Mediator complex subunit 1 architects a tumorigenic Treg cell program independent of inflammation.

While immunotherapy has revolutionized cancer treatment, its safety has been hampered by immunotherapy-related adverse events. Unexpectedly, we show that Mediator complex subunit 1 (MED1) is required for T regulatory (Treg) cell function specifically in the tumor microenvironment. Treg cell-specific MED1 deletion does not predispose mice to autoimmunity or excessive inflammation. In contrast, MED1 is required for Treg cell promotion of tumor growth because MED1 is required for the terminal differentiation of effector Treg cells in the tumor. Suppression of these terminally differentiated Treg cells is sufficient for eliciting antitumor immunity. Both human and murine Treg cells experience divergent paths of differentiation in tumors and matched tissues with non-malignant inflammation. Collectively, we identify a pathway promoting the differentiation of a Treg cell effector subset specific to tumors and demonstrate that suppression of a subset of Treg cells is sufficient for promoting antitumor immunity in the absence of autoimmune consequences.

Trends in blood transfusion, hydroxyurea use, and iron overload among children with sickle cell disease enrolled in Medicaid, 2004-2019.

BACKGROUND: There have been significant changes in clinical guidelines for sickle cell disease (SCD) over the past two decades, including updated indications for hydroxyurea, transfusions, and iron overload management. In practice however, there are few studies that examine SCD care utilization over time. METHODS: We conducted a serial cross-sectional cohort study of pediatric SCD patients from 2004 to 2019 using Georgia Medicaid claims data. For each year, we reported receipt of any transfusion, chronic transfusion, or three or more filled hydroxyurea prescriptions. For children receiving chronic transfusion (six or more annual transfusions), we evaluated iron overload diagnosis, monitoring, and chelation use. Among children with sickle cell anemia (SCA), we examined rates of transfusions and hydroxyurea use. The Cochran-Armitage test was used to assess trend. RESULTS: There were 5316 unique children 2-18 years old with SCD enrolled in Georgia Medicaid from 2004 to 2019. Children receiving any transfusion increased from 2004 to 2010, then stabilized. In SCA patients, chronic transfusions initially increased from 2004 to 2010, then stabilized from 2010 to 2019. For chronically transfused children, monitoring of iron burden and filled chelator prescriptions both increased significantly. Hydroxyurea use in SCA patients increased from 12% to 37%, with increases noted within each age group, most notably from 21% to 60% in the 13-18-year-old cohort. CONCLUSION: We demonstrated changes in SCD care utilization over time, including increased hydroxyurea use, changes in transfusion rates, and increased attention to iron overload management. While trends in clinical management do follow updates in treatment guidelines, there is still delayed and suboptimal uptake of guideline recommendations in pediatric SCD patients.

A New Class of Cell Wall-Recycling l,d-Carboxypeptidase Determines ß-Lactam Susceptibility and Morphogenesis in Acinetobacter baumannii.

The hospital-acquired pathogen Acinetobacter baumannii possesses a complex cell envelope that is key to its multidrug resistance and virulence. The bacterium, however, lacks many canonical enzymes that build the envelope in model organisms. Instead, A. baumannii contains a number of poorly annotated proteins that may allow alternative mechanisms of envelope biogenesis. We demonstrated previously that one of these unusual proteins, ElsL, is required for maintaining a characteristic short rod shape and for withstanding antibiotics that attack the septal cell wall. Curiously, ElsL is composed of a leaderless YkuD-family domain usually found in secreted, cell wall-modifying l,d-transpeptidases (LDTs). Here, we show that, rather than being an LDT, ElsL is actually a new class of cytoplasmic l,d-carboxypeptidase (LDC) that provides a critical step in cell wall recycling previously thought to be missing from A. baumannii. Absence of ElsL impairs cell wall integrity, morphology, and intrinsic resistance due to buildup of murein tetrapeptide precursors, toxicity of which is bypassed by preventing muropeptide recycling. Multiple pathways in the cell become sites of vulnerability when ElsL is inactivated, including l,d-cross-link formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on envelope physiology. We thus reveal a novel class of cell wall-recycling LDC critical to growth and homeostasis of A. baumannii and likely many other bacteria. IMPORTANCE To grow efficiently, resist antibiotics, and control the immune response, bacteria recycle parts of their cell wall. A key step in the typical recycling pathway is the reuse of cell wall peptides by an enzyme known as an l,d-carboxypeptidase (LDC). Acinetobacter baumannii, an "urgent-threat" pathogen causing drug-resistant sepsis in hospitals, was previously thought to lack this enzymatic activity due to absence of a known LDC homolog. Here, we show that A. baumannii possesses this activity in the form of an enzyme class not previously associated with cell wall recycling. Absence of this protein intoxicates and weakens the A. baumannii cell envelope in multiple ways due to the accumulation of dead-end intermediates. Several other organisms of importance to health and disease encode homologs of the A. baumannii enzyme. This work thus reveals an unappreciated mechanism of cell wall recycling, manipulation of which may contribute to enhanced treatments targeting the bacterial envelope.

Essential Gene Analysis in Acinetobacter baumannii by High-Density Transposon Mutagenesis and CRISPR Interference.

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Development of rationally designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii, and we developed a CRISPR interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. IMPORTANCE New approaches are urgently needed to control A. baumannii, one of the most drug-resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

Author Correction: Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20098-z.

Antibiotic susceptibility signatures identify potential antimicrobial targets in the Acinetobacter baumannii cell envelope.

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.

Constitutive Interferon Attenuates RIPK1/3-Mediated Cytokine Translation.

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are well known for their capacity to drive necroptosis via mixed-lineage kinase-like domain (MLKL). Recently, RIPK1/3 kinase activity has been shown to drive inflammation via activation of MAPK signaling. However, the regulatory mechanisms underlying this kinase-dependent cytokine production remain poorly understood. In the present study, we establish that the kinase activity of RIPK1/3 regulates cytokine translation in mouse and human macrophages. Furthermore, we show that this inflammatory response is downregulated by type I interferon (IFN) signaling, independent of type I IFN-promoted cell death. Specifically, low-level constitutive IFN signaling attenuates RIPK-driven activation of cap-dependent translation initiation pathway components AKT, mTORC1, 4E-BP and eIF4E, while promoting RIPK-dependent cell death. Altogether, these data characterize constitutive IFN signaling as a regulator of RIPK-dependent inflammation and establish cap-dependent translation as a crucial checkpoint in the regulation of cytokine production.

Constitutive interferon signaling maintains critical threshold of MLKL expression to license necroptosis.

Interferons (IFNs) are critical determinants in immune-competence and autoimmunity, and are endogenously regulated by a low-level constitutive feedback loop. However, little is known about the functions and origins of constitutive IFN. Recently, lipopolysaccharide (LPS)-induced IFN was implicated as a driver of necroptosis, a necrotic form of cell death downstream of receptor-interacting protein (RIP) kinase activation and executed by mixed lineage kinase like-domain (MLKL) protein. We found that the pre-established IFN status of the cell, instead of LPS-induced IFN, is critical for the early initiation of necroptosis in macrophages. This pre-established IFN signature stems from cytosolic DNA sensing via cGAS/STING, and maintains the expression of MLKL and one or more unknown effectors above a critical threshold to allow for MLKL oligomerization and cell death. Finally, we found that elevated IFN-signaling in systemic lupus erythematosus (SLE) augments necroptosis, providing a link between pathological IFN and tissue damage during autoimmunity.

Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.

Cell death and inflammation are intimately linked during Yersinia infection. Pathogenic Yersinia inhibits the MAP kinase TGFß-activated kinase 1 (TAK1) via the effector YopJ, thereby silencing cytokine expression while activating caspase-8-mediated cell death. Here, using Yersinia pseudotuberculosis in corroboration with costimulation of lipopolysaccharide and (5Z)-7-Oxozeaenol, a small-molecule inhibitor of TAK1, we show that caspase-8 activation during TAK1 inhibition results in cleavage of both gasdermin D (GSDMD) and gasdermin E (GSDME) in murine macrophages, resulting in pyroptosis. Loss of GsdmD delays membrane rupture, reverting the cell-death morphology to apoptosis. We found that the Yersinia-driven IL-1 response arises from asynchrony of macrophage death during bulk infections in which two cellular populations are required to provide signal 1 and signal 2 for IL-1α/ß release. Furthermore, we found that human macrophages are resistant to YopJ-mediated pyroptosis, with dampened IL-1ß production. Our results uncover a form of caspase-8-mediated pyroptosis and suggest a hypothesis for the increased sensitivity of humans to Yersinia infection compared with the rodent reservoir.

Expression Atlas update--a database of gene and transcript expression from microarray- and sequencing-based functional genomics experiments.

Expression Atlas (http://www.ebi.ac.uk/gxa) is a value-added database providing information about gene, protein and splice variant expression in different cell types, organism parts, developmental stages, diseases and other biological and experimental conditions. The database consists of selected high-quality microarray and RNA-sequencing experiments from ArrayExpress that have been manually curated, annotated with Experimental Factor Ontology terms and processed using standardized microarray and RNA-sequencing analysis methods. The new version of Expression Atlas introduces the concept of 'baseline' expression, i.e. gene and splice variant abundance levels in healthy or untreated conditions, such as tissues or cell types. Differential gene expression data benefit from an in-depth curation of experimental intent, resulting in biologically meaningful 'contrasts', i.e. instances of differential pairwise comparisons between two sets of biological replicates. Other novel aspects of Expression Atlas are its strict quality control of raw experimental data, up-to-date RNA-sequencing analysis methods, expression data at the level of gene sets, as well as genes and a more powerful search interface designed to maximize the biological value provided to the user.