Gluten induces rapid reprogramming of natural memory αß and γδ intraepithelial T cells to induce cytotoxicity in celiac disease.

Celiac disease (CD) is an autoimmune disease in which intestinal inflammation is induced by dietary gluten. The means through which gluten-specific CD4+ T cell activation culminates in intraepithelial T cell (T-IEL)-mediated intestinal damage remain unclear. Here, we performed multiplexed single-cell analysis of intestinal and gluten-induced peripheral blood T cells from patients in different CD states and healthy controls. Untreated, active, and potential CD were associated with an enrichment of activated intestinal T cell populations, including CD4+ follicular T helper (TFH) cells, regulatory T cells (Tregs), and natural CD8+ αß and γδ T-IELs. Natural CD8+ αß and γδ T-IELs expressing activating natural killer cell receptors (NKRs) exhibited a distinct TCR repertoire in CD and persisted in patients on a gluten-free diet without intestinal inflammation. Our data further show that NKR-expressing cytotoxic cells, which appear to mediate intestinal damage in CD, arise from a distinct NKR-expressing memory population of T-IELs. After gluten ingestion, both αß and γδ T cell clones from this memory population of T-IELs circulated systemically along with gluten-specific CD4+ T cells and assumed a cytotoxic and activating NKR-expressing phenotype. Collectively, these findings suggest that cytotoxic T cells in CD are rapidly mobilized in parallel with gluten-specific CD4+ T cells after gluten ingestion.

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.

Analysis of respiratory and systemic immune responses in COVID-19 reveals mechanisms of disease pathogenesis.

Immune responses to respiratory viruses like SARS-CoV-2 originate and function in the lung, yet assessments of human immunity are often limited to blood. Here, we conducted longitudinal, high-dimensional profiling of paired airway and blood samples from patients with severe COVID-19, revealing immune processes in the respiratory tract linked to disease pathogenesis. Survival from severe disease was associated with increased CD4 + T cells and decreased monocyte/macrophage frequencies in the airway, but not in blood. Airway T cells and macrophages exhibited tissue-resident phenotypes and activation signatures, including high level expression and secretion of monocyte chemoattractants CCL2 and CCL3 by airway macrophages. By contrast, monocytes in blood expressed the CCL2-receptor CCR2 and aberrant CD163 + and immature phenotypes. Extensive accumulation of CD163 + monocyte/macrophages within alveolar spaces in COVID-19 lung autopsies suggested recruitment from circulation. Our findings provide evidence that COVID-19 pathogenesis is driven by respiratory immunity, and rationale for site-specific treatment and prevention strategies.

Distinct epigenetic profiles in children with perinatally-acquired HIV on antiretroviral therapy.

Perinatally-acquired HIV has persistent effects on long-term health outcomes, even after early treatment. We hypothesize that epigenetic indicators, such as DNA methylation, may elucidate cellular processes that explain these effects. Here, we compared DNA methylation profiles in whole blood from 120 HIV-infected children on antiretroviral therapy (ART) and 60 frequency age-matched HIV-uninfected children aged 4-9 years in Johannesburg, South Africa. Using an individual CpG site approach, we found 1,309 differentially-methylated (DM) CpG sites between groups, including 1,271 CpG sites that were hyper-methylated in the HIV-infected group and 38 CpG sites that were hypo-methylated in the HIV-infected group. Six hyper-methylated CpG sites were in EBF4, which codes for a transcription factor involved in B-cell maturation. The top hypomethylated site was in the promoter region of NLRC5, encoding a transcription factor that regulates major histocompatibility complex (MHC) class I molecule expression. Using a differentially-methylated region (DMR) approach, we found 315 DMRs between groups, including 28 regions encompassing 686 CpG sites on chromosome 6. A large number of the genes identified in both the CpG site and DMR approaches were located in the MHC region on chromosome 6, which plays an important role in the adaptive immune system. This study provides the first evidence that changes in the epigenome are detectable in children with perinatally-acquired HIV infection on suppressive ART started at an early age.

Breast cancer family history and allele-specific DNA methylation in the legacy girls study.

Family history, a well-established risk factor for breast cancer, can have both genetic and environmental contributions. Shared environment in families as well as epigenetic changes that also may be influenced by shared genetics and environment may also explain familial clustering of cancers. Epigenetic regulation, such as DNA methylation, can change the activity of a DNA segment without a change in the sequence; environmental exposures experienced across the life course can induce such changes. However, genetic-epigenetic interactions, detected as methylation quantitative trait loci (mQTLs; a.k.a. meQTLs) and haplotype-dependent allele-specific methylation (hap-ASM), can also contribute to inter-individual differences in DNA methylation patterns. To identify differentially methylated regions (DMRs) associated with breast cancer susceptibility, we examined differences in white blood cell DNA methylation in 29 candidate genes in 426 girls (ages 6-13 years) from the LEGACY Girls Study, 239 with and 187 without a breast cancer family history (BCFH). We measured methylation by targeted massively parallel bisulfite sequencing (bis-seq) and observed BCFH DMRs in two genes: ESR1 (Δ4.9%, P = 0.003) and SEC16B (Δ3.6%, P = 0.026), each of which has been previously implicated in breast cancer susceptibility and pubertal development. These DMRs showed high inter-individual variability in methylation, suggesting the presence of mQTLs/hap-ASM. Using single nucleotide polymorphisms data in the bis-seq amplicon, we found strong hap-ASM in SEC16B (with allele specific-differences ranging from 42% to 74%). These findings suggest that differential methylation in genes relevant to breast cancer susceptibility may be present early in life, and that inherited genetic factors underlie some of these epigenetic differences.

Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation.

Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A(∗)-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders.

Distress During Pregnancy: Epigenetic Regulation of Placenta Glucocorticoid-Related Genes and Fetal Neurobehavior.

OBJECTIVE: Increased risk of psychopathology is observed in children exposed to maternal prenatal distress, and elevated maternal cortisol and epigenetic regulation of placental glucocorticoid-pathway genes are potential mechanisms. The authors examined maternal distress and salivary cortisol in relation to fetal movement and heart rate ("coupling") and DNA methylation of three glucocorticoid pathway genes-HSD11B2, NR3C1, and FKBP5-in term placentas. METHOD: Mood questionnaires and salivary cortisol were collected from 61 women between 24-27 gestational weeks, and fetal assessment was conducted at 34-37 weeks. Placental CpG methylation in the three genes was analyzed using 450K Beadchips and bisulfite sequencing; correlations between maternal and fetal variables and DNA methylation were tested; and maternal distress effects on fetal behavior via DNA methylation were investigated. RESULTS: Perceived stress (Perceived Stress Scale), but not cortisol, was associated with altered CpG methylation in placentas. In the highest tertile of the Perceived Stress Scale, the Beadchip data revealed modestly elevated methylation of HSD11B2, associated with lower fetal coupling (ß=-0.51), and modestly elevated methylation of FKBP5, also with lower fetal coupling (ß=-0.47). These increases in methylation were validated by bisulfite sequencing, where they occurred in a minority of clones. CONCLUSIONS: This is the first study to link the effects of pregnant women's distress on the fetus and epigenetic changes in placental genes. Since increased DNA methylation in HSD11B2 and FKBP5 are seen in a minority of bisulfite sequencing clones, these epigenetic changes, and functional consequences, may affect subpopulations of placental cells.

Eyeing the Cyr61/CTGF/NOV (CCN) group of genes in development and diseases: highlights of their structural likenesses and functional dissimilarities.

"CCN" is an acronym referring to the first letter of each of the first three members of this original group of mammalian functionally and phylogenetically distinct extracellular matrix (ECM) proteins [i.e., cysteine-rich 61 (CYR61), connective tissue growth factor (CTGF), and nephroblastoma-overexpressed (NOV)]. Although "CCN" genes are unlikely to have arisen from a common ancestral gene, their encoded proteins share multimodular structures in which most cysteine residues are strictly conserved in their positions within several structural motifs. The CCN genes can be subdivided into members developmentally indispensable for embryonic viability (e.g., CCN1, 2 and 5), each assuming unique tissue-specific functions, and members not essential for embryonic development (e.g., CCN3, 4 and 6), probably due to a balance of functional redundancy and specialization during evolution. The temporo-spatial regulation of the CCN genes and the structural information contained within the sequences of their encoded proteins reflect diversity in their context and tissue-specific functions. Genetic association studies and experimental anomalies, replicated in various animal models, have shown that altered CCN gene structure or expression is associated with "injury" stimuli--whether mechanical (e.g., trauma, shear stress) or chemical (e.g., ischemia, hyperglycemia, hyperlipidemia, inflammation). Consequently, increased organ-specific susceptibility to structural damages ensues. These data underscore the critical functions of CCN proteins in the dynamics of tissue repair and regeneration and in the compensatory responses preceding organ failure. A better understanding of the regulation and mode of action of each CCN member will be useful in developing specific gain- or loss-of-function strategies for therapeutic purposes.

The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.