Role of MDA5 in regulating CXCL10 expression induced by TLR3 signaling in human rheumatoid fibroblast-like synoviocytes

AbstractC-X-C motif chemokine 10 (CXCL10) is an inflammatory chemokine and a key molecule in the pathogenesis of rheumatoid arthritis (RA). Melanoma differentiation-associated gene 5 (MDA5) is an RNA helicase that plays a role in innate immune and inflammatory reactions. The details of the regulatory mechanisms of CXCL10 production and the precise role of MDA5 in RA synovitis have not been fully elucidated. The aim of this study was to examine the role of MDA5 in regulating CXCL10 expression in cultured human rheumatoid fibroblast-like synoviocytes (RFLS). RFLS was stimulated with Toll-like receptor 3 (TLR3) ligand polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA mimetic. Expression of interferon beta (IFN-β), MDA5, and CXCL10 was measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assay. A neutralizing antibody of IFN-β and siRNA-mediated MDA5 knockdown were used to determine the role of these molecules in regulating CXCL10 expression downstream of TLR3 signaling in RFLS. Poly I:C induced IFN-β, MDA5, and CXCL10 expression in a concentration- and time-dependent manner. IFN-β neutralizing antibody suppressed the expression of MDA5 and CXCL10, and knockdown of MDA5 decreased a part of CXCL10 expression (p < 0.001). The TLR3/IFN-β/CXCL10 axis may play a crucial role in the inflammatory responses in RA synovium, and MDA5 may be partially involved in this axis.

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D-Lactate Increases Cytokine Production in Bovine Fibroblast-Like Synoviocytes via MCT1 Uptake and the MAPK, PI3K/Akt, and NFκB Pathways

Animals ◽

10.3390/ani10112105 ◽

2020 ◽

Vol 10 (11) ◽

pp. 2105

Author(s):

Carolina Manosalva ◽

John Quiroga ◽

Stefanie Teuber ◽

Sebastián Cárdenas ◽

María Daniella Carretta ◽

...

Keyword(s):

Cytokine Production ◽

Inflammatory Responses ◽

Monocarboxylate Transporter ◽

Dependent Manner ◽

Monocarboxylate Transporter 1 ◽

Ruminal Acidosis ◽

Excessive Intake ◽

Fermentable Carbohydrates ◽

Fibroblast Like Synoviocytes

Acute ruminal acidosis (ARA) is caused by the excessive intake of highly fermentable carbohydrates, followed by the massive production of D-lactate and the appearance of neutrophilic aseptic polysynovitis. Bovines with ARA develop different lesions, such as ruminitis, polioencephalomalacia (calves), liver abscess and lameness. Lameness in cattle with ARA is closely associated with the presence of laminitis and polysynovitis. However, despite decades of research in bovine lameness as consequence of ruminal acidosis, the aetiology and pathogenesis remain unclear. Fibroblast-like synoviocytes (FLSs) are components of synovial tissue, and under pathological conditions, FLSs increase cytokine production, aggravating inflammatory responses. We hypothesized that D-lactate could induce cytokine production in bovine FLSs. Analysis by qRT-PCR and ELISA revealed that D-lactate, but not L-lactate, increased the expression of IL-6 and IL-8 in a monocarboxylate transporter-1-dependent manner. In addition, we observed that the inhibition of the p38, ERK1/2, PI3K/Akt, and NF-κB pathways reduced the production of IL-8 and IL-6. In conclusion, our results suggest that D-lactate induces an inflammatory response; this study contributes to the literature by revealing a potential key role of D-lactate in the polysynovitis of cattle with ARA.

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TLR3-Dependent Activation of TLR2 Endogenous Ligands via the MyD88 Signaling Pathway Augments the Innate Immune Response

Cells ◽

10.3390/cells9081910 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1910

Author(s):

Hellen S. Teixeira ◽

Jiawei Zhao ◽

Ethan Kazmierski ◽

Denis F. Kinane ◽

Manjunatha R. Benakanakere

Keyword(s):

Signaling Pathway ◽

Innate Immune ◽

Poly I:C ◽

Innate Immune Responses ◽

Endogenous Ligands ◽

Polyinosinic:Polycytidylic Acid ◽

Tlr3 Signaling ◽

Myd88 Signaling

The role of the adaptor molecule MyD88 is thought to be independent of Toll-like receptor 3 (TLR3) signaling. In this report, we demonstrate a previously unknown role of MyD88 in TLR3 signaling in inducing endogenous ligands of TLR2 to elicit innate immune responses. Of the various TLR ligands examined, the TLR3-specific ligand polyinosinic:polycytidylic acid (poly I:C), significantly induced TNF production and the upregulation of other TLR transcripts, in particular, TLR2. Accordingly, TLR3 stimulation also led to a significant upregulation of endogenous TLR2 ligands mainly, HMGB1 and Hsp60. By contrast, the silencing of TLR3 significantly downregulated MyD88 and TLR2 gene expression and pro-inflammatory IL1β, TNF, and IL8 secretion. The silencing of MyD88 similarly led to the downregulation of TLR2, IL1β, TNF and IL8, thus suggesting MyD88 to somehow act downstream of TLR3. Corroborating in vitro data, Myd88−/− knockout mice downregulated TNF, CXCL1; and phospho-p65 and phospho-IRF3 nuclear localization, upon poly I:C treatment in a mouse model of skin infection. Taken together, we identified a previously unknown role for MyD88 in the TLR3 signaling pathway, underlying the importance of TLRs and adapter protein interplay in modulating endogenous TLR ligands culminating in pro-inflammatory cytokine regulation.

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Interleukin-6 via Toll-Like Receptor 3 Signaling Attenuates the Expression of Proinflammatory Chemokines in Human Podocytes

Kidney & Blood Pressure Research ◽

10.1159/000514589 ◽

2021 ◽

Vol 46 (2) ◽

pp. 207-218

Author(s):

Hidenori Umetsu ◽

Shojiro Watanabe ◽

Tadaatsu Imaizumi ◽

Tomomi Aizawa ◽

Koji Tsugawa ◽

...

Keyword(s):

Rna Interference ◽

Molecular Mechanisms ◽

Kidney Diseases ◽

Enzyme Linked Immunosorbent Assay ◽

Toll Like Receptor ◽

Induced Expression ◽

Inflammatory Reactions ◽

Monocyte Chemoattractant Protein 1 ◽

Polycytidylic Acid ◽

Tlr3 Signaling

Background: Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. Methods: We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-β and IL-6. Results: We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-β in human podocytes. RNA interference experiments revealed that IFN-β was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. Conclusion: IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.

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Immunomodulatory role of paliperidone in the poly(I:C) model of schizophrenia

European Psychiatry ◽

10.1016/j.eurpsy.2016.01.541 ◽

2016 ◽

Vol 33 (S1) ◽

pp. s220-s221

Author(s):

K. MacDowell ◽

E. Munarriz-Cuezva ◽

D. Martín-Hernández ◽

A. Sayd ◽

B. García-Bueno ◽

...

Keyword(s):

Antioxidant Enzymes ◽

Inflammatory Responses ◽

Poly I:C ◽

Mrna Levels ◽

Behavioral Test ◽

Inflammatory Pathways ◽

Anti Inflammatory ◽

Cellular Markers ◽

Endogenous Antioxidant

IntroductionAlterations on the innate inflammatory response may underlie the pathophysiology of psychiatric diseases, but the mechanisms implicated remain elusive. Current antipsychotics modulate pro/anti-inflammatory pathways, but the specific mechanisms involved remain elusive. One attractive possibility is the regulation of the intracellular signalling pathways of the innate immune receptors Toll-like 3 (TLR3), which triggers antiviral and inflammatory responses.AimsTo elucidate the regulatory role of paliperidone on maternal immune activation (MIA) induced alterations on TLR3 pathway and on the two emerging endogenous antiinflammatory/antioxidant mechanisms NRF2/antioxidant enzymes pathway and the cytokine milieu regulating M1/M2 polarization in microglia.MethodsPregnant mice were treated with the synthetic Toll-like Receptor 3 (TLR3) agonist Poly(I:C) in gestational day 9 and chronically treated with paliperidone (0,05 mg/kg i.p.) in adult offspring. Animals were sacrificed one day after treatment and behavioral test. Inflammation oxidative stress-related mediators were analysed at mRNA and protein level in prefrontal cortex samples. In addition, behavioral test t-maze was conducted.ResultsPaliperidone prevented TLR3 pathway activation and the subsequent MIA-induced neuroinflammatory response. Also, paliperidone induced an increment in the activity and protein expression of nuclear NRF2, as well as increased mRNA levels of the antioxidant enzymes HO1, SOD and catalase in the MIA model. Otherwise, paliperidone increases the antiinflammatory cytokines levels TGFβ and IL-10 in favour of a M2 microglia profile and increased the levels of the M2 cellular markers ArgI and FOLR2.ConclusionsThe modulation of neuroinflammation and enhancement of endogenous antioxidant/anti-inflammatory pathways by current and new antipsychotics could represent an interesting therapeutic strategy for the future.Disclosure of interestThe authors have not supplied their declaration of competing interest.

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Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner

Infection and Immunity ◽

10.1128/iai.00361-09 ◽

2009 ◽

Vol 77 (9) ◽

pp. 3686-3695 ◽

Cited By ~ 30

Author(s):

Hany M. Ibrahim ◽

Hiroshi Bannai ◽

Xuenan Xuan ◽

Yoshifumi Nishikawa

Keyword(s):

Nitric Oxide ◽

Toxoplasma Gondii ◽

Inflammatory Responses ◽

Interleukin 12 ◽

Dependent Manner ◽

Independent Manner ◽

Necrosis Factor Alpha ◽

Parasite Multiplication ◽

Factor Alpha

ABSTRACT Toxoplasma gondii modulates pro- and anti-inflammatory responses to regulate parasite multiplication and host survival. Pressure from the immune response causes the conversion of tachyzoites into slowly dividing bradyzoites. The regulatory mechanisms involved in this switch are poorly understood. The aim of this study was to investigate the immunomodulatory role of T. gondii cyclophilin 18 (TgCyp18) in macrophages and the consequences of the cellular responses on the conversion machinery. Recombinant TgCyp18 induced the production of nitric oxide (NO), interleukin-12 (IL-12), and tumor necrosis factor alpha through its binding with cysteine-cysteine chemokine receptor 5 (CCR5) and the production of gamma interferon and IL-6 in a CCR5-independent manner. Interestingly, the treatment of macrophages with TgCyp18 resulted in the inhibition of parasite growth and an enhancement of the conversion into bradyzoites via NO in a CCR5-dependent manner. In conclusion, T. gondii possesses sophisticated mechanisms to manipulate host cell responses in a TgCyp18-mediated process.

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cAMP metabolism controls caspase-11 inflammasome activation and pyroptosis in sepsis

Science Advances ◽

10.1126/sciadv.aav5562 ◽

2019 ◽

Vol 5 (5) ◽

pp. eaav5562 ◽

Cited By ~ 16

Author(s):

Ruochan Chen ◽

Ling Zeng ◽

Shan Zhu ◽

Jiao Liu ◽

Herbert J. Zeh ◽

...

Keyword(s):

Metabolic Pathways ◽

Inflammatory Responses ◽

Poly I:C ◽

Inflammasome Activation ◽

Proof Of Concept ◽

Potential Therapeutic Target ◽

Camp Metabolism ◽

Camp Hydrolysis ◽

Insight Into

The ability of cytosolic lipopolysaccharide (LPS) to activate caspase-11–dependent nonclassical inflammasome is intricately controlled to avoid excessive inflammatory responses. However, very little is known about the regulatory role of various metabolic pathways in the control of caspase-11 activation. Here, we demonstrate that l-adrenaline can act on receptor ADRA2B to inhibit the activation of the caspase-11 inflammasome by cytosolic LPS or Escherichia coli infection in macrophages. l-adrenaline–induced cAMP production via the enzyme ADCY4 promotes protein kinase A (PKA) activation, which then blocks the caspase-11–mediated proteolytic maturation of interleukin-1β, gasdermin D (GSDMD) cleavage, and consequent DAMP release. Inhibition of PDE8A-mediated cAMP hydrolysis limits caspase-11 inflammasome activation and pyroptosis in macrophages. Consequently, pharmacological modulation of the ADRA2B-ADCY4-PDE8A-PKA axis, knockout of caspase-11 (Casp11−/−), or Gsdmd inactivation (GsdmdI105N/I105N) similarly protects against LPS-induced lethality in poly(I:C)-primed mice. Our results provide previously unidentified mechanistic insight into immune regulation by cAMP and represent a proof of concept that immunometabolism constitutes a potential therapeutic target in sepsis.

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Macrophagic Stabilin-1 Restored Disruption of Vascular Integrity Caused by Sepsis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1669477 ◽

2018 ◽

Vol 118 (10) ◽

pp. 1776-1789 ◽

Cited By ~ 11

Author(s):

Wonhwa Lee ◽

Seung-Yoon Park ◽

Youngbum Yoo ◽

Soon-Young Kim ◽

Jung-Eun Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Survival Rate ◽

Vascular Permeability ◽

Inflammatory Responses ◽

Vascular Endothelial Cells ◽

High Mobility ◽

Neutralizing Antibody ◽

Dependent Manner ◽

Vascular Integrity ◽

In The Wild

AbstractSepsis develops because of overwhelming inflammatory responses to bacterial infection, and disrupts vascular integrity. Stabilin-1 (STAB-1) is a phagocytic receptor, which mediates efferocytosis in a phosphatidylserine (PS)-dependent manner. STAB-1 is expected to play important roles in efferocytosis during sepsis. Here, we determined the role of STAB-1 in maintaining and restoring vascular integrity. Macrophages and vascular endothelial cells were used to assess the effect of STAB-1 on survival rate, phagocytic activity, vascular permeability and transendothelial migration (TEM). Additionally, we investigated whether the high-mobility group box 1 (HMGB1)-receptor for advanced glycated end products complex interfered with the binding of Stab1 to PS. Mortality rate was higher in the Stab1-knockout mice than in the wild-type mice, and STAB-1 deficiency was related to reduced macrophage-mediated efferocytosis and the disruption of vascular integrity, which increased vascular permeability, and enhanced TEM. STAB-1 deficiency promoted lung injury, and elevated the expression of sepsis markers. The exogenous application of the anti-HMGB1 neutralizing antibody improved efferocytosis, vascular integrity and survival rate in sepsis. Collectively, our findings indicated that STAB-1 regulated and maintained vascular integrity through the clearance of infected apoptotic endothelial cells. Moreover, our results suggested that interventions targeting vascular integrity by STAB-1 signalling are promising therapeutic approaches to sepsis.

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GPR110 (ADGRF1) mediates anti-inflammatory effects of N-docosahexaenoylethanolamine

Journal of Neuroinflammation ◽

10.1186/s12974-019-1621-2 ◽

2019 ◽

Vol 16 (1) ◽

Cited By ~ 8

Author(s):

Taeyeop Park ◽

Huazhen Chen ◽

Hee-Yong Kim

Keyword(s):

Inflammatory Cytokine ◽

Inflammatory Responses ◽

Cytokine Expression ◽

Regulatory Function ◽

Enzyme Linked Immunosorbent Assay ◽

Inflammatory Condition ◽

Wild Type ◽

Knock Out ◽

The Brain

Abstract Background Neuroinflammation is a widely accepted underlying condition for various pathological processes in the brain. In a recent study, synaptamide, an endogenous metabolite derived from docosahexaenoic acid (DHA, 22:6n-3), was identified as a specific ligand to orphan adhesion G-protein-coupled receptor 110 (GPR110, ADGRF1). Synaptamide has been shown to suppress lipopolysaccharide (LPS)-induced neuroinflammation in mice, but involvement of GPR110 in this process has not been established. In this study, we investigated the possible immune regulatory role of GPR110 in mediating the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. Methods For in vitro studies, we assessed the role of GPR110 in synaptamide effects on LPS-induced inflammatory responses in adult primary mouse microglia, immortalized murine microglial cells (BV2), primary neutrophil, and peritoneal macrophage by using quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) as well as neutrophil migration and ROS production assays. To evaluate in vivo effects, wild-type (WT) and GPR110 knock-out (KO) mice were injected with LPS intraperitoneally (i.p.) or TNF intravenously (i.v.) followed by synaptamide (i.p.), and expression of proinflammatory mediators was measured by qPCR, ELISA, and western blot analysis. Activated microglia in the brain and NF-kB activation in cells were examined microscopically after immunostaining for Iba-1 and RelA, respectively. Results Intraperitoneal (i.p.) administration of LPS increased TNF and IL-1β in the blood and induced pro-inflammatory cytokine expression in the brain. Subsequent i.p. injection of the GPR110 ligand synaptamide significantly reduced LPS-induced inflammatory responses in wild-type (WT) but not in GPR110 knock-out (KO) mice. In cultured microglia, synaptamide increased cAMP and inhibited LPS-induced proinflammatory cytokine expression by inhibiting the translocation of NF-κB subunit RelA into the nucleus. These effects were abolished by blocking synaptamide binding to GPR110 using an N-terminal targeting antibody. GPR110 expression was found to be high in neutrophils and macrophages where synaptamide also caused a GPR110-dependent increase in cAMP and inhibition of LPS-induced pro-inflammatory mediator expression. Intravenous injection of TNF, a pro-inflammatory cytokine that increases in the circulation after LPS treatment, elicited inflammatory responses in the brain which were dampened by the subsequent injection (i.p.) of synaptamide in a GPR110-dependent manner. Conclusion Our study demonstrates the immune-regulatory function of GPR110 in both brain and periphery, collectively contributing to the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. We suggest GPR110 activation as a novel therapeutic strategy to ameliorate inflammation in the brain as well as periphery.

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Binding of Brucella protein, Bp26, to select extracellular matrix molecules

BMC Molecular and Cell Biology ◽

10.1186/s12860-019-0239-7 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 4

Author(s):

Yasmin ElTahir ◽

Amna Al-Araimi ◽

Remya R. Nair ◽

Kaija J. Autio ◽

Hongmin Tu ◽

...

Keyword(s):

Extracellular Matrix ◽

Type I Collagen ◽

Host Cells ◽

Mouse Serum ◽

Enzyme Linked Immunosorbent Assay ◽

Target Cells ◽

Type I ◽

Dependent Manner ◽

Biolayer Interferometry

Abstract Background Brucella is a facultative intracellular pathogen responsible for zoonotic disease brucellosis. Little is known about the molecular basis of Brucella adherence to host cells. In the present study, the possible role of Bp26 protein as an adhesin was explored. The ability of Brucella protein Bp26 to bind to extracellular matrix (ECM) proteins was determined by enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). Results ELISA experiments showed that Bp26 bound in a dose-dependent manner to both immobilized type I collagen and vitronectin. Bp26 bound weakly to soluble fibronectin but did not bind to immobilized fibronectin. No binding to laminin was detected. Biolayer interferometry showed high binding affinity of Bp26 to immobilized type I collagen and no binding to fibronectin or laminin. Mapping of Bp26 antigenic epitopes by biotinylated overlapping peptides spanning the entire sequence of Bp26 using anti Bp26 mouse serum led to the identification of five linear epitopes. Collagen and vitronectin bound to peptides from several regions of Bp26, with many of the binding sites for the ligands overlapping. The strongest binding for anti-Bp26 mouse serum, collagen and vitronectin was to the peptides at the C-terminus of Bp26. Fibronectin did not bind to any of the peptides, although it bound to the whole Bp26 protein. Conclusions Our results highlight the possible role of Bp26 protein in the adhesion process of Brucella to host cells through ECM components. This study revealed that Bp26 binds to both immobilized and soluble type I collagen and vitronectin. It also binds to soluble but not immobilized fibronectin. However, Bp26 does not bind to laminin. These are novel findings that offer insight into understanding the interplay between Brucella and host target cells, which may aid in future identification of a new target for diagnosis and/or vaccine development and prevention of brucellosis.

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Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.694675 ◽

2021 ◽

Vol 9 ◽

Author(s):

Xudong Pu ◽

Xin Li ◽

Lili Cao ◽

Kaiming Yue ◽

Panpan Zhao ◽

...

Keyword(s):

Inflammatory Response ◽

Signaling Pathways ◽

Proinflammatory Cytokines ◽

Giardia Duodenalis ◽

Erk Signaling ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Mouse Macrophages ◽

Host Inflammatory Response

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

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