Monocyte state 1 (MS1) cells in critically ill patients with sepsis or non-infectious conditions: association with disease course and host response.

BACKGROUND: Sepsis is a life-threatening condition arising from an aberrant host response to infection. Recent single-cell RNA sequencing investigations identified an immature bone-marrow-derived CD14+ monocyte phenotype with immune suppressive properties termed "monocyte state 1" (MS1) in patients with sepsis. Our objective was to determine the association of MS1 cell profiles with disease presentation, outcomes, and host response characteristics. METHODS: We used the transcriptome deconvolution method (CIBERSORTx) to estimate the percentage of MS1 cells from blood RNA profiles of patients with sepsis admitted to the intensive care unit (ICU). We compared these profiles to ICU patients without infection and to healthy controls. Host response dysregulation was further studied by gene co-expression network and gene set enrichment analyses of blood leukocytes, and measurement of 15 plasma biomarkers indicative of pathways implicated in sepsis pathogenesis. RESULTS: Sepsis patients (n = 332) were divided into three equally-sized groups based on their MS1 cell levels (low, intermediate, and high). MS1 groups did not differ in demographics or comorbidities. The intermediate and high MS1 groups presented with higher disease severity and more often had shock. MS1 cell abundance did not differ between survivors and non-survivors, or between patients who did or did not acquire a secondary infection. Higher MS1 cell percentages were associated with downregulation of lymphocyte-related and interferon response genes in blood leukocytes, with concurrent upregulation of inflammatory response pathways, including tumor necrosis factor signaling via nuclear factor-κB. Previously described sepsis host response transcriptomic subtypes showed different MS1 cell abundances, and MS1 cell percentages positively correlated with the "quantitative sepsis response signature" and "molecular degree of perturbation" scores. Plasma biomarker levels, indicative of inflammation, endothelial cell activation, and coagulation activation, were largely similar between MS1 groups. In ICU patients without infection (n = 215), MS1 cell percentages and their relation with disease severity, shock, and host response dysregulation were highly similar to those in sepsis patients. CONCLUSIONS: High MS1 cell percentages are associated with increased disease severity and shock in critically ill patients with sepsis or a non-infectious condition. High MS1 cell abundance likely indicates broad immune dysregulation, entailing not only immunosuppression but also anomalies reflecting exaggerated inflammatory responses.

C/EBPδ Suppresses Motility-Associated Gene Signatures and Reduces PDAC Cell Migration.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive human cancers and occurs globally at an increasing incidence. Metastases are the primary cause of cancer-related death and, in the majority of cases, PDAC is accompanied by metastatic disease at the time of diagnosis, making it a particularly lethal cancer. Regrettably, to date, no curative treatment has been developed for patients with metastatic disease, resulting in a 5-year survival rate of only 11%. We previously found that the protein expression of the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) negatively correlates with lymph node involvement in PDAC patients. To better comprehend the etiology of metastatic PDAC, we explored the role of C/EBPδ at different steps of the metastatic cascade, using established in vitro models. We found that C/EBPδ has a major impact on cell motility, an important prerequisite for tumor cells to leave the primary tumor and to reach distant sites. Our data suggest that C/EBPδ induces downstream pathways that modulate actin cytoskeleton dynamics to reduce cell migration and to induce a more epithelial-like cellular phenotype. Understanding the mechanisms dictating epithelial and mesenchymal features holds great promise for improving the treatment of PDAC.

Intracellular pyruvate levels positively correlate with cytokine production capacity in tolerant monocytes from patients with pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is responsible for a high morbidity and mortality worldwide. Monocytes are essential for pathogen recognition and the initiation of an innate immune response. Immune cells induce intracellular glycolysis upon activation to support several functions. OBJECTIVE: To obtain insight in the metabolic profile of blood monocytes during CAP, with a focus on glycolysis and branching metabolic pathways, and to determine a possible association between intracellular metabolite levels and monocyte function. METHODS: Monocytes were isolated from blood of patients with CAP within 24 h of hospital admission and from control subjects matched for age, sex and chronic comorbidities. Changes in glycolysis, oxidative phosphorylation (OXPHOS), tricarboxylic acid (TCA) cycle and the pentose phosphate pathway were investigated through RNA sequencing and metabolomics measurements. Monocytes were stimulated ex vivo with lipopolysaccharide (LPS) to determine their capacity to produce tumor necrosis factor (TNF), interleukin (IL)-1ß and IL-10. RESULTS: 50 patients with CAP and 25 non-infectious control subjects were studied. When compared with control monocytes, monocytes from patients showed upregulation of many genes involved in glycolysis, including PKM, the gene encoding pyruvate kinase, the rate limiting enzyme for pyruvate production. Gene set enrichment analysis of OXPHOS, the TCA cycle and the pentose phosphate pathway did not reveal differences between monocytes from patients and controls. Patients' monocytes had elevated intracellular levels of pyruvate and the TCA cycle intermediate α-ketoglutarate. Monocytes from patients were less capable of producing cytokines upon LPS stimulation. Intracellular pyruvate (but not α-ketoglutarate) concentrations positively correlated with IL-1ß and IL-10 levels released by patients' (but not control) monocytes upon exposure to LPS. CONCLUSION: These results suggest that elevated intracellular pyruvate levels may partially maintain cytokine production capacity of hyporesponsive monocytes from patients with CAP.

Human alveolar macrophages do not rely on glucose metabolism upon activation by lipopolysaccharide.

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [13C]-glucose revealed no differences in [13C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.

HIVEP1 Is a Negative Regulator of NF-κB That Inhibits Systemic Inflammation in Sepsis.

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

CEBPD Potentiates the Macrophage Inflammatory Response but CEBPD Knock-Out Macrophages Fail to Identify CEBPD-Dependent Pro-Inflammatory Transcriptional Programs.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

Role of Myeloid Tet Methylcytosine Dioxygenase 2 in Pulmonary and Peritoneal Inflammation Induced by Lipopolysaccharide and Peritonitis Induced by Escherichia coli.

Tet methylcytosine dioxygenase 2 (Tet2) mediates demethylation of DNA. We here sought to determine the expression and function of Tet2 in macrophages upon exposure to lipopolysaccharide (LPS), and in the host response to LPS induced lung and peritoneal inflammation, and during Escherichia (E.) coli induced peritonitis. LPS induced Tet2 expression in mouse macrophages and human monocytes in vitro, as well as in human alveolar macrophages after bronchial instillation in vivo. Bone marrow-derived macrophages from myeloid Tet2 deficient (Tet2fl/flLysMCre) mice displayed enhanced production of IL-1ß, IL-6 and CXCL1 upon stimulation with several Toll-like receptor agonists; similar results were obtained with LPS stimulated alveolar and peritoneal macrophages. Histone deacetylation was involved in the effect of Tet2 on IL-6 production, whilst methylation at the Il6 promoter was not altered by Tet2 deficiency. Tet2fl/flLysMCre mice showed higher IL-6 and TNF levels in bronchoalveolar and peritoneal lavage fluid after intranasal and intraperitoneal LPS administration, respectively, whilst other inflammatory responses were unaltered. E. coli induced stronger production of IL-1ß and IL-6 by Tet2 deficient peritoneal macrophages but not in peritoneal lavage fluid of Tet2fl/flLysMCre mice after in vivo intraperitoneal infection. Tet2fl/flLysMCre mice displayed enhanced bacterial growth during E. coli peritonitis, which was associated with a reduced capacity of Tet2fl/flLysMCre peritoneal macrophages to inhibit the growth of E. coli in vitro. Collectively, these data suggest that Tet2 is involved in the regulation of macrophage functions triggered by LPS and during E. coli infection.

Transcriptome sequencing of human breast cancer reveals aberrant intronic transcription in amplicons and dysregulation of alternative splicing with major therapeutic implications.

Advances in genomic and transcriptome sequencing are revealing the massive scale of previously unrecognised alterations occurring during neoplastic transformation. Breast cancers are genetically and phenotypically heterogeneous. Each of the three major subtypes [ERBB2 amplified, estrogen receptor (ESR)-positive and triple-negative] poses diagnostic and therapeutic challenges. Here we show, using high-resolution next-generation transcriptome sequencing, that in all three breast cancer subtypes, but not matched controls, there was significant overexpression of transcripts from intronic and untranslated regions in addition to exons from specific genes, particularly amplified oncogenes and hormone receptors. For key genes ERBB2 and ESR1, we demonstrate that overexpression is linked to the production of highly modified and truncated splice variants in tumours, but not controls, correlated with tumour subtype. Translation of these tumour-specific splice variants generates truncated proteins with altered subcellular locations and functions, modifying the phenotype, affecting tumour biology, and targeted antitumour therapies. In contrast, tumour suppressors TP53, BRCA1/2 and NF1 did not show intronic overexpression or truncated splice variants in cancers. These findings emphasize the detection of intronic as well as exonic changes in the transcriptional landscapes of cancers have profound therapeutic implications.

A missense variant in CST3 exerts a recessive effect on susceptibility to age-related macular degeneration resembling its association with Alzheimer's disease.

Age-related macular degeneration (AMD) and Alzheimer's disease (AD) are degenerative, multifactorial diseases involving age-related accumulation of extracellular deposits linked to dysregulation of protein homeostasis. Here, we strengthen the evidence that an nsSNP (p.Ala25Thr) in the cysteine proteinase inhibitor cystatin C gene CST3, previously confirmed by meta-analysis to be associated with AD, is associated with exudative AMD. To our knowledge, this is the first report highlighting a genetic variant that increases the risk of developing both AD and AMD. Furthermore, we demonstrate that the risk associated with the mutant allele follows a recessive model for both diseases. We perform an AMD-CST3 case-control study genotyping 350 exudative AMD Caucasian individuals. Bringing together our data with the previously reported AMD-CST3 association study, the evidence of a recessive effect on AMD risk is strengthened (OR = 1.89, P = 0.005). This effect closely resembles the AD-CST3 recessive effect (OR = 1.73, P = 0.005) previously established by meta-analysis. This resemblance is substantiated by the high correlation between CST3 genotype and effect size across the two diseases (R(2) = 0.978). A recessive effect is in line with the known function of cystatin C, a potent enzyme inhibitor. Its potency means that, in heterozygous individuals, a single functional allele is sufficient to maintain its inhibitory function; only homozygous individuals will lack this form of proteolytic regulation. Our findings support the hypothesis that recessively acting variants account for some of the missing heritability of multifactorial diseases. Replacement therapy represents a translational opportunity for individuals homozygous for the mutant allele.