Intrathymic dendritic cell-biased precursors promote human T cell lineage specification through IRF8-driven transmembrane TNF.

The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.

Spns2/S1P: it takes two to tango with inflammation and metabolic rewiring during sepsis.

Sepsis is the result of a dysregulated host response to an infection and causes high morbidity and mortality at the intensive care units worldwide. Despite intensive research, the current management of sepsis is supportive rather than curative. Therefore, new therapeutic interventions for sepsis and septic shock patients are urgently needed. In this issue of EMBO Reports, Fang et al have used rat sepsis models to show that macrophage-expressed SPNS2, a major transporter of S1P, is a crucial mediator of metabolic reprogramming of macrophages during sepsis which regulates inflammation via the lactate-ROS axis.

Mousepost 2.0, a major expansion of the resource.

The Mousepost 1.0 online search tool, launched in 2017, allowed to search for variations in all protein-coding gene sequences of 36 sequenced mouse inbred strains, compared to the reference strain C57BL/6J, which could be linked to strain-specific phenotypes and modifier effects. Because recently these genome sequences have been significantly updated and sequences of 16 extra strains added by the Mouse Genomes Project, a profound update, correction and expansion of the Mousepost 1.0 database has been performed and is reported here. Moreover, we have added a new class of protein disturbing sequence polymorphisms (besides stop codon losses, stop codon gains, small insertions and deletions, and missense mutations), namely start codon mutations. The current version, Mousepost 2.0 (https://mousepost.be), therefore is a significantly updated and invaluable tool available to the community and is described here and foreseen by multiple examples.

Passenger Mutations Confound Interpretation of All Genetically Modified Congenic Mice.

Targeted mutagenesis in mice is a powerful tool for functional analysis of genes. However, genetic variation between embryonic stem cells (ESCs) used for targeting (previously almost exclusively 129-derived) and recipient strains (often C57BL/6J) typically results in congenic mice in which the targeted gene is flanked by ESC-derived passenger DNA potentially containing mutations. Comparative genomic analysis of 129 and C57BL/6J mouse strains revealed indels and single nucleotide polymorphisms resulting in alternative or aberrant amino acid sequences in 1,084 genes in the 129-strain genome. Annotating these passenger mutations to the reported genetically modified congenic mice that were generated using 129-strain ESCs revealed that nearly all these mice possess multiple passenger mutations potentially influencing the phenotypic outcome. We illustrated this phenotypic interference of 129-derived passenger mutations with several case studies and developed a Me-PaMuFind-It web tool to estimate the number and possible effect of passenger mutations in transgenic mice of interest.

Reprogramming of glucocorticoid receptor function by hypoxia.

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance.

Comparative Analysis of Hepatic Gene Expression Profiles in Murine and Porcine Sepsis Models.

Sepsis remains a huge unmet medical need for which no approved drugs, besides antibiotics, are on the market. Despite the clinical impact of sepsis, its molecular mechanism remains inadequately understood. Recent insights have shown that profound hepatic transcriptional reprogramming, leading to fatal metabolic abnormalities, might open a new avenue to treat sepsis. Translation of experimental results from rodents to larger animal models of higher relevance for human physiology, such as pigs, is critical and needs exploration. We performed a comparative analysis of the transcriptome profiles in murine and porcine livers using the following sepsis models: cecal ligation and puncture (CLP) in mice and fecal instillation (FI) in pigs, both of which induce polymicrobial septic peritonitis, and lipopolysaccharide (LPS)-induced endotoxemia in pigs, inducing sterile inflammation. Using bulk RNA sequencing, Metascape pathway analysis, and HOMER transcription factor motif analysis, we were able to identify key genes and pathways affected in septic livers. Conserved upregulated pathways in murine CLP and porcine LPS and FI generally comprise typical inflammatory pathways, except for ER stress, which was only found in the murine CLP model. Conserved pathways downregulated in sepsis comprise almost exclusively metabolic pathways such as monocarboxylic acid, steroid, biological oxidation, and small-molecule catabolism. Even though the upregulated inflammatory pathways were equally induced in the two porcine models, the porcine FI model more closely resembles the metabolic dysfunction observed in the CLP liver compared to the porcine LPS model. This comprehensive comparison focusing on the hepatic responses in mouse CLP versus LPS or FI in pigs shows that the two porcine sepsis models generally resemble quite well the mouse CLP model, with a typical inflammatory signature amongst the upregulated genes and metabolic dysfunction amongst the downregulated genes. The hepatic ER stress observed in the murine model could not be replicated in the porcine models. When studying metabolic dysfunction in the liver upon sepsis, the porcine FI model more closely resembles the mouse CLP model compared to the porcine LPS model.

Point mutation I634A in the glucocorticoid receptor causes embryonic lethality by reduced ligand binding.

The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GRdim/dim mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GRD/D) have previously helped to define the functions of GR monomers and dimers. Since GRD/D retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GRD+L/D+L mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GRL/L mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and Kd values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.

The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation.

Whereas dimerization of the DNA-binding domain of the androgen receptor (AR) plays an evident role in recognizing bipartite response elements, the contribution of the dimerization of the ligand-binding domain (LBD) to the correct functioning of the AR remains unclear. Here, we describe a mouse model with disrupted dimerization of the AR LBD (ARLmon/Y ). The disruptive effect of the mutation is demonstrated by the feminized phenotype, absence of male accessory sex glands, and strongly affected spermatogenesis, despite high circulating levels of testosterone. Testosterone replacement studies in orchidectomized mice demonstrate that androgen-regulated transcriptomes in ARLmon/Y mice are completely lost. The mutated AR still translocates to the nucleus and binds chromatin, but does not bind to specific AR binding sites. In vitro studies reveal that the mutation in the LBD dimer interface also affects other AR functions such as DNA binding, ligand binding, and co-regulator binding. In conclusion, LBD dimerization is crucial for the development of AR-dependent tissues through its role in transcriptional regulation in vivo. Our findings identify AR LBD dimerization as a possible target for AR inhibition.

Coding variants in mouse and rat model organisms: mousepost and ratpost.

Mice and rats are the most commonly used vertebrate model organisms in biomedical research. The availability of a reference genome in both animals combined with the deep sequencing of several doze of popular inbred lines also provides rich sequence variation data in these species. In some cases, such sequence variants can be linked directly to a distinctive phenotype. In previous work, we created the mouse and rat online searchable databases ("Mousepost" and "Ratpost") where small variant information for protein coding transcripts in mouse and rat inbred strains can be easily retrieved at the amino acid level. These tools are directly useful in forward genetics strategies or as a repository of existing sequence variations. Here, we perform a comparison between the "Mousepost" and "Ratpost" databases and we couple these two tools to a database of human sequence variants ClinVar. We investigated the level of redundancy and complementarity of known variants in protein coding transcripts and found that the large majority of variants is species-specific. However, a small set of positions is conserved in an inbred line between both species. We conclude that both databases are highly complementary, but this may change with further sequencing efforts in both species.

An extracellular microRNA can rescue lives in sepsis.

Sepsis, or blood poisoning, is a savage response of the body to infection. It can lead to organ failure, blood pressure decline, heart failure, and coma. Between 20 and 30 million people suffer from sepsis each year, leading to 8 million deaths. Although certain people are more at risk than others (young children, elderly), anyone can develop sepsis. Patients are resuscitated and treated with antibiotics, and their organ functions are supported. Despite the investment in sepsis research during the previous decades, successful clinical trials are scarce and sepsis remains one of the most difficult and deadly unmet medical needs of today. A study in this issue now provides new insight into sepsis and points to a therapeutic future [1 ].

Glucocorticoids limit lipopolysaccharide-induced lethal inflammation by a double control system.

Lipopolysaccharides (LPS) can lead to a lethal endotoxemia, which is a systemic inflammatory response syndrome (SIRS) characterized by a systemic release of cytokines, such as TNF. Endotoxemia is studied intensely, as a model system of Gram-negative infections. LPS- and TNF-induced SIRS involve a strong induction of interferon-stimulated genes (ISGs), some of which cause cell death in the intestinal epithelium cells (IECs). It is well known that glucocorticoids (GCs) protect against endotoxemia. By applying numerous mutant mouse lines, our data support a model whereby GCs, via their glucocorticoid receptor (GR), apply two key mechanisms to control endotoxemia, (i) at the level of suppression of TNF production in a GR monomer-dependent way in macrophages and (ii) at the level of inhibition of TNFR1-induced ISG gene expression and necroptotic cell death mediators in IECs in a GR dimer-dependent way. Our data add new important insights to the understanding of the role of TNF in endotoxemia and the two separate key roles of GCs in suppressing TNF production and activity.

TNF-α inhibits glucocorticoid receptor-induced gene expression by reshaping the GR nuclear cofactor profile.

Glucocorticoid resistance (GCR) is defined as an unresponsiveness to the therapeutic effects, including the antiinflammatory ones of glucocorticoids (GCs) and their receptor, the glucocorticoid receptor (GR). It is a problem in the management of inflammatory diseases and can be congenital as well as acquired. The strong proinflammatory cytokine TNF-alpha (TNF) induces an acute form of GCR, not only in mice, but also in several cell lines: e.g., in the hepatoma cell line BWTG3, as evidenced by impaired Dexamethasone (Dex)-stimulated direct GR-dependent gene up- and down-regulation. We report that TNF has a significant and broad impact on this transcriptional performance of GR, but no impact on nuclear translocation, dimerization, or DNA binding capacity of GR. Proteome-wide proximity-mapping (BioID), however, revealed that the GR interactome was strongly modulated by TNF. One GR cofactor that interacted significantly less with the receptor under GCR conditions is p300. NFκB activation and p300 knockdown both reduced direct transcriptional output of GR whereas p300 overexpression and NFκB inhibition reverted TNF-induced GCR, which is in support of a cofactor reshuffle model. This hypothesis was supported by FRET studies. This mechanism of GCR opens avenues for therapeutic interventions in GCR diseases.

Acute Endotoxemia-Induced Respiratory and Intestinal Dysbiosis.

Systemic inflammatory response syndrome (SIRS) is a severe condition characterized by systemic inflammation, which may lead to multiple organ failure, shock and death. SIRS is common in burn patients, pancreatitis and sepsis. SIRS is often accompanied by intestinal dysbiosis. However, the mechanism, role and details of microbiome alterations during the early phase of acute SIRS are not completely understood. The current study aimed to characterize the dynamic alterations of both the intestinal and respiratory microbiome at two timepoints during the early phase of acute SIRS (4 and 8 h after LPS) and link these to the host response in a mouse model of a LPS-induced lethal SIRS. Acute SIRS had no effect on the microbiome in the large intestine but induced a rapid dysbiosis in the small intestine, which resembled the microbiome alterations commonly observed in SIRS patients. Later in the disease progression, a dysbiosis of the respiratory microbiome was observed, which was associated with the MMP9 expression in the lungs. Although similar bacteria were increased in both the lung and the small intestine, no evidence for a gut-lung translocation was observed. Gut dysbiosis is commonly observed in diseases involving inflammation in the gut. However, whether the inflammatory response associated with SIRS and sepsis can directly cause gut dysbiosis was still unclear. In the current study we provide evidence that a LPS-induced SIRS can directly cause dysbiosis of the small intestinal and respiratory microbiome.

Easy Access to and Applications of the Sequences of All Protein-Coding Genes of All Sequenced Mouse Strains.

An easily accessible and searchable overview of all protein sequences in the 36 genome-sequenced mouse strains, compared to those in the reference strain C57BL/6J, is now available, as well as an overview of the aberrant proteins in this reference strain. We provide an insight into the advantages of using these databases.

GILZ in sepsis: "Poor is the pupil who does not surpass his master".

With the legendary saying of Leonardo da Vinci in the title, we suggest that Glucocorticoid Induced Leucine Zipper (GILZ) may have more promising effects against polymicrobial sepsis, than glucocorticoids (GC). Indeed, the use of GCs in sepsis remains a matter of debate. The rationale for their use in sepsis is to modulate the exaggerated inflammatory response while maintaining innate immunity. However, GC resistance and side-effects limit their therapeutic value in sepsis. Hence, there is a growing interest in understanding the mechanisms by which GCs modulate immune responses upon infection. In this issue of the European Journal of Immunology, Ellouze et al. provide data demonstrating that deregulated expression of GILZ, a GC-induced protein, in monocytes/macrophages (M/M) recovered from septic shock patients may contribute to the pathogenesis. Furthermore, the authors demonstrate that GILZ overexpression in M/M improves outcome in septic animals by limiting systemic inflammation while increasing bacterial clearance. Overall, these data provide evidence that GCs may modulate immune responses via GILZ and that GILZ is a valuable alternative for GC therapy in sepsis.

Ratpost: a searchable database of protein-inactivating sequence variations in 40 sequenced rat-inbred strains.

Rat-inbred strains are essential as scientific tools. We have analyzed the publicly available genome sequences of 40 rat-inbred strains and provide an overview of sequence variations leading to amino acid changes in protein-coding genes, premature STOP codons or loss of STOP codons, and short in-frame insertions and deletions of all protein-coding genes across all these inbred lines. We provide an overview of the predicted impact on protein function of all these affected proteins in the database, by comparing their sequence with the sequences of the rat reference strain BN/SsNHsdMcwi. We also investigate the flaws of the protein-coding sequences of this reference strain itself, by comparing them with a consensus genome. These data can be retrieved via a searchable website (Ratpost.be) and allow a global, better interpretation of genetic background effects and a source of naturally defective alleles in these 40 sequenced classical and high-priority rat-inbred strains.

Genetic mapping of species differences via in vitro crosses in mouse embryonic stem cells.

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate "in vitro crosses" in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between the Mus musculus laboratory mouse and Mus spretus (∼1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine-guanine phosphoribosyltransferase (Hprt) in as few as 21 d through "flow mapping" by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids.

RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome.

Engagement of tumor necrosis factor receptor 1 signals two diametrically opposed pathways: survival-inflammation and cell death. An additional switch decides, depending on the cellular context, between caspase-dependent apoptosis and RIP kinase (RIPK)-mediated necrosis, also termed necroptosis. We explored the contribution of both cell death pathways in TNF-induced systemic inflammatory response syndrome (SIRS). Deletion of apoptotic executioner caspases (caspase-3 or -7) or inflammatory caspase-1 had no impact on lethal SIRS. However, deletion of RIPK3 conferred complete protection against lethal SIRS and reduced the amounts of circulating damage-associated molecular patterns. Pretreatment with the RIPK1 kinase inhibitor, necrostatin-1, provided a similar effect. These results suggest that RIPK1-RIPK3-mediated cellular damage by necrosis drives mortality during TNF-induced SIRS. RIPK3 deficiency also protected against cecal ligation and puncture, underscoring the clinical relevance of RIPK kinase inhibition in sepsis and identifying components of the necroptotic pathway that are potential therapeutic targets for treatment of SIRS and sepsis.

Complete overview of protein-inactivating sequence variations in 36 sequenced mouse inbred strains.

Mouse inbred strains remain essential in science. We have analyzed the publicly available genome sequences of 36 popular inbred strains and provide lists for each strain of protein-coding genes that acquired sequence variations that cause premature STOP codons, loss of STOP codons and single nucleotide polymorphisms, and short in-frame insertions and deletions. Our data give an overview of predicted defective proteins, including predicted impact scores, of all these strains compared with the reference mouse genome of C57BL/6J. These data can also be retrieved via a searchable website (mousepost.be) and allow a global, better interpretation of genetic background effects and a source of naturally defective alleles in these 36 sequenced classical and high-priority mouse inbred strains.

IFN-γ stimulates CpG-induced IL-10 production in B cells via p38 and JNK signalling pathways.

The production of IL-10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN-γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL-10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN-γ on Toll-like receptor (TLR)-induced IL-10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN-γ inhibited IL-10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL-10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN-γ on TLR9-induced IL-10 was restricted to B cells. In line with the increased IL-10, B cells stimulated with CpG and IFN-γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen-activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 - an inhibitor of p38 and JNK activity - is downregulated after combined stimulation with IFN-γ and CpG. Our data may represent a novel immunoregulatory role of IFN-γ in B cells after triggering of TLR9, by stimulating IL-10 production.