An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis.

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.

Tissue-resident memory T cell reactivation by diverse antigen-presenting cells imparts distinct functional responses.

CD8+ tissue-resident memory T cells (TRM cells) are poised at the portals of infection and provide long-term protective immunity. Despite their critical roles, the precise mechanics governing TRM cell reactivation in situ are unknown. Using a TCR-transgenic Nur77-GFP reporter to distinguish "antigen-specific" from "bystander" reactivation, we demonstrate that lung CD8+ TRM cells are reactivated more quickly, yet less efficiently, than their counterparts in the draining LNs (TLN cells). Global profiling of reactivated memory T cells revealed tissue-defined and temporally regulated recall response programs. Unlike the reactivation of CD8+ TLN cells, which is strictly dependent on CD11c+XCR1+ APCs, numerous antigen-presenting partners, both hematopoietic and non-hematopoietic, were sufficient to reactivate lung CD8+ TRM cells, but the quality of TRM cell functional responses depended on the identity of the APCs. Together, this work uncovers fundamental differences in the activation kinetics, mechanics, and effector responses between CD8+ memory T cells in peripheral vs. lymphoid organs, revealing a novel tissue-specific paradigm for the reactivation of memory CD8+ T cells.

mRNA destabilization by BTG1 and BTG2 maintains T cell quiescence.

T cells maintain a quiescent state prior to activation. As inappropriate T cell activation can cause disease, T cell quiescence must be preserved. Despite its importance, the mechanisms underlying the "quiescent state" remain elusive. Here, we identify BTG1 and BTG2 (BTG1/2) as factors responsible for T cell quiescence. BTG1/2-deficient T cells show an increased proliferation and spontaneous activation due to a global increase in messenger RNA (mRNA) abundance, which reduces the threshold to activation. BTG1/2 deficiency leads to an increase in polyadenylate tail length, resulting in a greater mRNA half-life. Thus, BTG1/2 promote the deadenylation and degradation of mRNA to secure T cell quiescence. Our study reveals a key mechanism underlying T cell quiescence and suggests that low mRNA abundance is a crucial feature for maintaining quiescence.

Author Correction: Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity.

Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.

Effector TH17 Cells Give Rise to Long-Lived TRM Cells that Are Essential for an Immediate Response against Bacterial Infection.

Adaptive immunity provides life-long protection by generating central and effector memory T cells and the most recently described tissue resident memory T (TRM) cells. However, the cellular origin of CD4 TRM cells and their contribution to host defense remain elusive. Using IL-17A tracking-fate mouse models, we found that a significant fraction of lung CD4 TRM cells derive from IL-17A-producing effector (TH17) cells following immunization with heat-killed Klebsiella pneumonia (Kp). These exTH17 TRM cells are maintained in the lung by IL-7, produced by lymphatic endothelial cells. During a memory response, neither antibodies, γδ T cells, nor circulatory T cells are sufficient for the rapid host defense required to eliminate Kp. Conversely, using parabiosis and depletion studies, we demonstrated that exTH17 TRM cells play an important role in bacterial clearance. Thus, we delineate the origin and function of airway CD4 TRM cells during bacterial infection, offering novel strategies for targeted vaccine design.

The translation of non-canonical open reading frames controls mucosal immunity.

The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.

The long non-coding RNA Morrbid regulates Bim and short-lived myeloid cell lifespan.

Neutrophils, eosinophils and 'classical' monocytes collectively account for about 70% of human blood leukocytes and are among the shortest-lived cells in the body. Precise regulation of the lifespan of these myeloid cells is critical to maintain protective immune responses and minimize the deleterious consequences of prolonged inflammation. However, how the lifespan of these cells is strictly controlled remains largely unknown. Here we identify a long non-coding RNA that we termed Morrbid, which tightly controls the survival of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. To control the lifespan of these cells, Morrbid regulates the transcription of the neighbouring pro-apoptotic gene, Bcl2l11 (also known as Bim), by promoting the enrichment of the PRC2 complex at the Bcl2l11 promoter to maintain this gene in a poised state. Notably, Morrbid regulates this process in cis, enabling allele-specific control of Bcl2l11 transcription. Thus, in these highly inflammatory cells, changes in Morrbid levels provide a locus-specific regulatory mechanism that allows rapid control of apoptosis in response to extracellular pro-survival signals. As MORRBID is present in humans and dysregulated in individuals with hypereosinophilic syndrome, this long non-coding RNA may represent a potential therapeutic target for inflammatory disorders characterized by aberrant short-lived myeloid cell lifespan.

Epithelial IL-18 Equilibrium Controls Barrier Function in Colitis.

The intestinal mucosal barrier controlling the resident microbiome is dependent on a protective mucus layer generated by goblet cells, impairment of which is a hallmark of the inflammatory bowel disease, ulcerative colitis. Here, we show that IL-18 is critical in driving the pathologic breakdown of barrier integrity in a model of colitis. Deletion of Il18 or its receptor Il18r1 in intestinal epithelial cells (Δ/EC) conferred protection from colitis and mucosal damage in mice. In contrast, deletion of the IL-18 negative regulator Il18bp resulted in severe colitis associated with loss of mature goblet cells. Colitis and goblet cell loss were rescued in Il18bp(-/-);Il18r(Δ/EC) mice, demonstrating that colitis severity is controlled at the level of IL-18 signaling in intestinal epithelial cells. IL-18 inhibited goblet cell maturation by regulating the transcriptional program instructing goblet cell development. These results inform on the mechanism of goblet cell dysfunction that underlies the pathology of ulcerative colitis.

Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA.

The mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a proinflammatory type of cell death.

miR-181 and metabolic regulation in the immune system.

Regulation of metabolism is emerging as a central mechanism to control cellular identity and function. Extensive research in the last few years has revealed that the PI3K pathway is at the forefront of establishing metabolic changes required for immune cell growth, proliferation, migration, and differentiation. However, we currently have a limited understanding of how signaling through the PI3K pathway is tightly regulated during immune responses and immune cell development. Although a growing number of miRNAs have been shown to target important metabolic pathways, including the PI3K pathway itself, almost nothing is known regarding metabolic regulation by miRNAs in the context of the immune system. Recently, we revealed that the miR-181 family is a metabolic rheostat in vivo through the nonredundant regulation of PTEN. Over the next few years, additional miRNAs with the capacity to regulate various aspects of metabolism in immune cells are likely to be identified. We propose that these miRNAs will form a network to finely tune cellular metabolic status and that miR-181 will function as the primary metabolic rheostat of this network.

Human liver cocaine esterases: ethanol-mediated formation of ethylcocaine.

A new, pharmacologically active metabolite of cocaine, ethylcocaine, has been reported in individuals after concurrent use of cocaine and ethanol. Formation of ethylcocaine may contribute to the common coabuse of these two drugs and the apparent danger of this practice. We have identified a nonspecific carboxyl-esterase that catalyzes the ethyl transesterification of cocaine to ethylcocaine in the presence of ethanol. In the absence of ethanol, this human liver esterase catalyzes the hydrolysis of cocaine to benzoylecgonine, a metabolite that is inactive as a psychomotor stimulant. A second human liver esterase is also described. This enzyme catalyzes hydrolysis of cocaine to ecgonine methyl ester, also inactive as a stimulant. These two liver esterases may play important roles in regulating the metabolic inactivation of cocaine.

Effects of influenza virus vaccine on hepatic drug metabolism.

Experimental and more limited clinical studies have suggested that influenza vaccination may depress the oxidative hepatic metabolism of various drugs and lead to drug toxicity. The alleged mechanism is the formation of interferon and the resulting decrease in cytochrome P-450 available for drug oxidation. Because of the clinical and basic science implications of these reports, we undertook to study the effects of influenza vaccine on the metabolism of three commonly used drugs: chlordiazepoxide, theophylline, and lorazepam. Our healthy male subjects were studied just before and 1 and 7 days after vaccination. As expected, lorazepam metabolism, which proceeds by glucuronidation and not oxidation, was not altered by vaccination. Surprisingly, however, the oxidation of chlordiazepoxide was also not depressed by the vaccine. Theophylline oxidation, which proceeds primarily by microsomal oxidation (demethylation), was significantly decreased 1 day, but not 7 days, after vaccination. Serum alpha-interferon levels rose after vaccination for only about 8 hours, and levels of gamma-interferon rose to about 500 IU/ml at 24 hours, peaked at 72 hours, and returned to normal by 100 hours after dosing. It appeared that the higher the theophylline clearance before vaccination, the greater the degree of clearance depression after vaccination. Thus the inhibition of drug oxidation after influenza vaccination is selective and each drug should be studied individually. The degree of depression of theophylline clearance is small and transient and appears to be greater in subjects with higher prevaccination clearance.

Cimetidine inhibits renal procainamide clearance.

Procainamide and cimetidine are eliminated in large part by the kidneys. Both are secreted by an active transport mechanism in the proximal tubule and each inhibits secretion of the other in the isolated, perfused rabbit tubule. In this study in man, cimetidine inhibited renal clearance of oral procainamide by 36%. This was associated with a 28% decrease in the ratio of systemic clearance of procainamide to bioavailability, an 18% decrease in the elimination rate constant, and a 24% prolongation of elimination t1/2. These results suggest that cimetidine increases plasma t1/2 and decreases systemic clearance of procainamide in part by inhibiting its active secretion by the kidneys.

Poor-prognosis gestational trophoblastic disease: an update.

Ten patients with poor-prognosis gestational trophoblastic neoplasia were treated from 1979 to 1983 primarily with the modified Bagshawe chemotherapy regimen. Four patients were noted to have brain metastases, and one patient had liver metastases. In addition, etoposide (VP-16) was used in seven patients, bleomycin in six, cisplatin in five, 5-fluorouracil in two, and cis-retinoic acid in four patients. Thoracotomy, hysterectomy, and splenectomy were performed after chemotherapy was initiated for isolated resistant disease. At present, nine of the ten patients (90%) are in complete remission for four to 44 months. Significant myelosuppression was encountered in seven patients and life-threatening toxicity was noted in two patients. Currently, poor-prognosis patients are treated with the modified Bagshawe regimen, alternating every other week with etoposide, bleomycin, and cisplatin.

Follicular fluid treatment during the follicular versus luteal phase of the menstrual cycle: effects on corpus luteum function.

Administration of charcoal-extracted porcine follicular fluid (pFF) to rhesus monkeys at the time of menses impairs the subsequent function of the corpus luteum of the menstrual cycle. The following studies were performed: 1) to characterize the luteal phase defect induced by pFF treatment at menses, and 2) to determine whether pFF treatment in the luteal phase alters corpus luteum function. Adult, female rhesus monkeys were injected sc for 3 days with pFF (10, 5, and 5 ml) beginning on day 1 (n = 5) or day 18 (n = 4) of the menstrual cycle. Femoral venous blood was collected daily throughout the treatment cycle and during the posttreatment cycle of day 18 to 20-treated monkeys. Serum LH, FSH, 17 beta-estradiol (E2), and progesterone (P) were measured by RIA. After pFF treatment on days 1-3, FSH and E2 levels in the early follicular phase were less (P less than 0.05) than those of control cycles (n = 7). Serum LH was not suppressed by pFF treatment. Moreover, the preovulatory rise in circulating E2 and the amplitude of the LH/FSH surge were similar in control and pFF-treated monkeys. Although timely midcycle gonadotropin surges occurred in four of five pFF-treated monkeys, serum P was markedly reduced (P less than 0.05) during the first half of the luteal phase. Circulating P increased to control levels during the late luteal phase before normal onset of menses 16.3 +/- 1.0 (SE) days after the LH surge. Treatment with pFF on days 18-20 of the cycle reduced the levels of circulating FSH, but serum LH, E2, P, and the length of the luteal phase remained comparable to control cycles. Moreover, the hormonal patterns and the length of the follicular and luteal phases in the posttreatment cycle indicated normal ovarian function. Thus, pFF treatment at menses results in an aberrant ovarian cycle characterized by an insufficient, rather than short, luteal phase. Whereas pFF treatment in the early follicular phase vitiates development of the dominant follicle and the related corpus luteum, similar treatment at midluteal phase does not suppress concurrent luteal function or subsequent folliculogenesis.

Sex ratio in ectopic gestations.

Sex assignment was determined in 80 cases of ectopic pregnancy. Tissue sections of chorionic villi were stained with quinacrine for demonstration of either X or Y chromatin. Forty-one (51.2%) were X-chromatin (Barr body)-positive; 38 (47.5%) were Y-chromatin-positive, and 1 (1.3%) was Barr- and Y-positive in the same nuclei. The sex ratio of ectopic gestations in this study was 0.92 male to 1 female. Based on these data, there appears to be no predilection for ectopic implantation by either sex.

Diphenhydramine disposition in chronic liver disease.

Diphenhydramine (DPHM) disposition was examined in nine patients with chronic alcohol-related liver disease and in eight normal subjects. Sleep of 1 to 2 hr duration was induced in all subjects by a 0.8 mg/kg iv dose without an apparent increase in cerebral sensitivity in the patients with cirrhosis. Protein binding as determined by equilibrium dialysis (3H-DPHM) revealed a 15% decrease in the cirrhotic patients, while recovery of unchanged DPHM in urine (2%) was of the same order in the two groups. Computerized biexponential curve analysis was used to compare the plasma profiles for five of the patients and six of the normal subjects. Monoexponential curve analysis of the terminal beta-phase, including all subjects, was also used to compare the two groups. The means of plasma clearance and apparent volume of distribution in cirrhotic patients were respectively less and greater than in normal subjects, but these differences were not significant. The t1/2 for the beta-phase (t1/2 beta), which reflects this reciprocal trend, was increased in the patients (15.2 +/- 1.5 and 9.3 +/- 0.9 hr). This correlated in part with severity of disease, with r = 0.723 between t1/2 beta and the serum bilirubin levels. In conclusion, a single intravenous dose of DPHM provided safe and effective sedation in patients with cirrhosis.

Fatty tumor of the liver in a patient with Cushing's syndrome.

Cushing's syndrome is occasionally associated with unusual deposition of fat. A case is reported of fatty mass of the liver in a patient with nodular adrenal hyperplasia.