Showing papers in "Journal of Inflammation in 2018"

The role of adipokines in skeletal muscle inflammation and insulin sensitivity

Abstract: There is currently an unmet clinical need to develop better pharmacological treatments to improve glucose handling in Type II Diabetes patients with obesity. To this end, determining the effect of obesity-associated adipokines on skeletal muscle insulin sensitivity has emerged as an important area of drug discovery research. This review draws together the data on the functional role of adipokines on skeletal muscle insulin signalling, highlights several understudied novel adipokines and provides a perspective on the direction of future research. The adipokines leptin, resistin, visfatin and adiponectin have all been shown to affect skeletal muscle insulin sensitivity by impacting on the activity of components within insulin signalling pathways, affecting GLUT4 translocation and modulating insulin-mediated skeletal muscle glucose uptake. Furthermore, proteomic analysis of the adipose tissue secretome has recently identified several novel adipokines including vaspin, chemerin and pref-1 that are associated with obesity and insulin resistance in humans and functionally impact on insulin signalling pathways. However, predominantly, these functional findings are the result of studies in rodents, with in vitro studies utilising either rat L6 or murine C2C12 myoblasts and/or myotubes. Despite the methodology to isolate and culture human myoblasts and to differentiate them into myotubes being established, the use of human muscle in vitro models for the functional validation of adipokines on skeletal muscle insulin sensitivity is limited. Understanding the mechanism of action and function of adipokines in mediating insulin sensitivity in skeletal muscle may lead to the development of novel therapeutics for patients with type 2 diabetes. However, to date, studies conducted in human skeletal muscle cells and tissues are limited. Such human in vitro studies should be prioritised in order to reduce the risk of candidate drugs failing in the clinic due to the assumption that rodent skeletal muscle target validation studies will to translate to human.

Contribution of the inflammasome to inflammaging

Abstract: Inflammation is a natural part of the aging process. This process is referred to as inflammaging. Inflammaging has been associated with deleterious outcomes in the aging brain in diseases such as Alzheimer’s disease and Parkinson’s disease. The inflammasome is a multi-protein complex of the innate immune response involved in the activation of caspase-1 and the processing of the inflammatory cytokines interleukin (IL)-1β and IL-18. We have previously shown that the inflammasome plays a role in the aging process in the brain. In this study, we analyzed the brain of young (3 months old) and aged (18 months old) mice for the expression of inflammasome proteins. Our findings indicate that the inflammasome proteins NLRC4, caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and IL-18 are elevated in the cytosol of cortical lysates in aged mice when compared to young. In addition, in the cytosolic fraction of hippocampal lysates in aged mice, we found an increase in NLRC4, caspase-1, caspase-11, ASC and IL-1β. Moreover, we found higher levels of ASC in the mitochondrial fraction of aged mice when compared to young, consistent with higher levels of the substrate of pyroptosis gasdermin-D (GSDM-D) and increased pyroptosome formation (ASC oligomerization). Importantly, in this study we obtained fibroblasts from a subject that donated his cells at three different ages (49, 52 and 64 years old (y/o)) and found that the protein levels of caspase-1 and ASC were higher at 64 than at 52 y/o. In addition, the 52 y/o cells were more susceptible to oxidative stress as determined by lactose dehydrogenase (LDH) release levels. However, this response was ameliorated by inhibition of the inflammasome with Ac-Tyr-Val-Ala-Asp-Chloromethylketone (Ac-YVAD-CMK). In addition, we found that the protein levels of ASC and IL-18 are elevated in the serum of subjects over the age of 45 y/o when compared to younger subjects, and that ASC was higher in Caucasians than Blacks and Hispanics, whereas IL-18 was higher in Caucasians than in blacks, regardless of age. Taken together, our data indicate that the inflammasome contributes to inflammaging and that the inflammasome-mediated cell death mechanism of pyroptosis contributes to cell demise in the aging brain.

Cannabinoid 2 receptor attenuates inflammation during skin wound healing by inhibiting M1 macrophages rather than activating M2 macrophages

Abstract: The anti-inflammatory properties of the cannabinoid 2 receptor (CB2R) in injury and inflammatory diseases have been widely substantiated. Specifically, the anti-inflammatory effect of CB2R may be achieved by regulating macrophage polarisation. Several research findings suggested that the activation of CB2R could attenuate inflammation by reducing pro-inflammatory M1 macrophage polarisation and promoting anti-inflammatory M2 polarisation. However, considering CB2R inhibits fibrosis and M2 promotes fibrosis, that the activation of CB2R may lead to an increase in M2 macrophages seems contradictory. Therefore, we hypothesised that the activation of CB2R to attenuate inflammation is not achieved by up-regulating M2 macrophages. We established an incised wound model using mouse skin and used this to evaluate the effect of CB2R agonists (JWH133 or GP1a) and an antagonist (AM630) on wound healing. At various post-injury intervals, we used western blot analysis, immunofluorescence staining, enzyme-linked immunosorbent assay and quantitative reverse transcription polymerase chain reaction assays to determine CB2R protein expression, M1/M2 macrophage infiltration, and the protein and gene expression of M1/M2-associated markers and cytokines in skin lesions. Activation of CB2R significantly reduced M1 macrophage infiltration and slightly increased M2 macrophage infiltration. Similarly, gene expression and protein levels of M1-associated markers and cytokines (interleukin [IL]-6, IL-12, CD86 and inducible nitric oxide synthase) were significantly down-regulated after CB2R agonist administration; in contrast, markers and cytokines were increased in the CB2R antagonist–treated group. Conversely, the administration of agonists slightly increased gene expression and protein levels of M2-associated markers and cytokines (IL-4, IL-10, CD206 and arginase-1 [Arg-1]); however, a statistical significance at most time points post-injury was not noted. In summary, our findings suggested that during incised skin wound healing in mice, increased levels of CB2R may affect inflammation by regulating M1 rather than M2 macrophage subtype polarisation. These results offer a novel understanding of the molecular mechanisms involved in the inhibition of inflammation by CBR2 that may lead to new treatments for cutaneous inflammation.

Expression and clinical significance of the NEK7-NLRP3 inflammasome signaling pathway in patients with systemic lupus erythematosus

Abstract: The aim of the study was to investigate the expression of the NEK7-NLRP3 inflammasome signaling pathway in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), as well as its clinical significance A total of 38 SLE patients and 33 healthy volunteers were recruited Real time PCR and western blotting were performed to determine mRNA and protein levels of NEK7, NLRP3 inflammasome components (NLRP3, ASC, and Caspase-1), and downstream cytokines (IL-1b and IL-18) in PBMCs from the two groups ELISA was used to detect serum levels of IL-1b and IL-18 The same methods were used to detect changes in the above indices in the 25 SLE patients after treatment Correlations between clinical and laboratory parameters were also analyzed Compared to those in healthy controls, levels of NEK7, NLPR3, and ASC were lower in SLE patients; however, Caspase-1, IL-1b, and IL-18 were expressed at higher levels mRNA levels of NEK7, NLRP3, and ASC were inversely correlated with disease activity, whereas a positive correlation was observed with IL-1b and IL-18 After treatment, mRNA levels of NEK7 and NLRP3 increased, whereas Caspase-1, IL-1b, and IL-18 decreased significantly Compared to those in SLE patients without renal damage, patients with lupus nephritis (LN) exhibited lower mRNA levels of NEK7, NLRP3, and ASC but higher levels of Caspase-1, IL-1b, and IL-18 Results indicate that the expression of the NEK7-NLRP3 complex might play a protective role in the pathogenesis of SLE and is inversely correlated with disease activity A positive effect of NEK7 on NLRP3 was observed, and the low expression of NLRP3 in SLE patients might be related to the low expression of NEK7 Overexpression of Caspase-1 in SLE patients mediates the maturation and release of IL-1b and IL-18, and contributes to the pathogenesis of SLE and LN

Interleukin-10 attenuates impairment of the blood-brain barrier in a severe acute pancreatitis rat model.

Abstract: Impairment of the blood-brain barrier (BBB) in severe acute pancreatitis (SAP) could result in life-threatening pancreatic encephalopathy. Interleukin-10 (IL-10) is a classical cytokine that is well-known for its strong immunoregulatory and anti-inflammatory abilities. However, whether and how IL-10 protects the BBB in SAP are still unclear. This study includes in vivo experiments using a SAP rat model and in vitro experiments using an in vitro BBB model consisting of a monolayer of brain microvascular endothelial cells (BMECs). The study groups are divided into the control, SAP (in vivo)/TNF-α (in vitro), IL-10 treatment, IL-10 + signal transducer and activator of transcription 3 (STAT3) inhibitor S3I-201 treatment groups. Pancreatic pathological scores, serum amylase, serum TNF-α levels and BBB permeability by Evan’s blue assay in SAP rat models were evaluated. BMEC apoptosis in SAP rats or induced by TNF-αin vitro was detected by terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and flow cytometry, separately. Expression levels of claudin-5 and proteins involved in the STAT3 signaling pathway were measured by Western blotting. Location and changes of junctional structure of claudin-5 on BMECs were assessed by immunohistochemistry and immunofluorescence. In vivo, IL-10 alleviated the severity of inflammation, attenuated the increased BBB permeability in SAP rat models by reducing BMEC apoptosis via the STAT3 pathway and ameliorated the down-regulation of claudin-5 expression in BMECs; in vitro, IL-10 improved BBB integrity against TNF-α by attenuating BMEC apoptosis via the STAT3 pathway, the impairment of tight junction structure and the down-regulation of claudin-5 expression in BMECs. IL-10 improves BBB properties in SAP by attenuating the down-regulation of claudin-5 expression and the impairment of tight junctions and by STAT3 pathway-mediated anti-apoptotic effects on BMECs.

Histone deacetylase 2 (HDAC2) attenuates lipopolysaccharide (LPS)-induced inflammation by regulating PAI-1 expression

Abstract: Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection, and is primarily characterized by an uncontrolled systemic inflammatory response. In the present study, we developed an effective adjunct therapy mediated by a novel mechanism, to attenuate overt inflammation. LPS-treated macrophages were adopted as an in vitro model of endotoxin-induced inflammation during sepsis. Experiments were carried out using primary mouse peritoneal macrophages and the murine macrophage cell line RAW264.7, to elucidate the mechanisms by which HDAC2 modulates endotoxin-induced inflammation. Results revealed that PAI-1, TNF, and MIP-2 expression were inhibited by theophylline, an HDAC2 enhancer, in a RAW macrophage cell line, following LPS-induced inflammation. Thus, HDAC2 plays an important role in immune defense by regulating the expression of inflammatory genes via the c-Jun/PAI-1 pathway. During LPS-induced inflammation, overexpression of HDAC2 was found to inhibit PAI-1, TNF, and MIP-2 expression. Following LPS stimulation, HDAC2 knockdown increased nuclear translocation and DNA binding of c-Jun to the PAI-1 gene promoter, thereby activating PAI-1 gene transcription. Furthermore, inhibition of PAI-1 by TM5275 alone or in combination with theophylline notably suppressed TNF and MIP-2 expression. HDAC2 can attenuate lipopolysaccharide-induced inflammation by regulating c-Jun and PAI-1 expression in macrophages.

Plasma vascular non-inflammatory molecule 3 is associated with gastrointestinal acute graft-versus-host disease in mice

Abstract: Gastrointestinal acute graft-versus-host disease (GI aGVHD) is a lethal complication following allogeneic hematopoietic stem cell transplantation (HSCT). However, it is still very difficult to make a diagnosis of GI aGVHD in practice. To date, no consensus plasma biomarker of GI aGVHD can be used to help make a diagnosis. Here, we attempted to identify GI aGVHD associated plasma proteins in murine model, which can help make a diagnosis of GI aGVHD. We used 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) to screen out proteins in plasma samples taken from murine models before and after allogeneic HSCT. Next mRNA expressions were validated by quantitative real-time polymerase chain reaction in mouse intestinal epithelial samples. We found that five proteins were increased at least 2-fold in the allogeneic group at day 7 compared with days 0, 3 and 15 (after Cyclosporin A treatment) and the syngeneic group at day 7. These 5 proteins were VNN3, ZNF746, C4BP, KNG1 and FETUB, and they were consistent with results from negative labeling with 8-plex iTRAQ. Furthermore, increase in mRNA level of VNN3 was confirmed in murine intestinal epithelial samples with aGVHD. Our results demonstrate that plasma VNN3 protein is associated with GI aGVHD in murine model.

Anti-inflammatory effect of hesperidin enhances chondrogenesis of human mesenchymal stem cells for cartilage tissue repair.

Abstract: Articular cartilage diseases are considered a major health problem, and tissue engineering using human mesenchymal stem cells (MSCs) have been shown as a promising solution for cartilage tissue repair. Hesperidin is a flavonoid extract from citrus fruits with anti-inflammatory properties. We aimed to investigate the effect of hesperidin on MSCs for cartilage tissue repair. MSCs were treated by hesperidin, and colony formation and proliferation assays were performed to evaluate self-renewal ability of MSCs. Alcian blue staining and Sox9 expression were measured to evaluate chondrogenesis of MSCs. Secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and expression of nuclear factor kappa B (NF-κB) subunit p65 were also assessed. Hesperidin improved self-renewal ability and chondrogenesis of MSCs, inhibited secretion of pro-inflammatory cytokines IFN-γ, IL-2, IL-4 and IL-10, and suppressed the expression of p65. Overexpression of p65 was able to reverse the hesperidin inhibited secretions of pro-inflammatory cytokines, and abolish the enhancing effect of hesperidin on chondrogenesis of MSCs. Hesperidin could serve as a therapeutic agent to effectively enhance chondrogenesis of human MSCs by inhibiting inflammation to facilitate cartilage tissue repair.

MBD2 regulates differentiation and function of Th17 cells in neutrophils- dominant asthma via HIF-1α

Abstract: T helper 17 (Th17) cells have proven to be crucial in the pathogenesis of neutrophils-dominant asthma. Hypoxia inducible factor-1α (HIF-1α) is involved in allergic responses in asthma. Our previous studies indicated that Methtyl-CpG binding domain protein 2 (MBD2) expression was increased in asthma patients. The aim of the present study is to understand how MBD2 interacts with HIF-1α to regulate Th17 cell differentiation and IL-17 expression in neutrophils-dominant asthma. A neutrophils-dominant asthma mouse model was established using female C57BL/6 mice to investigate Th17 cell differentiation and MBD2 and HIF-1α expression regulation using flow cytometry, western blot or qRT-PCR. MBD2 and HIF-1α genes were silenced or overexpressed through lentiviral transduction to explore the roles of MBD2 in Th17 cell differentiation and IL-17 release in neutrophils-dominant asthma. A neutrophilic inflammatory asthma phenotype model was established successfully. This was characterized by airway hyperresponsiveness (AHR), increased BALF neutrophil granulocytes, activated Th17 cell differentiation, and high IL-17 levels. MBD2 and HIF-1α expression were significantly increased in the lung and spleen cells of mice with neutrophils-dominant asthma. Through overexpression or silencing of MBD2 and HIF-1α genes, we have concluded that MBD2 and HIF-1α regulate Th17 cell differentiation and IL-17 secretion. Moreover, MBD2 was also found to regulate HIF-1α expression. Our findings have uncovered new roles for MBD2 and HIF-1α, and provide novel insights into the epigenetic regulation of neutrophils-dominant asthma.

The severity of LPS induced inflammatory injury is negatively associated with the functional liver mass after LPS injection in rat model.

Abstract: High levels of serum lipopolysaccharide (LPS) were observed in sepsis patients with liver injury and high mortality. However, the role of liver in modulation LPS induced inflammatory injury was ill investigated. In the present study, the severity of LPS induced inflammatory response was observed after liver resection or portal branch occlusion to decreasing functional liver mass. The local and systemic damage was observed to investigate the role of liver in modulation inflammatory injury. First, 30%, 70%, and 90% partial hepatectomy (PH) were performed, and serum TNF-α, survival rate, and hepatic LPS uptake was observed. Second, LPS-exposure of the functional liver mass was decreased by selectively blocking the RL prior to LPS-injection, which was given 30 min before a 70% PH, and the inflammatory response was compared in the occluded and the non-occluded liver. The control group was subjected to LPS injection 30 min prior to liver resection without blocking the RL transiently. The serum TNF-α, ALT, AST, creatinine levels, and urea levels, survival rate, hepatic LPS uptake, and hepatic inflammatory cytokines was observed. The decreasing of functional liver mass after 90%, 70%, and 30% PH was associated with decreased serum TNF-α, survival rate, and increased hepatic LPS uptake after LPS injection. Occluding the right lobes (RL) prior to LPS administration reversed the liver injury caused by 70% PH, indicated by 100% survival rate and decreased liver and kidney injury, and systemic inflammatory response. The induction of inflammatory response in occluding liver lobes were lower than un-occluding liver lobes. The severity of the LPS-induced systemic inflammatory injury is determined by functional liver volume. This observation suggests that the liver is the central organ for the initiation of the inflammatory response, and is involved in causing a severe SIRS with systemic damage and death.

Transient receptor potential ankyrin 1 (trpa1) mediates il-1β-induced apoptosis in rat chondrocytes via calcium overload and mitochondrial dysfunction

Abstract: Chondrocyte apoptosis is a central feature in the progression of osteoarthritis (OA), and would be triggered by sustained elevation of intracellular calcium ion (Ca2+), also known as a cellular second messenger. Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, and the activation of which causes an influx of cation ions, in particularly Ca2+, into the activated cells. Therefore, we investigate the potential role of TRPA1 in mediating Ca2+ influx to promote chondrocyte apoptosis in OA. The expression of TRPA1 in interleukin (IL)-1β-treated rat chondrocytes was assessed by Polymerase chain reaction (PCR) and Western blot (WB), and the functionality of TRPA1 channel by Ca2+ influx measurements. Meanwhile, the chondrocyte apoptosis in IL-1β-treated cells was measured by TUNEL assay and flow cytometry. The measurement of mitochondrial membrane potential and apoptosis-associated proteins after inhibition of TRPA1 were also performed in IL-1β-treated rat chondrocytes. After being induced by IL-1β, the gene and protein expression of TRPA1 was increased in the dose-dependent manner. Meanwhile, Ca2+ influx mediated by TRPA1 in rat chondrocytes was also enhanced. Pharmacological inhibition of TRPA1 downregulated the apoptotic rate in IL-1β-treated rat chondrocytes. In addition, the membrane potential depolarization was improved and significantly increased expression of apoptosis-associated proteins also reduced by the TRPA1 antagonist. We found the IL-1β caused the increased functional expression of TRPA1, the activation of which involved IL-1β-induced apoptosis in rat chondrocytes. The potential mechanism may be linked to the intracellular calcium overload mediated by TRPA1 and attendant mitochondrial dysfunction.

Atorvastatin and diacerein reduce insulin resistance and increase disease tolerance in rats with sepsis

Abstract: Sepsis is one of the leading causes of death among hospitalized patients. At the onset of this condition, there is an over-production of pro-inflammatory mediators that contribute to organ failure and death. The excess production of pro-inflammatory mediators also impairs insulin signaling, which may be a pathophysiological tissue marker of proinflammatory cytokine action before organ failure. Statins and diacerein have pleiotropic effects, such as the blockage of inflammatory signaling pathways, suggesting that these drugs may be an attractive therapeutic or prophylactic strategy against sepsis. The aim of the present study was to investigate whether a statin or diacerein can improve insulin signaling, disease tolerance and survival in sepsis by inhibiting inflammatory pathways. We investigated the effect of these drugs on survival, tissue insulin signaling and inflammatory pathways in the liver and muscle of rats with sepsis induced by cecal ligation and puncture (CLP). The results showed that administration of medications, with anti-inflammatory ability, to septic animals increased survival and improved disease tolerance and insulin resistance in the liver and muscle. The treatment also attenuated ER stress, NF-κB, JNK activation and restored glucose-6-phosphatase (G6Pase) levels in the liver. Our results indicate that atorvastatin and diacerein treatment can modulate inflammatory pathways and, in parallel, attenuate insulin resistance in sepsis. Since these two drugs have safety profiles and minimal side effects, we suggest that these drugs may be alternative therapies for the prevention or therapies for the treatment of insulin resistance in sepsis, which could potentially reduce mortality in patients with sepsis.

Caspase recruitment domain (CARD) family (CARD9, CARD10, CARD11, CARD14 and CARD15) are increased during active inflammation in patients with inflammatory bowel disease

Abstract: The CARD family plays an important role in innate immune response by the activation of NF-κB. The aim of this study was to determine the gene expression and to enumerate the protein-expressing cells of some members of the CARD family (CARD9, CARD10, CARD11, CARD14 and CARD15) in patients with IBD and normal controls without colonic inflammation. We included 48 UC patients, 10 Crohn’s disease (CD) patients and 18 non-inflamed controls. Gene expression was performed by RT-PCR and protein expression by immunohistochemistry. CARD-expressing cells were assessed by estimating the positively staining cells and reported as the percentage. The CARD9 and CARD10 gene expression was significantly higher in UC groups compared with CD (P < 0.001). CARD11 had lower gene expression in UC than in CD patients (P < 0.001). CARD14 gene expression was higher in the group with active UC compared to non-inflamed controls (P < 0.001). The low expression of CARD14 gene was associated with a benign clinical course of UC, characterized by initial activity followed by long-term remission longer than 5 years (P = 0.01, OR = 0.07, 95%CI:0.007–0.70). CARD15 gene expression was lower in UC patients versus CD (P = 0.004). CARD9 protein expression was detected in inflammatory infiltrates; CARD14 in parenchymal cells, while CARD15 in inflammatory and parenchymal cells. CARD9−, CARD14− and CARD15 − expressing cells were significantly higher in patients with active UC versus non-inflamed controls (P < 0.05). The CARD family is involved in the inflammatory process and might be involved in the IBD pathophysiology.

CXCR-4 expression by circulating endothelial progenitor cells and SDF-1 serum levels are elevated in septic patients

Abstract: Endothelial progenitor cell (EPC) numbers are increased in septic patients and correlate with survival. In this study, we investigated, whether surface expression of chemokine receptors and other receptors important for EPC homing is upregulated by EPC from septic patients and if this is associated with clinical outcome. Peripheral blood mononuclear cells from septic patients (n = 30), ICU control patients (n = 11) and healthy volunteers (n = 15) were isolated by Ficoll density gradient centrifugation. FACS-analysis was used to measure the expression of the CXC motif chemokine receptors (CXCR)-2 and − 4, the receptor for advanced glycation endproducts (RAGE) and the stem cell factor receptor c-Kit. Disease severity was assessed via the Simplified Acute Physiology Score (SAPS) II. The serum concentrations of vascular endothelial growth factor (VEGF), stromal cell-derived factor (SDF)-1α and angiopoietin (Ang)-2 were determined with Enzyme linked Immunosorbent Assays. EPC from septic patients expressed significantly more CXCR-4, c-Kit and RAGE compared to controls and were associated with survival-probability. Significantly higher serum concentrations of VEGF, SDF-1α and Ang-2 were found in septic patients. SDF-1α showed a significant association with survival. Our data suggest that SDF-1α and CXCR-4 signaling could play a crucial role in EPC homing in the course of sepsis.

The roles of IL-19 and IL-20 in the inflammation of degenerative lumbar spondylolisthesis

Abstract: Degenerative lumbar spondylolisthesis (DLS) is a major cause of spinal canal stenosis and is often related to lower back pain. IL-20 is emerging as a potent angiogenic, chemotactic, and proinflammatory cytokine related to several chronic inflammatory bone disorders likes intervertebral disc herniation, rheumatoid arthritis (RA), osteoporosis, and bone fracture. IL-19 also acts as a proinflammatory cytokine in RA. The aim of the present study was to investigate whether IL-19 and IL-20 are involved in DLS and compare three different tissues including disc, facet joint, and ligamentum flavum of patients with DLS to verify which tissue is affected more by inflammation. Disc, facet joint and ligamentum flavum from 13 patients with DLS was retrieved, and the expression pattern of IL-19, IL-20, IL-20R1, IL-20R2, TNF-α, IL-1β, and MCP-1 was evaluated using immunohistochemical staining with specific antibodies. The disc cells were isolated and incubated with IL-19 and IL-20 under CoCl2-mimicked hypoxic conditions to analyze the proinflammatory cytokine expression pattern using real-time quantitative PCR with specific primers. IL-19 and IL-20 were positively stained and accompanied by abundant expression of TNF-α, IL-1β, and MCP-1 in facet joints of DLS patients. IL-19 and IL-20’s receptors (IL-20R1 and IL-20R2) were expressed on chondrocytes and fibrocytes/fibroblasts in facet joint and ligamentum flavum tissues from patients with DLS. There was a significant correlation between the expression of IL-20 and IL-1β in facet joint. In vitro assay, IL-19 and IL-20 upregulated the expression of IL-1β, IL-6, TNF-α, IL-8, VEGF, and MCP-1 in primary cultured DLS disc cells under CoCl2-mimicked hypoxic conditions. IL-19, IL-20, and their receptors as well as proinflammatory cytokines (TNF-α, IL-1β, and MCP-1) were expressed more in facet joints than the other tissues in patients with DLS; therefore, the etiology of inflammation might be more facet-centric. IL-19 and IL-20 induced proinflammatory cytokine expression in disc cells and might play a role in the pathogenesis of DLS.

The role of N-glycosylation of CD200-CD200R1 interaction in classical microglial activation

Abstract: Microglial inflammatory activation is the common feature of the central nervous system (CNS) diseases. Microglia can be activated and particularly polarized toward a dual role in the injured CNS. The CD200 receptor 1 (CD200R1) inhibits inflammatory microglia activation as illustrated by studies. Publications show abnormal activation of microglia secondary to the deficient inhibit of CD200-CD200R interaction. In the present study, we established a neuronal-microglia co-culture system to investigate the association between CD200R1 engagement and classical microglial activation. We analyzed the glycosylation of CD200R1 and the CD200 binding. Secretion of pro-inflammatory cytokines were measured. CD200R1 was N-glycosylated at Asparagine 44 (Asn44, N44). Mutation of this site disrupted CD200-CD200R1 interaction and up-regulated the expression of cytokines iNOS, CD86, IL-1β and TNF-α. N44 of CD200R1 is a significant binding site for CD200-CD200R1 interaction and play a critical role in the maintenance of microglia. The N-glycosylation of CD200R1 could serve as a therapeutic agent for CNS inflammation.

Suppression of hyperexcitability of trigeminal nociceptive neurons associated with inflammatory hyperalgesia following systemic administration of lutein via inhibition of cyclooxygenase-2 cascade signaling

Abstract: Lutein is a dietary constituent known to inhibit inflammation; however, its effect on nociceptive neuron-associated hyperalgesia remains to be determined. The present study therefore investigated under in vivo conditions whether administration of lutein attenuates the inflammation-induced hyperexcitability of trigeminal spinal nucleus caudalis (SpVc) neurons that is associated with mechanical hyperalgesia. Complete Freund’s adjuvant (CFA) was injected into the whisker pads of rats to induce inflammation, and then mechanical stimulation was applied to the orofacial area to assess the threshold of escape. The mechanical threshold was significantly lower in inflamed rats compared to uninjected naive rats, and this lowered threshold was returned to control levels by 3 days after administration of lutein (10 mg/Kg, i.p.) Also the lutein administration, inflammation-induced thickness of edema was returned to control levels. The mean increased number of cyclooxygenase-2 (Cox-2)-immunoreactive cells in the whisker pads of inflamed rats was also returned to control levels by administration with lutein. The mean discharge frequency of SpVc wide-dynamic range (WDR) neurons to both nonnoxious and noxious mechanical stimuli in inflamed rats was significantly decreased after lutein administration. In addition, the increased mean spontaneous discharge of SpVc WDR in inflamed rats was significantly decreased after lutein administration. Similarly, lutein significantly diminished noxious pinch-evoked mean after discharge frequency and occurrence in inflamed rats. Finally, lutein restored the expanded mean size of the receptive field in inflamed rats to control levels. These results together suggest that administration of lutein attenuates inflammatory hyperalgesia associated with hyperexcitability of nociceptive SpVc WDR neurons via inhibition of the peripheral Cox-2 signaling cascade. These findings support the proposed potential of lutein as a therapeutic agent in complementary alternative medicine strategies for preventing inflammatory mechanical hyperalgesia.

Zymosan attenuates melanoma growth progression, increases splenocyte proliferation and induces TLR-2/4 and TNF-α expression in mice.

Abstract: Melanoma is one of the most common types of skin malignancies. Since current therapies are suboptimal, considerable interest has focused on novel natural-based treatments. Toll-like receptors (TLRs) play an important role in evoking innate immunity against cancer cells. Zymosan, a known TLR-2 agonist, is a glucan derived from yeast cell walls with promising immunomodulatory effects. The aim of this study was to evaluate whether Saccharomyces cerevisiae-derived zymosan-modulated skin melanoma progression by regulation of TLR-2 and TLR-4 expression in peritoneal macrophages and serum TNF-α level. Male C57BL/6 mice were divided into four groups: i) zymosan-treated (Z), ii) Melanoma-bearing mice (M), iii) Melanoma-bearing mice treated with zymosan (ZM) and iv) a healthy control group (negative control). 15 days after melanoma induction, mice were injected i.p. with zymosan (10 μg) daily for 4 consecutive days. Mice were CO2-euthanized and serum TNF-α level, TLR-2 and TLR-4 expression in peritoneal macrophages and tumor growth measured. Splenocytes were treated ex-vivo with zymosan to determine viability and proliferation. Tumor weight significantly decreased following therapeutic dosing with zymosan (P < 0.05). This was associated with zymosan-induced upregulation of TLR-2, TLR-4 and TNF-α mRNA in peritoneal macrophages and enhanced serum TNF-α levels (P < 0.05). Splenocyte number and viability were increased in a concentration-dependent manner by zymosan. Our study suggests that zymosan-induced upregulation of TLR-2, TLR-4 and TNF-α gene expression and of TNF-α release; together with increased level of lymphocyte proliferation may play a role in the inhibition of melanoma progression.

Functional role of microRNA-135a in colitis.

Abstract: Inflammatory bowel disease (IBD) is one of the chronic gastrointestinal diseases with increasing risk of colon cancer development in the future. Apoptosis and inflammation play an important role in the etiology of this disease. MicroRNAs are associated with etiology of different diseases including IBD. In this study, we aimed to explore the role of miR-135a in the etiology of colitis in murine model of DSS-induced colitis. The results showed that expression of miR-135a in colonic cells was suppressed and up-regulating miR-135a inhibited apoptosis and inflammation of colonic epithelial cells. Additionally, Hif1α was identified as the target gene of miR-135a which promoted apoptosis and inflammation as knockdown of Hif1α led to the suppression of both apoptosis and inflammation. Overexpression of miR-135a might be beneficial in IBD due to its anti-apoptosis and anti-inflammation effects in vitro.

Dynamics of soluble vascular endothelial growth factor receptors and their ligands in aqueous humour during ranibizumab for age-related macular degeneration.

Abstract: Intravitreal ranibizumab injection (IRI) is effective for patients with exudative age-related macular degeneration (AMD) and decreases intraocular levels of vascular endothelial growth factor (VEGF), but VEGF receptor intraocular dynamics after IRI are unclear. Therefore, we evaluated changes in the aqueous humor levels of soluble vascular endothelial growth factor receptor (sVEGFR)-1, sVEGFR-2, and their ligands for these receptors (VEGF) patients with AMD receiving IRI. The subjects were 24 patients with AMD (24 eyes) who received 3 doses of IRI at monthly intervals. Aqueous humor samples were obtained when each IRI dose was given (visits 0, 1, and 2 at 4-week intervals). Then the suspension array method was employed to measure sVEGFR-1, sVEGFR-2, VEGF, and placental growth factor (PlGF) in aqueous humor samples from the 24 AMD patients and 13 cataract patients (as controls). Best corrected visual acuity (BCVA; logMAR) chart and central macular thickness (CMT; optical coherence tomography) were also assessed over time. At baseline, the aqueous humor levels of sVEGFR-1, sVEGFR-2, VEGF, and PlGF were significantly higher in the AMD group than in the control group. There was a significant correlation between VEGF and PlGF or between sVEGFR-1 and sVEGFR-2. BCVA and CMT both improved significantly after IRI, and the aqueous humor levels of VEGF, PlGF, and sVEGFR-1 also decreased significantly. VEGFRs may be involved in the pathogenesis of AMD. IRI improves clinical parameters in AMD patients by suppressing intraocular levels of VEGF, PlGF, and sVEGFR-1.

ZNF580 – a brake on Interleukin-6

Abstract: Zinc finger protein 580 (ZNF580) was reported to modulate angiogenesis, endothelial homeostasis and blood pressure control. ZNF580 regulated genes include VEGF-A and IL-8. However, it is unknown if ZNF580 could play a role during inflammation. The aim of this study was to find out if ZNF580 affects the expression of IL-6, if it occurs in monocytic cells and responds to inflammatory mediators. Overexpression of ZNF580 reduced LPS-induced promotor activity of IL-6. Consistently, overexpression of ZNF580 reduced by half the LPS-induced expression of IL-6. ZNF580 was strongly expressed in the nucleus of MonoMac6, a human monocytic cell line. LPS-stimulated IL-6 secretion increased when ZNF580 was suppressed with siRNA. After stimulation of MonoMac6 with LPS for 24 h, ZNF580 negatively correlated with the amount of secreted IL-6. In response to LPS, ZNF580 was increased within the first 8 h, followed by a marked decrease after 16 h. This decrease coincided with sustained IL-6 production. This study demonstrated that ZNF580 inhibits LPS-induced expression of IL-6. ZNF580 was highly expressed in monocytic cells and therefore may contribute to the modulation of its IL-6 production, at least in response to LPS. This suggests cooperation between ZNF580 and NFκB, which could play a role during sepsis.

Circulating natural antibodies to inflammatory cytokines are potential biomarkers for atherosclerosis.

Abstract: Inflammatory cytokines contribute to the development of atherosclerosis. Natural antibodies in the circulation have protective effects on common diseases including atherosclerosis-related conditions. The present study aimed to investigate the possible involvement of circulating IgG antibodies against inflammatory cytokines in atherosclerosis. A total of 220 patients with diagnosis of atherosclerosis and 200 healthy controls were recruited. Seven linear peptide antigens were used to develop an enzyme-linked immunosorbent assay in-house for detection of plasma IgG antibodies against interleukin 1β (fragments IL1β-1 and IL1β-2), IL6, IL8, tumor necrosis factor alpha (fragments TNFα-1 and TNFα-2) and IL1α. Atherosclerotic patients had an increase in the levels of circulating IgG to TNFα-1(adjusted r2 = 0.038, p < 0.001) and IL1α (adjusted r2 = 0.025, p = 0.002) compared with control subjects. Female patients mainly contributed to increased anti-TNFα-1 IgG levels (adjusted r2 = 0.073, p < 0.001) and anti-IL1α IgG levels (adjusted r2 = 0.044, p = 0.003). In addition, female patients showed higher anti-IL1β-2 IgG levels than controls (adjusted r2 = 0.023, p = 0.026). There was no significant change of circulating IgG antibodies to other cytokines. ROC curve analysis showed an AUC of 0.564 for anti-TNFα-1 IgG assay with 22.8% sensitivity against a specificity of 90.0%, and an AUC of 0.539 for anti-IL1α IgG assay with 17.8% sensitivity against a specificity of 90.0%; the anti-IL1β-2 IgG assay had an AUC of 0.580 with 26.3% sensitivity against a specificity of 89.8% in female patients. There was no correlation between plasma IgG levels and carotid intima-media thickness. Natural antibodies to inflammatory cytokines may be potential biomarkers for atherosclerosis.

In vitro suppression of inflammatory cytokine response by methionine sulfoximine

Abstract: The glutamine synthetase inhibitor methionine sulfoximine (MSO), shown previously to prevent death caused by an inflammatory liver response in mice, was tested on in vitro production of cytokines by mouse peritoneal macrophages triggered with lipopolysaccharide (LPS). MSO significantly reduced the production of Interleukin 6 (IL-6) and Tumor Necrosis Factor Alpha (TNFα) at 4 and 6 h after LPS-treatment. This reduction did not result from decreased transcription of IL-6 and TNFα genes, and therefore appeared to result from post-transcriptional inhibition of synthesis of these cytokines. MSO treatment did not inhibit total protein synthesis and did not reduce the production of a third LPS-triggered cytokine CXCL1, so the effect was not a toxic or global downregulation of the LPS response. The anti-inflammatory effects of a glutamine synthetase inhibitor were seen even though the medium contained abundant (2 mM) glutamine, suggesting that the target for this activity was not glutamine synthetase. In agreement with this hypothesis, the L,R isomer of MSO, which does not inhibit glutamine synthetase and was previously thought to be inert, both significantly reduced IL-6 secretion in isolated macrophages and increased survival in a mouse model for inflammatory liver failure. Our findings provide evidence for a novel target of MSO. Future attempts to identify the additional target would therefore also provide a target for therapies to treat diseases involving damaging cytokine responses.

Anti-inflammatory cellular targets on neutrophils elucidated using a novel cell migration model and confocal microscopy: a clinical supplementation study.

Abstract: In vivo studies have shown grape seed-derived polyphenols (GSP) to benefit in recovery from muscle injury by modulation of neutrophil infiltration into damaged tissue, thereby reducing secondary damage, as well as by facilitating an early anti-inflammatory macrophage phenotype shift. The current study aimed to provide data in this context from human models and to elucidate specific molecular targets of GSP. Using a placebo-controlled, double-blind study design, eighteen normally healthy volunteers between the ages of 18–35 years old (13 female and 5 male) were orally supplemented with 140 mg/day of GSP for 2 weeks. Blood samples (days 0 and 14) were comprehensively analysed for in vitro neutrophil chemokinetic capacity towards a chemotaxin (fMLP) using a novel neutrophil migration assay, in combination with live cell tracking, as well as immunostaining for neutrophil polarisation factors (ROCK, PI3K) at migration endpoint. Macrophage phenotype marker expression was assessed using flow cytometry. fMLP induced significant chemokinesis (P < 0.01), validating our model. GSP did not exert a significant effect on neutrophil chemokinesis in this non-compromised population, but tended to decrease overall ROCK expression in fMLP-stimulated neutrophils (P = 0.06). Macrophage phenotype markers CD274 and MPO – indicators of a pro-inflammatory M1 phenotype – seemed to be normalised relative to baseline expression levels after GSP treatment. Current data suggest that GSP may have a modulatory effect on the ROCK-PI3K-PTEN system, but results in this normal population is not conclusive and should be confirmed in a larger, more inflamed population. Potential modulation of macrophage phenotype by GSP should be investigated further.

The impact of septic stimuli on the systemic inflammatory response and physiologic insult in a preclinical non-human primate model of polytraumatic injury.

Abstract: Established animal trauma models are limited in recapitulating the pathophysiology of human traumatic injury. Herein, we characterize the physiologic insult and inflammatory response in two clinically relevant non-human primate (NHP) trauma models. Mauritian Cynomolgus Macaques underwent either a laparoscopic closed abdomen liver injury (laparoscopic 60% left-lobe hepatectomy) in an established uncontrolled severe hemorrhage model (THM), or a polytrauma hemorrhage model (PHM) involving combined liver and bowel injury, uncontrolled severe hemorrhage as well as an open full-thickness cutaneous flank wound. Fixed volume resuscitation strategies were employed in the THM and goal directed resuscitation was used in the PHM. Complete peripheral blood and critical clinical chemistry parameters, serum biomarkers of systemic inflammation, tissue perfusion parameters, as well as survival, were compared between the models throughout the 2-week study period. NHPs in both the THM (n = 7) and the PHM (n = 21) demonstrated tissue hypoperfusion (peak lactate 6.3 ± 0.71 mmol/L) with end organ injury (peak creatinine 3.08 ± 0.69 mg/dL) from a similar liver injury (60% left hemi-hepatectomy), though the PHM NHPs had a significantly higher blood loss (68.1% ± 12.7% vs. 34.3% ± 2.3%, p = 0.02), lower platelet counts (59 ± 25 vs. 205 ± 46 K/uL, p = 0.03) and a trend towards higher mortality (90.5% vs. 33.3%, p = 0.09). The inflammatory response was robust in both models with peak cytokine (IL-6 > 6000-fold above baseline) and peak leukocyte values (WBC 27 K/uL) typically occurring around t = 240 min from the time of hepatic injury. A more robust systemic inflammatory response was appreciated in the PHM resulting in marked elevations in peak serum IL-6 (7887 ± 2521 pg/mL vs.1076 ± 4833 pg/mL, p = 0.02), IL-1ra (34,499 ± 5987 pg/mL vs. 2511 ± 1228 pg/mL, p < 0.00), and IL-10 (13,411 pg/mL ± 5598 pg/mL vs. 617 pg/mL ± 252 pg/mL, p = 0.03). This comparative analysis provides a unique longitudinal perspective on the post-injury inflammatory response in two clinically relevant models, and demonstrates that the addition of septic stimuli to solid organ injury increases both the hemorrhagic insult and inflammatory response.

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages

Abstract: Breakdown of the alveolo-capillary wall is pathognomonic for Acute Lung Injury (ALI). Angiopoietins, vascular-specific growth factors, are linked to endothelial barrier dysfunction, and elevated Angiopoietin-2 (ANG2) levels are associated with poor outcome of ALI patients. Specialized immune cells, referred to as ‘TIE2-expressing monocytes and macrophages’ (TEM), were shown to specifically respond to ANG2 binding. However, their involvement in acute inflammatory processes is so far completely undescribed. Thus, our aim was to assess the dynamics of TEMs in a murine model of ALI. Intratracheal instillation of LPS induced a robust pulmonary pro-inflammatory response with endothelial barrier dysfunction and significantly enhanced ANG2 expression. The percentage number of TEMs, assessed by FACS analysis, was more than trebled compared to controls, with TEM count in lungs reaching more than 40% of all macrophages. Such distinct dynamic was absent in all other analyzed compartments (alveolar space, spleen, blood). Incubation of the monocytic cell line THP-1 with LPS or TNF-α resulted in a dose-dependent, significant upregulation of TIE2, suggesting that not recruitment from extra-pulmonary compartments but TIE2 upregulation in resident macrophages accounts for increased lung TEM frequencies. For the first time, our data provide evidence that the activity of TEMs changes at sites of acute inflammation.

Contribution of diminished kidney transplant GFR to increased circulating chemokine ligand 27 level

Abstract: Inflammatory chemokine ligands (CCLs) play an important role in cardiovascular disease and allograft injury. CCLs may independently associate with diminished estimated glomerular filtration rate (eGFR) in stable renal transplant recipients (RTR). Plasma levels of 19 CCLs (1, 2, 3, 4, 5, 8, 11, 13, 15, 17, 21, 24, 26, 27, CXCL5, 8, 10, 12 and 13) were measured in a cohort of 101 RTR. The cohort was divided according to CKD-EPI equation into three groups; group 1: eGFR ≥ 60 ml/min, group 2: eGFR 30–59.9 ml/min and group 3 eGFR ≤ 29.9 ml/min. ANOVA, Krusklwallis, Mann- Whitney Spearman correlation and regression analysis tests were used to determine association between reduced eGFR and inflammatory CCLs plasma levels measured by multiplex techniques. 20 healthy subjects with eGFR above 90 ml/min were included as control. Significance was sat at < 0.05. Levels of CCLs 1, 4, 15, 27, CXCL8 and CXCL10 were significantly different among the four studied groups. Multivariate regression analysis (MVA) between eGFR and all CCLs demonstrated that CCL27 was the only ligand to remain significantly associated with diminished eGFR {P = 0.021 and r = − 0.35,(P = 0.001)}. In a second MVA between CCL 27 and patient’s demographics and laboratory variables, diminished eGFR, and elevated PTH, out of the twenty one available variables remained significantly associated with elevated CCL27levels. Diminished eGFR in stable RTR is associated with elevated plasma levels of CCL27. This association may explain, at least in part, the independent contribution of reduced eGFR to enhanced inflammation in RTR.