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High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

ABSTRACT: Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage-associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box-1 (HMGB1) protein has been implicated as a potent DAMP and pro-inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL-6 levels increase in patients with advanced DD in comparison to early DD. This study further tested the hypothesis that HMGB1 promotes inflammatory signaling driving DD in human nucleus pulposus (NP) cells and tissue. Immunofluorescence and western blot analysis confirmed the expression of HMGB1 and its extracellular release by NP cells under cell stress. Gene expression and protein quantification indicate that HMGB1 stimulates the expression IL-6 and MMP-1 in a dose-dependent manner. The contributions of toll-like receptor (TLR) -2, -4 and receptor for advanced glycation end products (RAGE) as receptors mediating HMGB1 signaling was examined using small molecule inhibitors. Inhibition of TLR-4 signaling, with TAK-242, completely abrogated HMGB1 induced IL-6 and MMP-1 expression, whereas inhibition of TLR-2, with O-vanillin, or RAGE, with FPS-ZM1, had mild inhibitory effects. HMGB1 stimulation activated NF-?B signaling while TAK-242 co-treatment abrogated it. Lastly, effects of HMGB1 on matrix deposition was evaluated in a 3D culture system of human NP cells. These results implicate HMGB1 as a potent DAMP that promotes inflammation in NP cells and degradation of NP tissues. TLR4-HMGB1 axis is a potential major pathway to alleviate disc inflammation and mitigate DD. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

SUBMITTER: Shah BS

PROVIDER: S-EPMC7401857 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

Shah Bhranti S BS Burt Kevin G KG Jacobsen Timothy T Fernandes Tiago D TD Alipui Didier Olivier DO Weber Kathryn T KT Levine Mitchell M Chavan Sangeeta S SS Yang Huan H Tracey Kevin J KJ Chahine Nadeen O NO

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20181029 1

Intervertebral disc (IVD) degeneration (DD) is associated with low back pain, the leading cause of disability worldwide. Damage-associated molecular patterns (DAMPs) that contribute to inflammation and trigger DD have not been well characterized. Extracellular high mobility group box-1 (HMGB1) protein has been implicated as a potent DAMP and pro-inflammatory stimulus in the immune system. In this study, we show that HMGB1 and IL-6 levels increase in patients with advanced DD in comparison to ear ...[more]

PMID: 30273982

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Project description:IntroductionHigh mobility group box 1 (HMGB1) is released by necrotic cells or secreted in response to inflammatory stimuli. Extracellular HMGB1 may act as a pro-inflammatory cytokine in rheumatoid arthritis. We have recently reported that HMGB1 is released by osteoarthritic synoviocytes after activation with interleukin-1beta (IL-1?) The present study investigated the role of HMGB1 in synovial inflammation in osteoarthritis (OA).MethodsHMGB1 was determined in human synovium using immunohistochemistry, comparing normal to OA. OA synoviocytes were incubated with HMGB1 at 15 or 25 ng/ml in the absence or presence of IL-1? (10 ng/ml). Gene expression was analyzed by quantitative PCR and protein expression by Western Blot and ELISA. Matrix metalloproteinase (MMP) activity was studied by fluorometric procedures and nuclear factor (NF)-?B activation by transient transfection with a NF-?B-luciferase plasmid.ResultsIn the normal synovium, HMGB1 was found in the synovial lining cells, sublining cells, and in the vascular wall cells. The distribution of HMGB1 in OA synovium was similar but the number of HMGB1 positive cells was higher and HMGB1 was also present in infiltrated cells. In normal synovial membrane cells, HMGB1 was found mostly in the nuclei, whereas in OA, HMGB1 was generally found mostly in the cytoplasm. In OA synoviocytes, HMGB1 alone at concentrations of 15 or 25 ng/ml did not affect the production of IL-6, IL-8, CCL2, CCL20, MMP-1 or MMP-3, but in the presence of IL-1?, a significant potentiation of protein and mRNA expression, as well as MMP activity was observed. HMGB1 also enhanced the phosphorylated ERK1/2 and p38 levels, with a lower effect on phosphorylated Akt. In contrast, JNK1/2 phosphorylation was not affected. In addition, HMGB1 at 25 ng/ml significantly potentiated NF-?B activation in the presence of IL-1?.ConclusionsOur results indicate that HMGB1 is overexpressed in OA synovium and mostly present in extracellular form. In OA synoviocytes, HMGB1 cooperates with IL-1? to amplify the inflammatory response leading to the production of a number of cytokines, chemokines and MMPs. Our data support a pro-inflammatory role for this protein contributing to synovitis and articular destruction in OA.

Project description:Trauma is common in dogs and causes significant morbidity and mortality, but it remains challenging to predict the prognosis of dogs with traumatic injuries. This study aimed to quantify plasma high-mobility group box-1 (HMGB-1) and cytokine concentrations in dogs with moderate-to-severe trauma, and to evaluate the association between these biomarkers and the injury severity and survival to discharge. Using a prospective, observational case-control study design, 49 dogs with an animal trauma triage (ATT) score ≥3 were consecutively enrolled from 07/2015 to 10/2017 and followed to hospital discharge. Dogs <3 kg and those with pre-existing coagulopathies were excluded. Thirty three healthy control dogs were also enrolled. Illness and injury severity scores including the acute patient physiologic and laboratory evaluation (APPLE) were calculated using at-presentation data. Plasma HMGB-1 concentrations were measured by ELISA; concentrations of 13 cytokines were measured using multiplex bead-based assays and separately concentrations of 4 cytokines were measured using a multiplex canine-specific ELISA. All biomarkers were measured in duplicate. Mann-Whitney U tests were used to compare biomarker concentrations between groups and between survivors and non-survivors. Associations between biomarkers were evaluated using Spearman's correlation coefficients. Independent predictors of survival were identified using multivariable logistic regression. Alpha was set at 0.05. Plasma concentrations of HMGB-1, interleukin-6, C-X-C motif chemokine-8, keratinocyte chemoattractant-like, and C-C chemokine ligand-2 were significantly greater in injured dogs vs. controls (all P ≤ 0.011). In univariate analyses, HMGB-1 was significantly greater in non-survivors 46.67 ng/mL (8.94-84.73) compared to survivors 6.03 ng/mL (3.30-15.75), (P = 0.003). Neither HMGB-1 or the cytokines were associated with survival independent of illness severity as measured by the APPLE score, however.

Project description:Initiation of intervertebral disc degeneration is thought to be biologically driven. This reflects a process, where biochemical and mechanical stimuli affect cell activity (CA) that compromise the tissue strength over time. Experimental research enhanced our understanding about the effect of such stimuli on different CA, such as protein synthesis or mRNA expression. However, it is still unclear how cells respond to their native environment that consists of a "cocktail" of different stimuli that might locally vary. This work presents an interdisciplinary approach of experimental and in silico research to approximate Nucleus Pulposus CA within multifactorial biochemical environments. Thereby, the biochemical key stimuli glucose, pH, and the proinflammatory cytokines TNF-α and IL1β were considered that were experimentally shown to critically affect CA. To this end, a Nucleus Pulposus multicellular system was modelled. It integrated experimental findings from in vitro studies of human or bovine Nucleus Pulposus cells, to relate the individual effects of targeted stimuli to alterations in CA. Unknown stimulus-CA relationships were obtained through own experimental 3D cultures of bovine Nucleus Pulposus cells in alginate beads. Translation of experimental findings into suitable parameters for network modelling approaches was achieved thanks to a new numerical approach to estimate the individual sensitivity of a CA to each stimulus type. Hence, the effect of each stimulus type on a specific CA was assessed and integrated to approximate a multifactorial stimulus environment. Tackled CA were the mRNA expressions of Aggrecan, Collagen types I & II, MMP3, and ADAMTS4. CA was assessed for four different proinflammatory cell states; non-inflamed and inflamed for IL1β, TNF-α or both IL1β&TNF-α. Inflamed cell clusters were eventually predicted in a multicellular 3D agent-based model. Experimental results showed that glucose had no significant impact on proinflammatory cytokine or ADAMTS4 mRNA expression, whereas TNF-α caused a significant catabolic shift in most explored CA. In silico results showed that the presented methodology to estimate the sensitivity of a CA to a stimulus type importantly improved qualitative model predictions. However, more stimuli and/or further experimental knowledge need to be integrated, especially regarding predictions about the possible progression of inflammatory environments under adverse nutritional conditions. Tackling the multicellular level is a new and promising approach to estimate manifold responses of intervertebral disc cells. Such a top-down high-level network modelling approach allows to obtain information about relevant stimulus environments for a specific CA and could be shown to be suitable to tackle complex biological systems, including different proinflammatory cell states. The development of this methodology required a close interaction with experimental research. Thereby, specific experimental needs were derived from systematic in silico approaches and obtained results were directly used to enhance model predictions, which reflects a novelty in this research field. Eventually, the presented methodology provides modelling solutions suitable for multiscale approaches to contribute to a better understanding about dynamics over multiple spatial scales. Future work should focus on an amplification of the stimulus environment by integrating more key relevant stimuli, such as mechanical loading parameters, in order to better approximate native physiological environments.

Dataset Information

High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

Publications

High mobility group box-1 induces pro-inflammatory signaling in human nucleus pulposus cells via toll-like receptor 4-dependent pathway.

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