Type IIA Secreted Phospholipase A2 in Host Defense against Bacterial Infections.

The enzyme type IIA secreted phospholipase A2 (sPLA2-IIA) is crucial for mammalian innate host defense against bacterial pathogens. Most studies have investigated the role of sPLA2-IIA in systemic bacterial infections, identifying molecular pathways of bacterial resistance against sPLA2-IIA-mediated killing, and providing insight into sPLA2-IIA mechanisms of action. Sensitization of (antibiotic-resistant) bacteria to sPLA2-IIA action by blocking bacterial resistance or by applying sPLA2-IIA to treat bacterial infections might represent a therapeutic option in the future. Because sPLA2-IIA is highly expressed at mucosal barriers, we also discuss how sPLA2-IIA is likely to be an important driver of microbiome composition; we anticipate that future research in this area may bring new insights into the role of sPLA2-IIA in health and disease.

Lipopolysaccharide reduces USP13 stability through c-Jun N-terminal kinase activation in Kupffer cells.

Protein ubiquitination regulates protein stability, cellular localization, and enzyme activity. Deubiquitinases catalyze the removal of ubiquitin from target proteins and reverse ubiquitination. USP13, a deubiquitinase, has been shown to regulate a variety of cellular responses including inflammation; however, the molecular regulation of USP13 has not been demonstrated. In this study, we revealed that USP13 is degraded in response to lipopolysaccharide (LPS) in Kupffer cells. USP13 levels are significantly decreased in inflamed organs, including liver tissues from septic mice. LPS reduces USP13 protein stability, not transcription, in Kupffer cells. Furthermore, LPS increases USP13 polyubiquitination. Inhibition of proteasome, but not lysosome or immunoproteasome, attenuates LPS-induced USP13 degradation, suggesting USP13 degradation is mediated by the ubiquitin-proteasome system. A catalytically inactive form of USP13 exhibits similar degree of degradation compared with USP13 wild-type, suggesting that USP13 degradation is not dependent on its activity. Furthermore, USP13 degradation is dependent on new protein synthesis. Inhibition of c-Jun N-terminal kinase (JNK) attenuates USP13 degradation, indicating that JNK-dependent new protein synthesis is necessary for USP13 degradation. This study reveals a molecular mechanism of regulation of USP13 degradation in Kupffer cells in response to bacterial endotoxin.

Chrysin Ameliorates Sepsis-Induced Cardiac Dysfunction Through Upregulating Nfr2/Heme Oxygenase 1 Pathway.

ABSTRACT: The incidence of myocardial dysfunction caused by sepsis is high, and the mortality of patients with sepsis can be significantly increased. During sepsis, oxidative stress and inflammation can lead to severe organ dysfunction. Flavone chrysin is one of the indispensable biological active ingredients for different fruits and vegetables and has antioxidant and anti-inflammatory properties. However, it is not clear whether chrysin is an effective treatment for heart dysfunction caused by sepsis. We found that it had protective effects against the harmful effects caused by LPS, manifested in improved survival, normalized cardiac function, improved partial pathological scores of myocardial tissue, and remission of apoptosis, as well as reduced oxidative stress and inflammation. Mechanism studies have found that chrysin is an important antioxidant protein, a key regulator of heme oxygenase 1 (HO-1). We found that HO-1 levels were increased after LPS intervention, and chrysin further increased HO-1 levels, along with the addition of Nrf2, a regulator of antioxidant proteins. Pretreatment with PD98059, an extracellular signal-regulated kinase-specific inhibitor, blocked chrysin-mediated phosphorylation of Nrf2 and the nuclear translocation of Nrf2. The protective effect of chrysin on sepsis-induced cardiac dysfunction was blocked by ZnPP, which is a HO-1 blocker. Chrysin increased antioxidant activity and reduced markers of oxidative stress (SOD and MDA) and inflammation (MPO and IL-1ß), all of which were blocked by ZnPP. This indicates that HO-1 is the upstream molecule regulating the protective effect of chrysin. Thus, by upregulation of HO-1, chrysin protects against LPS-induced cardiac dysfunction and inflammation by inhibiting oxidative stress.

Prekallikrein inhibits innate immune signaling in the lung and impairs host defense during pneumosepsis in mice.

Prekallikrein (PKK, also known as Fletcher factor and encoded by the gene KLKB1 in humans) is a component of the contact system. Activation of the contact system has been implicated in lethality in fulminant sepsis models. Pneumonia is the most frequent cause of sepsis. We sought to determine the role of PKK in host defense during pneumosepsis. To this end, mice were infected with the common human pathogen Klebsiella pneumoniae via the airways, causing an initially localized infection of the lungs with subsequent bacterial dissemination and sepsis. Mice were treated with a selective PKK-directed antisense oligonucleotide (ASO) or a scrambled control ASO for 3 weeks prior to infection. Host response readouts were determined at 12 or 36 h post-infection, including genome-wide messenger RNA profiling of lungs, or mice were followed for survival. PKK ASO treatment inhibited constitutive hepatic Klkb1 mRNA expression by >80% and almost completely abolished plasma PKK activity. Klkb1 mRNA could not be detected in lungs. Pneumonia was associated with a progressive decline in PKK expression in mice treated with control ASO. PKK ASO administration was associated with a delayed mortality, reduced bacterial burdens, and diminished distant organ injury. While PKK depletion did not influence lung pathology or neutrophil recruitment, it was associated with an upregulation of multiple innate immune signaling pathways in the lungs already prior to infection. Activation of the contact system could not be detected, either during infection in vivo or at the surface of Klebsiella in vitro. These data suggest that circulating PKK confines pro-inflammatory signaling in the lung by a mechanism that does not involve contact system activation, which in the case of respiratory tract infection may impede early protective innate immunity. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Global Lysine Acetylome Analysis of LPS-Stimulated HepG2 Cells Identified Hyperacetylation of PKM2 as a Metabolic Regulator in Sepsis.

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.

Transglutaminase 2 as a Marker for Inflammation and Therapeutic Target in Sepsis.

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

WNK1-TAK1 signaling suppresses lipopolysaccharide-induced cytokine production and classical activation in macrophages.

With-no-lysine kinase (WNK) plays important roles in regulating electrolyte homeostasis, cell signaling, survival, and proliferation. It has been recently demonstrated that WNK1, a member of the WNK family, modifies the function of immune cells. Here we report that in macrophages, WNK1 has suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses via TGFß-activated kinase 1 (TAK1)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. We found that WNK1 heterozygous (WNK1+/-) mice produced excessive proinflammatory cytokines in an experimental LPS-induced sepsis model, and peritoneal macrophages isolated from WNK1+/- mice produced higher levels of LPS-induced cytokines and NOS2 expression as canonical proinflammatory M1 macrophage markers. We confirmed that small hairpin RNA (shRNA)-mediated knockdown of WNK1 activated LPS-induced cytokine production and NOS2 expression in RAW 264.7 macrophages. Moreover, we demonstrated that WNK1 knockdown increased the nuclear translocation of NF-κB and activated the p38 and Jun N-terminal kinase (JNK) MAPK signaling pathway and that a TAK1 inhibitor diminished these effects of WNK1 knockdown. These results suggest that WNK1 acts as a physiologic immune modulator via interactions with TAK1. WNK1 may be a therapeutic target against the cytokine storm caused by sepsis.

miR-128-3p enhances the protective effect of dexmedetomidine on acute lung injury in septic mice by targeted inhibition of MAPK14.

To investigate the role of miR-128-3p and MAPK14 in the dexmedetomidine treatment of acute lung injury in septic mice. SPF C57BL/6 mice were divided into 8 groups. The pathological changes and wet/dry weight ratio (W/D), PaO2, PaCO2, MDA, SOD and MPO levels in lung tissue and the serum levels of inflammation factors were observed. Dual luciferase reporter assay was used to detect the targeting relationship of miR-128-3p and MAPK14, and qPCR and WB were used to detect the expression of miR-128-3p and MAPK14. Compared with the Normal group, other groups had lower MDA, MPO, inflammatory factors levels and the expression level of MAPK14, while the content of SOD and the expression level of miR-128-3p was significantly decreased (all p < 0.05). Compared with the Model group, the contents of MDA, MPO, inflammatory factors in the DEX group and miR-128-3p mimic group were significantly decreased, and the content SOD was significantly increased, however, opposite results were occurred in oe-MAPK14 group (all p < 0.05). Compared with the DEX group, all the indicators in miR-128-3p mimic+DEX group showed significant improvement (all p < 0.05). Compared with the miR-128-3p mimic group, all the indicators were deteriorated in the miR-128-3p mimic+oe-MAPK14 group (all p < 0.05). The combination of DEX and oe-MAPK14 blocked the protective effect of dexmedetomidine on acute lung injury in septic mice. miR-128-3p can further enhance the protective effect of dexmedetomidine on acute lung injury in septic mice by targeting and inhibiting MAPK14 expression.

Loss of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels is coupled to persistent neuroinflammation and behavioral deficits in late sepsis.

Sepsis is a host response to systemic inflammation and infection that may lead to multi-organ dysfunction and eventual death. While acute brain dysfunction is common among all sepsis patients, chronic neurological impairment is prevalent among sepsis survivors. The brain microvasculature has emerged as a major determinant of sepsis-associated brain dysfunction, yet the mechanisms that underlie its associated neuroimmune perturbations and behavioral deficits are not well understood. An emerging body of data suggests that inhibition of tissue-nonspecific alkaline phosphatase (TNAP) enzyme activity in cerebral microvessels may be associated with changes in endothelial cell barrier integrity. The objective of this study was to elucidate the connection between alterations in cerebrovascular TNAP enzyme activity and brain microvascular dysfunction in late sepsis. We hypothesized that the disruption of TNAP enzymatic activity in cerebral microvessels would be coupled to the sustained loss of brain microvascular integrity, elevated neuroinflammatory responses, and behavioral deficits. Male mice were subjected to cecal ligation and puncture (CLP), a model of experimental sepsis, and assessed up to seven days post-sepsis. All mice were observed daily for sickness behavior and underwent behavioral testing. Our results showed a significant decrease in brain microvascular TNAP enzyme activity in the somatosensory cortex and spinal cord of septic mice but not in the CA1 and CA3 hippocampal regions. Furthermore, we showed that loss of cerebrovascular TNAP enzyme activity was coupled to a loss of claudin-5 and increased perivascular IgG infiltration in the somatosensory cortex. Analyses of whole brain myeloid and T-lymphoid cell populations also revealed a persistent elevation of infiltrating leukocytes, which included both neutrophil and monocyte myeloid derived suppressor cells (MDSCs). Regional analyses of the somatosensory cortex, hippocampus, and spinal cord revealed significant astrogliosis and microgliosis in the cortex and spinal cord of septic mice that was accompanied by significant microgliosis in the CA1 and CA3 hippocampal regions. Assessment of behavioral deficits revealed no changes in learning and memory or evoked locomotion. However, the hot plate test uncovered a novel anti-nociceptive phenotype in our septic mice, and we speculate that this phenotype may be a consequence of sustained GFAP astrogliosis and loss of TNAP activity in the somatosensory cortex and spinal cord of septic mice. Taken together, these results demonstrate that the loss of TNAP enzyme activity in cerebral microvessels during late sepsis is coupled to sustained neuroimmune dysfunction which may underlie, in part, the chronic neurological impairments observed in sepsis survivors.

Heme Oxygenase-1 in Macrophages Drives Septic Cardiac Dysfunction via Suppressing Lysosomal Degradation of Inducible Nitric Oxide Synthase.

RATIONALE: To date, our understanding of the role of HO-1 (heme oxygenase-1) in inflammatory diseases has mostly been limited to its catalytic function and the potential for its heme-related catabolic products to suppress inflammation and oxidative stress. Whether and how HO-1 in macrophages plays a role in the development of septic cardiac dysfunction has never been explored. OBJECTIVE: Here, we investigated the role of macrophage-derived HO-1 in septic cardiac dysfunction. METHODS AND RESULTS: Intraperitoneal injection of lipopolysaccharide significantly activated HO-1 expression in cardiac infiltrated macrophages. Surprisingly, we found that myeloid conditional HO-1 deletion in mice evoked resistance to lipopolysaccharide-triggered septic cardiac dysfunction and lethality in vivo, which was accompanied by reduced cardiomyocyte apoptosis in the septic hearts and decreased peroxynitrite production and iNOS (inducible NO synthase) in the cardiac infiltrated macrophages, whereas proinflammatory cytokine production and macrophage infiltration were unaltered. We further demonstrated that HO-1 suppression abolished the lipopolysaccharide-induced iNOS protein rather than mRNA expression in macrophages. Moreover, we confirmed that the inhibition of HO-1 promoted iNOS degradation through a lysosomal rather than proteasomal pathway in macrophages. Suppression of the lysosomal degradation of iNOS by bafilomycin A1 drove septic cardiac dysfunction in myeloid HO-1-deficient mice. Mechanistically, we demonstrated that HO-1 interacted with iNOS at the flavin mononucleotide domain, which further prevented iNOS conjugation with LC3 (light chain 3) and subsequent lysosomal degradation in macrophages. These effects were independent of HO-1's catabolic products: ferrous ion, carbon monoxide, and bilirubin. CONCLUSIONS: Our results indicate that HO-1 in macrophages drives septic cardiac dysfunction. The mechanistic insights provide potential therapeutic targets to treat septic cardiac dysfunction.

Sirt3 is a novel target to treat sepsis induced myocardial dysfunction by acetylated modulation of critical enzymes within cardiac tricarboxylic acid cycle.

Sepsis induced myocardial dysfunction (SIMD) results in high morbidity and mortality. However, the effective therapeutic strategies for SIMD treatment remain limited. Sirt3 is the main mitochondrial Sirtuin member and is a key modulator of mitochondrial metabolism and function. In this study, we aimed to investigate the effect and mechanism of Sirt3 on SIMD. SIMD was induced by 20 mg/kg Lipopolysaccharides (LPS) injection for 6 h in mice. Sepsis could induce the reduction of cardiac Sirt3 expression and global deficiency of Sirt3 exacerbated cardiac function. Quantitative acetyl-proteomics and cardiac metabolomics analysis revealed that loss of Sirt3 led to hyper-acetylation of critical enzymes within cardiac tricarboxylic acid (TCA) cycle and generation of lactate and NADH, subsequently promotion of cardiac dysfunction after sepsis. Additionally, to evaluate whether Emodin could be utilized as a potential Sirt3 modulator to treat SIMD, male wild type mice (WT mice) or global Sirt3 deficient mice (Sirt3-/- mice) were intraperitoneally injected with 40 mg/kg Emodin for 5 days followed by 20 mg/kg LPS administration for another 6 h and observed that exogenous administration of Emodin could attenuate myocardial dysfunction in septic WT mice. However, septic Sirt3-/- mice can not gain benefit on cardiac performance from Emodin infusion. In conclusion, this study presented the protective role of Sirt3 targeting SIMD, which may provide a potential novel approach to maintain normal cardiac performance after sepsis.

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Klebsiella pneumoniae-Induced Pneumonia-Derived Sepsis.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site-coordinating core ß-sheet-containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

SIRT1-mediated HMGB1 deacetylation suppresses sepsis-associated acute kidney injury.

Sepsis is the leading cause of death in the intensive care unit and continues to lack effective treatment. It is widely accepted that high-mobility group box 1 (HMGB1) is a key inflammatory mediator in the pathogenesis of sepsis. Moreover, some studies indicate that the functions of HMGB1 depend on its molecular localization and posttranslational modifications. Our previous study confirms that sirtuin 1, silent information regulator 2-related enzyme 1 (SIRT1), a type III deacetylase, can ameliorate sepsis-associated acute kidney injury (SA-AKI). We explored the effect and mechanism of SIRT1 on HMGB1 using a mouse model of cecal ligation and puncture-induced sepsis and LPS-treated human kidney (HK-2) cell line. We found that HMGB1 is elevated in the serum but is gradually reduced in kidney cells in the later stages of septic mice. The acetylation modification of HMGB1 is a key process before its nucleus-to-cytoplasm translocation and extracellular secretion in kidney cells, accelerating the development of SA-AKI. Moreover, SIRT1 can physically interact with HMGB1 at the deacetylated lysine sites K28, K29, and K30, subsequently suppressing downstream inflammatory signaling. Thus the SIRT1-HMGB1 signaling pathway is a crucial mechanism in the development of SA-AKI and presents a novel experimental perspective for future SA-AKI research.

Acute downregulation of miR-199a attenuates sepsis-induced acute lung injury by targeting SIRT1.

MicroRNA-199a (miR-199a) is a novel gene regulator with an important role in inflammation and lung injury. However, its role in the pathogenesis of sepsis-induced acute respiratory distress syndrome (ARDS) is currently unknown. Our study explored the role of miR-199a in sepsis-induced ARDS and its mechanism of action. First, we found that LPS could upregulate miR-199a in alveolar macrophages. Downregulation of miR-199a inhibited the upregulation of inflammatory cytokines in alveolar macrophages and induced the remission of histopathologic changes, the reduction of proinflammatory cytokines, and the upregulation of apoptosis protein expression in an ARDS lung, showing a protective role for miR-199a. We further identified sirtuin 1 (SIRT1) as a direct target of miR-199a in alveolar macrophages, and the expression of SIRT1 was negatively correlated with the level of miR-199a. The protective role of miR-199a downregulation in LPS-stimulated alveolar macrophages and sepsis-induced ARDS could be attenuated by SIRT1 inhibitor. Taken together, these results indicate that downregulation of miR-199a might protect lung tissue against sepsis-induced ARDS by upregulation of SIRT1 through the suppression of excessive inflammatory responses and the inhibition of cellular apoptosis in lung tissue, suggesting its potential therapeutic effects on sepsis-induced ARDS.

Therapeutic potency of mTOR signaling pharmacological inhibitors in the treatment of proinflammatory diseases, current status, and perspectives.

Mammalian target of rapamycin (mTOR) signaling pathway controls cell energy metabolism. There is an interplay between mTOR and proinflammatory signaling pathways, supporting the role of the pathway in the pathogenesis of inflammatory diseases. Inhibition of mTOR signaling using specific pharmacological inhibitors could offer therapeutic promise in several inflammatory-associated diseases. In this review, we summarize recent findings on the regulatory effects of mTOR signaling on inflammation and the therapeutic potency of mTOR pharmacological inhibitors in the treatment of inflammatory diseases including cancer, neurodegenerative diseases, atherosclerosis, sepsis, and rheumatoid arthritis for a better understanding and hence a better management of these diseases.

Age-dependent cardiac function during experimental sepsis: effect of pharmacological activation of AMP-activated protein kinase by AICAR.

Age represents a major risk factor for multiple organ failure, including cardiac dysfunction, in patients with sepsis. AMP-activated protein kinase (AMPK) is a crucial regulator of energy homeostasis that controls mitochondrial biogenesis by activation of peroxisome proliferator-activated receptor-γ coactivator-1α and disposal of defective organelles by autophagy. We investigated whether AMPK dysregulation contributes to age-dependent cardiac injury in young (2-3 mo) and mature adult (11-13 mo) male mice subjected to sepsis by cecal ligation and puncture and whether AMPK activation by 5-amino-4-imidazole carboxamide riboside affords cardioprotective effects. Plasma proinflammatory cytokines and myokine follistatin were similarly elevated in vehicle-treated young and mature adult mice at 18 h after sepsis. However, despite equivalent troponin I and T levels compared with similarly treated young mice, vehicle-treated mature adult mice exhibited more severe cardiac damage by light and electron microscopy analyses with more marked intercellular edema, inflammatory cell infiltration, and mitochondrial derangement. Echocardiography revealed that vehicle-treated young mice exhibited left ventricular dysfunction after sepsis, whereas mature adult mice exhibited a reduction in stroke volume without apparent changes in load-dependent indexes of cardiac function. At molecular analysis, phosphorylation of the catalytic subunits AMPK-α1/α2 was associated with nuclear translocation of peroxisome proliferator-activated receptor-γ coactivator-1α in vehicle-treated young but not mature adult mice. Treatment with 5-amino-4-imidazole carboxamide riboside ameliorated cardiac architecture derangement in mice of both ages. These cardioprotective effects were associated with attenuation of the systemic inflammatory response and amelioration of cardiac dysfunction in young mice only, not in mature adult animals. NEW & NOTEWORTHY Our data suggest that sepsis-induced cardiac dysfunction manifests with age-dependent characteristics, which are associated with a distinct regulation of AMP-activated protein kinase-dependent metabolic pathways. Consistent with this age-related deterioration, pharmacological activation of AMP-activated protein kinase may afford cardioprotective effects allowing a partial recovery of cardiac function in young but not mature age.

NADPH oxidases as potential pharmacological targets against increased seizure susceptibility after systemic inflammation.

BACKGROUND: Systemic inflammation associated with sepsis can induce neuronal hyperexcitability, leading to enhanced seizure predisposition and occurrence. Brain microglia are rapidly activated in response to systemic inflammation and, in this activated state, release multiple cytokines and signaling factors that amplify the inflammatory response and increase neuronal excitability. NADPH oxidase (NOX) enzymes promote microglial activation through the generation of reactive oxygen species (ROS), such as superoxide anion. We hypothesized that NOX isoforms, particularly NOX2, are potential targets for prevention of sepsis-associated seizures. METHODS: To reduce NADPH oxidase 2-derived ROS production, mice with deficits of NOX regulatory subunit/NOX2 organizer p47phox (p47phox-/-) or NOX2 major subunit gp91phox (gp91phox-/-) were used or the NOX2-selective inhibitor diphenyleneiodonium (DPI) was used to treat wild-type (WT) mice. Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS). Seizure susceptibility was compared among mouse groups in response to intraperitoneal injection of pentylenetetrazole (PTZ). Brain tissues were assayed for proinflammatory gene and protein expression, and immunofluorescence staining was used to estimate the proportion of activated microglia. RESULTS: Increased susceptibility to PTZ-induced seizures following sepsis was significantly attenuated in gp91phox-/- and p47phox-/- mice compared with WT mice. Both gp91phox-/- and p47phox-/- mice exhibited reduced microglia activation and lower brain induction of multiple proconvulsive cytokines, including TNFα, IL-1ß, IL-6, and CCL2, compared with WT mice. Administration of DPI following LPS injection significantly attenuated the increased susceptibility to PTZ-induced seizures and reduced both microglia activation and brain proconvulsive cytokine concentrations compared with vehicle-treated controls. DPI also inhibited the upregulation of gp91phox transcripts following LPS injection. CONCLUSIONS: Our results indicate that NADPH oxidases contribute to the development of increased seizure susceptibility in mice after sepsis. Pharmacologic inhibition of NOX may be a promising therapeutic approach to reducing sepsis-associated neuroinflammation, neuronal hyperexcitability, and seizures.

Heme oxygenase-1 deficiency promotes severity of sepsis in a non-surgical preterm mouse model.

BACKGROUND: Sepsis in preterm infants is associated with systemic inflammatory responses. The stress-response protein heme oxygenase-1 (HO-1) has protective anti-inflammatory properties. Recently, we reported a protective role of HO-1 using our non-surgical cecal slurry (CS) model in wild-type (WT) mouse pups. Here, we extend these findings to investigate the association of HO-1 deficiency with sepsis severity. METHODS: Adapting the Wynn model, we induced sepsis in 4-day-old HO-1-deficient (HO-1+/-, Het) pups to determine if HO-1 deficiency affected survival rates at the LD40 (2.0 mg/g) of WT pups. To see if HO-1 induction affected sepsis severity, we gave 30-µmol heme/kg subcutaneously to 3-day-old mice 24 h prior to sepsis induction. RESULTS: Post-sepsis induction, Het pups had a mortality of 85.0% (n = 20) and increased expression of the pro-inflammatory gene in the livers and affected hematologic profiles. Heme treatment 24 h prior to sepsis induction significantly increased liver HO activity, reduced mortality to 24.5% (n = 17), attenuated inflammatory responses, reduced spleen bacterial counts, and significantly increased peripheral neutrophils. CONCLUSIONS: A partial deficiency in HO-1 increased the progression and mortality in sepsis. Furthermore, induction of HO-1 significantly reduced the mortality even in Het pups. Thus, we conclude that HO-1 plays an important role in the protection against preterm sepsis.