Mechanism of UVB-Induced Suppression of the Immune Response to Mycobacterium bovis Bacillus Calmette-Guerin: Role of Cytokines on Macrophage Function

Previously we demonstrated that treatment of mice with either UVB radiation or supernatants derived from UVB-irradiated PAM 212 keratinocytes decreased the induction of the delayed-type hypersensitivity (DTH) response to Mycobacterium bovis bacillus Calmette-Guerin (BCG), impaired the clearance of bacteria from their lymphoid organs and also altered macrophage functions. In order to characterize the cytokines involved in these phenomena, UV-irradiated mice were injected with antibodies to interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1), or tumor necrosis factor-α (TNF-α). Injection of UVB-irradiated mice with anti-IL-10 immediately after UV irradiation restored the DTH response and reversed the UV-induced inhibition of bacterial clearance. Injection of UV-irradiated mice with anti-TGF-β only partially restored the DTH response although it allowed a better clearance of BCG than injection of mice with the control antibody. In contrast, injection of anti-TNF-α did not affect the UVB-induced suppression of DTH or impaired bacterial clearance. Similarly, the ability of macrophages to phagocytose BCG and kill the intracellular organisms was restored to almost normal levels after injecting UV-irradiated mice with antibodies specific for IL-10 or TGF-β. Injection of mice with either recombinant IL-10 or TGF-β mimicked the effect of whole-body UV irradiation on immune function. These results suggest that IL-10 has a major role in UV-induced suppression of both DTH to BCG and impairment in the clearance of bacteria and that TGF-β has a more significant role in blocking bacterial clearance. Furthermore, these cytokines seem to modulate immune responses by altering macrophage functions in UVB-irradiated mice.     

Effect of Photobiomodulation on C2C12 Myoblasts Cultivated in M1 Macrophage-conditioned Media

Moderate levels of a proinflammatory macrophages phenotype are indispensable and play an important role in the skeletal muscle repair process since this response depends on their secreted products concentration to influence and modulate muscle inflammation as well as the differentiation of myoblasts. This study investigated the effects of photobiomodulation (PBM) on undifferentiated and differentiation-induced C2C12 myoblasts cultivated in different concentrations of M1 phenotype macrophage-conditioned media of J774 cells (MCM1) also submitted to PBM using the same irradiation parameters. Irradiation was performed once with low-level laser (780 nm, 70 mW, 1 J) and was evaluated cell viability, proliferation and differentiation, nitric oxide (NO) synthesis and IL-6 and TNF-α protein levels 24 and 48 h after C2C12 irradiation. PBM treatment in undifferentiated myoblasts exhibited lower IL-6 levels in the presence of nonirradiated MCM1 at both concentrations. Myoblasts in proliferation condition cultivated with irradiated MCM1 showed lower IL-6 and TNF-α levels after 48 h in the presence of both concentrations evaluated. PBM induced a decrease in the synthesis of NO on undifferentiated and differentiation-induced myoblasts. PBM was able to reduce the level of proinflammatory protein and markers, which are important to allow the differentiation of myoblasts during the muscle repair process.     

Photobiomodulation Enhances Cisplatin Cytotoxicity in a Culture Model with Oral Cell Lineages

Cisplatin plays a central role in cancer chemotherapy, but resistance to this drug remains a major obstacle in treatment. Drawbacks related to cisplatin failure may be associated with cell energy metabolism. This study investigated whether photobiomodulation (PBM) can potentiate the effects of cisplatin on keratinocytes (HaCat) and cancer cells (SCC25 and HN12). Cells were treated with laser (GaAlAs; 660 nm; 60 mW; 0.33 J; 2.14 W cm⁻²; 11.7 J cm⁻² and 6 s) and cisplatin (7.8 μg mL⁻¹) to evaluate cell viability, Ki-67, VEGF, TGF-β1, EGF expression and ROS production. Observations were validated in the SCC25 cell lineage, where the type of cell death (necrosis/apoptosis) and the amount of ATP were assessed. Cell lineages showed increased sensitivity to cisplatin associated with PBM (Cis-PBM). Ki-67 was augmented in all cell lineages treated with Cis-PBM when compared to cisplatin alone (Cis). Cis or Cis-PBM significantly decreased VEGF expression in cancer cells, while no changes were seen in the expression of TGF-β1 or EGF compared to control. ROS levels were similar in the Cis and Cis-PBM groups. Cells treated with Cis-PBM died by apoptosis, leading to greater consumption of ATP. These observations suggest that PBM may potentiate the effects of cisplatin, leading to increased drug cytotoxicity and enhanced cell death.     

Tilianin alleviates airway inflammation in ovalbumin-induced allergic asthma in mice through the regulation of Th2 cytokines and TGF-β1/Smad markers

BackgroundAllergic asthma is a inflammatory disease defined as a condition of chronic airway inflammation. Asthma can be provoked by various stimuli like allergens inhalation like dust particles, pollen, and pollutants in the air.ObjectiveThis exploration was dedicated to investigate the anti-asthmatic properties of tilianin against the ovalbumin (OVA)-initiated asthma in mice.MethodologyThe asthma was provoked to the mice via administering 100 μl of aluminum hydroxide containing 20 μg of OVA and treated with the 10 and 20 mg/kg of tilianin, respectively. The levels of Th2 cytokines, OVA-specific IgE, eotaxin, pro-inflammatory mediators, antioxidants, and other markers were inspected by marker specific assay kits. The mRNA expressions of TGF-β1, Smad, iNOS, and COX-2 was assessed using RT-PCR analysis. The lung histology was analyzed microscopically to detect the histological changes.ResultsTilianin treatment remarkably suppressed the IL-4, IL-5, and IL-13, IFN-γ, eotaxin, and IgE levels. The NO, MPO, and inflammatory makers TNF-α, IL-6, IL-12, and TXB2 was substantially diminished by the tilianin treatment. The TGF-β1, iNOS, and COX-2 expressions were appreciably suppressed by the tilianin. The histological findings proved that the tilianin treatment alleviated the OVA-provoked histopathological changes in the lung tissues.ConclusionOur findings proved that tilianin effectively alleviated the OVA-provoked asthma in animals and it could be a talented anti-asthmatic candidate.     

Inhibition of inflammatory mediators by neobavaisoflavone in activated RAW264.7 macrophages

Flavonoids and coumarins are the major bioactive constituents identified in Psoralea corylifolia. The active fraction isolated from fruits, seeds and roots possesses antibacterial, antioxidative and immunomodulatory properties. Neobavaisoflavone is one of the flavonoids found in Psoralea corylifolia. In the present study we investigated in vitro the anti-inflammatory activity of neobavaisoflavone. Macrophages play an important role in inflammation through the release of inflammatory mediators involved in the immune response. Inappropriate and prolonged macrophage activation is largely responsible for the pathology of acute and chronic inflammatory conditions. Neobavaisoflavone significantly inhibited the production of reactive oxygen species (ROS), reactive nitrogen species (RNS) and cytokines: IL-1β, IL-6, IL-12p40, IL-12p70, TNF-α in LPS+IFN-γ- or PMA- stimulated RAW264.7 macrophages.     

TGF-β-signaling with small molecule FKBP12 antagonists that bind myristoylated FKBP12-TGF-β type I receptor fusion proteins

Background: Growth arrest in many cell types is triggered by transforming growth factor beta (TGF-β), which signals through two TGF-β receptors (type I, TGF-βRI, and type II, TGF-βRII). In the signaling pathway, TGF-β binds to the extracellular domain of TGF-βRII, which can then transphosphorylate TGF-βRI in its glycine/serine (GS)-rich box. Activated TGF-βRI phosphorylates two downstream effectors, Smad2 and Smad3, leading to their translocation into the nucleus. Cell growth is arrested and plasminogen activator inhibitor 1 (PAI-1) is upregulated. We investigated the role of the immunophilin FKBP12, which can bind to the GS box of TGF-βRI, in TGF-β signaling.Results: Overexpression of myristoylated TGF-βRI and TGF-βRII cytoplasmic tails caused constitutive nuclear translocation of a green-fluorescent-protein-Smad2 construct in COS-1 cells, and constitutive activation of a PAI-1 reporter plasmid in mink lung cells. Fusing FKBP12 to TGF-βRI resulted in repression of autosignaling that could be alleviated by FK506M or rapamycin (two small molecules that can bind to FKBP12). Mutation of the FKBP12-binding site in the FKBP12-TGF-βRI fusion protein restored constitutive signaling. An acidic mutation in the FKBP12-TGFβRI protein allowed FKBP12 antagonists to activate signaling in the absence of TGF-βRII. Further mutations in the TGF-βRI FKBP12-binding site resulted in TGF-β signaling that was independent of both TGF-βRII and FKBP12 antagonists.Conclusions: Fusing FKBP12 to TGF-βRI results in a novel receptor that is activated by small molecule FKBP12 antagonists. These results suggest that FKBP12 binding to TGF-βRI is inhibitory and that FKBP12 plays a role in inhibiting TGF-β superfamily signals.     

Lipid length and iso-branching of trehalose diesters influences Mincle agonist activity

We report on the efficient synthesis of linear trehalose diesters (TDEs) and iso-branched TDEs (maradolipids or iso-TDEs) and their ability to activate bone marrow-derived macrophages (BMDMs) as determined by cytokine (IL-1β, IL-12, IL-6, IL-10) and chemokine (MIP-2) production. Both classes of TDEs were found to activate BMDMs in a Mincle-dependent manner, with longer-chain (≥C18) lipids leading to a robust inflammatory response. On the whole, the iso-branched TDEs led to greater cytokine production and a faster immune response when compared to their linear counterparts. Moreover, C12-TDE and iso-C12-TDE elicited the production of MIP-2 by BMDMs, thereby providing the first example of TDEs with a chain length of ≤ C12 leading to a Mincle-dependent immune response and one that is less inflammatory in nature.     

Inhibition of corneal neovascularization by rapamycin

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.     

mTHPC-mediated photodynamic treatment up-regulates the cytokines VEGF and IL-1alpha

Photodynamic therapy (PDT) of cancer induces oxidative stress, which intervenes in the expression of cytokines by tumor cells. The cytokines might have either a positive or a negative impact on tumor eradication. Here, we studied the expression of cytokines vascular endothelial growth factor (VEGF) and interleukin-1alpha (IL-1alpha) in the human epidermoid carcinoma A-431 cells following m-tetra(3-hydroxyphenyl)-chlorin (mTHPC)-mediated PDT in vitro and assessed the IL-1alpha effect on VEGF expression. Quantitative polymerase chain reaction and enzyme-linked immunosorbent assay revealed the enhanced production of VEGF and IL-1alpha both on mRNA and protein levels by mTHPC-loaded cells after light exposure. The silencing of IL1A by small interfering RNA resulted in decreased production of IL-1alpha and a reduced amount of VEGF. Furthermore, exogenous recombinant IL-1alpha stimulated the VEGF expression after PDT. Thus, in addition to the cytotoxic action on the A-431 cells, mTHPC-mediated PDT stimulated the production of VEGF and IL-1alpha, and IL-1alpha contributed to the VEGF overexpression. These data establish IL-1alpha as a possible target of combined cancer treatment.     

Asiatic Acid Glucosamine Salt Alleviates Ultraviolet B-induced Photoaging of Human Dermal Fibroblasts and Nude Mouse Skin

Herbal extracts including asiatic acid (AA) have become popular candidates of anti-photoaging agents due to their anti-inflammatory and antioxidant properties and minimal side effect. Nevertheless, low bioavailability due to poor solubility limits their practical application. In this study, a highly bioavailable form of AA called AAGS (compounded by asiatic acid and glucosamine) was investigated for its anti-photoaging effect using both in vitro and in vivo models along with UVB irradiation. The results showed that AAGS alleviated UVB-induced cell proliferation inhibition by reducing G2 phase arrest and cell apoptosis rate as well as the gene expressions of P53, BAX, CASPASE 3 and CASPASE 9, but enhancing BCL-2 expression. It also reduced the production of reactive oxygen species along with increased gene expression of GPX-1 and downregulated the gene expression of IL-1β, IL-6, IL-8, IL-17 and TNF-α compared to nontreated cells. In vivo results demonstrated the antiphotodamaging effects by restoring skin thickness, collagen content and reducing MMPs expression, which are also supported by reduced MMPs gene expression and enhanced collagen I and TGF-β1 gene expression in vitro. Thus, AAGS may become a potential anti-photoaging agent for topical use due to its capability of self-assembling into a water gel.     

Bilirubin protects grafts against nonspecific inflammation-induced injury in syngeneic intraportal islet transplantation

Nonspecific inflammatory response is the major cause for failure of islet grafts at the early phase of intraportal islet transplantation (IPIT). Bilirubin, a natural product of heme catabolism, has displayed anti-oxidative and anti-inflammatory activities. The present study has demonstrated that bilirubin protected islet grafts by inhibiting nonspecific inflammatory response in a syngeneic rat model of IPIT. The inflammation-induced cell injury was mimicked by exposing cultured rat insulinoma INS-1 cells to cytokines (IL-1β, TNF-α and IFN-γ) in in vitro assays. At appropriate lower concentrations, bilirubin significantly attenuated the reduced cell viability and enhanced cell apoptosis induced by cytokines, and protected the insulin secretory function of INS-1 cells. Diabetic inbred male Lewis rats induced by streptozotocin underwent IPIT at different islet equivalents (IEQs) (optimal dose of 1000, and suboptimal doses of 750 or 500), and bilirubin was administered to the recipients every 12 h, starting from one day before transplantation until 5 days after transplantation. Administration of bilirubin improved glucose control and enhanced glucose tolerance in diabetic recipients, and reduced the serum levels of inflammatory mediators including IL-1β, TNF-α, soluble intercellular adhesion molecule 1, monocyte chemoattractant protein-1 and NO, and inhibited the infiltration of Kupffer cells into the islet grafts, and restored insulin-producing ability of transplanted islets.     

Receptor activator of NF-kappaB ligand enhances the activity of macrophages as antigen presenting cells

Receptor activator of NFkappaB ligand (RANKL) is known as a key regulator of osteoclastogenesis. However, the fact that fibroblasts and periodontal ligament cells express RANKL in response to bacterial substances, suggests that RANKL may have evolved as a part of the immunity to infection. As RANKL increases the survival and activity of dendritic cells, it may have similar effects on macrophages. To address this issue, we studied the effect of RANKL on various functions of macrophages using mouse bone marrow derived macrophages. RANKL enhanced the survival of macrophages and up-regulated the expression of CD86. RANKL-treated macrophages showed increased allogeneic T cell activation and phagocytic activity compared to control cells. In addition, RANKL increased the expression of TNFalpha, MCP-1, and IL-6 but not of IL-10, IL-12, IFN-gamma, and iNOS. Collectively, RANKL augmented the activity of macrophages especially as antigen presenting cells, suggesting its new role in immune regulation.     

Modulation effect of Semen Ziziphi Spinosae extracts on IL-1β, IL-4, IL-6, IL-10, TNF-α and IFN-γ in mouse serum

This study aims to explore and evaluate the effects of Semen Ziziphi Spinosae extracts on the serum levels of interleukin (IL)-6, IFN-γ, IL-1β, TNF-α, IL-10 and IL-4 in mice, and the regulative effect of Semen Ziziphi Spinosae on the cytokine system. Using an ELISA assay, the serum levels of IL-6, IFN-γ, IL-1β, TNF-α, IL-10 and IL-4 were examined in mice after intraperitoneal injection (i.p.) with Semen Ziziphi Spinosae extracts. The results showed that the levels of IL-6 and IL-1β were significantly increased compared with the control groups (p<0.01), while the level of tumour necrosis factor-α (TNF-α) was significantly decreased (p<0.01). Semen Ziziphi Spinosae possesses certain modulation effects on cytokines, and the immuno-regulating function and hypnosis effects of Semen Ziziphi Spinosae may be relevant to these modulation effects on cytokines.     

两个基于2-丙基-4，5-咪唑二甲酸的锶和钡配合物的合成，结构及性质研究

基于2-丙基-4,5-咪唑二甲酸配体合成了2个新型锶和钡配合物,分别为[Sr(H₂pimda)₂(H₂O)₂]_n (1)和{[Ba(H₂pimda)₂(H₂O)₃]H₂O}_n (2),(H₃pimda=2-丙基-4,5-咪唑二甲酸),用元素分析、红外光谱、热重分析对产物进行表征,用单晶X-射线衍射方法测定了配合物1和2的晶体结构。配合物1属于单斜晶系C2/c空间群,晶胞参数：a=1.307 18(12) nm,b=1.319 13(12) nm,c=1.168 24(11)nm,β=98.477 0(10)°。配合物2属于三斜晶系P1 空间群,晶胞参数：a=1.0316 0(10) nm,b=1.054 60(10) nm,c=1.098 11(11) nm,α=75.878 0(10)°,β=78.512 0(10)°,γ=84.399(2)°。并且研究了配合物1的荧光性质。     

Role of anti-inflammatory interventions in high-fat-diet-induced obesity

Lipotoxicity is defined as deposition of excess fat associated with an inflammatory response. Metabolomic analysis of fatty acids (FAs) can be a marker of silent inflammation. ω3-Enriched diet, celecoxib, and safranal may have a protective anti-inflammatory role. In this work, total FAs extracted from red blood cells and arachidonic acid-to-eicosapentaenoic acid (AA-to-EPA) ratios were assessed using GC–MS assay in single-ion monitoring mode. The study was conducted on 64 male rats divided into eight groups: I, controls; II, rats received high-fat diet (HFD), III, rats received ω-6-enriched HFD; IV, rats received ω-3-enriched HFD; V, rats received celecoxib with HFD; VI, rats received safranal with HFD; VII and VIII, rats received celecoxib and safranal with ω-3 HFD, respectively. GC–MS Gas chromatography Mass spectrometry was performed for analysis of fatty acid methyl ester. Enzyme-linked immunosorbent assay was used to analyze serum interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-β1) concentrations. A statistically significant decrease of AA-to-EPA ratio was observed in group VII when compared with the groups receiving HFDs. This group also showed the lowest serum IL-6 level and highest TGF-β1 level. In conclusion, ω3-enriched diet along with drugs (e.g. celecoxib) and herbal medications (e.g. safranal) may have an anti-inflammatory effect in lipotoxicity. GC–MS with single-ion monitoring is valid for the analysis of FAs.     

Elevated serum cytokine levels in mesothelioma patients who have undergone pleurectomy or extrapleural pneumonectomy and adjuvant intraoperative photodynamic therapy

Patients treated on a Phase-I clinical trial of photodynamic therapy (PDT) developed a systemic capillary leak syndrome that constituted the dose-limiting toxicity. We examined serum samples from patients treated at the maximally tolerated dose level for evidence of a systemic, cytokine-mediated inflammatory response. Patients underwent pleurectomy or extrapleural pneumonectomy (EPP) followed by intraoperative PDT of the thorax using Foscan at a dose of 0.1 mg/kg 6 days before surgery and 652 nm red light at a dose of 10 J/cm2. Levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12 were assayed before Foscan administration; after anesthetic induction, surgical resection and light delivery; in postoperative recovery and the day after the surgery. Of the analyzed patients, eight underwent a pleurectomy and one an EPP followed by PDT. IFN-gamma, TNF-alpha and IL-12 showed no elevation, but IL-1beta, IL-6, IL-8 and IL-10 levels were elevated after surgery and PDT. IL-1beta showed a statistically significant variation from baseline after surgery and IL-6, after PDT. The results suggest a systemically mediated inflammatory response resulting from thoracic surgery followed by PDT. Further investigation of specific mechanisms is warranted.     

In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors

The homing properties of adipose tissue-derived mesenchymal stem cells (AdMSCs) have stimulated intravenous applications for their use in stem cell therapy. However, the soluble factors and corresponding cellular receptors responsible for inducing chemotaxis of AdMSCs have not yet been reported. In the present study, the migration capacity of human AdMSCs (hAdMSCs) toward various cytokines or growth factors (GFs) and the expression of their receptors were determined. In a conventional migration assay, PDGF-AB, TGF-β1, and TNF-α showed the most effective chemoattractant activity. When AdMSCs were preincubated with various chemokines or GF, and then allowed to migrate toward medium containing 10% FBS, those preincubated with TNF-α showed the highest migratory activity. Next, hAdMSCs were either preincubated or not with TNF-α, and allowed to migrate in response to various GFs or chemokines. Prestimulation with TNF-α increased the migration activity of hAdMSCs compared to unstimulated hAdMSCs. When analyzed by FACS and RT-PCR methods, hAdMSCs were found to express C-C chemokine receptor type 1 (CCR1), CCR7, C-X-C chemokine receptor type 4 (CXCR4), CXCR5, CXCR6, EGF receptor, fibroblast growth factor receptor 1, TGF-β receptor 2, TNF receptor superfamily member 1A, PDGF receptor A and PDGF receptor B at both the protein and the mRNA levels. These results indicate that the migration capacity of hAdMSCs is controlled by various GFs and chemokines. Prior in vitro modulation of the homing capacity of hAdMSCs could stimulate their movement into injured sites in vivo when administered intravenously, thereby improving their therapeutic potential.