Validation of SeptiCyte RAPID to Discriminate Sepsis from Non-Infectious Systemic Inflammation.

(1) Background: SeptiCyte RAPID is a molecular test for discriminating sepsis from non-infectious systemic inflammation, and for estimating sepsis probabilities. The objective of this study was the clinical validation of SeptiCyte RAPID, based on testing retrospectively banked and prospectively collected patient samples. (2) Methods: The cartridge-based SeptiCyte RAPID test accepts a PAXgene blood RNA sample and provides sample-to-answer processing in ~1 h. The test output (SeptiScore, range 0-15) falls into four interpretation bands, with higher scores indicating higher probabilities of sepsis. Retrospective (N = 356) and prospective (N = 63) samples were tested from adult patients in ICU who either had the systemic inflammatory response syndrome (SIRS), or were suspected of having/diagnosed with sepsis. Patients were clinically evaluated by a panel of three expert physicians blinded to the SeptiCyte test results. Results were interpreted under either the Sepsis-2 or Sepsis-3 framework. (3) Results: Under the Sepsis-2 framework, SeptiCyte RAPID performance for the combined retrospective and prospective cohorts had Areas Under the ROC Curve (AUCs) ranging from 0.82 to 0.85, a negative predictive value of 0.91 (sensitivity 0.94) for SeptiScore Band 1 (score range 0.1-5.0; lowest risk of sepsis), and a positive predictive value of 0.81 (specificity 0.90) for SeptiScore Band 4 (score range 7.4-15; highest risk of sepsis). Performance estimates for the prospective cohort ranged from AUC 0.86-0.95. For physician-adjudicated sepsis cases that were blood culture (+) or blood, urine culture (+)(+), 43/48 (90%) of SeptiCyte scores fell in Bands 3 or 4. In multivariable analysis with up to 14 additional clinical variables, SeptiScore was the most important variable for sepsis diagnosis. A comparable performance was obtained for the majority of patients reanalyzed under the Sepsis-3 definition, although a subgroup of 16 patients was identified that was called septic under Sepsis-2 but not under Sepsis-3. (4) Conclusions: This study validates SeptiCyte RAPID for estimating sepsis probability, under both the Sepsis-2 and Sepsis-3 frameworks, for hospitalized patients on their first day of ICU admission.

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.

Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Genome-wide analyses in Lyme borreliosis: identification of a genetic variant associated with disease susceptibility and its immunological implications.

BACKGROUND: Genetic variation underly inter-individual variation in host immune responses to infectious diseases, and may affect susceptibility or the course of signs and symptoms. METHODS: We performed genome-wide association studies in a prospective cohort of 1138 patients with physician-confirmed Lyme borreliosis (LB), the most common tick-borne disease in the Northern hemisphere caused by the bacterium Borrelia burgdorferi sensu lato. Genome-wide variants in LB patients-divided into a discovery and validation cohort-were compared to two healthy cohorts. Additionally, ex vivo monocyte-derived cytokine responses of peripheral blood mononuclear cells to several stimuli including Borrelia burgdorferi were performed in both LB patient and healthy control samples, as were stimulation experiments using mechanistic/mammalian target of rapamycin (mTOR) inhibitors. In addition, for LB patients, anti-Borrelia antibody responses were measured. Finally, in a subset of LB patients, gene expression was analysed using RNA-sequencing data from the ex vivo stimulation experiments. RESULTS: We identified a previously unknown genetic variant, rs1061632, that was associated with enhanced LB susceptibility. This polymorphism was an eQTL for KCTD20 and ETV7 genes, and its major risk allele was associated with upregulation of the mTOR pathway and cytokine responses, and lower anti-Borrelia antibody production. In addition, we replicated the recently reported SCGB1D2 locus that was suggested to have a protective effect on B. burgdorferi infection, and associated this locus with higher Borrelia burgdorferi antibody indexes and lower IL-10 responses. CONCLUSIONS: Susceptibility for LB was associated with higher anti-inflammatory responses and reduced anti-Borrelia antibody production, which in turn may negatively impact bacterial clearance. These findings provide important insights into the immunogenetic susceptibility for LB and may guide future studies on development of preventive or therapeutic measures. TRIAL REGISTRATION: The LymeProspect study was registered with the International Clinical Trials Registry Platform (NTR4998, registration date 2015-02-13).

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.

Tracheal epithelial cell-exosome-derived MiR-21-5p inhibits alveolar macrophage pyroptosis to resist pulmonary bacterial infection through PIK3CD-autophagy pathway.

AIMS: Structural cells play an important role in regulating immune cells during infection. Our aim was to determine whether structural porcine tracheal epithelial cells (PTECs) can regulate alveolar macrophages (AMs) to prevent bacterial pneumonia, explore the underlying mechanism(s) and therapeutic target. MATERIALS AND METHODS: Actinobacillus pleuropneumoniae (APP) was used as the model strain for infection studies. Small RNA sequencing was used to identify differentially abundant exosome-derived miRNAs. The role of PTECs exosome-derived miR-21-5p in regulating AMs autophagy, pyroptosis, reactive oxygen species (ROS) was determined using RT-qPCR, western-blotting, flow cytometry, immunohistochemistry. Luciferase reporter assays were conducted to identify potential binding targets of miR-21-5p. The universality of miR-21-5p action on resistance to bacterial pulmonary infection was demonstrated using Klebsiella pneumoniae or Staphylococcus aureus in vitro and in vivo infection models. KEY FINDINGS: MiR-21-5p was enriched in PETCs-derived exosomes, which protected AMs against pulmonary bacterial infection. Mechanistically, miR-21-5p targeted PIK3CD, to promote autophagy of AMs, which reduced the pyroptosis induced by APP infection via inhibiting the over-production of ROS, which in turn suppressed the over-expression of pro-inflammatory cytokines, and increased bacterial clearance. Importantly, the protective effect and mechanism of miR-21-5p were universal as they also occurred upon challenge with Klebsiella pneumoniae and Staphylococcus aureus. SIGNIFICANCE: Our data reveals miR-21-5p can promote pulmonary resistance to bacterial infection by inhibiting pyroptosis of alveolar macrophages through the PIK3CD-autophagy-ROS pathway, suggesting PIK3CD may be a potential therapeutic target for bacterial pneumonia.

Profiling the dysregulated immune response in sepsis: overcoming challenges to achieve the goal of precision medicine.

Sepsis is characterised by a dysregulated host immune response to infection. Despite recognition of its significance, immune status monitoring is not implemented in clinical practice due in part to the current absence of direct therapeutic implications. Technological advances in immunological profiling could enhance our understanding of immune dysregulation and facilitate integration into clinical practice. In this Review, we provide an overview of the current state of immune profiling in sepsis, including its use, current challenges, and opportunities for progress. We highlight the important role of immunological biomarkers in facilitating predictive enrichment in current and future treatment scenarios. We propose that multiple immune and non-immune-related parameters, including clinical and microbiological data, be integrated into diagnostic and predictive combitypes, with the aid of machine learning and artificial intelligence techniques. These combitypes could form the basis of workable algorithms to guide clinical decisions that make precision medicine in sepsis a reality and improve patient outcomes.

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.

Myeloid miR-155 plays a limited role in antibacterial defense during Klebsiella-derived pneumosepsis and is dispensable for lipopolysaccharide- or Klebsiella-induced inflammation in mice.

MicroRNA-155 (miR-155) plays a crucial role in regulating host inflammatory responses during bacterial infection. Previous studies have shown that constitutive miR-155 deficiency alleviates inflammation while having varying effects in different bacterial infection models. However, whether miR-155 in myeloid cells is involved in the regulation of inflammatory and antibacterial responses is largely elusive. Mice with myeloid cell specific miR-155 deficiency were generated to study the in vitro response of bone marrow-derived macrophages (BMDMs), alveolar macrophages (AMs) and peritoneal macrophages (PMs) to lipopolysaccharide (LPS), and the in vivo response after intranasal or intraperitoneal challenge with LPS or infection with Klebsiella (K.) pneumoniae via the airways. MiR-155-deficient macrophages released less inflammatory cytokines than control macrophages upon stimulation with LPS in vitro. However, the in vivo inflammatory cytokine response to LPS or K. pneumoniae was not affected by myeloid miR-155 deficiency. Moreover, bacterial outgrowth in the lungs was not altered in myeloid miR-155-deficient mice, but Klebsiella loads in the liver of these mice were significantly higher than in control mice. These data argue against a major role for myeloid miR-155 in host inflammatory responses during LPS-induced inflammation and K. pneumoniae-induced pneumosepsis but suggest that myeloid miR-155 contributes to host defense against Klebsiella infection in the liver.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

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.

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.

An immune dysfunction score for stratification of patients with acute infection based on whole-blood gene expression.

Dysregulated host responses to infection can lead to organ dysfunction and sepsis, causing millions of global deaths each year. To alleviate this burden, improved prognostication and biomarkers of response are urgently needed. We investigated the use of whole-blood transcriptomics for stratification of patients with severe infection by integrating data from 3149 samples from patients with sepsis due to community-acquired pneumonia or fecal peritonitis admitted to intensive care and healthy individuals into a gene expression reference map. We used this map to derive a quantitative sepsis response signature (SRSq) score reflective of immune dysfunction and predictive of clinical outcomes, which can be estimated using a 7- or 12-gene signature. Last, we built a machine learning framework, SepstratifieR, to deploy SRSq in adult and pediatric bacterial and viral sepsis, H1N1 influenza, and COVID-19, demonstrating clinically relevant stratification across diseases and revealing some of the physiological alterations linking immune dysregulation to mortality. Our method enables early identification of individuals with dysfunctional immune profiles, bringing us closer to precision medicine in infection.

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.

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.

Patients with hypothermic sepsis have a unique gene expression profile compared to patients with fever and sepsis.

The pathophysiology of hypothermia during sepsis is unclear. Using genomic profiling of blood leukocytes, we aimed to determine if hypothermia is associated with a different gene expression profile compared to fever during sepsis. Patients with sepsis and either hypothermia or fever within 24 hours after ICU admission were included in the study (n = 168). Hypothermia was defined as body temperature below 36 °C. Fever was defined as body temperature equal to or above 38.3°C. We compared blood gene expression (whole-genome transcriptome in leukocytes) in hypothermic septic compared to febrile septic patients in an unmatched analysis and matched for APACHE IV score and the presence of shock. In total, 67 septic patients were hypothermic and 101 patients were febrile. Hypothermia was associated with a distinct gene expression profile in both unmatched and matched analyses. There were significant differences related to the up- and downregulation of canonical signalling pathways. In the matched analysis, the top upregulated gene was cold-inducible mRNA binding protein (CIRBP) which plays a role in cold-induced suppression of cell proliferation. In addition, we found three signalling pathways significantly upregulated in hypothermic patients compared to febrile patients; tryptophan degradation X, phenylalanine degradation IV and putrescine degradation III. In conclusion, there are distinct signalling pathways and genes associated with hypothermia, including tryptophan degradation and CIRBP expression, providing a possible link to the modulation of body temperature and early immunosuppression. Future studies may focus on the canonical signalling pathways presented in this paper to further investigate spontaneous hypothermia in sepsis.

The Long Non-Coding Antisense RNA JHDM1D-AS1 Regulates Inflammatory Responses in Human Monocytes.

Monocytes are key players in innate immunity, with their ability to regulate inflammatory responses and combat invading pathogens. There is a growing body of evidence indicating that long non-coding RNA (lncRNA) participate in various cellular biological processes, including the innate immune response. The immunoregulatory properties of numerous lncRNAs discovered in monocytes remain largely unexplored. Here, by RNA sequencing, we identified a lncRNA JHDM1D-AS1, which was upregulated in blood monocytes obtained from patients with sepsis relative to healthy controls. JHDM1D-AS1 expression was induced in primary human monocytes exposed to Toll-like receptor ligands, such as lipopolysaccharide (LPS), or bacteria. The inducibility of JHDM1D-AS1 expression in monocytes depended, at least in part, on nuclear factor-κB activation. JHDM1D-AS1 knockdown experiments in human monocyte-derived macrophages revealed significantly enhanced expression of inflammatory mediators, before and after exposure to LPS, relative to control cells. Specifically, genes involved in inflammatory responses were upregulated (e.g., CXCL2, CXCL8, IL1RN, TREM1, TNF, and IL6), whereas genes involved in anti-inflammatory pathways were downregulated (e.g., SOCS1 and IL10RA). JHDM1D-AS1 overexpression in a pro-monocytic cell line revealed diminished pro-inflammatory responses subsequent to LPS challenge. Collectively, these findings identify JHDM1D-AS1 as a potential anti-inflammatory mediator induced in response to inflammatory stimuli.

Immune suppression is associated with enhanced systemic inflammatory, endothelial and procoagulant responses in critically ill patients.

OBJECTIVE: Patients admitted to the Intensive Care Unit (ICU) oftentimes show immunological signs of immune suppression. Consequently, immune stimulatory agents have been proposed as an adjunctive therapy approach in the ICU. The objective of this study was to determine the relationship between the degree of immune suppression and systemic inflammation in patients shortly after admission to the ICU. Design: An observational study in two ICUs in the Netherlands. METHODS: The capacity of blood leukocytes to produce cytokines upon stimulation with lipopolysaccharide (LPS) was measured in 77 patients on the first morning after ICU admission. Patients were divided in four groups based on quartiles of LPS stimulated tumor necrosis factor (TNF)-α release, reflecting increasing extents of immune suppression. 15 host response biomarkers indicative of aberrations in inflammatory pathways implicated in sepsis pathogenesis were measured in plasma. RESULTS: A diminished capacity of blood leukocytes to produce TNF-α upon stimulation with LPS was accompanied by a correspondingly reduced ability to release of IL-1ß and IL-6. Concurrently measured plasma concentrations of host response biomarkers demonstrated that the degree of reduction in TNF-α release by blood leukocytes was associated with increasing systemic inflammation, stronger endothelial cell activation, loss of endothelial barrier integrity and enhanced procoagulant responses. CONCLUSIONS: In patients admitted to the ICU the strongest immune suppression occurs in those who simultaneously display signs of stronger systemic inflammation. These findings may have relevance for the selection of patients eligible for administration of immune enhancing agents. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01905033.

Myeloid DNA methyltransferase3b deficiency aggravates pulmonary fibrosis by enhancing profibrotic macrophage activation.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and severe disease characterized by excessive matrix deposition in the lungs. Macrophages play crucial roles in maintaining lung homeostasis but are also central in the pathogenesis of lung diseases like pulmonary fibrosis. Especially, macrophage polarization/activation seems to play a crucial role in pathology and epigenetic reprograming is well-known to regulate macrophage polarization. DNA methylation alterations in IPF lungs have been well documented, but the role of DNA methylation in specific cell types, especially macrophages, is poorly defined. METHODS: In order to determine the role of DNA methylation in macrophages during pulmonary fibrosis, we subjected macrophage specific DNA methyltransferase (DNMT)3B, which mediates the de novo DNA methylation, deficient mice to the bleomycin-induced pulmonary fibrosis model. Macrophage polarization and fibrotic parameters were evaluated at 21 days after bleomycin administration. Dnmt3b knockout and wild type bone marrow-derived macrophages were stimulated with either interleukin (IL)4 or transforming growth factor beta 1 (TGFB1) in vitro, after which profibrotic gene expression and DNA methylation at the Arg1 promotor were determined. RESULTS: We show that DNMT3B deficiency promotes alternative macrophage polarization induced by IL4 and TGFB1 in vitro and also enhances profibrotic macrophage polarization in the alveolar space during pulmonary fibrosis in vivo. Moreover, myeloid specific deletion of DNMT3B promoted the development of experimental pulmonary fibrosis. CONCLUSIONS: In summary, these data suggest that myeloid DNMT3B represses fibrotic macrophage polarization and protects against bleomycin induced pulmonary fibrosis.