Lymph node stromal cell responses to perinatal T cell waves, a temporal atlas.

The perinatal period is a critical time window in establishing T cell tolerance. Regulatory T cells (Tregs) made during the first 2 wk of life are key drivers of perinatal tolerance induction, but how these cells are generated and operate has not been established. To elucidate the unique environment murine perinatal Tregs encounter within the lymph nodes (LNs) as they first emerge from the thymus, and how it evolves over the succeeding days, we employed single-cell RNA sequencing to generate an atlas of the early LN niche. A highly dynamic picture emerged, the stromal cell compartment showing the most striking changes and putative interactions with other LN cell compartments. In particular, LN stromal cells showed increasing potential for lymphocyte interactions with age. Analogous studies on mice lacking α:ß T cells or enriched for autoreactive α:ß T cells revealed an acute stromal cell response to α:ß T cell dysfunction, largely reflecting dysregulation of Tregs. Punctual ablation of perinatal Tregs induced stromal cell activation that was dependent on both interferon-gamma signaling and activation of conventional CD4+ T cells. These findings elucidate some of the earliest cellular and molecular events in perinatal induction of T cell tolerance, providing a framework for future explorations.

A discrete 'early-responder' stromal-cell subtype orchestrates immunocyte recruitment to injured tissue.

Following acute injury, stromal cells promote tissue regeneration by a diversity of mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to acute injury identified an 'early-responder' subtype that spiked on day 1 and expressed a notable array of transcripts encoding immunomodulators. IL-1ß, TNF-α and oncostatin M each strongly and rapidly induced MmSCs transcribing this immunomodulatory program. Macrophages amplified the program but were not strictly required for its induction. Transfer of the inflammatory MmSC subtype, tagged with a unique surface marker, into healthy hindlimb muscle induced inflammation primarily driven by neutrophils and macrophages. Among the abundant inflammatory transcripts produced by this subtype, Cxcl5 was stroma-specific and highly upregulated with injury. Depletion of this chemokine early after injury revealed a substantial impact on recruitment of neutrophils, a prolongation of inflammation to later times and an effect on tissue regeneration. Mesenchymal stromal cell subtypes expressing a comparable inflammatory program were found in a mouse model of muscular dystrophy and in several other tissues and pathologies in both mice and humans. These 'early-responder' mesenchymal stromal cells, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators in response to injury.

Tregs in visceral adipose tissue up-regulate circadian-clock expression to promote fitness and enforce a diurnal rhythm of lipolysis.

Regulatory T cells (Tregs) in nonlymphoid organs provide critical brakes on inflammation and regulate tissue homeostasis. Although so-called "tissue Tregs" are phenotypically and functionally diverse, serving to optimize their performance and survival, up-regulation of pathways related to circadian rhythms is a feature they share. Yet the diurnal regulation of Tregs and its consequences are controversial and poorly understood. Here, we profiled diurnal variations in visceral adipose tissue (VAT) and splenic Tregs in the presence and absence of core-clock genes. VAT, but not splenic, Tregs up-regulated their cell-intrinsic circadian program and exhibited diurnal variations in their activation and metabolic state. BMAL1 deficiency specifically in Tregs led to constitutive activation and poor oxidative metabolism in VAT, but not splenic, Tregs. Disruption of core-clock components resulted in loss of fitness: BMAL1-deficient VAT Tregs were preferentially lost during competitive transfers and in heterozygous TregBmal1Δ females. After 16 weeks of high-fat diet feeding, VAT inflammation was increased in mice harboring BMAL1-deficient Tregs, and the remaining cells lost the transcriptomic signature of bona fide VAT Tregs. Unexpectedly, VAT Tregs suppressed adipocyte lipolysis, and BMAL1 deficiency specifically in Tregs abrogated the characteristic diurnal variation in adipose tissue lipolysis, resulting in enhanced suppression of lipolysis throughout the day. These findings argue for the importance of the cell-intrinsic clock program in optimizing VAT Treg function and fitness.

Distinct immunocyte-promoting and adipocyte-generating stromal components coordinate adipose tissue immune and metabolic tenors.

Regulatory T cells (Tregs) are key brakes on the visceral adipose tissue (VAT) inflammation that regulates local and systemic metabolic tenor. Breakdown of this regulation promotes type 2 diabetes. The cytokine IL-33 expands and sustains the unique Treg population residing within VAT. Here, relying on single-cell RNA sequencing, we identified the major IL-33 producers in VAT to be particular mesenchymal stromal cell subtypes, related to but distinct from adipocyte progenitor cells. We explored modulation of the VAT stromal cell landscape with physiologic variables such as age and sex, as well as its remodeling in pathogenic states like obesity. Last, we uncovered a VAT Treg:stromal cell negative regulatory loop that keeps the potent effect of IL-33 under rein.

ERVmap analysis reveals genome-wide transcription of human endogenous retroviruses.

Endogenous retroviruses (ERVs) are integrated retroviral elements that make up 8% of the human genome. However, the impact of ERVs on human health and disease is not well understood. While select ERVs have been implicated in diseases, including autoimmune disease and cancer, the lack of tools to analyze genome-wide, locus-specific expression of proviral autonomous ERVs has hampered the progress in the field. Here we describe a method called ERVmap, consisting of an annotated database of 3,220 human proviral ERVs and a pipeline that allows for locus-specific genome-wide identification of proviral ERVs that are transcribed based on RNA-sequencing data, and provide examples of the utility of this tool. Using ERVmap, we revealed cell-type-specific ERV expression patterns in commonly used cell lines as well as in primary cells. We identified 124 unique ERV loci that are significantly elevated in the peripheral blood mononuclear cells of patients with systemic lupus erythematosus that represent an IFN-independent signature. Finally, we identified additional tumor-associated ERVs that correlate with cytolytic activity represented by granzyme and perforin expression in breast cancer tissue samples. The open-source code of ERVmap and the accompanied web tool are made publicly available to quantify proviral ERVs in RNA-sequencing data with ease. Use of ERVmap across a range of diseases and experimental conditions has the potential to uncover novel disease-associated antigens and effectors involved in human health that is currently missed by focusing on protein-coding sequences.

Feasibility and acceptability of a text message intervention used as an adjunct tool by WIC breastfeeding peer counsellors: The LATCH pilot.

Breastfeeding rates among mothers in the Supplemental Nutrition Program for Women, Infants, and Children (WIC) are lower than for other mothers in the United States. The objective of this study was to test the acceptability and feasibility of the Lactation Advice thru Texting Can Help intervention. Mothers were enrolled at 18-30 weeks gestation from two WIC breastfeeding peer counselling (PC) programmes if they intended to breastfeed and had unlimited text messaging, more than fifth-grade literacy level, and fluency in English or Spanish. Participants were randomized to the control arm (PC support without texting) or the intervention arm (PC support with texting). The two-way texting intervention provided breastfeeding education and support from peer counsellors. Primary outcomes included early post-partum (PP) contact and exclusive breastfeeding (EBF) rates at 2 weeks PP. Feasibility outcomes included text messaging engagement and mother's satisfaction with texting platform. Fifty-eight women were enrolled, 52 of whom were available for intention-to-treat analysis (n = 30 texting, n = 22 control). Contact between mothers and PCs within 48 hr of delivery was greater in the texting group (86.6% vs. 27.3%, p < .001). EBF rates at 2 weeks PP among participants in the texting intervention was 50% versus 31.8% in the control arm (p = .197). Intervention group mothers tended to be more likely to meet their breastfeeding goals (p = .06). Participants were highly satisfied with the Lactation Advice thru Texting Can Help intervention, and findings suggest that it may improve early post-delivery contact and increase EBF rates among mothers enrolled in WIC who receive PC. A large, multicentre trial is feasible and warranted.

IRE1α promotes viral infection by conferring resistance to apoptosis.

The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-folding stresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1α (IRE1α) promote type I interferon (IFN) responses. We found that Xbp1-deficient mouse embryonic fibroblasts and macrophages had impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather an inability of Xbp1-deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infection in wild-type cells. Xbp1-deficient cells were generally resistant to the intrinsic pathway of apoptosis through an indirect mechanism involving activation of the nuclease IRE1α. We observed an IRE1α-dependent reduction in the abundance of the proapoptotic microRNA miR-125a and a corresponding increase in the amounts of the members of the antiapoptotic Bcl-2 family. The activation of IRE1α by the hepatitis C virus (HCV) protein NS4B in XBP1-proficient cells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1α activation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to those in the livers of healthy controls. Our results reveal a prosurvival role for IRE1α in virally infected cells and suggest a possible target for IFN-independent antiviral therapy.