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.

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.

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.

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.

Using the inbred mouse strain SPRET/EiJ to provide novel insights in inflammation and infection research.

Inbred mouse strains derived from the species Mus spretus have been very informative in the study of certain gene polymorphisms in inflammation and infection. Based on our interest in sepsis, we used SPRET/EiJ mice and mapped several critical loci that are linked to sensitivity to cytokine-induced inflammation and endotoxemia. These studies were based on prominent phenotypes that have never been observed in strains derived from Mus musculus and we mapped them at a resolution that enables us to draw conclusions on the mechanisms. Now that the genome of SPRET/EiJ has been sequenced, and other tools have become available, it is time to revisit this strain and emphasize its advantages and disadvantages as a research tool and a discovery platform.

Simultaneous Inhibition of Tumor Necrosis Factor Receptor 1 and Matrix Metalloproteinase 8 Completely Protects Against Acute Inflammation and Sepsis.

OBJECTIVES: Sepsis causes very high mortality and morbidity rates and remains one of the biggest medical challenges. This study investigates whether plasma levels of both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 are associated with sepsis severity and also investigates the therapeutic applicability of simultaneous inhibition of the two molecules in sepsis. DESIGN: Observational human pilot study-prospective controlled animal study. SETTING: University hospital and research laboratory. SUBJECTS: Sepsis patients and C57BL/6 mice deficient for matrix metalloproteinase 8 and/or tumor necrosis factor receptor 1. INTERVENTION: Plasma and whole blood RNA were collected from 13 sepsis patients for 7 consecutive days and within 24 hours of admission to ICU. Matrix metalloproteinase 8 and tumor necrosis factor receptor 1 plasma and expression levels were determined in these patients. Mice deficient for both matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were generated and subjected to endotoxemia and cecal ligation and puncture. Additionally, a bispecific Nanobody that simultaneously blocks matrix metalloproteinase 8 and tumor necrosis factor receptor 1 was created. MEASUREMENTS AND MAIN RESULTS: Plasma levels of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 were positively correlated with the Sequential Organ Failure Assessment score (r, 0.51 and 0.58) and interleukin 6 levels (r, 0.59 and 0.52) in 13 sepsis patients. Combined elimination of tumor necrosis factor receptor 1 and matrix metalloproteinase 8 in double knockout mice resulted in superior survival in endotoxemia and CLP compared with single knockouts and wild-type mice. Cotreatment with our bispecific Nanobody in CLP resulted in improved survival rates (28% vs 19%) compared with untreated mice. CONCLUSIONS: Inhibition of matrix metalloproteinase 8 and tumor necrosis factor receptor 1 might have therapeutic potential to treat sepsis and proof-of-principle was provided as therapeutics that inhibit both tumor necrosis factor receptor 1 and matrix metalloproteinase 8 are effective in CLP.

Microbial metabolome in septic shock: Bridging bench to bedside.

Due to its enormous incidence and mortality, sepsis is a huge health priority. In the latest issue of Cell, an innovative approach is provided that addresses the major sepsis hurdles of today, i.e., early recognition, prompt treatment, translation from experimental animals to human patients, and urgent need for novel therapeutics.

Sepsis-induced changes in pyruvate metabolism: insights and potential therapeutic approaches.

Sepsis is a heterogeneous syndrome resulting from a dysregulated host response to infection. It is considered as a global major health priority. Sepsis is characterized by significant metabolic perturbations, leading to increased circulating metabolites such as lactate. In mammals, pyruvate is the primary substrate for lactate production. It plays a critical role in metabolism by linking glycolysis, where it is produced, with the mitochondrial oxidative phosphorylation pathway, where it is oxidized. Here, we provide an overview of all cytosolic and mitochondrial enzymes involved in pyruvate metabolism and how their activities are disrupted in sepsis. Based on the available data, we also discuss potential therapeutic strategies targeting these pyruvate-related enzymes leading to enhanced survival.

A critical role for HNF4α in polymicrobial sepsis-associated metabolic reprogramming and death.

In sepsis, limited food intake and increased energy expenditure induce a starvation response, which is compromised by a quick decline in the expression of hepatic PPARα, a transcription factor essential in intracellular catabolism of free fatty acids. The mechanism upstream of this PPARα downregulation is unknown. We found that sepsis causes a progressive hepatic loss-of-function of HNF4α, which has a strong impact on the expression of several important nuclear receptors, including PPARα. HNF4α depletion in hepatocytes dramatically increases sepsis lethality, steatosis, and organ damage and prevents an adequate response to IL6, which is critical for liver regeneration and survival. An HNF4α agonist protects against sepsis at all levels, irrespectively of bacterial loads, suggesting HNF4α is crucial in tolerance to sepsis. In conclusion, hepatic HNF4α activity is decreased during sepsis, causing PPARα downregulation, metabolic problems, and a disturbed IL6-mediated acute phase response. The findings provide new insights and therapeutic options in sepsis.

Paneth cell TNF signaling induces gut bacterial translocation and sepsis.

The cytokine tumor necrosis factor (TNF) plays important roles in limiting infection but is also linked to sepsis. The mechanisms underlying these paradoxical roles are unclear. Here, we show that TNF limits the antimicrobial activity of Paneth cells (PCs), causing bacterial translocation from the gut to various organs. This TNF-induced lethality does not occur in mice with a PC-specific deletion in the TNF receptor, P55. In PCs, TNF stimulates the IFN pathway and ablates the steady-state unfolded protein response (UPR), effects not observed in mice lacking P55 or IFNAR1. TNF triggers the transcriptional downregulation of IRE1 key genes Ern1 and Ern2, which are key mediators of the UPR. This UPR deficiency causes a significant reduction in antimicrobial peptide production and PC antimicrobial activity, causing bacterial translocation to organs and subsequent polymicrobial sepsis, organ failure, and death. This study highlights the roles of PCs in bacterial control and therapeutic targets for sepsis.

The impact of hepatocyte-specific deletion of hypoxia-inducible factors on the development of polymicrobial sepsis with focus on GR and PPARα function.

Introduction: Polymicrobial sepsis causes acute anorexia (loss of appetite), leading to lipolysis in white adipose tissue and proteolysis in muscle, and thus release of free fatty acids (FFAs), glycerol and gluconeogenic amino acids. Since hepatic peroxisome proliferator-activated receptor alpha (PPARα) and glucocorticoid receptor (GR) quickly lose function in sepsis, these metabolites accumulate (causing toxicity) and fail to yield energy-rich molecules such as ketone bodies (KBs) and glucose. The mechanism of PPARα and GR dysfunction is not known. Methods & results: We investigated the hypothesis that hypoxia and/or activation of hypoxia inducible factors (HIFs) might play a role in these issues with PPARα and GR. After cecal ligation and puncture (CLP) in mice, leading to lethal polymicrobial sepsis, bulk liver RNA sequencing illustrated the induction of the genes encoding HIF1α and HIF2α, and an enrichment of HIF-dependent gene signatures. Therefore, we generated hepatocyte-specific knock-out mice for HIF1α, HIF2α or both, and a new HRE-luciferase reporter mouse line. After CLP, these HRE-luciferase reporter mice show signals in several tissues, including the liver. Hydrodynamic injection of an HRE-luciferase reporter plasmid also led to (liver-specific) signals in hypoxia and CLP. Despite these encouraging data, however, hepatocyte-specific HIF1α and/or HIF2α knock-out mice suggest that survival after CLP was not dependent on the hepatocyte-specific presence of HIF proteins, which was supported by measuring blood levels of glucose, FFAs, and KBs. The HIF proteins were also irrelevant in the CLP-induced glucocorticoid resistance, but we found indications that the absence of HIF1α in hepatocytes causes less inactivation of PPARα transcriptional function. Conclusion: We conclude that HIF1α and HIF2α are activated in hepatocytes in sepsis, but their contribution to the mechanisms leading to lethality are minimal.

Sepsis: a failing starvation response.

Sepsis is involved in ~ 20% of annual global deaths. Despite decades of research, the current management of sepsis remains supportive rather than curative. Clinical trials in sepsis have mainly been focused on targeting the inflammatory pathway, but without success. Recent data indicate that metabolic dysregulation takes place in sepsis, and targeting metabolic pathways might hold much promise for the management of sepsis. Sepsis yields a strong starvation response, including the release of high-energy metabolites such as lactate and free fatty acids. However, the activity of two major transcription factors, GR and PPARα, is downregulated in hepatocytes, leading to the accumulation and toxicity of metabolites that, moreover, fail to be transformed into useful molecules such as glucose and ketones. We review the literature and suggest mechanisms and potential therapeutic targets that might prevent or revert the fatal metabolic dysregulation in sepsis.

Dimerization of the Glucocorticoid Receptor and Its Importance in (Patho)physiology: A Primer.

The glucocorticoid receptor (GR) is a very versatile protein that comes in several forms, interacts with many proteins and has multiple functions. Numerous therapies are based on GRs' actions but the occurrence of side effects and reduced responses to glucocorticoids have motivated scientists to study GRs in great detail. The notion that GRs can perform functions as a monomeric protein, but also as a homodimer has raised questions about the underlying mechanisms, structural aspects of dimerization, influencing factors and biological functions. In this review paper, we are providing an overview of the current knowledge and insights about this important aspect of GR biology.

Hepatic Peroxisome Proliferator-Activated Receptor Alpha Dysfunction in Porcine Septic Shock.

Despite decades of research, sepsis remains one of the most urgent unmet medical needs. Mechanistic investigations into sepsis have mainly focused on targeting inflammatory pathways; however, recent data indicate that sepsis should also be seen as a metabolic disease. Targeting metabolic dysregulations that take place in sepsis might uncover novel therapeutic opportunities. The role of peroxisome proliferator-activated receptor alpha (PPARɑ) in liver dysfunction during sepsis has recently been described, and restoring PPARɑ signaling has proven to be successful in mouse polymicrobial sepsis. To confirm that such therapy might be translated to septic patients, we analyzed metabolic perturbations in the liver of a porcine fecal peritonitis model. Resuscitation with fluids, vasopressor, antimicrobial therapy and abdominal lavage were applied to the pigs in order to mimic human clinical care. By using RNA-seq, we detected downregulated PPARɑ signaling in the livers of septic pigs and that reduced PPARɑ levels correlated well with disease severity. As PPARɑ regulates the expression of many genes involved in fatty acid oxidation, the reduced expression of these target genes, concomitant with increased free fatty acids in plasma and ectopic lipid deposition in the liver, was observed. The results obtained with pigs are in agreement with earlier observations seen in mice and support the potential of targeting defective PPARɑ signaling in clinical research.

miR-511 Deficiency Protects Mice from Experimental Colitis by Reducing TLR3 and TLR4 Responses via WD Repeat and FYVE-Domain-Containing Protein 1.

Antimicrobial responses play an important role in maintaining intestinal heath. Recently we reported that miR-511 may regulate TLR4 responses leading to enhanced intestinal inflammation. However, the exact mechanism remained unclear. In this study we investigated the effect of miR-511 deficiency on anti-microbial responses and DSS-induced intestinal inflammation. miR-511-deficient mice were protected from DSS-induced colitis as shown by significantly lower disease activity index, weight loss and histology scores in the miR-511-deficient group. Furthermore, reduced inflammatory cytokine responses were observed in colons of miR-511 deficient mice. In vitro studies with bone marrow-derived M2 macrophages showed reduced TLR3 and TLR4 responses in miR-511-deficient macrophages compared to WT macrophages. Subsequent RNA sequencing revealed Wdfy1 as the potential miR-511 target. WDFY1 deficiency is related to impaired TLR3/TLR4 immune responses and the expression was downregulated in miR-511-deficient macrophages and colons. Together, this study shows that miR-511 is involved in the regulation of intestinal inflammation through downstream regulation of TLR3 and TLR4 responses via Wdfy1.