Anti-Inflammatory Potential of the Oleoresin from the Amazonian Tree Copaifera reticulata with an Unusual Chemical Composition in Rats

Copaifera reticulata Ducke is a popularly known species known as copaíba that is widely spread throughout the Amazon region. The tree yields an oleoresin which is extensively used in local traditional medicine mainly as an anti-inflammatory and antinociceptive agent. The aim of the present study was to assess the anti-inflammatory potential of this oleoresin obtained from a national forest in the central Amazon which presented an unusual chemical composition. The chemical composition of volatile compounds of oleoresin was analyzed by gas chromatography-mass spectrometry. The acute toxicity assay was performed with a single dose of 2000 mg/kg. The anti-inflammatory potential was evaluated by carrageenan-induced paw edema and air pouch assays using four different C. reticulata oleoresin concentrations (10, 100, and 400 mg/kg). The exudate was evaluated for nitrite concentration through the colorimetric method and for TNF-α, IL-1β, and PGE2 by ELISA. C. reticulata oleoresin collected in the Amazonian summer contained six major sesquiterpene compounds (β-bisabolene, cis-eudesma-6,11-diene, trans-α-bergamotene, β-selinene, α-selinene, and β-elemene) and was nontoxic at a dose of 2000 mg/kg, showing low acute toxicity. Different from oleoresin obtained from other sites of the Brazilian Amazon, the major volatile compound found was β-Bisabolene with 25.15%. This β-Bisabolene-rich oleoresin reduced the formation of paw edema induced by carrageenan and reduced the global number of cells in the air pouch assay, as well as exudate volume and nitrite, TNF-α, IL-1β, and prostaglandin E2 levels (p < 0.05). C. reticulata oleoresin with a high β-Bisabolene concentration showed anti-inflammatory activity, reducing vascular permeability and consequently edema formation, and thus reducing cell migration and the production of inflammatory cytokine, confirming its traditional use by local Amazonian communities.

Exosomes From Human Urine-Derived Stem Cells Encapsulated Into PLGA Nanoparticles for Therapy in Mice With Particulate Polyethylene-Induced Osteolysis

Background: Periprosthetic osteolysis is the primary reason for arthroplasty failure after total joint replacement because of the generation of wear particles and subsequent bone erosion around the prosthesis, which leads to aseptic loosening. Periprosthetic osteolysis is often treated with revision surgery because of the lack of effective therapeutic agents. As key messengers of intercellular interactions, exosomes can be independently used as therapeutic agents to promote tissue repair and regeneration. In this study, we fabricated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) that carry exosomes derived from human urine stem cells (USC-Exos) and explored their effects on polyethylene-induced osteolysis.Methods: USCs were identified by multipotent differentiation and flow cytometry analyses. USC-Exos were isolated and identified by transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting. PLGA microspheres containing USC-Exos were fabricated to synthesize NPs using the mechanical double-emulsion method. The obtained NPs were characterized in terms of stability, toxicity, exosome release, and cell uptake. Then, these NPs were implanted into the murine air pouch model, and their effects on polyethylene-induced osteolysis were evaluated by microcomputed tomography (micro-CT) and histological analyses.Results: The average NP diameter was ~282 ± 0.4 nm, and the zeta potential was −2.02 ± 0.03 mV. After long-term storage at room temperature and 4°C, the NP solution was stable without significant coaggregation. In vitro release profiles indicated sustained release of exosomes for 12 days. In vivo, injection of NPs into the murine air pouch caused less osteolysis than that of USC-Exos, and NPs significantly reduced bone absorption, as indicated by histology and micro-CT scanning.Conclusion: Our findings suggest that USC-Exo-based PLGA NPs can prevent particulate polyethylene-induced osteolysis and bone loss.

IL-33/ST2 Axis Plays a Protective Effect in Streptococcus pyogenes Infection through Strengthening of the Innate Immunity

Group A Streptococcus (GAS) causes invasive human diseases with the cytokine storm. Interleukin-33 (IL-33)/suppression of tumorigenicity 2 (ST2) axis is known to drive TH2 response, while its effect on GAS infection is unclear. We used an air pouch model to examine the effect of the IL-33/ST2 axis on GAS-induced necrotizing fasciitis. GAS infection induced IL-33 expression in wild-type (WT) C57BL/6 mice, whereas the IL-33- and ST2-knockout mice had higher mortality rates, more severe skin lesions and higher bacterial loads in the air pouches than those of WT mice after infection. Surveys of infiltrating cells in the air pouch of GAS-infected mice at the early stage found that the number and cell viability of infiltrating cells in both gene knockout mice were lower than those of WT mice. The predominant effector cells in GAS-infected air pouches were neutrophils. Absence of the IL-33/ST2 axis enhanced the expression of inflammatory cytokines, but not TH1 or TH2 cytokines, in the air pouch after infection. Using in vitro assays, we found that the IL-33/ST2 axis not only enhanced neutrophil migration but also strengthened the bactericidal activity of both sera and neutrophils. These results suggest that the IL-33/ST2 axis provided the protective effect on GAS infection through enhancing the innate immunity.

Insulin Derived Fibrils Induce Cytotoxicity in vitro and Trigger Inflammation in Murine Models

Background: Effective exogenous insulin delivery is the cornerstone of insulin dependent diabetes mellitus management. Recent literature indicates that commercial insulin-induced tissue reaction and cellular cytotoxicity may contribute to variability in blood glucose as well as permanent loss of injection or infusion site architecture and function. It is well accepted that insulin formulations are susceptible to mechanical and chemical stresses that lead to insulin fibril formation. This study aims to characterize in vitro and in vivo toxicity, as well as pro-inflammatory activity of insulin fibrils. Method: In vitro cell culture evaluated cytotoxicity and fibril uptake by macrophages and our modified murine air-pouch model quantified inflammatory activity. The latter employed FLOW cytometry and histopathology to characterize fibril-induced inflammation in vivo, which included fibril uptake by inflammatory phagocytes. Results: These studies demonstrated that insulin derived fibrils are cytotoxic to cells in vitro. Furthermore, inflammation is induced in the murine air-pouch model in vivo and in response, macrophages uptake fibrils both in vitro and in vivo. Conclusions: Administration of insulin fibrils can lead to cytotoxicity in macrophages. In vivo data demonstrate insulin fibrils to be pro-inflammatory which over time can lead to cumulative cell/tissue toxicity, inflammation, and destructive wound healing. Long term, these tissue reactions could contribute to loss of insulin injection site architecture and function.

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

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

Anti-inflammatory effects of DhHP-6 in LPS-induced RAW264.7 macrophages and carrageenan-induced air pouch model in rat

DhHP-6 (Deuterohemin-Ala-His-Thr-Val-Glu-Lys) is a novel peptide mimic of peroxidases that was previously designed in our laboratory. Here, we explored the anti-inflammatory potential of DhHP-6 against lipopolysaccharide(LPS)stimulated inflammatory response in RAW264.7 cells and carrageenan-induced air pouch model rats. DhHP-6 treatment dramatically attenuated the production of nitric oxide (NO), IL-6, andTNF-α in LPS induced RAW264.7 cells. Also, it blocked phosphorylation and degradation of IκBα and suppressed the nuclear translocation of p65. DhHP-6 (0.2, 0.6, and 2.0 mg/kg) significantly reduced the levels of total proteins and WBC counts in the exudates of the air pouch model rats. Moreover, MDA contents in the plasma of rats were reduced and SOD activities were enhanced in the DhHP-6-treatment group. Our results strongly show the effectiveness of DhHP-6 as an anti-inflammatory agent. The mechanism could be related to the reduction of Reactive oxygen species(ROS), inhibition of NF-κB nuclear translocation, and reduction of pro-inflammatory cytokines.

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

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.