RESUMO
Thymic stromal lymphopoietin (TSLP) is a primarily epithelial-derived cytokine that drives type 2 allergic immune responses. Early life viral respiratory infections elicit high TSLP production, which leads to the development of type 2 inflammation and airway hyperreactivity. The goal of this study was to examine in vivo and in vitro the human airway epithelial responses leading to high TSLP production during viral respiratory infections in early infancy. A total of 129 infants (<1-24 m, median age 10 m) with severe viral respiratory infections were enrolled for in vivo (n = 113), and in vitro studies (n = 16). Infants were classified as 'high TSLP' or 'low TSLP' for values above or below the 50th percentile. High versus low TSLP groups were compared in terms of type I-III IFN responses and production of chemokines promoting antiviral (CXCL10), neutrophilic (CXCL1, CXCL5, CXCL8), and type 2 responses (CCL11, CCL17, CCL22). Human infant airway epithelial cell (AEC) cultures were used to define the transcriptomic (RNAseq) profile leading to high versus low TSLP responses in vitro in the absence (baseline) or presence (stimulated) of a viral mimic (poly I:C). Infants in the high TSLP group had greater in vivo type III IFN airway production (median type III IFN in high TSLP 183.2 pg/mL vs. 63.4 pg/mL in low TSLP group, p = 0.007) and increased in vitro type I-III IFN AEC responses after stimulation with a viral mimic (poly I:C). At baseline, our RNAseq data showed that infants in the high TSLP group had significant upregulation of IFN signature genes (e.g., IFIT2, IFI6, MX1) and pro-inflammatory chemokine genes before stimulation. Infants in the high TSLP group also showed a baseline AEC pro-inflammatory state characterized by increased production of all the chemokines assayed (e.g., CXCL10, CXCL8). High TSLP responses in the human infant airways are associated with pre-activated airway epithelial IFN antiviral immunity and increased baseline AEC production of pro-inflammatory chemokines. These findings present a new paradigm underlying the production of TSLP in the human infant airway epithelium following early life viral exposure and shed light on the long-term impact of viral respiratory illnesses during early infancy and beyond childhood.
RESUMO
Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We show that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels significantly affect immune cell proportions in the babies. Thus, lack of prenatal vitamin D, particularly at the time of hematopoietic stem cell migration from the liver to the bone marrow, has long-lasting effects on immune cell proportions. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.
RESUMO
BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.