Increased Release Of TNF-α Research Articles

Microbiome-derived metabolite effects on intestinal barrier integrity and immune cell response to infection.

The gut microbiota exerts a significant influence on human health and disease. While compositional changes in the gut microbiota in specific diseases can easily be determined, we lack a detailed mechanistic understanding of how these changes exert effects at the cellular level. However, the putative local and systemic effects on human physiology that are attributed to the gut microbiota are clearly being mediated through molecular communication. Here, we determined the effects of gut microbiome-derived metabolites l-tryptophan, butyrate, trimethylamine (TMA), 3-methyl-4-(trimethylammonio)butanoate (3,4-TMAB), 4-(trimethylammonio)pentanoate (4-TMAP), ursodeoxycholic acid (UDCA), glycocholic acid (GCA) and benzoate on the first line of defence in the gut. Using in vitro models of intestinal barrier integrity and studying the interaction of macrophages with pathogenic and non-pathogenic bacteria, we could ascertain the influence of these metabolites at the cellular level at physiologically relevant concentrations. Nearly all metabolites exerted positive effects on barrier function, but butyrate prevented a reduction in transepithelial resistance in the presence of the pathogen Escherichia coli, despite inducing increased apoptosis and exerting increased cytotoxicity. Induction of IL-8 was unaffected by all metabolites, but GCA stimulated increased intra-macrophage growth of E. coli and tumour necrosis-alpha (TNF-α) release. Butyrate, 3,4-TMAB and benzoate all increased TNF-α release independent of bacterial replication. These findings reiterate the complexity of understanding microbiome effects on host physiology and underline that microbiome metabolites are crucial mediators of barrier function and the innate response to infection. Understanding these metabolites at the cellular level will allow us to move towards a better mechanistic understanding of microbiome influence over host physiology, a crucial step in advancing microbiome research.

8343 The Role Of Pancreatic Nerve Activity On The Development Of Beta Cell Dysfunction With The Macrophage-Beta Cell Interaction

Abstract Disclosure: J. Yu: None. Y. Wang: None. L. Sha: None. Islet inflammation is a vital factor to the beta cell dysfunction which is driven by inflammatory cytokines TNF-α and IL-1β released by resident macrophages. Pancreatic nerve innervates islet function and our previous study suggested that pancreatic nerve activity might involved in the development of pancreatic beta cell dysfunction. However, it is not known whether pancreatic nerve activity modulates macrophage activity and contributes to the macrophage - beta cell interaction. The aim of our study was to investigate the role of nerve activity on it and the possible mechanism. Methods: Sprague Dawley Rats and high fat diet-induced obese rates were used. An intrapancreatic nerve truck with its intact innervated pancreatic islets were used for the study. The nerve activity was evoked with electric nerve stimulation and insulin and ATP released by beta cells, TNF-α released by macrophages, and IL-1β released mainly by macrophages were measured with nerve activation. Results: Insulin secretion was induced significantly with the nerve activation (8 Hz). With nerve activation, ATP level was significantly increased by 25.5 ± 11.7%. The increased releases of insulin and ATP were abolished with cholinergic M receptor blocker atropine. With nerve activation, TNF-α and IL-1β levels were increased by 25.3±7.6% and 51.8±11.7%, respectively (p<0.05). Interestingly, the increases of TNF-α and IL-1β with nerve activation were also blocked by atropine. Since it is known that acetylcholine does not induce pro-inflammatory cytokine release in macrophages, we suspected that the nerve activity induced TNF-α and IL-1β releases were indirect. ATP is likely a candidate for the indirect effect as receptors. With applying Suramin, a ATP receptor blocker, nerve activation increased TNF-α release was inhibited significantly and IL-1β release was trended to inhibited. atropine, an M receptor blocker, compared with the Control group, decreased by 88.0±3.7% and 63.1±19.6%, respectively (p< 0.01). In obese rats, nerve activation increased TNF-α and IL-1β content by 68.1 ± 22.6% and 39.7 ± 7.4%, respectively, and suramin significantly inhibited the nerve activation induced increase of TNF-α release and trended to inhibit IL-1β release. Conclusion: Our results suggested that the pancreatic nerve activity contribute to the macrophage-beta cell interaction in the development of beta cells dysfunction by enhancing ATP release from islet beta cells. Presentation: 6/3/2024

The Effect of Static Magnetic Fields of Different Strengths and Polarities on Cytokine Production by Human Lymphocytes In Vitro.

In contrast to electromagnetic fields, static magnetic fields (SMFs) have not been extensively studied in terms of their potential health consequences. Although upward- and downward-oriented magnetic poles may cause various biological effects, only the pole with the upward orientation has been mainly investigated. Considering that the interaction of antigen-presenting cells (APCs) and T lymphocytes is crucial to trigger an immune response, we assessed the effect of long-term exposure of human T lymphocytes and dendritic cells (DCs) to moderate strength SMFs of different orientations focusing on the cytokine profile of activated T cells. Cultures of allogenic T lymphocytes and DCs (immature and matured by TLR3 and TLR7 agonists) were continuously exposed to four SMFs. The intensity of the applied field was 1 militesla (mT) or 56 mT of the upward- and downward-oriented pole of the SMF. Cell culture supernatants were assayed for IFN-γ, IL-4, IL-17, TNF-α, TNF-β, IL-1 β, IL-6, IL-8, and IL-10 by ELISA or flow cytometry. The upward-oriented 56 mT SMF significantly increased the release of IFN-γ and TNF-β (both p < 0.05) in the cell culture supernatants of T cells and immature DCs. In contrast, the same cultures exposed to the upward-oriented 1 mT SMF showed significantly elevated levels of IL-17 (p < 0.05). The levels of IL-4, TNF-α, IL-1 β, IL-6, IL-8, and IL-10 were not affected by the upward-oriented SMF. The downward-oriented 56 mT SMF increased TNF-α release when T cells were stimulated with mature DCs. The production of other cytokines was unchanged by the downward-oriented SMF. These findings demonstrate for the first time different in vitro biological effects of upward- and downward-oriented static magnetic fields on the cytokine production of T cells activated by DCs, helping to better understand SMF effects on the immune system and suggesting that the selective SMF effect on the immune response could have potential therapeutic effects in different immune-mediated disorders.

Chronotherapy involving rosiglitazone regulates the phenotypic switch of vascular smooth muscle cells by shifting the phase of TNF-α rhythm through triglyceride accumulation in macrophages

ObjectivesVascular diseases are often caused by the interaction between macrophages and vascular smooth muscle cells (VSMCs). This study aims to elucidate whether chronotherapy with rosiglitazone (RSG) can regulate the secretion rhythm of macrophages, thereby controlling the phenotypic switch of VSMCs and clarifying the potential molecular mechanisms, providing a chronotherapeutic approach for the treatment of vascular diseases. MethodsRAW264.7 cells and A7r5 cells were synchronized via a 50 % FBS treatment. M1-type macrophages were induced through Lipopolysaccharide (LPS) exposure. Additionally, siRNA and plasmids targeting PPARγ were transfected into macrophages. The assessment encompassed cell viability, migration, inflammatory factor levels, lipid metabolites, clock gene expression, and relative protein expression. ResultsWe revealed that, in alignment with core clock genes Bmal1 and CLOCK, RSG administration at ZT2 advanced the phase of TNF-α release rhythm, while ZT12 administration shifted it backward. Incubation with TNF-α at ZT2 significantly promoted the phenotype switch of VSMCs. This effect diminished when incubated at ZT12, implicating the involvement of the clock-MAPK pathway in VSMCs. Furthermore, RSG administration at ZT2 advanced the phases of PPARγ and Bmal1 genes, whereas ZT12 administration shifted them backward. Additionally, PPARγ overexpression significantly induced triglyceride (TG) accumulation in macrophages. Exogenous TG upregulated Bmal1 and CLOCK gene expression in macrophages and significantly increased TNF-α release. ConclusionChronotherapy involving RSG induces TG accumulation within macrophages, resulting in alterations in circadian gene rhythms. These changes, in turn, modulate the phase of rhythmic TNF-α release and play a regulatory role in VSMCs phenotype switch. Our study establishes a theoretical foundation for chronotherapy of PPARγ agonists.

A monocentric, randomized, double-blind, controlled crossover trial of nasturtium (Tropaeolum majus) on the lipid regulator prostaglandin E2.

As prostaglandin E2 (PGE2) has important roles in physiological and inflammatory functions, a double-blind randomized controlled crossover study to investigate the potential of nasturtium (Tropaeolum majus) for modulating PGE2 was conducted, aiming at clarifying the role of benzyl isothiocyanate (BITC). As secondary parameters leukotriene 4 (LTB4), and cytokine release (tumor necrosis factor alpha, TNF-α; interleukins IL-1β, IL-10, and IL-12) were quantified. Thirty-four healthy female participants consumed 1.5 g nasturtium containing BITC, (verum) or no BITC (control) twice a day for 2 weeks each. Nasturtium intervention resulted in an increase in mean PGE2 levels in serum samples (verum: 1.76-fold, p ≤ 0.05; control: 1.78-fold, p ≤ 0.01), and ex vivo stimulated peripheral blood mononuclear cells (PBMC) (verum: 1.71-fold, p ≤ 0.01; control: 1.43-fold). Using a pre-to-post responder analysis approach, 18 of 34 subjects showed a > 25% PGE2 increase in serum, while it was >25% decreased for 9 subjects (stimulated PBMC: 14 and 8 of 28, respectively). Under the selected conditions, the BITC content of nasturtium did not affect the observed changes in PGE2. Verum intervention also increased mean LTB4 serum level (1.24-fold, p ≤ 0.01), but not in LPS stimulated PBMC, and significantly increased TNF-α release in stimulated PBMC after 3 h (verum: 1.65-fold, p = 0.0032; control: 1.22-fold, p = 0.7818). No change was seen in the anti-inflammatory cytokine IL-10, or the pro-inflammatory cytokines IL-1β, and IL-12. In contrast to the previously reported in vitro results, on average, LPS activated PBMC and serum from both groups showed increased PGE2 levels. Further analyses suggest that PGE2 release after intervention could possibly depend on the baseline PGE2 level. Identification of phenotypes that respond differently to the nasturtium intervention could be useful to establish personalized approaches for dosing phytopharmaceuticals medicines.

Lipidomic Profiling of Bronchoalveolar Lavage Fluid Extracellular Vesicles Indicates Their Involvement in Lipopolysaccharide-Induced Acute Lung Injury

Emerging data support the pivotal role of extracellular vesicles (EVs) in normal cellular physiology and disease conditions. However, despite their abundance, there is much less information about the lipid mediators carried in EVs, especially in the context of acute lung injury (ALI). Our data demonstrate that C57BL/6 mice subjected to intranasal Escherichia coli lipopolysaccharide (LPS)-induced ALI release, a higher number of EVs into the alveolar space, compared to saline-treated controls. EVs released during ALI originated from alveolar epithelial cells, macrophages, and neutrophils and carry a diverse array of lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The eicosanoids in EVs correlated with cellular levels of arachidonic acid, expression of cytosolic phospholipase A2, cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome epoxygenase p450 proteins in pulmonary macrophages. Furthermore, EVs from LPS-toll-like receptor 4 knockout (TLR4^-/-) mice contained significantly lower amounts of COX and LOX catalyzed eicosanoids and ω-3 PUFA metabolites. More importantly, EVs from LPS-treated wild-type mice increased TNF-α release by macrophages and reduced alveolar epithelial monolayer barrier integrity compared to EVs from LPS-treated TLR4^−/− mice. In summary, our study demonstrates for the first time that the EV carried PUFA metabolite profile in part depends on the inflammatory status of the lung macrophages and modulates pulmonary macrophage and alveolar epithelial cell function during LPS-induced ALI.

Human monocytes respond to lipopolysaccharide (LPS) stimulation in a sex-dependent manner.

Monocytes play a critical role in inflammation and immune response, their activity being sex‐dependent. However, the basis of sex differences is not well understood. Therefore, we investigated the lipopolysaccharide (LPS) effects on tumor necrosis factor‐α (TNF‐α) release, autophagy, and chemotaxis in freshly isolated monocytes from healthy young men and women. In basal conditions, male and female monocytes had similar TNF‐α release, chemotaxis, and estrogen receptors (ER‐α) and ER‐β expression, while the LC3II/I ratio was significantly higher in males. LPS treatment induced qualitative and quantitative sex differences. It reduced autophagy and increased TNF‐α release only in male monocytes, while, chemotaxis was significantly influenced only in female cells. Moreover, it reduced the expression of ER‐α only in female cells, while ER‐β expression was reduced in both sexes, but more markedly in female cells. Finally, the interplay between LPS treatment and 17‐β‐estradiol (E2) was present only in female cells. Globally, these findings expand the concept that sex plays a role in regulating monocytes' functions, being sex differences cell‐ and parameter‐specific.

Administration of a 20-Hydroxyeicosatetraenoic Acid Synthesis Inhibitor Improves Outcome in a Rat Model of Pediatric Traumatic Brain Injury

The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. Here, we examined whether treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI). Male rats at postnatal day 9–10 underwent controlled cortical impact followed by intraperitoneal injection with vehicle or HET0016 (1 mg/kg, 5 min and 3 h post-injury). HET0016 decreased the lesion volume by over 50% at 3 days of recovery, and this effect persisted at 30 days as the brain matured. HET0016 decreased peri-lesion gene expression of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β]) at 1 day and increased reparative cytokine (IL-4, IL-10) expression at 3 days. It also partially preserved microglial ramified processes, consistent with less activation. HET0016 decreased contralateral hindlimb foot faults and improved outcome on the novel object recognition memory task 30 days after TBI. In cultured BV2 microglia, HET0016 attenuated the lipopolysaccharide-evoked increase in release of TNF-α. Our data show that HET0016 improves acute and long-term histologic and functional outcomes, in association with an attenuated neuroinflammatory response after contusion of an immature rat brain.

Exposure to particle debris generated from passenger and truck tires induces different genotoxicity and inflammatory responses in the RAW 264.7 cell line.

A number of studies have shown variable grades of cytotoxicity and genotoxicity in in vitro cell cultures, laboratory animals and humans when directly exposed to particle debris generated from tires. However, no study has compared the effects of particles generated from passenger tires with the effects of particles from truck tires. The aim of this study was to investigate and relate the cyto- and genotoxic effects of different types of particles (PP, passenger tire particles vs. TP, truck tire particles) in vitro using the phagocytic cell line RAW 264.7 (mouse leukaemic monocyte macrophage cell line). The viability of RAW 264.7 cells was determined by the 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay following exposure for 4, 24 and 48 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). The effects of particles of passenger and truck tires on cell proliferation and genotoxicity were evaluated by means of the cytokinesis-block micronucleus (CBMN) assay following exposure for 24 hours to different particle concentrations (10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml). In MTS assay, after 24 hours, it was found that PP induced a 30% decrease in metabolic activity at a concentration of 10 μg/ml, while TP caused reductions of 20% and 10% at concentrations of 10 μg/ml and 50 μg/ml, respectively. At 48 hours after the treatments, we observed increased metabolic activity at 50 μg/ml and 100 μg/ml for the PP while only at 50 μg/ml for the TP. The CBMN assay showed a significant increase in the number of micronuclei in the cells incubated with PP in all experimental conditions, while the cells treated with TP showed a meaningful increase only at 10 μg /ml. We utilized the TNF-α ELISA mouse test to detect the production of tumour necrosis factor-alpha (TNF-α) in RAW 264.7 cells. The effect of passenger and truck particles on TNF-α release was evaluated following exposure for 4 and 24 hours.After 4 hours of incubation, the cells treated with PP and TP at 100 μg / ml showed a slight but significant increase in TNF-α release, while there was a significant increase in the release of TNF-α after 24 hours of incubation with both tire samples in the cells treated with 50 and 100 μg / ml PP. The data obtained show a higher cytotoxic, clastogenic/genotoxic and inflammatory effects of passenger compared to the truck tire particles.

PM2.5 generated during rapid failure of fiber-reinforced concrete induces TNF-alpha response in macrophages

Failure of large, concrete structures can lead to the generation of very small fragments, including aerosols in the fine fraction, which have aerodynamic diameters of ≤2.5 μm (PM2.5). These aerosols can persist in the environment, pose exposure risks, and potentially cause negative health effects. New trends in construction favor the use of concrete reinforced with steel fibers, but little is known about the nature of the fragments generated during its failure. This study investigated the fragmentation of several steel-fiber reinforced concrete formulations using dynamic compression testing. The release of tumor necrosis factor alpha (TNF-α), an inflammatory marker widely used in both human and animal studies, was then analyzed to determine the effects of the fragments in the aerosol fine fraction on mouse macrophages (RAW 264.7). All concrete formulations studied showed statistically increased TNF-α release, which was inversely correlated with fiber length and fiber content (% weight). In addition, results from a select set of concrete formulations also showed a clear dose-response relationship. This paper postulates the fracture mechanisms by which concrete parameters (i.e., fiber length and content) lead to the generation of PM2.5, producing the observed TNF-α release.

Differential modulation of SIRT6 deacetylase and deacylase activities by lysine-based small molecules

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase regulating important functions: modulators of its enzymatic activity have been considered as possible therapeutic agents. Besides the deacetylase activity, SIRT6 also has NAD+-dependent deacylase activity, whereby it regulates the secretion of cytokines and proteins. We identified novel SIRT6 modulators with a lysine-based structure: compound 1 enhances SIRT6 deacylase while inhibiting the deacetylase activity. As expected based on the biological effects of SIRT6 deacetylase activity, compound 1 increased histone 3 lysine 9 acetylation and the activity of glycolytic enzymes. Moreover, the fact that compound 1 enhanced SIRT6 deacylase activity was accompanied by an increased TNF-α release. In conclusion, new SIRT6 modulators with a lysine-like structure were identified, with differential effects on specific SIRT6 activities. The novel SIRT6 modulator concomitantly inhibits deacetylase and enhances deacylase activity.

Does TNF-α regulate thymic T cell developmental block in mice with dysregulated expression of Ly-6A?

Abstract Ly-6A is a 12 kD GPI anchored member of the Ly-6 superfamily. Ly-6A and other gene family members show broad tissue expression, including expression on T lymphocytes. Ly-6A has been characterized as a cell adhesion molecule and shown to regulate cell signaling in T cells; specifically cytokine secretion. Published work indicates that this protein’s role in signaling regulation extends as far as mediated cell death pathways, resulting in growth arrest and ultimately cell death in T cell lines. Expression of Ly-6A on T cells in the thymus is developmentally regulated. Peak expression occurs during early stages of development, diminishes during maturation, and reappears at low levels on mature, CD4+ T cells. T lymphocytes in the thymus of Ly-6A transgenic (overexpressing) mice have been observed to experience increased instances of cell death and arrested early stage development. However, the underlying mechanisms of reduced cellularity in Ly-6A transgenic mice is unknown. I hypothesized that TNFα, a cytokine known to have cell death inducing properties, contributes to inhibited T cell development, lower cellularity and extensive cell death of developing T cells in thymi of Ly-6A transgenic mice. To address my hypothesis, I investigated the levels of TNFα in normal and Ly-6A transgenic mice. I find that there is decrease in cellularity in the developing thymus of Ly-6A transgenic mice compared to Ly-6A wildtype mice, consistent with published reports. On a per cell basis, this finding corresponds with increased TNFα release from Ly-6A transgenic T-lymphocytes. We plan to investigate development of T cells in Ly-6A transgenic mice that lack TNFα and specifically look for reversal of block in thymic T cell development.

G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses

ABSTRACTLeucine rich-repeat kinase 2 (LRRK2) is involved in the pathogenesis of Parkinson’s disease (PD). LRRK2 has kinase and GTPase activities, and mediates several cell functions, including vesicle trafficking, apoptosis, autophagy, mitochondrial dynamics, and neuroinflammation. G2019S (GS) is the most prevalent mutation of LRRK2. The mutation increases kinase activity, suggesting that this activity is crucial for PD pathogenesis. The activation and inhibition of LRRK2 kinase increases and reduces the levels of proinflammatory cytokines, respectively suggesting that the role of LRRK2 in neuroinflammation is critical for the pathology of PD. Previously, we demonstrated that microglial activation by lipopolysaccharide (LPS) increases mitochondrial fission via the activation of LRRK2 kinase, while LRRK2 kinase inhibition diminishes the fission morphology and release of tumor necrosis factor-alpha (TNFα) in BV2 or rat primary microglia and the brains of GS transgenic mice. In this study, the ectopic expression of GS LRRK2 in BV2 cells significantly elevated the expression of Drp1 along the fragmented mitochondria and decreased mitochondria size compared with controls. GS LRRK2-transfected BV2 cells displayed significantly increased TNFα release and neuronal death. Inhibition of LRRK2 kinase alleviated these features. TNFα levels in brains of GS mice were significantly increased compared to those in their littermates. These data further support our previous findings concerning LPS-induced neuroinflammation and mitochondrial fission in microglia via LRRK2 kinase activation.

Ceftaroline modulates the innate immune and host defense responses of immunocompetent cells exposed to cigarette smoke

BackgroundCigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system’s response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. Aims and methodsThe present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. ResultsThe constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. ConclusionCeftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients.

Serum amyloid A enrichment impairs the anti-inflammatory ability of HDL from diabetic nephropathy patients

AimsImpaired anti-inflammatory ability of high-density lipoprotein (HDL) has been demonstrated in patients with type-2 diabetes mellitus (T2DM). However, whether HDL from patients with diabetic nephropathy (DN) suffers additional damage remains unknown. This study compared the anti-inflammatory capacities of HDL from healthy controls, T2DM patients with normal renal function, and T2DM patients with DN. Materials and methodsHDL was isolated from healthy controls (n=33) and T2DM patients with normal renal function (n=21), chronic kidney disease (CKD) (n=27), and end-stage renal disease (ESRD) (n=27). Human peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pretreated with HDL (100μg/mL) for 1h, then incubated with lipopolysaccharide (LPS) (50ng/mL) for 24h. The anti-inflammatory ability of HDL was measured as the secretion of TNF-α in LPS-activated monocytes. ResultsThe anti-inflammatory ability of HDL was gradually impaired as kidney function declined. Serum amyloid A (SAA) concentration in HDLDN significantly increased and was positively correlated with the impaired anti-inflammatory ability of HDL (Pearson r=0.315, P=0.006). Furthermore, HDL supplemented with SAA significantly increased TNF-α release from PBMCs compared with that from control HDL. ConclusionsThese findings identified an impaired anti-inflammatory capacity of HDL from DN patients, which might be attributable to SAA enrichment.

Intracellular Ca2+ homeostasis and JAK1/STAT3 pathway are involved in the protective effect of propofol on BV2 microglia against hypoxia-induced inflammation and apoptosis.

BackgroundPerioperative hypoxia may induce microglial inflammation and apoptosis, resulting in brain injury. The neuroprotective effect of propofol against hypoxia has been reported, but the underlying mechanisms are far from clear. In this study, we explored whether and how propofol could attenuate microglia BV2 cells from CoCl2-induced hypoxic injury.MethodsMouse microglia BV2 cells were pretreated with propofol, and then stimulated with CoCl2. TNF-α level in the culture medium was measured by ELISA kit. Cell apoptosis and intracellular calcium concentration were measured by flow cytometry analysis. The effect of propofol on CoCl2-modulated expression of Ca2+/Calmodulin (CaM)-dependent protein kinase II (CAMKIIα), phosphorylated CAMKIIα (pCAMKIIα), STAT3, pSTAT3Y705, pSTAT3S727, ERK1/2, pERK1/2, pNFκB(p65), pro-caspase3, cleaved caspase 3, JAK1, pJAK1, JAK2, pJAK2 were detected by Western blot.ResultsIn BV2 cell, CoCl2 treatment time-dependently increased TNF-α release and induced apoptosis, which were alleviated by propofol. CoCl2 (500μmol/L, 8h) treatment increased intracellular Ca2+ level, and caused the phosphorylation of CAMKIIα, ERK1/2 and NFκB (p65), as well as the activation of caspase 3. More importantly, these effects could be modulated by 25μmol/L propofol via maintaining intracellular Ca2+ homeostasis and via up-regulating the phosphorylation of JAK1 and STAT3 at Tyr705.ConclusionPropofol could protect BV2 microglia from hypoxia-induced inflammation and apoptosis. The potential mechanisms may involve the maintaining of intracellular Ca2+ homeostasis and the activation of JAK1/STAT3 pathway.

Involvement of p38-βTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis.

Early release of tumor necrosis factor-alpha (TNF-α) during radiotherapy of thoracic cancers plays an important role in radiation pneumonitis, whose inhibition may provide lung radioprotection. We previously reported radiation inactivates Tristetraprolin (TTP), a negative regulator of TNF-α synthesis, which correlated with increased TNF-α release. However, the molecular events involved in radiation-induced TTP inactivation remain unclear. To determine if eliminating Ttp in mice resulted in a phenotypic response to radiation, Ttp-null mice lungs were exposed to a single dose of 15 Gy, and TNF-α release and lung inflammation were analyzed at different time points post-irradiation. Ttp−/− mice with elevated (9.5±0.6 fold) basal TNF-α showed further increase (12.2±0.9 fold, p<0.02) in TNF-α release and acute lung inflammation within a week post-irradiation. Further studies using mouse lung macrophage (MH-S), human lung fibroblast (MRC-5), and exogenous human TTP overexpressing U2OS and HEK293 cells upon irradiation (a single dose of 4 Gy) promoted p38-mediated TTP phosphorylation at the serine 186 position, which primed it to be recognized by an ubiquitin ligase (E3), beta transducing repeat containing protein (β-TrCP), to promote polyubiquitination-mediated proteasomal degradation. Consequently, a serine 186 to alanine (SA) mutant of TTP was resistant to radiation-induced degradation. Similarly, either a p38 kinase inhibitor (SB203580), or siRNA-mediated β-TrCP knockdown, or overexpression of dominant negative Cullin1 mutants protected TTP from radiation-induced degradation. Consequently, SB203580 pretreatment blocked radiation-induced TNF-α release and radioprotected macrophages. Together, these data establish the involvement of the p38-βTrCP-TTP-TNFα signaling axis in radiation-induced lung inflammation and identified p38 inhibition as a possible lung radioprotection strategy.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

Pharmacological interference with vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, is considered a potential strategy for cancer therapy. Macrophages are critically involved in tumor progression and may occur as pro-tumoral M2 phenotype, whereas classically-activated M1 can inhibit tumor development for example by releasing tumor-suppressing molecules, including tumor necrosis factor (TNF)α. Here, we show that targeting V-ATPase by selective inhibitors such as archazolid upregulates the expression and secretion of TNFα in lipopolysaccharide (LPS)- or LPS/interferon (INF)γ-activated M1-like macrophages derived from human blood monocytes. In contrast, archazolid failed to elevate TNFα production from uncommitted (M0) or interleukin (IL)-4-treated M2-like macrophages. Secretion of other relevant cytokines (i.e., IL-1β, IL-6, IL-10) or chemokines (i.e. IL-8 and monocyte chemotactic protein-1) from M1 was not affected by archazolid. Though V-ATPase inhibitors elevated the lysosomal pH in M1 comparable to chloroquine or ammonium chloride, the latter agents suppressed TNFα secretion. Archazolid selectively increased TNFα mRNA levels, which was abolished by dexamethasone. Interestingly, archazolid enhanced the phosphorylation and nuclear translocation of the p65 subunit of NFκB and stimulated phosphorylation of SAPK/JNK. In a microfluidically-supported human tumor biochip model, archazolid-treated M1 significantly reduced tumor cell viability. Together, our data show that V-ATPase inhibition selectively upregulates TNFα production in classically-activated macrophages along with NFκB and SAPK/JNK activation. Such increased TNFα release caused by V-ATPase inhibitors may contribute to tumor suppression in addition to direct targeting cancer cells.

Modulation of Human Airway Barrier Functions during Burkholderia thailandensis and Francisella tularensis Infection Running Title: Airway Barrier Functions during Bacterial Infections.

The bronchial epithelium provides protection against pathogens from the inhaled environment through the formation of a highly-regulated barrier. In order to understand the pulmonary diseases melioidosis and tularemia caused by Burkholderia thailandensis and Fransicella tularensis, respectively, the barrier function of the human bronchial epithelium were analysed. Polarised 16HBE14o- or differentiated primary human bronchial epithelial cells (BECs) were exposed to increasing multiplicities of infection (MOI) of B. thailandensis or F. tularensis Live Vaccine Strain and barrier responses monitored over 24–72 h. Challenge of polarized BECs with either bacterial species caused an MOI- and time-dependent increase in ionic permeability, disruption of tight junctions, and bacterial passage from the apical to the basolateral compartment. B. thailandensis was found to be more invasive than F. tularensis. Both bacterial species induced an MOI-dependent increase in TNF-α release. An increase in ionic permeability and TNF-α release was induced by B. thailandensis in differentiated BECs. Pretreatment of polarised BECs with the corticosteroid fluticasone propionate reduced bacterial-dependent increases in ionic permeability, bacterial passage, and TNF-α release. TNF blocking antibody Enbrel® reduced bacterial passage only. BEC barrier properties are disrupted during respiratory bacterial infections and targeting with corticosteroids or anti-TNF compounds may represent a therapeutic option.

Expression of proteinase-activated receptor (PAR)-2 in monocytes from allergic patients and potential molecular mechanism.

Serine proteases play an important role in inflammation via PARs. However, little is known of expression levels of PARs on monocytes of allergic patients, and influence of serine proteases and PARs on TNF-α secretion from monocytes. Using quantitative real-time PCR (qPCR) and flowcytometry techniques, we observed that the expression level of PAR-2 in monocytes of patients with allergic rhinitis and asthma was increased by 42.9 and 38.2%. It was found that trypsin, thrombin, and tryptase induced up to 200, 320, and 310% increase in TNF-α release from monocytes at 16h, respectively. PAR-1 agonist peptide, SFLLR-NH2, and PAR-2 agonist peptide tc-LIGRLO-NH2 provoked up to 210 and 240% increase in release of TNF-α. Since SCH 79797, a PAR-1 antagonist, and PD98059, an inhibitor of ERK inhibited thrombin- and SFLLR-NH2-induced TNF-α release, the action of thrombin is most likely through a PAR-1- and ERK-mediated signaling mechanism. Similarly, because FSLLRN-NH2, an inhibitor of PAR-2 diminished tryptase- and tc-LIGRLO-NH2-induced TNF-α release, the action of tryptase appears PAR-2 dependent. Moreover, in vivo study showed that both recombinant cockroachmajor allergens Per a 1 and Per a 7 provoked upregulation of PAR-2 and PAR-1 expression on CD14+ cells in OVA-sensitized mouse peritoneum. In conclusion, increased expression of PAR-2 in monocytes of AR and asthma implicates that PAR-2 likely play a role in allergy. PAR-2- and PAR-1-mediated TNF-α release from monocytes suggests that these unique protease receptors are involved in the pathogenesis of inflammation.