Pro-inflammatory effects of human apatite crystals extracted from patients suffering from calcific tendinopathy

Abstract Background Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1β, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. Results Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB, and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite. Conclusions As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.

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Pro-inflammatory effects of human apatite crystals extracted from patients suffering from calcific tendinopathy.

10.21203/rs.3.rs-156807/v1 ◽

2021 ◽

Author(s):

Julien Herman ◽

Benoit Le Goff ◽

Julien De Lima ◽

Régis Brion ◽

Catherine Chevalier ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Membrane Thickness ◽

Air Pouch ◽

Calcific Tendinopathy ◽

Caspase 1 ◽

Carbonated Apatite ◽

Monocytes And Macrophages ◽

Synthetic Hydroxyapatite

Abstract Background: Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystals-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved. Methods: Human calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1 and human tenocytes. The release of IL-1β, IL-6 and IL-8 by cells was quantified by ELISA. Gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement was assessed in THP-1 cells using a NF-kB inhibitor and a Caspase 1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. Results: Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL- 1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. Patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of Caspase 1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in air pouch led to significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite. Conclusions: As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape and protein content may explain this observation.

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POS0366 PRO-INFLAMMATORY EFFECTS OF HUMAN APATITE CRYSTALS EXTRACTED FROM PATIENTS SUFFERING FROM CALCIFIC TENDINOPATHY

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2624 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 413.1-413

Author(s):

J. Herman ◽

B. Le Goff ◽

J. De Lima ◽

R. Brion ◽

C. Chevalier ◽

...

Keyword(s):

Rotator Cuff ◽

Nlrp3 Inflammasome ◽

Membrane Thickness ◽

Dependent Manner ◽

Air Pouch ◽

Calcific Tendinopathy ◽

Caspase 1 ◽

Monocytes And Macrophages ◽

Synthetic Hydroxyapatite

Background:Calcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystals-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved.Objectives:The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved.Methods:Human calcifications were obtained from patients treated for their shoulder pain related to a calcific tendinopathy of the rotator cuff. Calcifications were extracted by ultrasound-guided lavage and aspiration as previously described [1]. Human calcifications and synthetic hydroxyapatite (sHA) were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1 and human tenocytes. The release of IL-1β, IL-6 and IL-8 by cells was quantified by ELISA. Gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement was assessed in THP-1 cells using a NF-kB inhibitor and a Caspase 1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model. The membrane thickness and infiltrate were assessed 6 and 24 hours after the injection of human calcifications or synthetic hydroxyapatite using hematoxylin and eosin staining. Macrophages, neutrophils and lymphocytes infiltrates were assessed by immunohistochemistry. Total RNA was extracted from the membranes and expression of IL-1β, IL-6 and TNFβ was quantified by PCR.Results:Human calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages and THP-1 only if they were first primed with LPS (lipopolysaccharide) for monocytes and macrophages or PMA (Phorbol 12-myristate 13-acetate) for THP-1. No IL-1β was detected in tenocytes’ supernatants. Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. IL-6 and IL-8 levels were not increased in the supernatants after crystal stimulation. Patient’s crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition NF-kB as well as Caspase 1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in air pouch led to significant increase in membrane thickness with an infiltrate mainly composed of macrophages. Significant overexpression of IL-1β was only observed in the synthetic hydroxyapatite group.Conclusion:As synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape and protein content may explain this observation.References:[1]Darrieutort-Laffite C, Arnolfo P, Garraud T, Adrait A, Couté Y, Louarn G, et al. Rotator Cuff Tenocytes Differentiate into Hypertrophic Chondrocyte-Like Cells to Produce Calcium Deposits in an Alkaline Phosphatase-Dependent Manner. J Clin Med. 2019 Sep 26;8(10):1544. doi: 10.3390/jcm8101544.Acknowledgements:Fondation Arthritis, Recherche et Rhumatismes and French Society for Rheumatology for their financial supportDisclosure of Interests:None declared

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The Essential Oil of Artemisia argyi H.Lév. and Vaniot Attenuates NLRP3 Inflammasome Activation in THP-1 Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.712907 ◽

2021 ◽

Vol 12 ◽

Author(s):

Pengxiao Chen ◽

Qi Bai ◽

Yanting Wu ◽

Qiongzhen Zeng ◽

Xiaowei Song ◽

...

Keyword(s):

Essential Oil ◽

Nlrp3 Inflammasome ◽

Therapeutic Potential ◽

Inflammasome Activation ◽

Caspase 1 ◽

Artemisia Argyi ◽

Nlrp3 Inflammasome Activation ◽

Long Time

Artemisia argyi H. Lév. and Vaniot is a traditional medical herb that has been used for a long time in China and other Asian counties. Essential oil is the main active fraction of Artemisia argyi H. Lév. and Vaniot, and its anti-inflammatory potential has been observed in vitro and in vivo. Here, we found that the essential oil of Artemisia argyi H. Lév. and Vaniot (EOAA) inhibited monosodium urate (MSU)- and nigericin-induced NLRP3 inflammasome activation. EOAA suppressed caspase-1 and IL-1β processing and pyroptosis. NF-κB p65 phosphorylation and translocation were also inhibited. In addition, EOAA suppressed nigericin-induced NLRP3 inflammasome activation without blocking ASC oligomerization, suggesting that it may inhibit NLRP3 inflammasome activation by preventing caspase-1 processing. Our study thus indicates that EOAA inhibits NLRP3 inflammasome activation and has therapeutic potential against NLRP3-driven diseases.

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Astragaloside IV Suppresses High Glucose-Induced NLRP3 Inflammasome Activation by Inhibiting TLR4/NF-κB and CaSR

Mediators of Inflammation ◽

10.1155/2019/1082497 ◽

2019 ◽

Vol 2019 ◽

pp. 1-16 ◽

Cited By ~ 10

Author(s):

Bin Leng ◽

Yingjie Zhang ◽

Xinran Liu ◽

Zhen Zhang ◽

Yang Liu ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

High Glucose ◽

Inflammasome Activation ◽

Astragaloside Iv ◽

Caspase 1 ◽

Endothelial Inflammation ◽

Nlrp3 Inflammasome Activation ◽

Anti Inflammatory

Long-term exposure to high glucose induces vascular endothelial inflammation that can result in cardiovascular disease. Astragaloside IV (As-IV) is widely used for anti-inflammatory treatment of cardiovascular diseases. However, its mechanism of action is still not fully understood. In this study, we investigated the effect of As-IV on high glucose-induced endothelial inflammation and explored its possible mechanisms. In vivo, As-IV (40 and 80 mg/kg/d) was orally administered to rats for 8 weeks after a single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg). In vitro, human umbilical vein endothelial cells (HUVECs) were treated with high glucose (33 mM glucose) in the presence or absence of As-IV, NPS2143 (CaSR inhibitor), BAY 11-7082 (NF-κB p65 inhibitor), and INF39 (NLRP3 inhibitor), and overexpression of CaSR was induced by infection of CaSR-overexpressing lentiviral vectors to further discuss the anti-inflammatory property of As-IV. The results showed that high glucose increased the expression of interleukin-18 (IL-18), interleukin-1β (IL-1β), NLRP3, caspase-1, and ASC, as well as the protein level of TLR4, nucleus p65, and CaSR. As-IV can reverse these changes in vivo and in vitro. Meanwhile, NPS2143, BAY 11-7082, and INF39 could significantly abolish the high glucose-enhanced NLRP3, ASC, caspase-1, IL-18, and IL-1β expression in vitro. In addition, both NPS2143 and BAY 11-7082 attenuated high glucose-induced upregulation of NLRP3, ASC, caspase-1, IL-18, and IL-1β expression. In conclusion, this study suggested that As-IV could inhibit high glucose-induced NLRP3 inflammasome activation and subsequent secretion of proinflammatory cytokines via inhibiting TLR4/NF-κB signaling pathway and CaSR, which provides new insights into the anti-inflammatory activity of As-IV.

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Hairy Platelet-Derived Extracellular Vesicles Promote Lung Vaso-Occlusion in Sickle Cell Disease

Blood ◽

10.1182/blood.v130.suppl_1.958.958 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 958-958

Author(s):

Prithu Sundd ◽

Maritza Ann Jimenez ◽

Margaret F. Bennewitz ◽

Tomasz Brzoska ◽

Egemen Tutuncuoglu ◽

...

Keyword(s):

Blood Flow ◽

Sickle Cell Disease ◽

Extracellular Vesicles ◽

Human Blood ◽

Nlrp3 Inflammasome ◽

Cell Disease ◽

Caspase 1 ◽

Neutrophil Aggregation

Abstract Background: Acute chest syndrome (ACS) is a type of acute lung injury and the leading cause of mortality in Sickle Cell Disease (SCD). Current treatments for ACS are primarily supportive, and there is a critical need for rescue therapies. ACS is often a sequela of acute systemic vaso-occlusive crisis and preceded by thrombocytopenia. However, the role of platelets in the pathogenesis of ACS remains largely unknown. Methods: We used our validated model of vaso-occlusive crisis in transgenic, humanized SCD mice, which is triggered by intravenous challenge with nanogram levels of the TLR4 ligand, lipopolysaccharide (LPS). Platelet-neutrophil aggregates and blood flow in the lung microcirculation was visualized in real time in vivo, using multi-photon-excitation microscopy of intact lung in live SCD mice. SCD or control human blood was perfused through microfluidic channels in vitro and neutrophil-platelet aggregation was visualized using fluorescence microscopy. Platelet derived extracellular vesicles were characterized using nanoparticle tracking and biochemical approaches. Results: We have made a novel finding that the arrest of blood flow and injury in the lung is secondary to blockade of pulmonary arterioles by platelet-neutrophil aggregates. Using in vitro microfluidic studies, we confirmed that platelet-neutrophil aggregation is higher in LPS-treated SCD patient blood compared with healthy controls, and this correlates with increased numbers of platelet-derived extracellular vesicles (EVs) that express IL-1β. Our studies also reveal that platelet-neutrophil aggregation in pulmonary arterioles of SCD mice is associated with an increase in peripheral blood levels of platelet-derived EVs containing IL-1β. Remarkably, inhibition of TLR4 or TLR4/NLRP3-inflammasome activated caspase-1, or inhibition of IL-1β signaling, attenuated release of platelet EVs and platelet-neutrophil aggregation in the lung arterioles of SCD mice in vivo and SCD human blood in vitro . Conclusions: TLR4 and NLRP3-inflammasome-mediated caspase-1 activation in platelets during vaso-occlusive crisis leads to release of IL-1β-containing EVs into the circulation. These circulating platelet EVs promote platelet-neutrophil aggregation in pulmonary arterioles, which results in arrest of blood flow in the lung, leading to ACS. Therapeutic inhibition of TLR4/NLRP3-caspase-1 signaling in platelets or IL-1β signaling is a potential therapy for ACS in SCD patients. Acknowledgments: This study was supported by 1R01HL128297-01 (P.S.) and VMI startup funds (P.S.). Disclosures No relevant conflicts of interest to declare.

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Curcumin Ameliorates White Matter Injury after Ischemic Stroke by Inhibiting Microglia/Macrophage Pyroptosis through NF-κB Suppression and NLRP3 Inflammasome Inhibition

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/1552127 ◽

2021 ◽

Vol 2021 ◽

pp. 1-25

Author(s):

Yuanyuan Ran ◽

Wei Su ◽

Fuhai Gao ◽

Zitong Ding ◽

Shuiqing Yang ◽

...

Keyword(s):

White Matter ◽

Nlrp3 Inflammasome ◽

Functional Outcomes ◽

Sensorimotor Function ◽

Curcumin Treatment ◽

Caspase 1 ◽

Cell Death Pathway ◽

The Impact

NLRP3 inflammasome-mediated pyroptosis is a proinflammatory programmed cell death pathway, which plays a vital role in functional outcomes after stroke. We previously described the beneficial effects of curcumin against stroke-induced neuronal damage through modulating microglial polarization. However, the impact of curcumin on microglial pyroptosis remains unknown. Here, stroke was modeled in mice by middle cerebral artery occlusion (MCAO) for 60 minutes and treated with curcumin (150 mg/kg) intraperitoneally immediately after reperfusion, followed by daily administrations for 7 days. Curcumin ameliorated white matter (WM) lesions and brain tissue loss 21 days poststroke and improved sensorimotor function 3, 10, and 21 days after stroke. Furthermore, curcumin significantly reduced the number of gasdermin D+ (GSDMD+) Iba1+ and caspase-1+Iba1+ microglia/macrophage 21 days after stroke. In vitro, lipopolysaccharide (LPS) with ATP treatment was used to induce pyroptosis in primary microglia. Western blot revealed a decrease in pyroptosis-related proteins, e.g., GSDMD-N, cleaved caspase-1, NLRP3, IL-1β, and IL-18, following in vitro or in vivo curcumin treatment. Mechanistically, both in vivo and in vitro studies confirmed that curcumin inhibited the activation of the NF-κB pathway. NLRP3 knocked down by siRNA transfection markedly increased the inhibitory effects of curcumin on microglial pyroptosis and proinflammatory responses, both in vitro and in vivo. Furthermore, stereotaxic microinjection of AAV-based NLRP3 shRNA significantly improved sensorimotor function and reduced WM lesion following curcumin treatment in MCAO mice. Our study suggested that curcumin reduced stroke-induced WM damage, improved functional outcomes, and attenuated microglial pyroptosis, at least partially, through suppression of the NF-κB/NLRP3 signaling pathway, further supporting curcumin as a potential therapeutic drug for stroke.

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Green Synthesis of Silver Nanoparticles Using Annona muricata Extract as an Inducer of Apoptosis in Cancer Cells and Inhibitor for NLRP3 Inflammasome via Enhanced Autophagy

Nanomaterials ◽

10.3390/nano11020384 ◽

2021 ◽

Vol 11 (2) ◽

pp. 384

Author(s):

Majid S. Jabir ◽

Yasmin M. Saleh ◽

Ghassan M. Sulaiman ◽

Nahi Y. Yaseen ◽

Usama I. Sahib ◽

...

Keyword(s):

Silver Nanoparticles ◽

Nlrp3 Inflammasome ◽

Proliferative Activity ◽

P53 Protein ◽

Adaptor Protein ◽

Inflammasome Activation ◽

Annona Muricata ◽

Caspase 1

Annona muricata is one of the most important traditional medicinal plants which contains numerous chemicals that exhibit various pharmacological properties. In this study, silver nanoparticles were prepared using A. muricata peel extract as a reducing agent and the effect was enhanced through A. muricata like pharmaceutical activity. AgNPs formation was confirmed by color changes, UV-visible spectroscopy, SEM, DLS, and XRD. The anti-proliferative activity of AgNPs against THP-1, AMJ-13, and HBL cell lines was studied. Apoptotic markers were tested using AO/EtBr staining assay, cell cycle phases using flowcytometry, and the expression of P53. Autophagy takes an essential part in controlling inflammasome activation by primary bone marrow-derived macrophages (BMDMs). We report novel functions for AgNPs-affected autophagy, represented by the control of the release of IL-1β, caspase-1, adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and NLRP3 in BMDMs following treatment with LPS+ATP. The current study revealed that the AgNPs inhibited THP-1 and AMJ-13 cell proliferation. Meanwhile, the AgNPs significantly increased autophagy and reduced IL-1b and NLRP3 levels in both in vivo and in vitro models. The secretion of IL-1β was reduced whereas the degradation of NLRP3 inflammasome was enhanced. These findings propose that AgNPs apply an anti-proliferative activity against THP-1 and AMJ-13 cells through the stimulation of apoptosis via mitochondrial damage and induction of p53 protein pathway. In addition, AgNP-induced autophagy reduced the levels of IL-1β and NLRP3 inflammasome activation. This indicated that the AgNPs augment autophagy controlled by the IL-1β pathway via two different novel mechanisms. The first one is regulating activation of the IL-1 β, caspase-1, and ASC, while the second is NLRP3 targeting for lysosomal degradation. Overall, this study suggests that AgNPs could be a potent therapy for various types of cancer and an alternative treatment for preventing inflammation via enhancing autophagy.

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Suppression of lncRNA NLRP3 inhibits NLRP3-triggered inflammatory responses in early acute lung injury

Cell Death and Disease ◽

10.1038/s41419-021-04180-y ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Deqiang Luo ◽

Wei Dai ◽

Xiaojin Feng ◽

Chengzhi Ding ◽

Qiang Shao ◽

...

Keyword(s):

Acute Lung Injury ◽

Lung Injury ◽

Inflammatory Response ◽

Nlrp3 Inflammasome ◽

Receptor Protein ◽

Luciferase Reporter ◽

Lung Pathology ◽

Caspase 1

AbstractAcute lung injury (ALI) is a common lung pathology that is accompanied by alveolar macrophage (AM) activation and inflammatory response. This study investigated the role of the long non-coding RNA NONRATT004344 (hereafter named lncRNA NLRP3) in regulating the Nod-like receptor protein 3 (NLRP3)-triggered inflammatory response in early ALI and the underlying mechanism as well. We established LPS-induced ALI models to explore their interactive mechanisms in vitro and in vivo. Luciferase reporter assays were performed to determine that miR-138-5p could bind to lncRNA NLRP3 and NLRP3. We observed increased lncRNA NLRP3 expression, decreased miR-138-5p expression, NLRP3 inflammasome activation, and upregulated caspase-1, IL-1β, and IL-18 expression in the LPS-induced ALI model. Furthermore, lncRNA NLRP3 overexpression activated the NLRP3 inflammasome and promoted IL-1β and IL-18 secretion; the miR-138-5p mimic abolished these effects in vivo and in vitro. Consistently, miR-138-5p inhibition reversed the effects of lncRNA NLRP3 silencing on the expression of NLRP3-related molecules and inhibition of the NLRP3/caspase-1/IL-1β signalling pathway. Mechanistically, lncRNA NLRP3 sponging miR-138-5p facilitated NLRP3 activation through a competitive endogenous RNA (ceRNA) mechanism. In summary, our results suggested that lncRNA NLRP3 binding miR-138-5p promotes NLRP3-triggered inflammatory response via lncRNA NLRP3/miR-138-5p/NLRP3 ceRNA network (ceRNET) and provides insights into the treatment of early ALI.

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NLRP3 Inflammasome: A New Target for Prevention and Control of Osteoporosis?

Frontiers in Endocrinology ◽

10.3389/fendo.2021.752546 ◽

2021 ◽

Vol 12 ◽

Author(s):

Na Jiang ◽

Jinyang An ◽

Kuan Yang ◽

Jinjin Liu ◽

Conghui Guan ◽

...

Keyword(s):

Bone Resorption ◽

Inflammatory Response ◽

Nlrp3 Inflammasome ◽

Osteoclast Differentiation ◽

Inflammatory Factor ◽

Inflammatory Microenvironment ◽

Risk Of Death ◽

Caspase 1

Osteoporosis is a systemic bone metabolism disease that often causes complications, such as fractures, and increases the risk of death. The nucleotide-binding oligomerization domain-like-receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates the maturation and secretion of Caspase-1 dependent proinflammatory cytokines interleukin (IL)-1β and IL-18, mediates inflammation, and induces pyroptosis. The chronic inflammatory microenvironment induced by aging or estrogen deficiency activates the NLRP3 inflammasome, promotes inflammatory factor production, and enhances the inflammatory response. We summarize the related research and demonstrate that the NLRP3 inflammasome plays a vital role in the pathogenesis of osteoporosis by affecting the differentiation of osteoblasts and osteoclasts. IL-1β and IL-18 can accelerate osteoclast differentiation by expanding inflammatory response, and can also inhibit the expression of osteogenic related proteins or transcription factors. In vivo and in vitro experiments showed that the overexpression of NLRP3 protein was closely related to aggravated bone resorption and osteogenesis deficiency. In addition, abnormal activation of NLRP3 inflammasome can not only produce inflammation, but also lead to pyroptosis and dysfunction of osteoblasts by upregulating the expression of Caspase-1 and gasdermin D (GSDMD). In conclusion, NLRP3 inflammasome overall not only accelerates bone resorption, but also inhibits bone formation, thus increasing the risk of osteoporosis. Thus, this review highlights the recent studies on the function of NLRP3 inflammasome in osteoporosis, provides information on new strategies for managing osteoporosis, and investigates the ideal therapeutic target to treat osteoporosis.

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Heme Induces NLRP3 Inflammasome Formation in Primary Human Macrophages and May Propagate Hemolytic Inflammatory Processes By Inducing S100A8 Expression

Blood ◽

10.1182/blood.v128.22.1256.1256 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1256-1256 ◽

Cited By ~ 2

Author(s):

Angelica A. Silveira ◽

Clare Cunningham ◽

Emma Corr ◽

Wilson Alves Ferreira ◽

Fernando F. Costa ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Research Funding ◽

Inflammatory Responses ◽

Saline Control ◽

Intravascular Hemolysis ◽

Human Macrophages ◽

Caspase 1 ◽

Tnf Α

Abstract Intravascular hemolysis results in the release of damaging hemoglobin and free heme into the circulation. A role for heme as a danger associated molecular pattern (DAMP), with a function in sterile inflammatory responses, is becoming increasingly recognized. Whilst heme has known effects on leukocytes, activating their migration, adhesion molecule expression and cytokine expression, more recent data demonstrate that this molecule can induce NLRP3 inflammasome formation in murine bone marrow macrophages, with consequent interleukin (IL)-1β processing and neutrophil recruitment (Dutra et al., Proc. Natl Acad Sci. 111: E4110, 2014). We aimed to investigate whether heme can also induce inflammasome activation in primary human macrophages (hMACs) and to further characterize the pathways by which heme-induced inflammatory responses may be amplified under sterile conditions. CD14+ cells were separated from human peripheral blood (using anti-CD14 magnetic beads) and differentiated into hMACs under M-CSF media supplementation and in the presence of 10% fetal bovine serum. In vitro results are expressed as means ± SEM for triplicate cultures and are representative of three independent experiments. Priming of hMACs with lipopolysaccharide (LPS; 100 ng/mL; 3h) alone induced low level secretion of IL-1β (14.11±9.2 pg/106 cells, as measured by ELISA), while heme (50 µM), in the absence of pre-stimulation with LPS, was unable to induce significant IL-1β secretion within 3h (2.46±1.4 pg/106 cells). In contrast, co-incubation of hMACs with both LPS and heme for 3h significantly enhanced hMAC IL-1β release (490.3±36.3 pg/106 cells; P<0.05 compared to LPS alone). The inflamassome pathway inhibitors, MCC950 (5 µM; a specific inhibitor of NLRP3) and YVAD (40 µM; a caspase-1 inhibitor) significantly inhibited IL-1β secretion in LPS-primed hMACs stimulated with heme (reduced to 35.12±3.9; 184±30.4 pg/106 cells, respectively; 3h; P<0.05 compared to LPS/heme). Co-incubation of the LPS-primed cells with varying concentrations of heme, under the conditions employed, did not induce TNF-α production (data not shown), consistent with the hypothesis that IL-1β processing in heme-induced LPS-primed hMAC was mediated by inflammasome formation. Interestingly, qPCR showed that incubation of hMACs (1x106 cells/mL) with heme (50 µM) for 24h stimulated an approximately 10-fold increase (P<0.01) in the expression of the gene encoding, S100A8, another DAMP known to act as a TLR-4 agonist and to contribute to ischemia/reperfusion injury. Priming of hMACs with 1 µg/ml recombinant S100A8 for 3h and subsequent activation with heme (50 or 100 µM, 14h) significantly augmented the release of IL-1β (42.1±0.4 and 89.4±32.4 pg/106 cells for 50 and 100 µM heme, respectively; P<0.05), compared with S100A8 alone (20.6±3.5 pg/106 cells), without any modulation in TNF-α secretion (P>0.05). Using a model of acute intravascular hemolysis, we confirmed an association between heme release and S100A8 secretion, in vivo. Plasma heme levels increased significantly from 26.3±5 µM (i.v. saline control; N=4) to 87±18 µM in C57BL/6 mice at 1h after receiving i.v. water (150 µl; N=4, P=0.04). A concomitant increase in plasma S100A8 levels was also observed within 1h of the hemolytic stimulus (986±102 pg/mL, compared to 694.2±102 pg/ml in control mice; N=4, P=0.05), which was maintained for 3h (P<0.05). Thus, we present data to demonstrate that heme can induce IL-1β processing in LPS-primed human macrophages under in vitro conditions, probably via formation of the NLRP3/caspase-1 inflammasome machinery. In the absence of LPS, heme-stimulated hMACs can express the S100A8 DAMP; furthermore, a hemolytic stimulus induced mouse S100A8 production in vivo. As such, S100A8 may amplify heme-dependent inflammasome formation in an autocrine fashion, even under sterile conditions. Data provide new insights into the mechanisms by which heme may induce and potentiate inflammatory responses in hemolytic diseases, such as sickle cell disease, and suggest S100A8, together with heme, as potential therapeutic targets for reducing inflammation in these diseases. Disclosures Ferreira: Bayer AG: Research Funding. Almeida:Jassen & Cilag: Other: Currently employed with. Conran:Bayer AG: Research Funding.

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