scholarly journals Unmethylated CpG motif-containing genomic DNA fragment of Bacillus calmette-guerin promotes macrophage functions through TLR9-mediated activation of NF-κB and MAPKs signaling pathways

2019 ◽  
Vol 26 (3) ◽  
pp. 183-203 ◽  
Author(s):  
Junli Li ◽  
Lili Fu ◽  
Guozhi Wang ◽  
Selvakumar Subbian ◽  
Chuan Qin ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Immune Cell ◽  
Ex Vivo ◽  
Effector Functions ◽  
Cell Functions ◽  
Cpg Motif ◽  
Pro Inflammatory Cytokines

The potency of synthetic CpG-oligo-deoxynucleotides (CpG-ODNs) adjuvants in modulating the immune cell functions through the TLR pathway has been tested and reported previously. However, the cellular signaling involved in the stimulation of macrophages by natural, CpG motif-containing adjuvant and the effector functions modulated by such stimulation has not been well studied. Here, we used in vitro and ex vivo murine macrophage assay systems, and mouse model of in vivo stimulation to explore the signaling pathway and the effector functions mediated by BC01. Results show that BC01 can induce the production of TNF-α and MCP-1 in macrophages by up-regulating the activation of NF-κB and MAPKs signaling pathway, and elevated the expression of MHC-II, CD40, CD80, and CD86. Upon stimulation with BC01, the peritoneal macrophages isolated from TLR9−/− mice produced significantly low levels of pro-inflammatory cytokines, attenuated the activation of NF-κB and MAPKs signaling pathways, and showed reduced phagocytosis. Following in vivo stimulation with BC01, the TLR9−/− mice produced significantly lower levels of pro-inflammatory cytokines in the serum and lymph nodes showed reduced cell proliferation. These results indicate that BC01 is an efficient agonist of TLR9 that can significantly enhance the host-protective immune functions of macrophages.

scholarly journals A novel application of bubble-eye strain of Carassius auratus for ex vivo fish immunological studies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Ex Vivo ◽  
Large Bubble ◽  
Suitable Model ◽  
Bacterial Cells ◽  
Functional Impairments ◽  
Gene Expressions ◽  
Pro Inflammatory Cytokines

AbstractIn this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at elevated temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and elevated rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

scholarly journals A Novel Application of Bubble-eye Strain of Carassius Auratus for Ex Vivo Fish Immunological Studies

2021 ◽  
Author(s):  
Hiroto Nakajima ◽  
Atsushi Miyashita ◽  
Hiroshi Hamamoto ◽  
Kazuhisa Sekimizu
Keyword(s):  
Inflammatory Cytokines ◽  
Immune Cells ◽  
Ex Vivo ◽  
Large Bubble ◽  
Suitable Model ◽  
Bacterial Cells ◽  
Functional Impairments ◽  
Gene Expressions ◽  
Pro Inflammatory Cytokines

Abstract In this study, we investigated a new application of bubble-eye goldfish (commercially available strain with large bubble-shaped eye sacs) for immunological studies in fishes utilizing the technical advantage of examining immune cells in the eye sac fluid ex vivo without sacrificing animals. As known in many aquatic species, the common goldfish strain showed an increased infection sensitivity at high temperature, which we demonstrate may be due to an immune impairment using the bubble-eye goldfish model. Injection of heat-killed bacterial cells into the eye sac resulted in an inflammatory symptom (surface reddening) and increased gene expression of pro-inflammatory cytokines observed in vivo, and high rearing temperature suppressed the induction of pro-inflammatory gene expressions. We further conducted ex vivo experiments using the immune cells harvested from the eye sac and found that the induced expression of pro-inflammatory cytokines was suppressed when we increased the temperature of ex vivo culture, suggesting that the temperature response of the eye-sac immune cells is a cell autonomous function. These results indicate that the bubble-eye goldfish is a suitable model for ex vivo investigation of fish immune cells and that the temperature-induced infection susceptibility in the goldfish may be due to functional impairments of immune cells.

scholarly journals Tanshinone IIA attenuates neuroinflammation via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Bo Ding ◽  
Chengheng Lin ◽  
Qian Liu ◽  
Yingying He ◽  
John Bosco Ruganzu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Neuronal Loss ◽  
Tanshinone Iia ◽  
Morris Water Maze Test ◽  
Maze Test ◽  
U87 Cells ◽  
Pro Inflammatory Cytokines

Abstract Background Glial activation and neuroinflammation play a crucial role in the pathogenesis and development of Alzheimer’s disease (AD). The receptor for advanced glycation end products (RAGE)-mediated signaling pathway is related to amyloid beta (Aβ)-induced neuroinflammation. This study aimed to investigate the neuroprotective effects of tanshinone IIA (tan IIA), a natural product isolated from traditional Chinese herbal Salvia miltiorrhiza Bunge, against Aβ-induced neuroinflammation, cognitive impairment, and neurotoxicity as well as the underlying mechanisms in vivo and in vitro. Methods Open-field test, Y-maze test, and Morris water maze test were conducted to assess the cognitive function in APP/PS1 mice. Immunohistochemistry, immunofluorescence, thioflavin S (Th-S) staining, enzyme-linked immunosorbent assay (ELISA), real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blotting were performed to explore Aβ deposition, synaptic and neuronal loss, microglial and astrocytic activation, RAGE-dependent signaling, and the production of pro-inflammatory cytokines in APP/PS1 mice and cultured BV2 and U87 cells. Results Tan IIA treatment prevented spatial learning and memory deficits in APP/PS1 mice. Additionally, tan IIA attenuated Aβ accumulation, synapse-associated proteins (Syn and PSD-95) and neuronal loss, as well as peri-plaque microgliosis and astrocytosis in the cortex and hippocampus of APP/PS1 mice. Furthermore, tan IIA significantly suppressed RAGE/nuclear factor-κB (NF-κB) signaling pathway and the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in APP/PS1 mice and cultured BV2 and U87 cells. Conclusions Taken together, the present results indicated that tan IIA improves cognitive decline and neuroinflammation partly via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro. Thus, tan IIA might be a promising therapeutic drug for halting and preventing AD progression.

scholarly journals IMMU-07. TUMOR STROMA–TARGETING ANTIBODY-CYTOKINE CONJUGATES TO CONVERT THE IMMUNOLOGICALLY COLD GLIOMA MICROENVIRONMENT INTO A HOT ONE

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii106-ii106
Author(s):  
Tobias Weiss ◽  
Emanuele Puca ◽  
Manuela Silginer ◽  
Teresa Hemmerle ◽  
Shila Pazahr ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Targeted Delivery ◽  
Fusion Proteins ◽  
Tumor Necrosis ◽  
Ex Vivo ◽  
Systemic Administration ◽  
Target Antigen ◽  
Tumor Site ◽  
Pro Inflammatory Cytokines

Abstract Glioblastoma is an immunological “desert”. The administration of pro-inflammatory cytokines could shift the balance between tumor-associated immune suppression and anti-tumor immunity but the systemic administration of therapeutically active doses of pro-inflammatory cytokines is hampered by toxic side effects. We investigated different antibody-cytokine fusion products that enable a targeted delivery of interleukin (IL-) 2, IL-12 or tumor-necrosis factor (TNF)α to the tumor site upon systemic administration by binding to a tumor-specific epitope of fibronectin. We confirmed the target antigen expression in ex vivo sections of orthotopic syngeneic glioma mouse models and human glioblastoma samples and characterized the distribution of these antibody-conjugates in glioma-bearing mice upon systemic administration using in vivo imaging. Subsequently, we demonstrated potent anti-tumor activity of these antibody-fusion proteins in fully immunocompetent orthotopic mouse glioma models and characterized their mode of action. We also translated this immunotherapeutic strategy to treat patients with recurrent glioblastoma systemically with an antibody-fusion protein that enables the targeted delivery of TNFα to the tumor site. This was well tolerated, led to a treatment-associated tumor necrosis and increased the number of tumor-infiltrating T cells. This work builds a basis for future studies with antibody-cytokine fusion proteins as a promising treatment strategy for central nervous system tumors.

scholarly journals Immunohistochemical analysis of the inflammatory reaction within the implantation bed of a collagen hemostatic biomaterial

2021 ◽  
Author(s):  
◽  
Carlos Arturo Herrera Vizcaino
Keyword(s):  
Wound Healing ◽  
Inflammatory Response ◽  
Inflammatory Cytokines ◽  
Degradation Rate ◽  
Ex Vivo ◽  
The Body ◽  
Cellular Reaction ◽  
Intergroup Comparison ◽  
Pro Inflammatory Cytokines

Current research on medical biomaterials have shown that the physical and chemical characteristics of biomaterials determine the body inflammatory cellular reaction after their implantation. The aim of this study was to evaluate the individual effects of the physical characteristics over the initial biomaterial-cellular interaction and the inflammatory cellular reaction. For this purpose, an equine-derived collagen hemostatic sponge (E-CHS) was modified by pressing and evaluated using ex vivo, in vitro and in vivo methods. The E-CHS was pressed by applying constant pressure (6.47± 0.85 N) for 2 min using a sterile stainless-steel cylinder and cut in segments of 1cm2. Subsequently, E-CHS and the pressed equine-derived collagen hemostatic sponge (P-E-CHS) were studied as two independent biomaterials and compared to a control group (CG). A blood concentrate containing inflammatory cells known as platelet rich fibrin (PRF) was used to mimic the initial biomaterial-cell interaction and to measure the absorption coefficient of the biomaterials to liquid PRF (iPAC). Additionally, the biomaterials were cultivated together with PRF for 3 and 6 days to measure the induction of pro-inflammatory cytokines (TNF-α and IL-8). The results were obtained through enzyme-linked immunosorbent assay (ELISA) and histological methods. PRF cultivated without biomaterials served as the CG. Additionally, the biomaterials were evaluated in vivo using a subcutaneous model in Wistar rats and compared to sham operated animals (CG) representing physiologic wound healing. After 3, 15 and 30 days, the explanted samples were evaluated using histochemical and immunohistochemical (IHC) staining using the following markers: CD68 (pan macrophages), CCR7 (pro-inflammatory macrophages, M1), CD206 (pro-wound healing macrophages, M2) and α-Smooth Muscle Actin (α-SMA; vessel identification). After the mixture of liquid PRF with both biomaterials for 15 minutes, the ex vivo results showed that E-CHS was penetrated by cells, whereas P-E-CHS was cell-occlusive. Additionally, P-E-CHS induced a higher release of pro-inflammatory cytokines compared to liquid PRF alone (CG) and E-CHS after 3 days (P< 0.05). Although the biomaterial was pressed, the difference of the iPAC value did not show statistical differences. In vivo, the CG induced at day 3 a higher inflammatory response compared to the experimental groups (EG) (P< 0.05). The intergroup comparison showed that P-E-CHS induced a higher presence of macrophages (CD68+/CC7+) compared to E-CHS at day 3 (P< 0.05). Only CD68+/CCR7+ mononuclear cells (MNCs) were observed without multinucleated giant cells (MNGCs). After 15 days, the presence of macrophages (CD68+ P<0.01 /CCR7+ P<0.001 /CD206+ P<0.05) reduced considerably in the CG. On the contrary, the inflammatory response increased in the EGs (CD68+/CCR7+). The intergroup comparison showed that this increment was statistically significant when comparing E-CHS and P-E-CHS to the CG at day 15 (P<0.01 and P< 0.05 respectively). At this time point, a reduced number of MNGCs were observed in the EGs. In the CG no MNGCs were observed. Furthermore, E-CHS showed a faster degradation rate and was fully invaded by cells and vessels formed in its interior region. On the other hand, P-E-CHS remained occlusive to cell penetration and vessels were formed only in the periphery. After 30 days, the cellular reaction shifted to a higher number of M2 macrophages (CD260+) in all groups and a reduced presence of CD68+ and CCR7+ MNCs. Both biomaterials degraded and only small fragments were found in the implantation bed surrounded by MNGCs (CCR7+). These results are of high clinical relevance and show that changes in biomaterial properties have a significant impact on their interaction with the body. They also serve as insight into the possibility to develop versatile biomaterials with different applications. For example, E-CHs can be applied to support hemostasis in a bleeding alveolar socket and P-E-CHs by being cell occlusive and having a delayed degradation rate can be applied for guided bone and tissue regeneration.

scholarly journals Euphorbia Factor L2 ameliorates the Progression of K/BxN Serum-Induced Arthritis by Blocking TLR7 Mediated IRAK4/IKKβ/IRF5 and NF-kB Signaling Pathways

2021 ◽  
Vol 12 ◽  
Author(s):  
Jing Tang ◽  
Xiaolan Cheng ◽  
Shiyu Yi ◽  
Yuanyuan Zhang ◽  
Zhigang Tang ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Inflammatory Cells ◽  
Human Pbmcs ◽  
Inhibitory Effects ◽  
Murine Macrophages

Toll like receptor (TLR)s have a central role in regulating innate immunity and their activation have been highlighted in the pathogenesis of rheumatoid arthritis (RA). EFL2, one of diterpenoids derived from Euphorbia seeds, is nearly unknown expect for its improving effect on acute lung injury. Our present study aimed to investigate EFL2’s pharmacokinetic features, its therapeutic effect on rheumatoid arthritis, and explored the potential anti-arthritic mechanisms. K/BxN serum transfer arthritis (STA) murine model was used to assess EFL2’s anti-arthritic effects. We also applied UPLC-MS method to measure the concentrations of EFL2 in plasma. The inhibitory effects of this compound on inflammatory cells infiltration and activation were determined by flow cytometry analysis and quantitative real-time polymerase chain reaction (qRT-PCR) in vivo, and immunochemistry staining and ELISA in murine macrophages and human PBMCs in vitro, respectively. The mechanism of EFL2 on TLRs mediated signaling pathway was evaluated by PCR array, Western blot, plasmid transfection and confocal observation. Intraperitoneal (i.p.) injection of EFL2, instead of oral administration, could effectively ameliorate arthritis severity of STA mice. The inflammatory cells migration and infiltration into ankles were also significantly blocked by EFL2, accompanied with dramatically reduction of chemokines mRNA expression and pro-inflammatory cytokines production. In vivo PCR microarray indicated that EFL2 exerted anti-arthritis bioactivity by suppressing TLR7 mediated signaling pathway. In vitro study confirmed the inhibitory effects of EFL2 on TLR7 or TLR3/7 synergistically induced inflammatory cytokines secretion in murine macrophages and human PBMCs. In terms of molecular mechanism, we further verified that EFL2 robustly downregulated TLR7 mediated IRAK4-IKKβ-IRF5 and NF-κB signaling pathways activation, and blocked IRF5 and p65 phosphorylation and translocation activity. Taken together, our data indicate EFL2’s therapeutic potential as a candidate for rheumatoid arthritis and other TLR7-dependent diseases.

Curcumin alleviates LPS-induced retinal inflammation by inhibiting PI3k/Akt signaling pathway

2021 ◽  
Vol 2 (1) ◽  
pp. 6-13
Author(s):  
Yan Li ◽  
◽  
Xue Li ◽  
Shan-Bi Zhou ◽  
◽  
...  
Keyword(s):  
Anterior Chamber ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Leukocyte Adhesion ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Rt Pcr ◽  
Retinal Inflammation ◽  
Pro Inflammatory Cytokines

AIM: To investigate whether the curcumin reduce retinal inflammation in animal model and human retinal pigment epithelium (ARPE)-19 cells. METHODS: In vivo, male C57/B6 mice received intraperitoneal injections of curcumin for 3d before intraperitoneal injection of lipopolysaccharide (LPS; 10 mg/kg) to induce retinal inflammation. 24h after LPS application, the mRNA levels of pro-inflammatory cytokines were detected by real-time polymerase chain reaction (RT-PCR). Concanavalin A lectin perfusion-labeling technique evaluated leukocyte adhesion to the retinal vasculature. The protein concentration in the anterior chamber was measured with a protein quantification kit. In vitro, ARPE-19 cells were cultured. The optimum concentration of curcumin was detected by cell counting kit-8 (CCK-8) assay. Before stimulated with 5 μg/mL LPS, ARPE-19 cells were incubated with or without curcumin for 1h. Pro-inflammatory cytokines were measured by RT-PCR and ELISA. PI3K/Akt expression was analyzed by Western blotting. RESULTS: Curcumin pre-treatment led to significant inhibition of EIU-associated leukocyte adhesion to retinal blood vessels and anterior-chamber protein leakage. The mRNA expression level of inflammatory cytokines was also significantly reduced with application of curcumin in vivo, such as IL-1β, IL-6 and TNF-α. Meanwhile, Curcumin significantly attenuated the expression of IL-6, IL-8 and MCP-1 at both mRNA and protein levels in ARPE-19 cells. Curcumin suppressed PI3K/Akt phosphorylation as well as NF-κB activation in LPS-activated ARPE-19 cells. CONCLUSION: Curcumin plays a preventive effect on LPS-induced retinal inflammation. The beneficial effect appears associated with inhibiting of the PI3k/Akt signaling pathway.

scholarly journals microRNA-27b shuttled by mesenchymal stem cell-derived exosomes prevents sepsis by targeting JMJD3 and downregulating NF-κB signaling pathway

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jia Sun ◽  
Xuan Sun ◽  
Junhui Chen ◽  
Xin Liao ◽  
Yixuan He ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Cecal Ligation ◽  
Gene Promoter ◽  
In Silico Analysis ◽  
Underlying Mechanism ◽  
Nuclear Factor Κb ◽  
Pro Inflammatory Cytokines

Abstract Background Exosomal microRNAs (miRs) derived from mesenchymal stem cells (MSCs) have been shown to play roles in the pathophysiological processes of sepsis. Moreover, miR-27b is highly enriched in MSC-derived exosomes. Herein, we aimed to investigate the potential role and downstream molecular mechanism of exosomal miR-27b in sepsis. Methods Inflammation was induced in bone marrow-derived macrophages (BMDMs) by lipopolysaccharide (LPS), and mice were made septic by cecal ligation and puncture (CLP). The expression pattern of miR-27b in MSC-derived exosomes was characterized using RT-qPCR, and its downstream gene was predicted by in silico analysis. The binding affinity between miR-27b, Jumonji D3 (JMJD3), or nuclear factor κB (NF-κB) was characterized to identify the underlying mechanism. We induced miR-27b overexpression or downregulation, along with silencing of JMJD3 or NF-κB to examine their effects on sepsis. The production of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 was detected by ELISA. Results miR-27b was highly expressed in MSC-derived exosomes. Mechanistic investigations showed that miR-27b targeted JMJD3. miR-27b decreased expression of pro-inflammatory genes by inhibiting the recruitment of JMJD3 and NF-κB at gene promoter region. Through this, MSC-derived exosomal miR-27b diminished production of pro-inflammatory cytokines in LPS-treated BMDMs and septic mice, which could be rescued by upregulation of JMJD3 and NF-κB. Besides, in vitro findings were reproduced by in vivo findings. Conclusion These data demonstrated that exosomal miR-27b derived from MSCs inhibited the development of sepsis by downregulating JMJD3 and inactivating the NF-κB signaling pathway.

scholarly journals Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2794 ◽  
Author(s):  
Cao ◽  
Chen ◽  
Ren ◽  
Zhang ◽  
Tan ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Cytokines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Inflammatory Responses ◽  
Raw264.7 Macrophages ◽  
Tnf Α ◽  
Autophagy Inhibition ◽  
Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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