The Plasma Lipidomic Landscape in Patients with Sepsis due to Community-acquired Pneumonia.

Rationale: The plasma lipidome has the potential to reflect many facets of the host status during severe infection. Previous work is limited to specific lipid groups or was focused on lipids as prognosticators.Objectives: To map the plasma lipidome during sepsis due to community-acquired pneumonia (CAP) and determine the disease specificity and associations with clinical features.Methods: We analyzed 1,833 lipid species across 33 classes in 169 patients admitted to the ICU with sepsis due to CAP, 51 noninfected ICU patients, and 48 outpatient controls. In a paired analysis, we reanalyzed patients still in the ICU 4 days after admission (n = 82).Measurements and Main Results: A total of 58% of plasma lipids were significantly lower in patients with CAP-attributable sepsis compared with outpatient controls (6% higher, 36% not different). We found strong lipid class-specific associations with disease severity, validated across two external cohorts, and inflammatory biomarkers, in which triacylglycerols, cholesterol esters, and lysophospholipids exhibited the strongest associations. A total of 36% of lipids increased over time, and stratification by survival revealed diverging lipid recovery, which was confirmed in an external cohort; specifically, a 10% increase in cholesterol ester levels was related to a lower odds ratio (0.84; P = 0.006) for 30-day mortality (absolute mortality, 18 of 82). Comparison with noninfected ICU patients delineated a substantial common illness response (57.5%) and a distinct lipidomic signal for patients with CAP-attributable sepsis (37%).Conclusions: Patients with sepsis due to CAP exhibit a time-dependent and partially disease-specific shift in their plasma lipidome that correlates with disease severity and systemic inflammation and is associated with higher mortality.

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.

Adherence Affects Monocyte Innate Immune Function and Metabolic Reprogramming after Lipopolysaccharide Stimulation In Vitro.

Circulating nonadherent monocytes can migrate to extravascular sites by a process that involves adherence. Alterations in intracellular metabolism shape the immunological phenotype of phagocytes upon activation. To determine the effect of adherence on their metabolic and functional response human monocytes were stimulated with LPS under nonadherent and adherent conditions. Adherent monocytes (relative to nonadherent monocytes) produced less TNF and IL-1ß (proinflammatory) and more IL-10 (anti-inflammatory) upon LPS stimulation and had an increased capacity to phagocytose and produce reactive oxygen species. RNA sequencing analysis confirmed that adherence modified the LPS-induced response of monocytes, reducing expression of proinflammatory genes involved in TLR signaling and increasing induction of genes involved in pathogen elimination. Adherence resulted in an increased glycolytic response as indicated by lactate release, gene set enrichment, and [13C]-glucose flux analysis. To determine the role of glycolysis in LPS-induced immune responses, this pathway was inhibited by glucose deprivation or the glucose analogue 2-deoxy-d-glucose (2DG). Although both interventions equally inhibited glycolysis, only 2DG influenced monocyte functions, inhibiting expression of genes involved in TLR signaling and pathogen elimination, as well as cytokine release. 2DG, but not glucose deprivation, reduced expression of genes involved in oxidative phosphorylation. Inhibition of oxidative phosphorylation affected TNF and IL-10 release in a similar way as 2DG. Collectively, these data suggest that adherence may modify the metabolic and immunological profile of monocytes and that inhibition of glycolysis and oxidative phosphorylation, but not inhibition of glycolysis alone, has a profound effect on immune functions of monocytes exposed to LPS.

Association between age and the host response in critically ill patients with sepsis.

BACKGROUND: The association of ageing with increased sepsis mortality is well established. Nonetheless, current investigations on the influence of age on host response aberrations are largely limited to plasma cytokine levels while neglecting other pathophysiological sepsis domains like endothelial cell activation and function, and coagulation activation. The primary objective of this study was to gain insight into the association of ageing with aberrations in key host response pathways and blood transcriptomes in sepsis. METHODS: We analysed the clinical outcome (n = 1952), 16 plasma biomarkers providing insight in deregulation of specific pathophysiological domains (n = 899), and blood leukocyte transcriptomes (n = 488) of sepsis patients stratified according to age decades. Blood transcriptome results were validated in an independent sepsis cohort and compared with healthy individuals. RESULTS: Older age was associated with increased mortality independent of comorbidities and disease severity. Ageing was associated with lower endothelial cell activation and dysfunction, and similar inflammation and coagulation activation, despite higher disease severity scores. Blood leukocytes of patients ≥ 70 years, compared to patients < 50 years, showed decreased expression of genes involved in cytokine signaling, and innate and adaptive immunity, and increased expression of genes involved in hemostasis and endothelial cell activation. The diminished expression of gene pathways related to innate immunity and cytokine signaling in subjects ≥ 70 years was sepsis-induced, as healthy subjects ≥ 70 years showed enhanced expression of these pathways compared to healthy individuals < 50 years. CONCLUSIONS: This study provides novel evidence that older age is associated with relatively mitigated sepsis-induced endothelial cell activation and dysfunction, and a blood leukocyte transcriptome signature indicating impaired innate immune and cytokine signaling. These data suggest that age should be considered in patient selection in future sepsis trials targeting the immune system and/or the endothelial cell response.

RNA-seq analysis of ageing human retinal pigment epithelium: Unexpected up-regulation of visual cycle gene transcription.

Ageing presents adverse effects on the retina and is the primary risk factor for age-related macular degeneration (AMD). We report the first RNA-seq analysis of age-related transcriptional changes in the human retinal pigment epithelium (RPE), the primary site of AMD pathogenesis. Whole transcriptome sequencing of RPE from human donors ranging in age from 31 to 93 reveals that ageing is associated with increasing transcription of main RPE-associated visual cycle genes (including LRAT, RPE65, RDH5, RDH10, RDH11; pathway enrichment BH-adjusted P = 4.6 × 10-6 ). This positive correlation is replicated in an independent set of 28 donors and a microarray dataset of 50 donors previously published. LRAT expression is positively regulated by retinoid by-products of the visual cycle (A2E and all-trans-retinal) involving modulation by retinoic acid receptor alpha transcription factor. The results substantiate a novel age-related positive feedback mechanism between accumulation of retinoid by-products in the RPE and the up-regulation of visual cycle genes.

Identification of candidate protective variants for common diseases and evaluation of their protective potential.

BACKGROUND: Human polymorphisms with derived alleles that are protective against disease may provide powerful translational opportunities. Here we report a method to identify such candidate polymorphisms and apply it to common non-synonymous SNPs (nsSNPs) associated with common diseases. Our study also sought to establish which of the identified protective nsSNPs show evidence of positive selection, taking this as indirect evidence that the protective variant has a beneficial effect on phenotype. Further, we performed an analysis to quantify the predicted effect of each protective variant on protein function/structure. RESULTS: An initial analysis of eight SNPs previously identified as associated with age-related macular degeneration (AMD), revealed that two of them have a derived allele that is protective against developing the disease. One is in the complement component 2 gene (C2; E318D) and the other is in the complement factor B gene (CFB; R32Q). Then, combining genomewide ancestral allele information with known common disease-associated nsSNPs from the GWAS catalog, we found 32 additional SNPs which have a derived allele that is disease protective. Out of the total 34 identified candidate protective variants (CPVs), we found that 30 show stronger evidence of positive selection than the protective variant in lipoprotein lipase (LPL; S447X), which has already been translated into gene therapy. Furthermore, 11 of these CPVs have a higher probability of affecting protein structure than the lipoprotein lipase protective variant (LPL; S447X). CONCLUSIONS: We identify 34 CPVs from the human genome. Diseases they confer protection against include, but are not limited to, type 2 diabetes, inflammatory bowel disease, age-related macular degeneration, multiple sclerosis and rheumatoid arthritis. We propose that those 30 CPVs with evidence of stronger positive selection than the LPL protective variant, may be considered as priority candidates for therapeutic approaches. The next step towards translation will require testing the hypotheses generated by our analyses, specifically whether the CPV arose from a gain-of-function or a loss-of-function mutation.

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.

Association between butyrate-producing gut bacteria and the risk of infectious disease hospitalisation: results from two observational, population-based microbiome studies.

BACKGROUND: Microbiota alterations are common in patients hospitalised for severe infections, and preclinical models have shown that anaerobic butyrate-producing gut bacteria protect against systemic infections. However, the relationship between microbiota disruptions and increased susceptibility to severe infections in humans remains unclear. We investigated the relationship between gut microbiota and the risk of future infection-related hospitalisation in two large population-based cohorts. METHODS: In this observational microbiome study, gut microbiota were characterised using 16S rRNA gene sequencing in independent population-based cohorts from the Netherlands (HELIUS study; derivation cohort) and Finland (FINRISK 2002 study; validation cohort). HELIUS was conducted in Amsterdam, Netherlands, and included adults (aged 18-70 years at inclusion) who were randomly sampled from the municipality register of Amsterdam. FINRISK 2002 was conducted in six regions in Finland and is a population survey that included a random sample of adults (aged 25-74 years). In both cohorts, participants completed questionnaires, underwent a physical examination, and provided a faecal sample at inclusion (Jan 3, 2013, to Nov 27, 2015, for HELIUS participants and Jan 21 to April 19, 2002, for FINRISK participants. For inclusion in our study, a faecal sample needed to be provided and successfully sequenced, and national registry data needed to be available. Primary predictor variables were microbiota composition, diversity, and relative abundance of butyrate-producing bacteria. Our primary outcome was hospitalisation or mortality due to any infectious disease during 5-7-year follow-up after faecal sample collection, based on national registry data. We examined associations between microbiota and infection risk using microbial ecology and Cox proportional hazards. FINDINGS: We profiled gut microbiota from 10 699 participants (4248 [39·7%] from the derivation cohort and 6451 [60·3%] from the validation cohort). 602 (5·6%) participants (152 [3·6%] from the derivation cohort; 450 [7·0%] from the validation cohort) were hospitalised or died due to infections during follow-up. Gut microbiota composition of these participants differed from those without hospitalisation for infections (derivation p=0·041; validation p=0·0002). Specifically, higher relative abundance of butyrate-producing bacteria was associated with a reduced risk of hospitalisation for infections (derivation cohort cause-specific hazard ratio 0·75 [95% CI 0·60-0·94] per 10% increase in butyrate producers, p=0·013; validation cohort 0·86 [0·77-0·96] per 10% increase, p=0·0077). These associations remained unchanged following adjustment for demographics, lifestyle, antibiotic exposure, and comorbidities. INTERPRETATION: Gut microbiota composition, specifically colonisation with butyrate-producing bacteria, was associated with protection against hospitalisation for infectious diseases in the general population across two independent European cohorts. Further studies should investigate whether modulation of the microbiome can reduce the risk of severe infections. FUNDING: Amsterdam UMC, Porticus, National Institutes of Health, Netherlands Organisation for Health Research and Development (ZonMw), and Leducq Foundation.

The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia.

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

Inflammatory and glycolytic programs underpin a primed blood neutrophil state in patients with pneumonia.

Neutrophils are potent immune cells with key antimicrobial functions. Previous in vitro work has shown that neutrophil effector functions are mainly fueled by intracellular glycolysis. Little is known about the state of neutrophils still in the circulation in patients during infection. Here, we combined flow cytometry, stimulation assays, transcriptomics, and metabolomics to investigate the link between inflammatory and metabolic pathways in blood neutrophils of patients with community-acquired pneumonia. Patients' neutrophils, relative to neutrophils from age- and sex- matched controls, showed increased degranulation upon ex vivo stimulation, and portrayed distinct upregulation of inflammatory transcriptional programs. This neutrophil phenotype was accompanied by a high-energy state with increased intracellular ATP content, and transcriptomic and metabolic upregulation of glycolysis and glycogenolysis. One month after hospital admission, these metabolic and transcriptomic changes were largely normalized. These data elucidate the molecular programs that underpin a balanced, yet primed state of blood neutrophils during pneumonia.

Uncovering heterogeneity in sepsis: a comparative analysis of subphenotypes.

PURPOSE: The heterogeneity in sepsis is held responsible, in part, for the lack of precision treatment. Many attempts to identify subtypes of sepsis patients identify those with shared underlying biology or outcomes. To date, though, there has been limited effort to determine overlap across these previously identified subtypes. We aimed to determine the concordance of critically ill patients with sepsis classified by four previously described subtype strategies. METHODS: This secondary analysis of a multicenter prospective observational study included 522 critically ill patients with sepsis assigned to four previously established subtype strategies, primarily based on: (i) clinical data in the electronic health record (α, ß, γ, and δ), (ii) biomarker data (hyper- and hypoinflammatory), and (iii-iv) transcriptomic data (Mars1-Mars4 and SRS1-SRS2). Concordance was studied between different subtype labels, clinical characteristics, biological host response aberrations, as well as combinations of subtypes by sepsis ensembles. RESULTS: All four subtype labels could be adjudicated in this cohort, with the distribution of the clinical subtype varying most from the original cohort. The most common subtypes in each of the four strategies were γ (61%), which is higher compared to the original classification, hypoinflammatory (60%), Mars2 (35%), and SRS2 (54%). There was no clear relationship between any of the subtyping approaches (Cramer's V = 0.086-0.456). Mars2 and SRS1 were most alike in terms of host response biomarkers (p = 0.079-0.424), while other subtype strategies showed no clear relationship. Patients enriched for multiple subtypes revealed that characteristics and outcomes differ dependent on the combination of subtypes made. CONCLUSION: Among critically ill patients with sepsis, subtype strategies using clinical, biomarker, and transcriptomic data do not identify comparable patient populations and are likely to reflect disparate clinical characteristics and underlying biology.

Platelets of COVID-19 patients display mitochondrial dysfunction, oxidative stress, and energy metabolism failure compatible with cell death.

Background: Alterations in platelet function have been implicated in the pathophysiology of COVID-19 since the beginning of the pandemic. While early reports linked hyperactivated platelets to thromboembolic events in COVID-19, subsequent investigations demonstrated hyporeactive platelets with a procoagulant phenotype. Mitochondria are important for energy metabolism and the function of platelets. Objectives: Here, we sought to map the energy metabolism of platelets in a cohort of noncritically ill COVID-19 patients and assess platelet mitochondrial function, activation status, and responsiveness to external stimuli. Methods: We enrolled hospitalized COVID-19 patients and controls between October 2020 and December 2021. Platelets function and metabolism was analyzed by flow cytometry, metabolomics, glucose fluxomics, electron and fluorescence microscopy and western blot. Results: Platelets from COVID-19 patients showed increased phosphatidylserine externalization indicating a procoagulant phenotype and hyporeactivity to ex vivo stimuli, associated with profound mitochondrial dysfunction characterized by mitochondrial depolarization, lower mitochondrial DNA-encoded transcript levels, an altered mitochondrial morphology consistent with increased mitochondrial fission, and increased pyruvate/lactate ratios in platelet supernatants. Metabolic profiling by untargeted metabolomics revealed NADH, NAD+, and ATP among the top decreased metabolites in patients' platelets, suggestive of energy metabolism failure. Consistently, platelet fluxomics analyses showed a strongly reduced utilization of 13C-glucose in all major energy pathways together with a rerouting of glucose to de novo generation of purine metabolites. Patients' platelets further showed evidence of oxidative stress, together with increased glutathione oxidation and synthesis. Addition of plasma from COVID-19 patients to normal platelets partially reproduced the phenotype of patients' platelets and disclosed a temporal relationship between mitochondrial decay and (subsequent) phosphatidylserine exposure and hyporeactivity. Conclusion: These data link energy metabolism failure in platelets from COVID-19 patients with a prothrombotic platelet phenotype with features matching cell death.

HIF-1α Stabilization in Flagellin-Stimulated Human Bronchial Cells Impairs Barrier Function.

The respiratory epithelium provides a first line of defense against pathogens. Hypoxia-inducible factor (HIF)1α is a transcription factor which is stabilized in hypoxic conditions through the inhibition of prolyl-hydroxylase (PHD)2, the enzyme that marks HIF1α for degradation. Here, we studied the impact of HIF1α stabilization on the response of primary human bronchial epithelial (HBE) cells to the bacterial component, flagellin. The treatment of flagellin-stimulated HBE cells with the PHD2 inhibitor IOX2 resulted in strongly increased HIF1α expression. IOX2 enhanced the flagellin-induced expression of the genes encoding the enzymes involved in glycolysis, which was associated with the intracellular accumulation of pyruvate. An untargeted pathway analysis of RNA sequencing data demonstrated the strong inhibitory effects of IOX2 toward key innate immune pathways related to cytokine and mitogen-activated kinase signaling cascades in flagellin-stimulated HBE cells. Likewise, the cell-cell junction organization pathway was amongst the top pathways downregulated by IOX2 in flagellin-stimulated HBE cells, which included the genes encoding claudins and cadherins. This IOX2 effect was corroborated by an impaired barrier function, as measured by dextran permeability. These results provide a first insight into the effects associated with HIF1α stabilization in the respiratory epithelium, suggesting that HIF1α impacts properties that are key to maintaining homeostasis upon stimulation with a relevant bacterial agonist.

Source-specific host response and outcomes in critically ill patients with sepsis: a prospective cohort study.

PURPOSE: There is limited knowledge on how the source of infection impacts the host response to sepsis. We aimed to compare the host response in sepsis patients with a single, known source at admission (< 24 h) to the intensive care unit. METHODS: From the molecular diagnosis and risk stratification of sepsis (MARS) prospective cohort, we measured 16 plasma host response biomarkers reflective of key host response pathways in 621 sepsis patients. In a subgroup (n = 335), blood leukocyte transcriptomes were compared between the sources. Differences in clinical patient profiles and survival were compared in the whole sepsis cohort (n = 2019). RESULTS: The plasma biomarker cohort was categorized into sepsis originating from the respiratory tract (n = 334, 53.8%), abdomen (n = 159, 25.6%), urinary tract (n = 44, 7.1%), cardiovascular (n = 41, 6.6%), central nervous system (CNS) (n = 18, 2.9%), or skin (n = 25, 4%). This analysis revealed stronger inflammatory and cytokine responses, loss of vascular integrity and coagulation activation in abdominal sepsis relative to respiratory. Endothelial cell activation was prominent in urinary, cardiovascular and skin infections, while CNS infection was associated with the least host response aberrations. The leukocyte transcriptional response showed the largest overlap between abdominal and pulmonary infections (76% in common); notable differences between the sources were detected regarding hemostasis, cytokine signaling, innate and adaptive immune, and metabolic transcriptional pathways. After adjustment for confounders, the source of infection remained an independent contributor to 30-day mortality (unadjusted p = 0.001, adjusted p = 0.028). CONCLUSION: Sepsis heterogeneity is partly explained by source-specific host response dysregulations and should be considered when selecting patients for trials testing immune modulatory drugs.

Rectal microbiota are coupled with altered cytokine production capacity following community-acquired pneumonia hospitalization.

Human studies describing the immunomodulatory role of the intestinal microbiota in systemic infections are lacking. Here, we sought to relate microbiota profiles from 115 patients with community-acquired pneumonia (CAP), both on hospital admission and following discharge, to concurrent circulating monocyte and neutrophil function. Rectal microbiota composition did not explain variation in cytokine responses in acute CAP (median 0%, IQR 0.0%-1.9%), but did one month following hospitalization (median 4.1%, IQR 0.0%-6.6%, p = 0.0035). Gene expression analysis of monocytes showed that undisrupted microbiota profiles following hospitalization were associated with upregulated interferon, interleukin-10, and G-protein-coupled-receptor-ligand-binding pathways. While CAP is characterized by profoundly distorted gut microbiota, the effects of these disruptions on cytokine responses and transcriptional profiles during acute infection were absent or modest. However, rectal microbiota were related to altered cytokine responses one month following CAP hospitalization, which may provide insights into potential mechanisms contributing to the high risk of recurrent infections following hospitalization.

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.

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.