechanisms underlying GBS ascension are poorly understood. Here, we describe the role of the GBS hyaluronidase in ascending infection and preterm birth. We show that clinical GBS strains associated with preterm labor or neonatal infections have increased hyaluronidase activity compared to commensal strains obtained from rectovaginal swabs of healthy women. Using a murine model of ascending infection, we show that hyaluronidase activity was associated with increased ascending GBS infection, preterm birth, and fetal demise. Interestingly, hyaluronidase activity reduced uterine inflammation but did not impact placental or fetal inflammation. Our study shows that hyaluronidase activity enables GBS to subvert uterine immune responses, leading to increased rates of ascending infection and preterm birth. These findings have important implications for the development of therapies to prevent in utero infection and preterm birth. GBS are a family of bacteria that frequently colonize the vagina of pregnant women. In some cases, GBS ascend from the vagina into the uterine space, leading to fetal injury and preterm birth. Unfortunately, little is known about the mechanisms underlying ascending GBS infection. In this study, we show that a GBS virulence factor, HylB, shows higher activity in strains isolated from cases of preterm birth than those isolates from rectovaginal swabs of healthy women. We discovered that GBS rely on HylB to avoid immune detection in uterine tissue, but not placental tissue, which leads to increased rates of fetal injury and preterm birth. These studies provide novel insight into the underlying mechanisms of ascending infection.

Project description:ObjectivePreterm birth (PTB) is a typical inflammatory disease with unclear pathogenesis. The studies investigating the relationship between anti-inflammatory factors IL-4 and IL-10 gene polymorphisms and PTB produced conflicting results. This systematic review and meta-analysis aimed to summarize the effects of IL-4 and IL-10 gene polymorphisms and clarify their possible association with PTB.MethodsA systematic literature review was conducted using PubMed, Web of Science, and Cochrane library (up to 02 April 2022). The MeSH terms, related entry terms, and other names in "Gene" database were used to find relevant articles. A fixed- or random-effects model was used to calculate the significance of IL-4 and IL-10 gene polymorphisms, depending on study heterogeneity. The odds ratios (OR) and 95% confidence intervals (CIs) were calculated in the allele, recessive, dominant, co-dominant, and over-dominant models. The Eggers publication bias plot was used to graphically represent the publication bias.ResultsPolymorphisms in two interleukins (IL-4-590C/T (rs2243250) = 5 and IL-10-592A/C (rs1800872), -819T/C (rs1800871) and -1082A/G (rs1800896) = 16) were found in 21 articles. Overall, only the over-dominant gene model AA + GG vs. AG revealed significant association between IL-10-1082A/G (rs1800896) and PTB (OR [95% CI] = 0.87 [0.76, 0.99], p = 0.04). However, in the allele model, recessive model, dominant model, co-dominant model, and over-dominant model, the polymorphisms for IL-4-590C/T (rs2243250), IL-10-592A/C (rs1800872), and IL-10-819T/C (rs1800871) were not found to be associated with the risk of PTB. In gene models, no statistically significant association was found between IL-4-590C/T (rs2243250), IL-10-592A/C (rs1800872), IL-10-819T/C (rs1800871), and IL-10-1082A/G (rs1800896) polymorphisms and PTB in subgroup analyses by racial or control group Hardy-Weinberg Equilibrium (HWE) p-value. Eggers's publication bias plot and heterogeneity test (I2<50%, p = 0.05) of IL-10-1082A/G (rs1800896) suggested that the funnel asymmetry could be due to publication bias rather than heterogeneity.ConclusionThe current study suggests that the over-dominant gene model AA + GG vs. AG of IL-10-1082A/G (rs1800896) polymorphism may be associated with genetic susceptibility to PTB and may have a protective function against PTB risk. There was unclear association found between IL-4-590C/T (rs2243250), IL-10-592A/C (rs1800872) and IL-10-819T/C (rs1800871) polymorphisms and PTB. Due to the limitations of included studies and the risk of publication bias, additional research is required to confirm our findings.Systematic review registrationhttps://inplasy.com/inplasy-2022-4-0044, identifier INPLASY202240044.

Project description:BackgroundMicrobial invasion of the intraamniotic cavity and intraamniotic inflammation are factors associated with spontaneous preterm birth. Understanding the route and kinetics of infection, sites of colonization, and mechanisms of host inflammatory response is critical to reducing preterm birth risk.ObjectivesThis study developed an animal model of ascending infection and preterm birth with live bacteria (E. coli) in pregnant CD-1 mice with the goal of better understanding the process of microbial invasion of the intraamniotic cavity and intraamniotic inflammation.Study designMultiple experiments were conducted in this study. To determine the dose of E. coli required to induce preterm birth, CD-1 mice were injected vaginally with four different doses of E. coli (103, 106, 1010, or 1011 colony forming units [CFU]) in 40 μL of nutrient broth or broth alone (control) on an embryonic day (E)15. Preterm birth (defined as delivery before E18.5) was monitored using live video. E. coli ascent kinetics were measured by staining the E. coli with lipophilic tracer DiD for visualization through intact tissue with an in vivo imaging system (IVIS) after inoculation. The E. coli were also directly visualized in reproductive tissues by staining the bacteria with carboxyfluorescein succinimidyl ester (CFSE) prior to administration and via immunohistochemistry (IHC) by staining tissues with anti-E. coli antibody. Each pup's amniotic fluid was cultured separately to determine the extent of microbial invasion of the intraamniotic cavity at different time points. Intraamniotic inflammation resulting from E. coli invasion was assessed with IHC for inflammatory markers (TLR-4, P-NF-κB) and neutrophil marker (Ly-6G) for chorioamnionitis at 6- and 24-h post-inoculation.ResultsVaginally administered E. coli resulted in preterm birth in a dose-dependent manner with higher doses causing earlier births. In ex vivo imaging and IHC detected uterine horns proximal to the cervix had increased E. coli compared to the distal uterine horns. E. coli were detected in the uterus, fetal membranes (FM), and placenta in a time-dependent manner with 6 hr having increased intensity of E. coli positive signals in pups near the cervix and in all pups at 24 hr. Similarly, E. coli grew from the cultures of amniotic fluid collected nearest to the cervix, but not from the more distal samples at 6 hr post-inoculation. At 24 hr, all amniotic fluid cultures regardless of distance from the cervix, were positive for E. coli. TLR-4 and P-NF-κB signals were more intense in the tissues where E. coli was present (placenta, FM and uterus), displaying a similar trend toward increased signal in proximal gestational sacs compared to distal at 6 hr. Ly-6G+ cells, used to confirm chorioamnionitis, were increased at 24 hr compared to 6 hr post-inoculation and control.ConclusionWe report the development of mouse model of ascending infection and the associated inflammation of preterm birth. Clinically, these models can help to understand mechanisms of infection associated preterm birth, determine targets for intervention, or identify potential biomarkers that can predict a high-risk pregnancy status early in pregnancy.

Project description:Spontaneous preterm birth (SPTB) is the leading cause of neonatal death and morbidity worldwide. Both maternal and fetal genetic factors likely contribute to SPTB. We performed a genome-wide association study (GWAS) on a population of Finnish origin that included 247 infants with SPTB (gestational age [GA] < 36 weeks) and 419 term controls (GA 38-41 weeks). The strongest signal came within the gene encoding slit guidance ligand 2 (SLIT2; rs116461311, minor allele frequency 0.05, p = 1.6×10-6). Pathway analysis revealed the top-ranking pathway was axon guidance, which includes SLIT2. In 172 very preterm-born infants (GA <32 weeks), rs116461311 was clearly overrepresented (odds ratio 4.06, p = 1.55×10-7). SLIT2 variants were associated with SPTB in another European population that comprised 260 very preterm infants and 9,630 controls. To gain functional insight, we used immunohistochemistry to visualize SLIT2 and its receptor ROBO1 in placentas from spontaneous preterm and term births. Both SLIT2 and ROBO1 were located in villous and decidual trophoblasts of embryonic origin. Based on qRT-PCR, the mRNA levels of SLIT2 and ROBO1 were higher in the basal plate of SPTB placentas compared to those from term or elective preterm deliveries. In addition, in spontaneous term and preterm births, placental SLIT2 expression was correlated with variations in fetal growth. Knockdown of ROBO1 in trophoblast-derived HTR8/SVneo cells by siRNA indicated that it regulate expression of several pregnancy-specific beta-1-glycoprotein (PSG) genes and genes involved in inflammation. Our results show that the fetal SLIT2 variant and both SLIT2 and ROBO1 expression in placenta and trophoblast cells may be correlated with susceptibility to SPTB. SLIT2-ROBO1 signaling was linked with regulation of genes involved in inflammation, PSG genes, decidualization and fetal growth. We propose that this receptor-ligand couple is a component of the signaling network that promotes SPTB.

Dataset Information

Extracellular Vesicles-mediated recombinant IL-10 protects against ascending infection-associated preterm birth by reducing fetal inflammatory response.

Background

Objective

Study design

Results

Conclusions

Publications

Extracellular Vesicles-mediated recombinant IL-10 protects against ascending infection-associated preterm birth by reducing fetal inflammatory response.

Similar Datasets

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