BackgroundGout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. The aim of this study was to evaluate the oxidative stress in human fibroblast-like synoviocytes (FLS) exposed to monosodium urate (MSU) crystals, which trigger an inflammatory process.MethodsHuman FLS isolated from synovial tissue explants were stimulated with MSU crystals (75 μg/mL) for 24 h. Cellular viability was evaluated by crystal violet staining, apoptosis was assessed using Annexin V, and the cellular content of reactive oxygen species (ROS) and nitrogen species (RNS) (O2-, H2O2, NO) was assessed with image-based cytometry and fluorometric methods. In order to determine protein oxidation levels, protein carbonyls were detected through oxyblot analysis, and cell ultrastructural changes were assessed by transmission electron microscopy.ResultsThe viability of FLS exposed to MSU crystals decreased by 30 % (P < 0.05), while apoptosis increased by 42 % (P = 0.01). FLS stimulated with MSU crystals exhibited a 2.1-fold increase in H2O2 content and a 1.5-fold increase in O2- and NO levels. Oxyblots revealed that the spots obtained from FLS protein lysates exposed to MSU crystals exhibited protein carbonyl immunoreactivity, which reflects the presence of oxidatively modified proteins. Concomitantly, MSU crystals triggered the induction of changes in the morphostructure of FLS, such as the thickening and discontinuity of the endoplasmic reticulum, and the formation of vacuoles and misfolded glycoproteins.ConclusionsOur results prove that MSU crystals induce the release of ROS and RNS in FLS, subsequently oxidizing proteins and altering the cellular oxidative state of the endoplasmic reticulum, which results in FLS apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1012-3) contains supplementary material, which is available to authorized users.

Synovial cells play a crucial part in gouty arthritis, with different features for the inflammation within the joint. However, there is no information about how the synoviocytes can mediate the activation of inflammation. We hypothesized that the process of monosodium urate (MSU) crystal uptake alters the inflammatory response of synoviocytes through regulation of unknown mechanisms. Synoviocytes were stimulated with MSU crystals, and the phagocytosis index (PhIx) was evaluated by counting of cells with MSU ingested using polarized light microscopy. Additionally, transmission electron microscopy and flow cytometry were performed. Secretion of cytokines was measured by a panel of immunoassays. Changes in gene expression of hypoxia-inducible factor-1 ( HIF1A), von Hippel-Lindau ( VHL), and vascular endothelial growth factor ( VEGF) were evaluated by quantitative real-time PCR (qRT-PCR). Protein levels were detected by ELISA. MSU crystals induced a time-dependent increase in PhIx and the formation of numerous secretory vesicles and cavities located in the cytoplasm. Culture supernatants of MSU-treated cells had high levels of the cytokines IL-1β, IL-6, IL-8, TNF-α, and MCP-1, and the growth factors NGF and HGF. The decrease in HIF1A gene expression was 0.58-fold, and overexpression of VHL and VEGF genes was 1.98- and 4-fold, respectively, in MSU-treated synoviocytes compared to untreated cells. Additionally, VEGF levels were increased. The identification of phagocytosis of MSU crystals triggering an inflammatory cellular state in synoviocytes suggests a possible mechanism of synovial activation in the pathogenesis of crystal-induced arthritis. Impact statement Gout is distinguished by an inflammatory process that is mediated by phagocytosis of monosodium urate (MSU) crystals in synoviocytes by regulation of unknown mechanisms. Here we suggest that the synovial cells play a crucial role in gouty arthritis by activating inflammation by MSU uptake and increasing the secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, TNF-α, MCP-1, and the growth factors NGF and HGF. We discuss some co-existing features in synoviocytes, including anomalous morphologies of the cells, and microvesicle formation, dysregulation in VEGF gene expression. We provide evidence that phagocytosis of MSU crystals triggers an inflammatory cellular state in synoviocytes in the pathogenesis of crystal-induced arthritis.

Influenza A virus (IAV) increases the presentation of class I human leukocyte antigen (HLA) proteins that limit antiviral responses mediated by natural killer (NK) cells, but molecular mechanisms for these processes have not yet been fully elucidated. We observed that infection with A/Fort Monmouth/1/1947(H1N1) IAV significantly increased the presentation of HLA-B, -C, and -E on lung epithelial cells. Virus entry was not sufficient to induce HLA upregulation because UV-inactivated virus had no effect. Aberrant internally deleted viral RNAs (vRNAs) known as mini viral RNAs (mvRNAs) and defective interfering RNAs (DI RNAs) expressed from an IAV minireplicon were sufficient for inducing HLA upregulation. These defective RNAs bind to retinoic acid-inducible gene I (RIG-I) and initiate mitochondrial antiviral signaling (MAVS) protein-dependent antiviral interferon (IFN) responses. Indeed, MAVS was required for HLA upregulation in response to IAV infection or ectopic mvRNA/DI RNA expression. The effect was partially due to paracrine signaling, as we observed that IAV infection or mvRNA/DI RNA-expression stimulated production of IFN-β and IFN-λ1 and conditioned media from these cells elicited a modest increase in HLA surface levels in naive epithelial cells. HLA upregulation in response to aberrant viral RNAs could be prevented by the Janus kinase (JAK) inhibitor ruxolitinib. While HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein; we determined that NS1 limits cell-intrinsic and paracrine mechanisms of HLA upregulation. Taken together, our findings indicate that aberrant IAV RNAs stimulate HLA presentation, which may aid viral evasion of innate immunity. IMPORTANCE Human leukocyte antigens (HLAs) are cell surface proteins that regulate innate and adaptive immune responses to viral infection by engaging with receptors on immune cells. Many viruses have evolved ways to evade host immune responses by modulating HLA expression and/or processing. Here, we provide evidence that aberrant RNA products of influenza virus genome replication can trigger retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS)-dependent remodeling of the cell surface, increasing surface presentation of HLA proteins known to inhibit the activation of an immune cell known as a natural killer (NK) cell. While this HLA upregulation would seem to be advantageous to the virus, it is kept in check by the viral nonstructural 1 (NS1) protein, which limits RIG-I activation and interferon production by the infected cell.

BackgroundCurrently, two pathogenic pathways describe the role of obesity in osteoarthritis (OA); one through biomechanical stress, and the other by the contribution of systemic inflammation. The aim of this study was to evaluate the effect of free fatty acids (FFA) in human chondrocytes (HC) expression of proinflammatory factors and reactive oxygen species (ROS).MethodsHC were exposed to two different concentrations of FFA in order to evaluate the secretion of adipokines through cytokines immunoassays panel, quantify the protein secretion of FFA-treated chondrocytes, and fluorescent cytometry assays were performed to evaluate the reactive oxygen species (ROS) production.ResultsHC injury was observed at 48 h of treatment with FFA. In the FFA-treated HC the production of reactive oxygen species such as superoxide radical, hydrogen peroxide, and the reactive nitrogen species increased significantly in a at the two-dose tested (250 and 500 μM). In addition, we found an increase in the cytokine secretion of IL-6 and chemokine IL-8 in FFA-treated HC in comparison to the untreated HC.ConclusionIn our in vitro model of HC, a hyperlipidemia microenvironment induces an oxidative stress state that enhances the inflammatory process mediated by adipokines secretion in HC.

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We hypothesized that the secretion of inflammatory mediators from synoviocytes affects the chondrocyte homeostasis of articular cartilage. This study was a preliminary attempt to elucidate the molecular mechanisms by which soluble mediators obtained from activated synoviocytes induce oxidative stress and inflammation in chondrocytes. We measured the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (NGF), superoxide anion (O 2•− ), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO • ) from articular human cells. First, we created a conditional basal medium by exposing synoviocytes (HS) to monosodium urate crystals (CBM). The chondrocytes were exposed to either CBM (CCM), urate crystals directly (CMSU), or remained untreated (CC) as a negative control. Data were analyzed by ANOVA tests; Bonferroni test was performed for multiple comparisons between groups. Interestingly, we observed that mediators of inflammation and oxidative stress were significantly higher in CCM than CMSU and CC groups (P<0.01). The specific concentrations were as follows: 19.85 ng/mL of IL-6, 9.79 ng/mL of IL-8, 5.17 ng/mL of NGF, and 11.91 ng/mL of MCP-1. Of note, we observed the same trend for reactive oxygen and nitrogen species (P<0.001). Soluble mediators secreted by synoviocytes after being activated with MSU crystals (as observed in individuals who present gout attacks) trigger chondrocyte activation intensifying the articular inflammatory, oxidative, and pain states that damage cartilage in OA; this damage is more severe even when compared to HC directly exposed to monosodium urate crystals.

Alveolar macrophages (AM) hold lung homeostasis intact. In addition to the defense against inhaled pathogens and deleterious inflammation, AM also maintain pulmonary surfactant homeostasis, a vital lung function that prevents pulmonary alveolar proteinosis. Signals transmitted between AM and pneumocytes of the pulmonary niche coordinate these specialized functions. However, the mechanisms that guide the metabolic homeostasis of AM remain largely elusive. We show that the NK cell-associated receptor, NKR-P1B, is expressed by AM and is essential for metabolic programming. Nkrp1b−/− mice are vulnerable to pneumococcal infection due to an age-dependent collapse in the number of AM and the formation of lipid-laden AM. The AM of Nkrp1b−/− mice show increased uptake but defective metabolism of surfactant lipids. We identify a physical relay between AM and alveolar type-II pneumocytes that is dependent on pneumocyte Clr-g expression. These findings implicate the NKR-P1B:Clr-g signaling axis in AM-pneumocyte communication as being important for maintaining metabolism in AM.

Cholesterol overloads not only trigger hepatic damage, but also affect the gallbladder significantly.