Maresin-1 reduces the pro-inflammatory response of bronchial epithelial cells to organic dust

Background

Exposure to organic dust causes detrimental airway inflammation. Current preventative and therapeutic measures do not adequately treat resulting disease, necessitating novel therapeutic interventions. Recently identified mediators derived from polyunsaturated fatty acids exhibit anti-inflammatory and pro-resolving actions. We tested the potential of one of these mediators, maresin-1 (MaR1), in reducing organic dust-associated airway inflammation.

Methods

As bronchial epithelial cells (BECs) are pivotal in initiating organic dust-induced inflammation, we investigated the in vitro effects of MaR1 on a human BEC cell line (BEAS-2B). Cells were pretreated for 1 hour with 0–200 nM MaR1, followed by 1–24 hour treatment with 5% hog confinement facility-derived organic dust extract (HDE). Alternatively, a mouse lung slice model was utilized in supportive cytokine studies. Supernatants were harvested and cytokine levels determined via enzyme-linked immunosorbent assays. Epithelial cell protein kinase C (PKC) isoforms α and ϵ, and PKA activities were assessed via radioactivity assays, and NFκB and MAPK-related signaling mechanisms were investigated using luciferase vector reporters.

Results

MaR1 dose-dependently reduced IL-6 and IL-8 production following HDE treatment of BECs. MaR1 also reduced HDE-stimulated cytokine release including TNF-α in a mouse lung slice model when given before or following HDE treatment. Previous studies have established that HDE sequentially activates epithelial PKCα and PKCϵ at 1 and 6 hours, respectively that regulated TNF-α, IL-6, and IL-8 release. MaR1 pretreatment abrogated these HDE-induced PKC activities. Furthermore, HDE treatment over a 24-hour period revealed temporal increases in NFκB, AP-1, SP-1, and SRE DNA binding activities, using luciferase reporter assays. MaR1 pretreatment did not alter the activation of NFκB, AP-1, or SP-1, but did reduce the activation of DNA binding at SRE.

Conclusions

These observations indicate a role for MaR1 in attenuating the pro-inflammatory responses of BECs to organic dust extract, through a mechanism that does not appear to rely on reduced NFκB, AP-1, or SP-1-related signaling, but may be mediated partly through SRE-related signaling. These data offer insights for a novel mechanistic action of MaR1 in bronchial epithelial cells, and support future in vivo studies to test MaR1’s utility in reducing the deleterious inflammatory effects of environmental dust exposures.     

Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

Introduction

Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment.

Methods

An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling.

Results

Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment.

Conclusions

These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity.     

Salusin-β, but Not Salusin-α, Promotes Human Umbilical Vein Endothelial Cell Inflammation via the p38 MAPK/JNK-NF-κB Pathway

Objective

Recently, salusin-β has been reported to have pro-atherosclerotic effects, but salusin-α has anti-atherosclerotic effects. Our previous study has shown that salusin-β but not salusin-α promotes vascular inflammation in apoE-deficient mice. However, the underlying mechanism remains unknown. In this study, we observed the effect of salusins on inflammatory responses and the MAPK-NF-κB signaling pathway in human umbilical vein endothelial cells (HUVECs).

Methods and Results

HUVECs were incubated with different concentrations of salusin-α and salusin-β. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were quantified using quantitative real-time polymerase chain reaction (PCR). The protein expressions of VCAM-1, MCP-1, I-κBα, NF-κB, p-JNK and p-p38 MAPK were measured using western blotting analysis. Our results showed that in HUVECs, salusin-β could up-regulate the levels of IL-6, TNF-α, VCAM-1 and MCP-1, promote I-κBα degradation and NF-κB activation, and increase the phosphorylation of JNK and p38 MAPK. These effects could be inhibited by p38 MAPK inhibitor SB203580 and/or JNK inhibitor SP600125. In contrast, salusin-α could selectively decrease VCAM-1 protein, but did not show any effect on the expressions of VCAM-1 mRNA, TNF-α, IL-6, MCP-1, I-κBα, NF-κB, p-JNK or p-p38 MAPK.

Conclusion

Salusin-β was able to promote inflammatory responses in HUVECs via the p38 MAPK-NF-κB and JNK-NF-κB pathways. In contrast, salusin-α failed to show any significant effects on the inflammatory responses in HUVECs. These results provide further insight into the mechanisms behind salusins in vascular inflammation and offer a potential target for the prevention and treatment of atherosclerosis.     

The Association between Polymorphisms in the MRPL4 and TNF-α Genes and Susceptibility to Allergic Rhinitis

Background

Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa, involving a complex interaction between genetic and environmental factors. Evidence suggests that polymorphisms in the gene coding for mitochondrial ribosomal protein L4 (MRPL4), located in close proximity to intercellular adhesion molecule-1 (ICAM-1) gene on chromosome location 19p13.2, may influence the risk factor for the development of AR.

Objective

The aim of our study was to investigate any association between AR susceptibility and polymorphisms in ICAM-1 gene, as well as associations between AR risk and polymorphisms in MRPL4, nuclear factor-kappaB (NF-κB) and tumor necrosis factor alpha(TNF-α) genes, associated with ICAM-1 expression.

Methods

A cohort of 414 patients with AR and 293 healthy controls was enrolled from the Han Chinese population in Beijing, China. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 14 single nucleotide polymorphisms (SNPs) in ICAM-1, NF-κB, TNF-α, and MRPL4 genes were selected using the CHB genotyping data from the International Haplotype Mapping (HapMap) and assessed for differences in frequencies of the alleles and genotypes between the AR patients and control subjects.

Results

TNF-α SNP rs1799964 and MRPL4 SNP rs11668618 were found to occur in significantly greater frequencies in the AR group compared to control group. There were no significant associations between SNPs in NF-κB, ICAM-1 and AR. The SNP-SNP interaction information analysis further indicated that there were no synergistic effects among the selected sets of polymorphisms.

Conclusions

Our results suggest a strong association between AR risk and polymorphisms of MRPL4 and TNF-α genes in Han Chinese population.     

Resolvin D1 attenuates inflammation in lipopolysaccharide-induced acute lung injury through a process involving the PPARγ/NF-κB pathway

Background

Docosahexaenoic acid (DHA) and DHA-derived lipid mediators have recently been shown to possess anti-inflammatory and pro-resolving properties. In fact, DHA can down-regulate lipolysaccharide (LPS)-induced activation of NF-κB via a PPARγ-dependent pathway. We sought to investigate the effects of the novel DHA-derived mediator resolvin D1 (RvD1) on LPS-induced acute lung injury and to determine whether these effects occur via a PPARγ-dependent pathway.

Methods

BALB/c mice aged 6–8 weeks were randomly divided into seven groups: two control groups receiving saline or RvD1 (600 ng) without LPS; a control group receiving LPS only; an experimental group receiving RvD1 (300 ng) or RvD1 (600 ng), followed by LPS; a group receiving the PPARγ antagonist GW9662; and a group receiving GW9662, then RvD1 (600 ng) and finally LPS. LPS (50 μM) and saline were administered intratracheally. RvD1 was injected intravenously 24 h and 30 min before LPS, while GW9662 was injected intravenously 30 min before RvD1. Mice were killed at 6, 12, and 24 h. Samples of bronchoalveolar lavage fluid (BALF) were analyzed for cell counts and cytokine analysis. Lung tissues were collected for histology, Western blotting and electrophoretic mobility shift assays (EMSAs).

Results

At all three time points, groups receiving either dose of RvD1 followed by LPS had significantly lower total leukocyte counts and levels of TNF-α and IL-6 levels in BALF than did the group given only LPS. RvD1 markedly attenuated LPS-induced lung inflammation at 24 h, based on hematoxylin-eosin staining of histology sections. RvD1 activated PPARγ and suppressed IκBα degradation and NF-κB p65 nuclear translocation, based on Western blots and EMSAs. The PPARγ inhibitor GW9662 partially reversed RvD1-induced suppression of IκBα degradation and p65 nuclear translocation.

Conclusions

These results suggest that RvD1 may attenuate lung inflammation of LPS-induced acute lung injury by suppressing NF-κB activation through a mechanism partly dependent on PPARγ activation.     

Persistent Inflammation and Endothelial Activation in HIV-1 Infected Patients after 12 Years of Antiretroviral Therapy

Objective

The study investigated markers of inflammation and endothelial activation in HIV infected patients after 12 years of successful combination antiretroviral treatment (cART).

Methods

Inflammation and endothelial activation were assessed by measuring levels of immunoglobulins, β2-microglobulin, interleukin (IL) 8, tumor necrosis factor α (TNFα), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1), sE-Selectin, and sP-Selectin.

Results

HIV infected patients had higher levels of β2-microglobulin, IL-8, TNFα, and sICAM-1 than uninfected controls, and HIV infected patients lacked correlation between platelet counts and sP-Selectin levels found in uninfected controls.

Conclusion

Discrete signs of systemic and vascular inflammation persist even after very long term cART.     

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells

Background

Dexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis.

Methods

Analysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10⁻⁸–10⁻⁶ M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10⁻⁶ M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay.

Results

Dexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC.

Conclusions

Dexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways.     

WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) Negatively Regulates TLR4-Mediated TNF-α and IL-6 Production by Proteasomal Degradation of TNF Receptor Associated Factor 6 (TRAF6)

Background

Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.

Methodology/Results

Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.

Conclusions/Significance

We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.     

Anti-Inflammatory Pharmacotherapy with Ketoprofen Ameliorates Experimental Lymphatic Vascular Insufficiency in Mice

Background

Disruption of the lymphatic vasculature causes edema, inflammation, and end-tissue destruction. To assess the therapeutic efficacy of systemic anti-inflammatory therapy in this disease, we examined the impact of a nonsteroidal anti-inflammatory drug (NSAID), ketoprofen, and of a soluble TNF-α receptor (sTNF-R1) upon tumor necrosis factor (TNF)-α activity in a mouse model of acquired lymphedema.

Methods and Findings

Lymphedema was induced by microsurgical ablation of major lymphatic conduits in the murine tail. Untreated control mice with lymphedema developed significant edema and extensive histopathological inflammation compared to sham surgical controls. Short-term ketoprofen treatment reduced tail edema and normalized the histopathology while paradoxically increasing TNF-α gene expression and cytokine levels. Conversely, sTNF-R1 treatment increased tail volume, exacerbated the histopathology, and decreased TNF-α gene expression. Expression of vascular endothelial growth factor-C (VEGF-C), which stimulates lymphangiogenesis, closely correlated with TNF-α expression.

Conclusions

Ketoprofen therapy reduces experimental post-surgical lymphedema, yet direct TNF-α inhibition does not. Reducing inflammation while preserving TNF-α activity appears to optimize the repair response. It is possible that the observed favorable responses, at least in part, are mediated through enhanced VEGF-C signaling.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background

Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.

Methodology/Principal Findings

PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.

Conclusions/Significance

These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.     

Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

Background

The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-κB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-α and MIP-2.

Methods

Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-κB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-κB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-α and MIP-2 release by ELISA.

Results

Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1–2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56°C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 μg/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-κB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-α and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-α and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression.

Conclusion

The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca²⁺-mediated NF-κB activity, necessary for maintenance of TLR4 expression and TNF-α and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.