Lipopolysaccharide-induced Proinflammatory Cytokine Production Research Articles

Modulating the RAGE-Induced Inflammatory Response: Peptoids as RAGE Antagonists.

While the primary pathology of Alzheimer's disease (AD) is defined by brain deposition of amyloid-β (Aβ) plaques and tau neurofibrillary tangles, chronic inflammation has emerged as an important factor in AD etiology. Upregulated cell surface expression of the receptor for advanced glycation end-products (RAGE), a key receptor of innate immune response, is reported in AD. In parallel, RAGE ligands, including Aβ aggregates, HMGB1, and S100B, are elevated in AD brain. Activation of RAGE by these ligands triggers release of inflammatory cytokines and upregulates cell surface RAGE. Despite such observation, there are currently no therapeutics that target RAGE for treatment of AD-associated neuroinflammation. Peptoids, a novel class of potential AD therapeutics, display low toxicity, facile blood-brain barrier permeability, and resistance to proteolytic degradation. In the current study, peptoids were designed to mimic Aβ, a ligand that binds the V-domain of RAGE, and curtail RAGE inflammatory activation. We reveal the nanomolar binding capability of peptoids JPT1 and JPT1a to RAGE and demonstrate their ability to attenuate lipopolysaccharide-induced pro-inflammatory cytokine production as well as upregulation of RAGE cell surface expression. These results support RAGE antagonist peptoid-based mimics as a prospective therapeutic strategy to counter neuroinflammation in AD and other neurodegenerative diseases.

Anti-inflammatory and wound healing properties of lactic acid bacteria and its peptides.

Recent studies manifest an increase of inflammatory diseases at an alarming rate due to gut microbiota dysbiosis, genetic and other environmental factors. Lactic acid bacteria (LAB) are known for their antimicrobial properties and their extensive applications in food and pharmaceutical industries. Cyclic peptides are receiving increased attention due to their remarkable stability to withstand variations in temperature and pH. LAB produces anti-inflammatory that can inhibit lipopolysaccharide-induced production of proinflammatory cytokines in macrophages. The structural backbones of cyclic peptides offer a promising approach for the treatment of chronic inflammatory conditions. The current review aims to present the overview of anti-inflammatory and wound healing properties of LAB-derived cyclic peptides.

Cardiolipin coordinates inflammatory metabolic reprogramming through regulation of Complex II disassembly and degradation.

Macrophage metabolic plasticity enables repurposing of electron transport from energy generation to inflammation and host defense. Altered respiratory complex II function has been implicated in cancer, diabetes, and inflammation, but regulatory mechanisms are incompletely understood. Here, we show that macrophage inflammatory activation triggers Complex II disassembly and succinate dehydrogenase subunit B loss through sequestration and selective mitophagy. Mitochondrial fission supported lipopolysaccharide-stimulated succinate dehydrogenase subunit B degradation but not sequestration. We hypothesized that this Complex II regulatory mechanism might be coordinated by the mitochondrial phospholipid cardiolipin. Cardiolipin synthase knockdown prevented lipopolysaccharide-induced metabolic remodeling and Complex II disassembly, sequestration, and degradation. Cardiolipin-depleted macrophages were defective in lipopolysaccharide-induced pro-inflammatory cytokine production, a phenotype partially rescued by Complex II inhibition. Thus, cardiolipin acts as a critical organizer of inflammatory metabolic remodeling.

Hericium erinaceus ethanol extract and ergosterol exert anti-inflammatory activities by neutralizing lipopolysaccharide-induced pro-inflammatory cytokine production in human monocytes

Edible mushrooms are known to exert anti-inflammatory effects. In this study, the effects of ethanol extracts from edible mushrooms, such as Hericium erinaceus, and other edible mushrooms on inflammatory responses were investigated. Experiments were conducted using the inflammatory responses of human monocytes induced by lipopolysaccharide (LPS), a bacterial component, that provokes inflammation. Notably, we demonstrated that LPS mixed with ethanol and hot water extracts derived from edible mushrooms attenuated the production of inflammatory cytokines, such as interleukin (IL)-1β, −6, and −8, induced by LPS in human monocytic cell cultures. Moreover, we found that the ethanol extract of H. erinaceus contained ergosterol, which attenuated IL-8 production in LPS-stimulated cells. Subsequent component analysis of the ethanol extract of H. erinaceus revealed that ergosterol binds to lipid A to attenuate LPS-induced inflammation. Together, our findings suggest that ergosterol in ethanol extracts from edible mushrooms can prevent the induction of inflammation by binding to LPS.

S-1-Propenylcysteine promotes IL-10-induced M2c macrophage polarization through prolonged activation of IL-10R/STAT3 signaling

Atherosclerosis is a chronic inflammatory disease that may lead to the development of serious cardiovascular diseases. Aged garlic extract (AGE) has been reported to ameliorate atherosclerosis, although its mode of action remains unclear. We found that AGE increased the mRNA or protein levels of arginase1 (Arg1), interleukin-10 (IL-10), CD206 and hypoxia-inducible factor 2α (HIF2α) and decreased that of CD68, HIF1α and inducible nitric oxide synthase in the aorta and spleen of apolipoprotein E knockout mice. We also found that S-1-propenylcysteine (S1PC), a characteristic sulfur compound in AGE, increased the level of IL-10-induced Arg1 mRNA and the extent of M2c-like macrophage polarization in vitro. In addition, S1PC increased the population of M2c-like macrophages, resulting in suppressed the population of M1-like macrophages and decreased lipopolysaccharide-induced production of pro-inflammatory cytokines. These effects were accompanied by prolonged phosphorylation of the IL-10 receptor α (IL-10Rα) and signal transducer and activator of transcription 3 (STAT3) that inhibited the interaction between IL-10Rα and Src homology-2-containing inositol 5’-phosphatase 1 (SHIP1). In addition, administration of S1PC elevated the M2c/M1 macrophage ratio in senescence-accelerated mice. These findings suggest that S1PC may help improve atherosclerosis due to its anti-inflammatory effect to promote IL-10-induced M2c macrophage polarization.

Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model.

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

Pharmacological Properties of JTE-952, an Orally Available and Selective Colony Stimulating Factor 1 Receptor Kinase Inhibitor.

Colony stimulating factor 1 (CSF1) receptor (CSF1R) is a receptor protein-tyrosine kinase specifically expressed in monocyte-lineage cells, such as monocytes and macrophages. In this study, we characterized the pharmacological properties of an azetidine compound, JTE-952 ((2S)-3-{[2-({3-[4-(4-cyclopropylbenzyloxy)-3-methoxyphenyl]azetidine-1-yl}carbonyl)pyridin-4-yl]methoxy}propane-1,2-diol), which is a novel CSF1R tyrosine kinase inhibitor. JTE-952 potently inhibited human CSF1R kinase activity, with a half maximal inhibitory concentration of 11.1 nmol/L, and inhibited the phosphorylation of CSF1R in human macrophages and the CSF1-induced proliferation of human macrophages. It also inhibited human tropomyosin-related kinase A activity, but only at concentrations 200-fold higher than that required to inhibit the activity of CSF1R in inducing the proliferation of human macrophages. JTE-952 displayed no marked inhibitory activity against other kinases. JTE-952 potently inhibited lipopolysaccharide-induced proinflammatory cytokine production by human macrophages and in whole blood. JTE-952 (≥3 mg/kg given orally) also significantly attenuated the CSF1-induced priming of lipopolysaccharide-induced tumor necrosis factor-alpha production in mice and arthritis severity in a mouse model of collagen-induced arthritis. Taken together, these results indicate that JTE-952 is an orally available compound with potent and specific inhibitory activity against CSF1R, both in vitro and in vivo. JTE-952 is a potentially clinically useful agent for various human inflammatory diseases, including rheumatoid arthritis.

Krill Oil-Incorporated Liposomes As An Effective Nanovehicle To Ameliorate The Inflammatory Responses Of DSS-Induced Colitis.

BackgroundPhosphatidylcholine (PC) and Omega-3 fatty acid (Omega-3) are promising therapeutic molecules for treating inflammatory bowel disease (IBD).PurposeBased on the IBD therapeutic potential of nanoparticles, we herein sought to develop Omega-3-incorporated PC nanoparticles (liposomes) as an orally administrable vehicle for treating IBD.MethodsLiposomes prepared with or without Omega-3 incorporation were compared in terms of colloidal stability and anitiinflammatory effects.ResultsThe incorporation of free Omega-3 (alpha-linolenic acid, eicosapentaenoic acid or docosahexaenoic acid) into liposomes induced time-dependent membrane fusion, resulting in particle size increase from nm to μm during storage. In contrast, krill oil incorporation into liposomes (KO liposomes) did not induce the fusion and the particle size maintained <250 nm during storage. KO liposomes also maintained colloidal stability in simulated gastrointestinal conditions and exhibited a high capacity to entrap the IBD drug, budesonide (BDS). KO liposomes greatly suppressed the lipopolysaccharide-induced production of pro-inflammatory cytokines in cultured macrophages and completely restored inflammation-impaired membrane barrier function in an intestinal barrier model. In mice subjected to dextran sulfate sodium-induced colitis, oral administration of BDS-entrapped KO liposomes suppressed tumor necrosis factor-α production (by 84.1%), interleukin-6 production (by 35.3%), and the systemic level of endotoxin (by 96.8%), and slightly reduced the macroscopic signs of the disease.ConclusionTaken together, KO liposomes may have great potential as a nanovehicle for oral delivery of IBD drugs.

Activation of TGR5 alleviates inflammation in rheumatoid arthritis peripheral blood mononuclear cells and in mice with collagen II‑induced arthritis.

Rheumatoid arthritis (RA) is characterized by chronic inflammatory synovitis resulting in progressive joint destruction. Persistent synovial inflammation is induced by activation of various inflammatory cells. G-protein-coupled bile acid receptor 1 (TGR5) is a G-protein-coupled receptor activated by various bile acids, which has been reported to act as a key adaptor in regulating various signaling pathways involved in inflammatory responses and a diverse array of physiological processes, including bile acid synthesis, lipid and carbohydrate metabolism, carcinogenesis, immunity and inflammation. In the present study, TGR5 expression was detected in RA peripheral blood mononuclear cells (PBMCs), and its association with clinical disease activity, histological synovitis severity and radiological joint destruction was analyzed. Subsequently, the role and potential underlying mechanisms of TGR5 in the PBMCs of patients with RA and mice with collagen II-induced arthritis (CIA) were investigated. PBMCs were obtained from 50 patients with RA and 40 healthy controls (HCs). The mRNA and protein expression levels of TGR5 were detected in PBMCs via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining, respectively. Additionally, the levels of proinflammatory cytokines were analyzed by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). The activation of nuclear factor-κB (NF-κB) and IκB kinase a was determined via western blot analysis. The anti-arthritic and anti-inflammatory effects of LCA on mice with CIA were then investigated. The arthritis score was assessed, and the protein levels of proinflammatory cytokines in the plasma of mice were detected via ELISA. TGR5 mRNA expression was significantly downregulated in the PBMCs of patients with RA compared with in those of the HCs (0.53±0.58 for patients vs. 1.49±0.83 for HCs; P<0.001); similar findings were observed at the protein level. The mRNA expression levels of TGR5 in the PBMCs of patients with RA with a high 28-Joint Disease Activity Score (DAS28) were significantly decreased compared with in patients with a low DAS28 (0.81±0.65 for low score vs. 0.35±0.46 for high score; P=0.002). Furthermore, TGR5 expression was significantly correlated with the levels of C-reactive protein (r=−0.429; P=0.002) and the DAS28 (r=−0.383; P=0.006). RT-qPCR and ELISA analyses indicated that lithocholic acid (LCA, 10 mg/kg/day) attenuated lipopolysaccharide-induced proinflammatory cytokine production via inhibition of NF-κB activity in the PBMCs of patients with RA. In addition, the arthritis score was significantly decreased in LCA-treated CIA mice compared with in non-treated CIA mice. The increased production of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8 was significantly reduced in the plasma of LCA-treated CIA mice compared with the control. In conclusion, TGR5 may contribute to the inflammation of PBMCs in patients with RA and mice with CIA.

Lipopolysaccharide-induced expression of astrocyte elevated gene-1 promotes degeneration and inflammation of chondrocytes via activation of nuclear factor-κB signaling

Osteoarthritis is an inflammatory disease characterized by joint degeneration and inflammation. Astrocyte elevated gene-1 (AEG-1) has been suggested as a novel inflammation-related factor in the pathological processes of various inflammatory diseases. To date, little is known about the role of AEG-1 in osteoarthritis. The aim of the present study was to explore the potential role of AEG-1 in the regulation of lipopolysaccharide-induced apoptosis and inflammation of chondrocytes. The results showed that AEG-1 expression was significantly upregulated in chondrocytes following exposure to lipopolysaccharide. Knockdown of AEG-1 increased the survival and decreased the expression of matrix metalloproteinases in chondrocytes treated with lipopolysaccharide. Moreover, silencing of AEG-1 restricted the lipopolysaccharide-induced production of proinflammatory cytokines. In contrast, AEG-1 overexpression caused opposite effects. Notably, we found that AEG-1 inhibition blocked the lipopolysaccharide-induced activation of nuclear factor-κB signaling through impeding the nuclear translocation of nuclear factor-κB p65 subunit. Additionally, inhibition of nuclear factor-κB partially reversed the AEG-1-mediated promotion of lipopolysaccharide-induced inflammatory injury in chondrocytes. In conclusion, our results demonstrate that inhibition of AEG-1 expression attenuates lipopolysaccharide-induced degeneration and inflammation of chondrocytes through suppressing the activation of nuclear factor-κB signaling. This work therefore highlights a potential role of AEG-1 in the pathogenesis of osteoarthritis, and indicates its potential as a therapeutic target.

Discovery and preclinical development of IIIM-160, a Bergenia ciliata-based anti-inflammatory and anti-arthritic botanical drug candidate

ObjectiveBergenia ciliata (Haw.) Sternb. is used in the Indian traditional system of medicine to treat various ailments including rheumatism and to heal wounds. The objective of the present study was to perform a preclinical characterization of the B. ciliata-based botanical extract IIIM-160. MethodsIIIM-160 was chemically standardized and analyzed for heavy metal content, aflatoxins, pesticides and microbial load. The in vitro and in vivo efficacies were determined in suitable models of inflammation, arthritis and nociception. An acute oral toxicity study was performed in Swiss albino mice. A suitable oral formulation was developed and characterized. ResultsBergenin was found to be the major component (9.1% w/w) of IIIM-160. The botanical lead displayed inhibition of lipopolysaccharide-induced production of proinflammatory cytokines in THP-1 cells, with selectivity toward interleukin-6 (IL-6) and had an excellent safety-window. It showed anti-inflammatory, anti-arthritic and antinociceptive activity in animal models and was not toxic at oral doses up to 2 g/kg in Swiss-albino mice. The gastroretentive, sustained-release capsule formulation showed sustained-release of the bergenin over the period of 24 h, resulting in improved plasma-exposure of bergenin in Sprague–Dawley rats. ConclusionThe dual-activity of IL-6 inhibition and antinociception marks the suitability of IIIM-160 for treating rheumatoid arthritis. This study will serve as the benchmark for further research on this botanical formulation.

Heat-killed Tsukamurella inchonensis reduces lipopolysaccharide-induced inflammatory responses in activated murine peritoneal macrophages

Tsukamurella inchonensis (T. inchonensis) is an aerobic species of Actinomycetales which has immunomodulatory activities when used as a suspension of killed bacilli. Here, the effects of T. inchonensis on lipopolysaccharide-induced inflammatory responses in mouse peritoneal macrophages have been examined. Peritoneal macrophages were harvested by lavaging with ice cold phosphate-buffered saline. Macrophages acquired from mice treated with different doses of T. inchonensis for seven days were cultured with 20 U/ml interferon-γ and 10 µg/ml lipopolysaccharide for in vivo assays. Nitrite levels were measured by using the diazotization method based on the Griess reaction, an indirect technique to determine nitric oxide (NO) production. T. inchonensis inhibited lipopolysaccharide-stimulated NO production in mouse peritoneal macrophages from mice previously exposed to concentrations of 108 and 5 × 10&lt;sup&gt;7&lt;/sup&gt; CFU per flask. Also, T. inchonensis decreased lipopolysaccharide-induced production of pro-inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α. Thus, it can be concluded that T. inchonensis is a powerful inhibitor of lipopolysaccharide-induced NO production in activated murine macrophages, and T. inchonensis may be useful as a novel agent for chemoprevention in inflammatory diseases.

Misoprostol Inhibits Lipopolysaccharide-Induced Pro-inflammatory Cytokine Production by Equine Leukocytes.

Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These receptors can be targeted by the EP2/4 agonist and prostaglandin E1 analog, misoprostol. Misoprostol is currently used as a gastroprotectant in horses but has not been evaluated as a cytokine-targeting therapeutic. Thus, we hypothesized that misoprostol treatment would inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-stimulated equine leukocytes in an in vitro inflammation model. To test this hypothesis, equine leukocyte-rich plasma (LRP) was collected from 12 healthy adult horses and used to model LPS-mediated inflammatory signaling. LRP was treated with varying concentrations of misoprostol either before (pretreated) or following (posttreated) LPS stimulation. LRP supernatants were assayed for 23 cytokines using an equine-specific multiplex bead immunoassay. Leukocytes were isolated from LRP, and leukocyte mRNA levels of four important cytokines were evaluated via RT-PCR. Statistical differences between treatments were determined using one-way RM ANOVA (Holm–Sidak post hoc testing) or Friedman’s RM ANOVA on Ranks (SNK post hoc testing), where appropriate (p < 0.05, n = 3–6 horses). These studies revealed that misoprostol pre- and posttreatment inhibited LPS-induced TNFα and IL-6 protein production in equine leukocytes but had no effect on IL-8 protein. Interestingly, misoprostol pretreatment enhanced IL-1β protein synthesis following 6 h of LPS stimulation, while misoprostol posttreatment inhibited IL-1β protein production after 24 h of LPS stimulation. At the mRNA level, misoprostol pre- and posttreatment inhibited LPS-induced TNFα, IL-1β, and IL-6 mRNA production but did not affect IL-8 mRNA. These results indicate that misoprostol exerts anti-inflammatory effects on equine leukocytes when applied before or after a pro-inflammatory stimulus. However, the effects we observed were cytokine-specific and sometimes differed at the mRNA and protein levels. Further studies are warranted to establish the inhibitory effects of misoprostol on equine cytokine production in vivo.

Docosahexaenoic acid induces M2 macrophage polarization through peroxisome proliferator-activated receptor γ activation

AimsImpaired resolution of acute inflammation results in development of chronic inflammatory disorders such as atherosclerosis, asthma and arthritis. Clearance of apoptotic neutrophils by M2 macrophages, the process termed efferocytosis, is critical for complete resolution of inflammation as it prevents secondary necrosis caused by disgorgement of toxic contents from apoptotic cells in the inflamed site. In the present study, we investigated the effect of docosahexaenoic acid (DHA) on efferocytosis. Main methodsTo determine the effect of DHA on efferocytosis, murine macrophage-like RAW264.7 cells were co-incubated with apoptotic Jurkat T cells, and efferocytosis was assessed by flow cytometry. The expression and production of M1 and M2 markers were determined by RT-PCR, ELISA and flow cytometry. To demonstrate the involvement of peroxisome proliferator-activated receptor γ (PPARγ) in DHA-mediated effects, siRNA against PPARγ was utilized. The expression of PPARγ was examined by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry. The PPARγ activation was measured by the electrophilic gel shift assay. Key findingsDHA enhanced the efferocytic ability of RAW264.7 cells, and induced their M2 polarization. Notably, knockdown of PPARγ abolished the stimulatory effect of DHA on M2 polarization as well as efferocytosis. Furthermore, lipopolysaccharide-induced production of pro-inflammatory cytokines was significantly inhibited by DHA, suggesting that DHA alters the macrophage phenotype in favor of M2 while it suppresses M1 polarization. SignificanceThese findings indicate that DHA can promote resolution of inflammation by facilitating efferocytosis through M2 macrophage polarization. Therefore, DHA may have a therapeutic potential in the management of inflammatory diseases which are related to impaired resolution of inflammation.

A novel mechanism for inhibition of lipopolysaccharide-induced proinflammatory cytokine production by valproic acid

The inhibitory effect of valproic acid (VPA) on lipopolysaccharide (LPS)-induced inflammatory response was studied by using mouse RAW 264.7 macrophage-like cells. VPA pretreatment attenuated LPS-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, but not nuclear factor (NF)-κB and mitogen-activated protein kinases. VPA reduced phosphorylation of MDM2, an ubiquitin ligase and then prevented LPS-induced p53 degradation, followed by enhanced p53 expression. Moreover, p53 small interfering RNA (siRNA) abolished the inhibitory action of VPA on LPS-induced NF-κB p65 transcriptional activation and further LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. VPA prevented LPS-induced degradation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and up-regulated the PTEN expression. Taken together, VPA was suggested to down-regulate LPS-induced NF-κB-dependent transcriptional activity via impaired PI3K/Akt/MDM2 activation and enhanced p53 expression. A detailed mechanism for inhibition of LPS-induced inflammatory response by VPA is discussed.

Ethyl linoleate from garlic attenuates lipopolysaccharide-induced pro-inflammatory cytokine production by inducing heme oxygenase-1 in RAW264.7 cells

In the present study, an essential fatty acid, ethyl linoleate (ELA), was isolated from the cloves of Allium sativum, and its structure was elucidated by NMR and GC-MS analyses. In vitro systems were used to evaluate the anti-inflammatory activity of ELA. Our results indicate that ELA down-regulates inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and thereby reduces nitric oxide (NO) and prostaglandin E2 production in lipopolysaccharide (LPS)-activated RAW 264.7 cells. Immunofluorescent microscopy and western blot analyses revealed that these effects were mediated by impaired translocation of nuclear factor (NF)-κB and inhibition of phosphorylation of mitogen activated protein kinases. Furthermore, ELA exerted its anti-inflammatory activity by inducing heme oxygenase-1 (HO-1) expression, as determined by HO-1 small interfering (Si) RNA system. Si RNA-mediated knock-down of HO-1 abrogated the inhibitory effects of ELA on the production of NO, TNF-α, IL-1β, and IL-6 in LPS-induced macrophages. These findings indicate the potential therapeutic use of ELA as an anti-inflammatory agent.

Pyroglutamated Apelin-13 Inhibits Lipopolysaccharide-Induced Production of Pro-Inflammatory Cytokines in Murine Macrophage J774.1 Cells

Apelin, recently identified as an endogenous ligand of the orphan G protein-coupled receptor APJ, has multiple pathophysiological properties. In the present study, we investigated whether pyroglutamated apelin-13 ([Pyr1]-apelin-13), the most highly active isoform among the mature apelin peptide family, modulates the effect of bacterial lipopolysaccharide (LPS) on cytokine induction in a murine macrophage-like cell line, J774.1 cells. J774.1 cells expressed the APJ protein in a stationary state, and the expression of APJ was not affected by LPS stimulation. No significant effect of [Pyr1]-apelin-13 treatment alone was observed on the proliferation or cytokine production of J774.1 cells in the stationary state. However, prior to LPS stimulation, pretreatment with [Pyr1]apelin-13 for 16 h significantly diminished mRNA expression and protein secretion of inflammatory cytokine interleukin-6, which was confirmed by RT-PCR and ELISA, respectively. Western blot analysis revealed that the phosphorylation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, but not extracellular signal-regulated kinase, which was induced by LPS, significantly decreased in [Pyr1]-apelin-13-pretreated J774.1 cells compared with untreated cells. These observations suggest that [Pyr1]-apelin-13 functions as a negative regulator of LPS-mediated pro-inflammatory responses in macrophages.

Expressive Writing and Wound Healing in Older Adults

To investigate whether expressive writing could speed wound reepithelialization in healthy, older adults. In this randomized controlled trial, 49 healthy older adults aged 64 to 97 years were assigned to write for 20 minutes a day either about upsetting life events (Expressive Writing) or about daily activities (Time Management) for 3 consecutive days. Two weeks postwriting, 4-mm punch biopsy wounds were created on the inner, upper arm. Wounds were photographed routinely for 21 days to monitor wound reepithelialization. Perceived stress, depressive symptoms, health-related behaviors, number of doctor visits, and lipopolysaccharide-stimulated proinflammatory cytokine production were also measured throughout the study. Participants in the Expressive Writing group had a greater proportion of fully reepithelialized wounds at Day 11 postbiopsy compared with the Time Management group, with 76.2% versus 42.1% healed, χ(2)(1, n = 40) = 4.83, p = .028. Ordinal logistic regression showed more sleep in the week before wounding also predicted faster healing wounds. There were no significant group differences in changes to perceived stress, depressive symptoms, health-related behaviors, lipopolysaccharide-induced proinflammatory cytokine production, or number of doctor visits over the study period. This study extends previous research by showing that expressive writing can improve wound healing in older adults and women. Future research is needed to better understand the underlying cognitive, psychosocial, and biological mechanisms contributing to improved wound healing from these simple, yet effective, writing exercises. Australian New Zealand Clinical Trials Registry (trial number 343095).

Inhibition of Neddylation Represses Lipopolysaccharide-induced Proinflammatory Cytokine Production in Macrophage Cells

Cullin-RING E3 ligases (CRLs) are a class of ubiquitin ligases that control the proteasomal degradation of numerous target proteins, including IκB, and the activity of these CRLs are positively regulated by conjugation of a Nedd8 polypeptide onto Cullin proteins in a process called neddylation. CRL-mediated degradation of IκB, which normally interacts with and retains NF-κB in the cytoplasm, permits nuclear translocation and transactivation of the NF-κB transcription factor. Neddylation occurs through a multistep enzymatic process involving Nedd8 activating enzymes, and recent studies have shown that the pharmacological agent, MLN4924, can potently inhibit Nedd8 activating enzymes, thereby preventing neddylation of Cullin proteins and preventing the degradation of CRL target proteins. In macrophages, regulation of NF-κB signaling functions as a primary pathway by which infectious agents such as lipopolysaccharides (LPSs) cause the up-regulation of proinflammatory cytokines. Here we have analyzed the effects of MLN4924, and compared the effects of MLN4924 with a known anti-inflammatory agent (dexamethasone), on certain proinflammatory cytokines (TNF-α and IL-6) and the NF-κB signaling pathway in LPS-stimulated macrophages. We also used siRNA to block neddylation to assess the role of this molecular process during LPS-induced cytokine responsiveness. Our results demonstrate that blocking neddylation, either pharmacologically or using siRNA, abrogates the increase in certain proinflammatory cytokines secreted from macrophages in response to LPS. In addition, we have shown that MLN4924 and dexamethasone inhibit LPS-induced cytokine up-regulation at the transcriptional level, albeit through different molecular mechanisms. Thus, neddylation represents a novel molecular process in macrophages that can be targeted to prevent and/or treat the LPS-induced up-regulation of proinflammatory cytokines and the disease processes associated with their up-regulation.

A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response

The jumonji (JMJ) family of histone demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context. Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance, as well as in development, physiology and disease. However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.