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The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Long-term success in beta-cell replacement remains limited by the toxic effects of calcineurin inhibitors (CNI) on beta-cells and renal function. We report a multi-modal approach including islet and pancreas-after-islet (PAI) transplant utilizing calcineurin-sparing immunosuppression. Ten consecutive non-uremic patients with Type 1 diabetes underwent islet transplant with immunosuppression based on belatacept (BELA; n = 5) or efalizumab (EFA; n = 5). Following islet failure, patients were considered for repeat islet infusion and/or PAI transplant. 70% of patients (four EFA, three BELA) maintained insulin independence at 10 years post-islet transplant, including four patients receiving a single islet infusion and three patients undergoing PAI transplant. 60% remain insulin independent at mean follow-up of 13.3 ± 1.1 years, including one patient 9 years after discontinuing all immunosuppression for adverse events, suggesting operational tolerance. All patients who underwent repeat islet transplant experienced graft failure. Overall, patients demonstrated preserved renal function, with a mild decrease in GFR from 76.5 ± 23.1 mL/min to 50.2 ± 27.1 mL/min (p = 0.192). Patients undergoing PAI showed the greatest degree of renal impairment following initiation of CNI (56% ± 18.7% decrease in GFR). In our series, repeat islet transplant is ineffective at maintaining long-term insulin independence. PAI results in durable insulin independence but is associated with impaired renal function secondary to CNI dependence.

The mutualistic relationship of gut-resident microbiota and the host immune system promotes homeostasis that ensures maintenance of the microbial community and of a largely non-aggressive immune cell compartment1,2. The consequences of disturbing this balance include proximal inflammatory conditions, such as Crohn's disease, and systemic illnesses. This equilibrium is achieved in part through the induction of both effector and suppressor arms of the adaptive immune system. Helicobacter species induce T regulatory (Treg) and T follicular helper (TFH) cells under homeostatic conditions, but induce inflammatory T helper 17 (TH17) cells when induced Treg (iTreg) cells are compromised3,4. How Helicobacter and other gut bacteria direct T cells to adopt distinct functions remains poorly understood. Here we investigated the cells and molecular components required for iTreg cell differentiation. We found that antigen presentation by cells expressing RORγt, rather than by classical dendritic cells, was required and sufficient for induction of Treg cells. These RORγt+ cells-probably type 3 innate lymphoid cells and/or Janus cells5-require the antigen-presentation machinery, the chemokine receptor CCR7 and the TGFβ activator αv integrin. In the absence of any of these factors, there was expansion of pathogenic TH17 cells instead of iTreg cells, induced by CCR7-independent antigen-presenting cells. Thus, intestinal commensal microbes and their products target multiple antigen-presenting cells with pre-determined features suited to directing appropriate T cell differentiation programmes, rather than a common antigen-presenting cell that they endow with appropriate functions.

Mammalian carotid body arterial chemoreceptors function as an early warning system for hypoxia, triggering acute life-saving arousal and cardiorespiratory reflexes. To serve this role, carotid body glomus cells are highly sensitive to decreases in oxygen availability. While the mitochondria and plasma membrane signaling proteins have been implicated in oxygen sensing by glomus cells, the mechanism underlying their mitochondrial sensitivity to hypoxia compared to other cells is unknown. Here, we identify HIGD1C, a novel hypoxia-inducible gene domain factor isoform, as an electron transport chain complex IV-interacting protein that is almost exclusively expressed in the carotid body and is therefore not generally necessary for mitochondrial function. Importantly, HIGD1C is required for carotid body oxygen sensing and enhances complex IV sensitivity to hypoxia. Thus, we propose that HIGD1C promotes exquisite oxygen sensing by the carotid body, illustrating how specialized mitochondria can be used as sentinels of metabolic stress to elicit essential adaptive behaviors.

Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Parathyroid allotransplantation is a burgeoning treatment for severe hypoparathyroidism. Deceased donor parathyroid gland (PTG) procurement can be technically challenging due to lack of normal intraoperative landmarks and exposure constraints in the neck of organ donors. In this study, we assessed standard 4-gland exposure in situ and en bloc surgical techniques for PTG procurement and ex vivo near-infrared autofluorescence (NIRAF) imaging for identification of PTGs during organ recovery.MethodsResearch tissue consent was obtained from organ donors or donor families for PTG procurement. All donors were normocalcemic, brain-dead, solid organ donors between 18 and 65 y of age. PTGs were procured initially using a standard 4-gland exposure technique in situ and subsequently using a novel en bloc resection technique after systemic organ preservation flushing. Parathyroid tissue was stored at 4 °C in the University of Wisconsin solution up to 48 h post-procurement. Fluoptics Fluobeam NIRAF camera and Image J software were utilized for quantification of NIRAF signal.ResultsThirty-one brain-dead deceased donor PTG procurements were performed by abdominal transplant surgeons. In the initial 8 deceased donors, a mean of 1.75 glands (±1.48 glands SD) per donor were recovered using the 4-gland in situ technique. Implementation of combined en bloc resection with ex vivo NIRAF imaging in 23 consecutive donors yielded a mean of 3.60 glands (±0.4 SD) recovered per donor (P < 0.0001). Quantification of NIRAF integrated density signal demonstrated >1-fold log difference in PTG (2.13 × 105 pixels) versus surrounding anterior neck structures (1.9 × 104 pixels; P < 0.0001). PTGs maintain distinct NIRAF signal from the time of recovery (1.88 × 105 pixels) up to 48 h post-procurement (1.55 × 105 pixels) in organ preservation cold storage (P = 0.34).ConclusionsThe use of an en bloc surgical technique with ex vivo NIRAF imaging significantly enhances the identification and recovery of PTG from deceased donors.

The autoimmune regulator (Aire), a well-defined transcriptional regulator in the thymus, is also found in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs are hematopoietic antigen-presenting cells and inducers of immune tolerance, but their precise identity has remained unclear. Here, we use single-cell multiomics, transgenic murine models, and functional approaches to define eTACs at the transcriptional, genomic, and proteomic level. We find that eTACs consist of two similar cell types: CCR7+ Aire-expressing migratory dendritic cells (AmDCs) and an Airehi population coexpressing Aire and retinoic acid receptor–related orphan receptor γt (RORγt) that we term Janus cells (JCs). Both JCs and AmDCs have the highest transcriptional and genomic homology to CCR7+ migratory dendritic cells. eTACs, particularly JCs, have highly accessible chromatin and broad gene expression, including a range of tissue-specific antigens, as well as remarkable homology to medullary thymic epithelium and RANK-dependent Aire expression. Transgenic self-antigen expression by eTACs is sufficient to induce negative selection and prevent autoimmune diabetes. This transcriptional, genomic, and functional symmetry between eTACs (both JCs and AmDCs) and medullary thymic epithelium—the other principal Aire-expressing population and a key regulator of central tolerance—identifies a core program that may influence self-representation and tolerance across the spectrum of immune development.

Naturally occurring cases of monogenic type 1 diabetes (T1D) help establish direct mechanisms driving this complex autoimmune disease. A recently identified de novo germline gain-of-function (GOF) mutation in the transcriptional regulator STAT3 was found to cause neonatal T1D. We engineered a novel knock-in mouse incorporating this highly diabetogenic human STAT3 mutation (K392R) and found that these mice recapitulated the human autoimmune diabetes phenotype. Paired single-cell TCR and RNA sequencing revealed that STAT3-GOF drives proliferation and clonal expansion of effector CD8+ cells that resist terminal exhaustion. Single-cell ATAC-seq showed that these effector T cells are epigenetically distinct and have differential chromatin architecture induced by STAT3-GOF. Analysis of islet TCR clonotypes revealed a CD8+ cell reacting against known antigen IGRP, and STAT3-GOF in an IGRP-reactive TCR transgenic model demonstrated that STAT3-GOF intrinsic to CD8+ cells is sufficient to accelerate diabetes onset. Altogether, these findings reveal a diabetogenic CD8+ T cell response that is restrained in the presence of normal STAT3 activity and drives diabetes pathogenesis.

Healthy pregnancy requires tolerance to fetal alloantigens as well as syngeneic embryonic and placental antigens. Given the importance of the autoimmune regulator (Aire) gene in self-tolerance, we investigated the role of Aire-expressing cells in maternal-fetal tolerance. We report that maternal ablation of Aire-expressing (Aire +) cells during early mouse pregnancy caused intrauterine growth restriction (IUGR) in both allogeneic and syngeneic pregnancies. This phenotype is immune mediated, as IUGR was rescued in Rag1-deficient mice, and involved a memory response, demonstrated by recurrence of severe IUGR in second pregnancies. Single-cell RNA sequencing demonstrated that Aire + cell depletion in pregnancy results in expansion of activated T cells, particularly T follicular helper cells. Unexpectedly, selective ablation of either Aire-expressing medullary thymic epithelial cells or extrathymic Aire-expressing cells (eTACs) mapped the IUGR phenotype exclusively to eTACs. Thus, we report a previously undescribed mechanism for the maintenance of maternal-fetal immune homeostasis and demonstrate that eTACs protect the conceptus from immune-mediated IUGR.

Background: Scoring systems have been proposed to select donation after circulatory death (DCD) donors and recipients for liver transplantation (LT). We hypothesized that complex scoring systems derived in large datasets might not predict outcomes locally. Methods: Based on 1-year DCD-LT graft survival predictors in multivariate logistic regression models, we designed, validated, and compared a simple index using the University of California, San Francisco (UCSF) cohort (n = 136) and a universal-comprehensive (UC)-DCD score using the United Network for Organ Sharing (UNOS) cohort (n = 5,792) to previously published DCD scoring systems. Results: The total warm ischemia time (WIT)-index included donor WIT (dWIT) and hepatectomy time (dHep). The UC-DCD score included dWIT, dHep, recipient on mechanical ventilation, transjugular-intrahepatic-portosystemic-shunt, cause of liver disease, model for end-stage liver disease, body mass index, donor/recipient age, and cold ischemia time. In the UNOS cohort, the UC-score outperformed all previously published scores in predicting DCD-LT graft survival (AUC: 0.635 vs. ≤0.562). In the UCSF cohort, the total WIT index successfully stratified survival and biliary complications, whereas other scores did not. Conclusion: DCD risk scores generated in large cohorts provide general guidance for safe recipient/donor selection, but they must be tailored based on non-/partially-modifiable local circumstances to expand DCD utilization.

Significant interest exists in water-tolerant 2D lead iodide perovskites owing to their stability and proven potential in photovoltaic and photonic applications. These materials have solid-state phase transitions that are accessible below 100 °C. Here, the study witnesses the multiple phase transitions of the last members of a series of organic–inorganic hybrid materials, [(CnH2n+1NH3)2PbI4], with even n as n = 14, 16, and 18, once again. By employing temperature-dependent steady-state photoluminescence (PL) and temperature-dependent time-resolved photoluminescence (TRPL) spectroscopy in the temperature range of −18 to +90 °C and at −196 °C, we explore the thermal responses of these materials. The investigation reveals reversible phase transitions occurring between room temperature (RT) and elevated temperatures, impacting the optical properties and emitting colors of the perovskite compounds. The longer the alkyl chain, the higher the phase transition temperature, attributed to increased conformational disorder and enhanced perovskite symmetry. The decay constants for all compounds are very close in value, which confirms the underlying excited-state dynamics, pointing to contributions primarily from inorganic components across different phases. We anticipate that our results on the detection of phase transitions in 2D perovskites will not only motivate the use of these techniques for detecting phase transitions but also would help to understand their excited states in more details to selectively use them for solar cell and next-generation display technologies. [ABSTRACT FROM AUTHOR]

Reactive flash sintering (RFS) enables the simultaneous synthesis and sintering of ceramics and has been shown to affect the reaction pathway of different materials. Herein, in situ synchrotron X‐ray diffraction (XRD) is used to investigate the (Mg,Ni,Co,Cu,Zn)O entropy‐stabilized oxide formation during: (i) conventional heating and (ii) RFS under current rate‐controlled mode. The same reaction pathway is verified in both instances: the starting rock‐salt (RS), spinel (Co3O4), tenorite (CuO), and wurtzite (ZnO) phases transform into a single RS phase with a (1 1 1) to (2 0 0) intensity ratio of 0.67, consistent with a random distribution of the cations into the structure. Pt lattice peak shift from the XRD patterns is used as standard to monitor the sample surface temperature, revealing a strong endothermic reaction during the RS single‐phase formation (Pt peaks shift toward higher angles while increasing sample temperature/current density). In RFS, the single‐phase RS structure is formed in just 60 s at a furnace temperature of 600°C and a current rate of 220 mA mm−2/min. Therefore, RFS greatly accelerates the synthesis of (Mg,Ni,Co,Cu,Zn)O, however, it does not play a role in the reaction pathway for this material formation. [ABSTRACT FROM AUTHOR]

Thymic epithelial cells in the medulla (mTECs) play a critical role in enforcing central tolerance through expression and presentation of tissue-specific antigens (TSAs) and deletion of autoreactive thymocytes. TSA expression requires autoimmune regulator (Aire), a transcriptional activator present in a subset of mTECs characterized by high CD80 and major histocompatibility complex II expression and a lack of potential for differentiation or proliferation. Here, using an Aire-DTR transgenic line, we show that short-term ablation specifically targets Aire(+) mTECs, which quickly undergo RANK-dependent recovery. Repeated ablation also affects Aire(-) mTECs, and using an inducible Aire-Cre fate-mapping system, we find that this results from the loss of a subset of mTECs that showed prior expression of Aire, maintains intermediate TSA expression, and preferentially migrates toward the center of the medulla. These results clearly identify a distinct stage of mTEC development and underscore the diversity of mTECs that play a key role in maintaining tolerance.

The autoimmune regulator (Aire) is essential for prevention of autoimmunity; its role is best understood in the thymus, where it promotes self-tolerance through tissue-specific antigen (TSA) expression. Recently, extrathymic Aire-expressing cells (eTACs) have been described in murine secondary lymphoid organs, but the identity of such cells and their role in immune tolerance remains unclear. Here we have shown that eTACs are a discrete major histocompatibility complex class II (MHC II)(hi), CD80(lo), CD86(lo), epithelial cell adhesion molecule (EpCAM)(hi), CD45(lo) bone marrow-derived peripheral antigen-presenting cell (APC) population. We also have demonstrated that eTACs can functionally inactivate CD4⁺ T cells through a mechanism that does not require regulatory T cells (Treg) and is resistant to innate inflammatory stimuli. Together, these findings further define eTACs as a distinct tolerogenic cell population in secondary lymphoid organs.

Foxp3(+) CD4(+) T helper cells called regulatory T (T reg) cells play a key role in controlling reactivity to self-antigens and onset of autoimmunity. T reg cells either arise in thymus and are called natural T reg (nT reg) cells or are generated in the periphery through induction of Foxp3 and are called inducible T reg (iT reg) cells. The relative contributions of iT reg cells and nT reg cells in peripheral tolerance remain unclear as a result of an inability to separate these two subsets of T reg cells. Using a combination of novel TCR transgenic mice with a defined self-antigen specificity and conventional mouse models, we demonstrate that a cell surface molecule, neuropilin-1 (Nrp-1), is expressed at high levels on nT reg cells and can be used to separate nT reg versus iT reg cells in certain physiological settings. In addition, iT reg cells generated through antigen delivery or converted under homeostatic conditions lack Nrp-1 expression. Nrp-1(lo) iT reg cells show similar suppressive activity to nT reg cells in controlling ongoing autoimmune responses under homeostatic conditions. In contrast, their activity might be compromised in certain lymphopenic settings. Collectively, our data show that Nrp-1 provides an excellent marker to distinguish distinct T reg subsets and will be useful in studying the role of nT reg versus iT reg cells in different disease settings.

Mutations in the gene encoding the zinc-finger transcription factor Ikaros (IKZF1) are found in patients with immunodeficiency, leukemia, and autoimmunity. Although Ikaros has a well-established function in modulating gene expression programs important for hematopoietic development, its role in other cell types is less well defined. Here, we uncover functions for Ikaros in thymic epithelial lineage development in mice and show that Ikzf1 expression in medullary thymic epithelial cells (mTECs) is required for both autoimmune regulator–positive (Aire+) mTEC development and tissue-specific antigen (TSA) gene expression. Accordingly, TEC-specific deletion of Ikzf1 in mice results in a profound decrease in Aire+ mTECs, a global loss of TSA gene expression, and the development of autoimmunity. Moreover, Ikaros shapes thymic mimetic cell diversity, and its deletion results in a marked expansion of thymic tuft cells and muscle-like mTECs and a loss of other Aire-dependent mimetic populations. Single-cell analysis reveals that Ikaros modulates core transcriptional programs in TECs that correlate with the observed cellular changes. Our findings highlight a previously undescribed role for Ikaros in regulating epithelial lineage development and function and suggest that failed thymic central tolerance could contribute to the autoimmunity seen in humans with IKZF1 mutations. Editor's summary: Within the thymus, a heterogeneous population of epithelial cells make proteins normally restricted to cell types found in other organs. By presenting antigens derived from these proteins, the epithelial cells contribute to the process of deleting T cells within the thymus, avoiding the release of autoreactive immune cells into the rest of the body. Sin et al. investigated what transcription factors, beyond those currently known, may control the development and function of thymic epithelial cells. Deletion of Ikaros in mice changed the development of specific subsets of thymic epithelial cells and decreased expression of tissue-specific antigens, resulting in the presence of autoreactive T cells within tissues. This function in central tolerance might, therefore, contribute to mutations in Ikaros in humans being associated with autoimmunity. —Sarah H. Ross [ABSTRACT FROM AUTHOR]