Edward Abraham

MicroRNA let-7c Regulates Macrophage Polarization

Macrophages demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. The role of microRNAs (miRNAs) in regulating macrophage polarization has been largely undefined. In this study, we found that miRNA let-7c is expressed at a higher level in M-BMM (M2 macrophages) than in GM-BMM (M1 macrophages). let-7c levels are also greater in alveolar macrophages from fibrotic lungs as compared with those from normal lungs. let-7c expression was decreased when M-BMM converted to GM-BMM, whereas it increased when GM-BMM converted to M-BMM. LPS stimulation reduced let-7c expression in M-BMM. We found that overexpression of let-7c in GM-BMM diminished M1 phenotype expression while promoting polarization to the M2 phenotype. In contrast, knockdown of let-7c in M-BMM promoted M1 polarization and diminished M2 phenotype expression. We found that let-7c targets C/EBP-δ, a transcriptional factor that plays an important role in inflammatory response. Furthermore, we found that let-7c regulates bactericidal and phagocytic activities of macrophages, two functional phenotypes implicated in macrophage polarization. Our data suggest that the miRNA let-7c plays an important role in regulating macrophage polarization.

HMGB1 promotes neutrophil extracellular trap formation through interactions with Toll-like receptor 4

Although neutrophil extracellular traps (NETs) form to prevent dissemination of pathogenic microorganisms, excessive release of DNA and DNA-associated proteins can also perpetuate sterile inflammation. In this study, we found that the danger-associated molecular pattern protein high-mobility group box 1 (HMGB1) can induce NET formation. NET formation was found after exposure of wild-type and receptor for advanced glycation end products-deficient neutrophil to HMGB1, whereas deficiency of Toll-like receptor (TLR)4 diminished the ability of neutrophils to produce NETs. Incubation of neutrophils with HMGB1 significantly increased the amount of DNA and histone 3 released as well as intracellular histone 3 citrullination, a signaling event that precedes chromatin decondensation. In vivo, neutrophils isolated from bronchoalveolar lavages of mice exposed to LPS and HMGB1 showed consistently greater ability to produce NETs compared with pulmonary neutrophils from mice that received LPS alone. In contrast, mice treated with LPS and neutralizing antibody to HMGB1 had decreased amounts of the inflammatory cytokines TNF-α and macrophage inflammatory protein 2, as well as of free DNA and histone 3 in bronchoalveolar lavage fluids. Airway neutrophils from LPS-exposed mice that had been treated with anti-HMGB1 antibodies showed decreased citrullination of histone 3. These results demonstrate that interactions between HMGB1 and TLR4 enhance the formation of NETs and provide a novel mechanism through which HMGB1 may contribute to the severity of neutrophil-associated inflammatory conditions.

The human long noncoding RNA lnc‐IL7R regulates the inflammatory response

Long noncoding RNAs (lncRNAs), once thought to be transcriptional noise, have been recently shown to regulate a variety of biological processes. However, there is not much knowledge regarding their roles in the inflammatory response. In this study, we performed human lncRNA microarray assays and identified a number of lncRNAs that demonstrated altered expression in response to LPS stimulation. Of these lncRNAs, lnc‐IL7R, which overlaps with the 3′untranslated region (3′UTR) of the human interleukin‐7 receptor α‐subunit gene (IL7R) gene, was significantly upregulated in LPS‐treated cells. Functionally, lnc‐IL7R was capable of diminishing the LPS‐induced inflammatory response, demonstrated by elevated expression of LPS‐induced E‐selectin, VCAM‐1, IL‐6, and IL‐8 in lnc‐IL7R knockdown cells. Mechanistically, we found that lnc‐IL7R knockdown diminished trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of silent transcription, at the proximal promoters of the inflammatory mediators. Our data suggest that lnc‐IL7R contributes another layer of complexity in regulation of the inflammatory response.

miR-125a-5p Regulates Differential Activation of Macrophages and Inflammation

Background: The role of miRNAs in macrophage polarization has not been well defined. Results: miR-125a-5p promotes M2, while it suppresses M1 macrophage polarization. Conclusion: miR-125a-5p is an important regulator of the differential activation of macrophages. Significance: Delineation of the role of miR-125a-5p in macrophage polarization provides insights into miRNA regulation of inflammation, tissue repair and tumor progression. Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.

Pyruvate Dehydrogenase Kinase 1 Participates in Macrophage Polarization via Regulating Glucose Metabolism

The M1 and M2 polarized phenotypes dictate distinctive roles for macrophages as they participate in inflammatory disorders. There has been growing interest in the role of cellular metabolism in macrophage polarization. However, it is currently unclear whether different aspects of a specific metabolic program coordinately regulate this cellular process. In this study, we found that pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, plays an important role in the differential activation of macrophages. Knockdown of PDK1 diminished M1, whereas it enhanced M2 activation of macrophages. Mechanistically, PDK1 knockdown led to diminished aerobic glycolysis in M1 macrophages, which likely accounts for the attenuated inflammatory response in these cells. Furthermore, we found that mitochondrial respiration is enhanced during and required by the early activation of M2 macrophages. Suppression of glucose oxidation, but not that of fatty acids, inhibits this process. Consistent with its inhibitory role in early M2 activation, knockdown of PDK1 enhanced mitochondrial respiration in macrophages. Our data suggest that two arms of the glucose metabolism synergistically regulate the differential activation of macrophages. Our findings also highlight the central role of PDK1 in this event via controlling glycolysis and glucose oxidation.

Pneumonia Prevention to Decrease Mortality in Intensive Care Unit: A Systematic Review and Meta-analysis

BACKGROUNDnTo determine the strategies of prevention of hospital-acquired pneumonia that reduce mortality in intensive care unit (ICU).nnnMETHODSnWe followed PRISMA (Preferred Reported Items for Systemic Reviews and Meta-Analyses) guidelines. We searched MEDLINE and the Cochrane Controlled Trials Register (through 10 June 2014) as well as reference lists of articles. We included all randomized controlled trials conducted in critically ill adult patients hospitalized in ICUs and evaluating digestive prophylactic methods (selective digestive decontamination [SDD], acidification of gastric content, early enteral feeding, prevention of microinhalation); circuit prophylactic methods (closed suctioning systems, early tracheotomy, aerosolized antibiotics, humidification, lung secretion drainage, silver-coated endotracheal tubes) or oropharyngeal prophylactic methods (selective oropharyngeal decontamination, patient position, sinusitis prophylaxis, subglottic secretion drainage, tracheal cuff monitoring). One reviewer extracted data that were checked by 3 others. The primary outcome was the mortality rate in the ICU.nnnRESULTSnWe identified 157 randomized trials to pool in a meta-analysis. The primary outcome was available in 145 studies (n = 37 156). The risk ratio (RR) for death was 0.95 (95% confidence interval [CI], .92-.99; P = .02) in the intervention groups. In subgroup analysis, only SDD significantly decreased mortality compared with control (n = 10 227; RR, 0.84 [95% CI, .76-.92; P < .001]). The RR for in-ICU death was 0.78 (95% CI, .69-.89; P < .001; I(2) = 33%) in trials investigating SDD with systemic antimicrobial therapy and 1.00 (.84-1.21; P = .96; I(2) = 0%) without systemic antimicrobial therapy.nnnCONCLUSIONSnSelective digestive decontamination with systemic antimicrobial therapy reduced mortality and should be considered in critically ill patients at high risk for death.

miR-27a Regulates Inflammatory Response of Macrophages by Targeting IL-10

Although microRNAs were shown to participate in innate immune responses, it is not completely understood how they regulate negative immunomodulatory events. IL-10 is an important anti-inflammatory mediator that prevents excessive inflammation and associated immunological pathologies. Although the regulation of IL-10 expression has been well studied at both the transcriptional and translational levels, it is less clear how microRNAs control IL-10 expression during inflammation. In this study, we found that miR-27a is downregulated in macrophages following stimulation through TLR2 and TLR4, but not TLR3. Upregulation of miR-27a enhanced the expression of proinflammatory cytokines in TLR2/4-activated macrophages. Conversely, knockdown of miR-27a diminished cytokine expression. Mechanistically, we found that miR-27a negatively regulates IL-10 expression; upregulation of miR-27a decreases, whereas downregulation of miR-27a increases, IL-10 expression in activated macrophages. Likely due to the decreased expression of IL-10, upregulation of miR-27a diminished IL-10–dependent STAT3 phosphorylation in TLR4-activated macrophages. Consistent with IL-10 being a potential mediator for the role of miR-27a in the immune response, blocking IL-10 abolished the enhancing effect of miR-27a on TLR4-activated inflammation. In conclusion, our study identified miR-27a downregulation as a negative-regulatory mechanism that prevents overly exuberant TLR2- and TLR4-driven inflammatory responses.

New Definitions for Sepsis and Septic Shock: Continuing Evolution but With Much Still to Be Done

The diagnosis of sepsis is not a new concern. Indeed, as early as 700 BCE, the Greeks recognized Σήψις (sepsis), referring to decomposition or rot, as a life-threatening condition associated with infection and high riskofdeath.Theprimarycriterion for sepsis has historically been progressive organ system dysfunction resulting frominfection.Because theonlyavailable therapies for this condition, antimicrobials and supportive care, are not specific, therewas little concern about developingmore detailed standards for diagnosis. Over the past 30 years, 2 major factors have led to a perceived need for better definitions. In particular, the increasing sophistication, at least in high-income countries, of modalities available for organ support in critical care units, including ventilators and dialysis, has resulted in growing numbers of patients with sepsis receiving care in intensive care units (ICUs) and enhanced awareness of the frequency and high costs associated with this condition. In addition, greater understanding of the underlying pathophysiologic mechanisms responsible for cellular dysfunction in experimental models and in patients with severe infection has accelerated the need for better entry criteria in clinical trials using therapies specifically directed toward molecular events thought to contribute to sepsis-associated morbidity and mortality. In this issue of JAMA, the Sepsis Definitions Task Force presents 3 articles: the updated definitions for sepsis and sepsis shock1 and 2 supporting reports with evidence for derivation and validation of these new definitions.2,3 In their Special Communication, Singer and colleagues1 describe the importance, process used, issues addressed, key findings from the evidence, and synthesis to develop the third iteration of consensus conference definitions for sepsis and septic shock and present the new definitions in detail. Previous versions of these definitions date from 1992 and 2003.4,5 A major underpinning for the present effort was the use of analyses in large cohorts to provide quantitative information in support of the revised criteria. The accompanying report by Seymour and colleagues2 assesses the predictive validity of the Sequential [Sepsisrelated] Organ Failure Assessment score (SOFA), systemic inflammatory response syndrome (SIRS) criteria, and the Logistic Organ Dysfunction System (LODS) score and derived a new score called quickSOFA (qSOFA) in a primary cohort that included 148907 patient encounters with suspected sepsis and a confirmatory analysis that included 706399 out-ofhospital and hospital patient encounters at 165 US and non-US hospitals. The investigators found that among ICU encounters with suspected infection (n = 7932), the predictive validity for in-hospital mortality of SOFA (area under the receiver operating characteristic curve [AUROC], 0.74 [95% CI, 0.73-0.76]) was not significantly different than that derived from the more complex LODS (AUROC, 0.75 [95% CI, 0.73-0.76]) but was superior to that from SIRS (AUROC, 0.64 [95% CI, 0.62-0.66]), supporting use of SOFA in clinical criteria for sepsis. Among patient encounters with suspected infection outside the ICU (n = 66522), qSOFA had high predictive validity for in-hospital mortality (AUROC, 0.81 [95% CI, 0.80-0.82]) that was statistically greater than that for SIRS (AUROC, 0.76 [95% CI, 0.75-0.77]), suggesting that it may have utility as a prompt to consider possible sepsis. In the other accompanying report, Shankar-Hari and colleagues3 describe the process of developing a new definition and clinical criteria for identifying septic shock in adults. The authors conducted a systematic review and meta-analysis of 92 studies informing a Delphi process that created the new definition, then tested the variables identified by the Delphi process in cohort studies (Surviving Sepsis Campaign [n = 28 150; University of Pittsburgh Medical Center [n = 1 309025], and Kaiser Permanente Northern California [n = 1847 165]). According to the new definitions, sepsis is now defined as evidence of infection plus life-threatening organ dysfunction, clinically characterized by an acute change of 2 points or greater in the SOFA score. The new clinical criteria for septic shock include sepsis with fluid-unresponsive hypotension, serum lactate level greater than 2 mmol/L, and the need for vasopressors to maintain mean arterial pressure of 65 mmHg or greater. A major change in the new definitions is the elimination of mention of SIRS. Components of SIRS include tachycardia, tachypnea, hyperthermia or hypothermia, and abnormalities in peripheral white blood cell count. Many studies have shown that the presence of SIRS is nearly ubiquitous in hospitalized patients and occurs in many benign conditions, both related and not related to infection, and thus is not adequately specific for the diagnosis of sepsis.6 It is a strength of the consensus definition that it no longer includes SIRS. Patients with infections and organ dysfunction are exceptionally heterogeneous in terms of demographic characteristics, underlying conditions, microbiology, and other clinically relevant factors.7 The updated definition for sepsis, Related articles pages 762, 775 and 801 Opinion

Toll-Like Receptor 4 Engagement Inhibits Adenosine 5′-Monophosphate–Activated Protein Kinase Activation through a High Mobility Group Box 1 Protein–Dependent Mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5′-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H2O2) to activate AMPK. Although ratios of AMP to adenosine 5′-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.

Clinical and translational medicine: Integrative and practical science.

Clinical and translational medicine plays a unique and critical role in fostering the flow of bidirectional information between basic and clinical scientists, optimizing new biotechnologies, improving clinical application of new therapeutic concepts, and ultimately improving the quality of life for patients. The term Clinical and Translational Medicine (CMT) is defined here as clinical potential and application of translational research and science to improve the understanding of mechanisms and therapies of human diseases, a new and important concept for the development of disease-specific biomarkers and therapeutic strategies to monitor and cure disease. Clinical and Translational Medicine as an international open-access journal publish articles focused on novel advances in clinical, translational, and clinically-relevant basic science, to accelerate the transition from preclinical research to clinical applications and the communication between basic and clinical scientists. Clinical and Translational Medicine is particularly interested in drug discovery and development, related regulatory processes, as well as broader topics related to public health and relevant policies. Clinical and Translational Medicine will also focus on new insights into molecular mechanisms, clinical questions and experience, as well as the discovery and development of personalized healthcare delivery.