Disruption of normal patterns of FOXF1 expression in a lethal disorder of lung development.

BACKGROUND: Alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) is a lethal disorder of lung development. ACDMPV is associated with haploinsufficiency of the transcription factor FOXF1, which plays an important role in the development of the lung and intestine. CNVs upstream of the FOXF1 gene have also been associated with an ACDMPV phenotype, but mechanism(s) by which these deletions disrupt lung development are not well understood. The objective of our study is to gain insights into the mechanisms by which CNVs contribute to an ACDMPV phenotype. METHODS: We analysed primary lung tissue from an infant with classic clinical and histological findings of ACDMPV and harboured a 340 kb deletion on chromosome 16q24.1 located 250 kb upstream of FOXF1. RESULTS: In RNA generated from paraffin-fixed lung sections, our patient had lower expression of FOXF1 than age-matched controls. He also had an abnormal pattern of FOXF1 protein expression, with a dramatic loss of FOXF1 expression in the lung. To gain insights into the mechanisms underlying these changes, we assessed the epigenetic landscape using chromatin immunoprecipitation, which demonstrated loss of histone H3 lysine 27 acetylation (H3K27Ac), an epigenetic mark of active enhancers, in the region of the deletion. CONCLUSIONS: Together, these data suggest that the deletion disrupts an enhancer responsible for directing FOXF1 expression in the developing lung and provide novel insights into the mechanisms underlying a fatal developmental lung disorder.

Pathogenetics of alveolar capillary dysplasia with misalignment of pulmonary veins.

Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is a lethal lung developmental disorder caused by heterozygous point mutations or genomic deletion copy-number variants (CNVs) of FOXF1 or its upstream enhancer involving fetal lung-expressed long noncoding RNA genes LINC01081 and LINC01082. Using custom-designed array comparative genomic hybridization, Sanger sequencing, whole exome sequencing (WES), and bioinformatic analyses, we studied 22 new unrelated families (20 postnatal and two prenatal) with clinically diagnosed ACDMPV. We describe novel deletion CNVs at the FOXF1 locus in 13 unrelated ACDMPV patients. Together with the previously reported cases, all 31 genomic deletions in 16q24.1, pathogenic for ACDMPV, for which parental origin was determined, arose de novo with 30 of them occurring on the maternally inherited chromosome 16, strongly implicating genomic imprinting of the FOXF1 locus in human lungs. Surprisingly, we have also identified four ACDMPV families with the pathogenic variants in the FOXF1 locus that arose on paternal chromosome 16. Interestingly, a combination of the severe cardiac defects, including hypoplastic left heart, and single umbilical artery were observed only in children with deletion CNVs involving FOXF1 and its upstream enhancer. Our data demonstrate that genomic imprinting at 16q24.1 plays an important role in variable ACDMPV manifestation likely through long-range regulation of FOXF1 expression, and may be also responsible for key phenotypic features of maternal uniparental disomy 16. Moreover, in one family, WES revealed a de novo missense variant in ESRP1, potentially implicating FGF signaling in the etiology of ACDMPV.

Developmentally determined reduction in CD31 during gestation is associated with CD8+ T cell effector differentiation in preterm infants.

Homeostatic T cell proliferation is more robust during human fetal development. In order to understand the relative effect of normal fetal homeostasis and perinatal exposures on CD8+ T cell behavior in PT infants, we characterized umbilical cord blood CD8+ T cells from infants born between 23-42weeks gestation. Subjects were recruited as part of the NHLBI-sponsored Prematurity and Respiratory Outcomes Program. Cord blood from PT infants had fewer naïve CD8+ T cells and lower regulatory CD31 expression on both naïve and effector, independent of prenatal exposures. CD8+ T cell in vitro effector function was greater at younger gestational ages, an effect that was exaggerated in infants with prior inflammatory exposures. These results suggest that CD8+ T cells earlier in gestation have loss of regulatory co-receptor CD31 and greater effector differentiation, which may place PT neonates at unique risk for CD8+ T cell-mediated inflammation and impaired T cell memory formation.

Preterm cord blood CD4⁺ T cells exhibit increased IL-6 production in chorioamnionitis and decreased CD4⁺ T cells in bronchopulmonary dysplasia.

BACKGROUND: Chorioamnionitis (CA) is associated with premature delivery and bronchopulmonary dysplasia (BPD). We hypothesize that preterm infants exposed to CA have reduced suppressive regulatory T cells (Treg) and increased non-regulatory T cell pro-inflammatory cytokines, increasing risk for BPD. OBJECTIVE: To evaluate cord blood CD4(+) T cell regulatory phenotype and pro-inflammatory cytokine production in CA and BPD groups. STUDY DESIGN: Cord blood mononuclear cells from infants (GA ⩽32 weeks), with or without placental histological evidence of CA (hChorio), were analyzed by flow cytometry. Clinical information was collected by retrospective chart review. Numbers of putative Treg (CD4(+)FoxP3(+)CD25(+)CD127Dim), CD4(+) non-Tregs, and CD4(+) T cell intracellular cytokine content following in vitro stimulation were compared with CA status and oxygen requirement at 36weeks postmenstrual age. RESULT: Absolute Treg numbers were not different in CA and non-CA exposed samples. However, the infants who developed BPD had a significant decrease in Treg and non-regulatory T cell numbers. Greater IL-6 production was observed in hCA group. CONCLUSION: A pro-inflammatory CD4(+) T cell status is noted in CA and BPD but the later disease is also associated with decrease in Tregs, suggesting that the development of BPD is marked by distinct inflammatory changes from those of CA exposed infants.

Stability of T cell phenotype and functional assays following heparinized umbilical cord blood collection.

Umbilical cord blood has been used for a wide variety of immunologic investigations including assessments of developmental perturbations by antenatal exposures. Recent advances in multiparameter flow cytometry have allowed finer characterization of lymphocyte phenotype and function, revealing important differences between the fetal and adult immune systems. The degree of variability between human subjects confounds the ability to draw firm conclusions. Artifacts resulting from processing techniques exacerbate this variability. The unpredictable nature of deliveries, especially of premature infants, makes it difficult to control variables such as timing of umbilical cord mononuclear cell (UCMC) isolation and method of collection. Additionally, in multicenter studies dependent on central processing, delays are inevitable. However, little available literature describes systematic testing of the degree to which processing variations affect UCMC phenotype and function. Using multiparameter flow cytometry, we tested the effect of collection technique and length of time prior to UCMC isolation on T cell phenotype and function, with the goal of creating a standardized operating procedure for a multicenter investigation. The study also provides a benchmark data set including extensive surface and functional phenotyping of umbilical cord T cells. UCMC isolation delay of up to 24 h produced similar T cell phenotype and function as tested by in vitro SEB stimulation. There were few statistically significant differences between time points based on data medians. We conclude that, for the purpose of immunologic investigations, a 24-h time delay from sample collection to mononuclear cell isolation does not introduce a significant degree of variation in T cell phenotype and function when adhering to strict standard operating procedures.