Silver nanoparticles (AgNPs) are commonly used in commercial and medical applications. However, AgNPs may induce toxicity, extracellular matrix (ECM) changes and inflammatory responses. Fibroblasts are key players in remodeling processes and major producers of the ECM. The aims of this study were to explore the effect of AgNPs on cell viability, both ex vivo in murine precision cut lung slices (PCLS) and in vitro in human lung fibroblasts (HFL-1), and immunomodulatory responses in fibroblasts. PCLS and HFL-1 were exposed to AgNPs with different sizes, 10 nm and 75 nm, at concentrations 2 µg/mL and 10 μg/mL. Changes in synthesis of ECM proteins, growth factors and cytokines were analyzed in HFL-1. Ag10 and Ag75 affected cell viability, with significantly reduced metabolic activities at 10 μg/mL in both PCLS and HFL-1 after 48 h. AgNPs significantly increased procollagen I synthesis and release of IL-8, prostaglandin E2, RANTES and eotaxin, whereas reduced IL-6 release was observed in HFL-1 after 72 h. Our data indicate toxic effects of AgNP exposure on cell viability ex vivo and in vitro with altered procollagen and proinflammatory cytokine secretion in fibroblasts over time. Hence, careful characterizations of AgNPs are of importance, and future studies should include timepoints beyond 24 h.
Platelets have recently emerged as important immune modulators in systemic lupus erythematosus (SLE), in addition to their role in thrombosis and cardiovascular disease. However, studies investigating mean platelet volume (MPV) in SLE are often scarce, conflicting and cross-sectional. In this study, MPV was measured in clinical routine throughout a defined time-period to quantify both individual MPV fluctuations and investigate if such variations are associated with disease activity and clinical phenotypes of SLE. Of our 212 patients, 34 patients had only one MPV value reported with the remaining 178 patients having between 2 and 19 visits with recorded MPV values. The intra-individual MPV variation was low, with a median variation of 0.7 fL. This was further supported by the finding that 84% of patients stayed within their reference interval category (i.e., small, normal or large) over time. In our cohort, no correlation between disease activity and MPV neither cross-sectionally nor longitudinally was found. Mean platelet volume values were significantly smaller in SLE patients (mean 10.5 fL) compared to controls (mean 10.8 fL), p < 0.0001. Based on the reference interval, 2.4% (n = 5) of patients had large-sized platelets, 84.4% (n = 179) had normal-sized and 13.2% (n = 28) had small-sized. A larger proportion (85.7%) of patients with small-sized platelets met the anti-dsDNA criterion (ACR10b; p = 0.003) compared to patients with normal and large (57.6%) sized platelets. In conclusion, the intra-individual MPV variation was of low magnitude and fluctuations in disease activity did not have any significant impact on MPV longitudinally. This lack of variability in MPV over time indicates that measuring MPV at any time-point is sufficient. Further studies are warranted to evaluate MPV as a possible biomarker in SLE, as well as to determine the underlying mechanisms influencing platelet size in SLE.
Background Neuropsychiatric (NP) involvement and fatigue are major problems in systemic lupus erythematosus (SLE). S100A8/A9 is a marker of inflammation and responds to therapy in SLE patients. S100A8/A9 has an immunopathogenic role in various neurological diseases. We investigated S100A8/A9 in relation to NP-involvement and fatigue in SLE. Methods 72 consecutive SLE outpatients at a tertiary centre and 26 healthy controls were included in this cross-sectional study. NPSLE was determined by specialists in rheumatology and neurology and defined according to three attribution models: “ACR”, “SLICC A” and “SLICC B”. Cerebral MRI was assessed by a neuroradiologist and neurocognitive testing by a neuropsychologist. The individuals were assessed by scores of pain (VAS), fatigue (VAS and FSS), and depression (MADRS-S). Concentrations of S100A8/A9 in serum and cerebrospinal fluid were measured with ELISA. Statistical calculations were performed using non-parametric methods. Results Serum concentrations of S100A8/A9 were higher in SLE patients compared with controls (medians 1230 ng/ml; 790 ng/ml, p = 0.023). The concentrations were higher in NPSLE patients compared with non-NPSLE patients when applying the SLICC A and ACR models, but not significant when applying the SLICC B model (medians 1400 ng/ml; 920 ng/ml, p = 0.011; 1560 ng/ml; 1090 ng/ml, p = 0.050; 1460 ng/ml; 1090 ng/ml, p = 0.083, respectively). No differences of CSF S100A8/A9 concentrations were observed between NPSLE and non-NPSLE patients. SLE patients with depression or cognitive dysfunction as an ACR NPSLE manifestation had higher serum S100A8/A9 concentrations than non-NPSLE patients (median 1460 ng/ml, p = 0.007 and 1380 ng/ml, p = 0.013, respectively). Higher serum S100A8/A9 correlated with higher VAS fatigue (r = 0.31; p = 0.008) and VAS pain (r = 0.27, p = 0.021) in SLE patients. Serum S100A8/A9 was not independently associated with NPSLE when adjusting for scores of fatigue (FSS) and pain (VAS) (OR 1.86, 95% CI 0.93–3.73, p = 0.08). Conclusions Serum S100A8/A9 concentrations may be associated with NPSLE and fatigue. S100A8/A9 may be of interest in evaluating NPSLE, although further investigations are needed.
Background SLE is a complex disease characterized by autoimmunity towards apoptotic cells, excessive amounts of circulating immune complexes and complement activation. Decreased platelet size has been observed in SLE and their non-hemostatic functions may play an active role in the disease. Our main objective in this study was to find clues that could explain their decreased size and functional role, analyzing the entire platelet proteome. Methods and patients Platelets were isolated from 23 patients with SLE. The five individuals with highest and lowest average platelet forward scatter were selected for further analysis. Platelet protein content was analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compared to platelets from five healthy controls (HC) Data are available via ProteomeXchange with identifier PXD031202. Results Out of 2572 proteins identified, 396 had significantly different levels (ANOVA q-value ≤ 0.01). Forty proteins, including immunoglobulin-, complement- and phosphatidylserine binding proteins had higher abundance in platelets from SLE patients, largely independent of size (fold difference of ≥ 1.5 and a t-test p-value of ≤ 0.05 as cut off). Functional characterization revealed increased degranulation and skewed hemostatic balance in platelets from SLE patients. In the SLE proteome, immunoglobulin proteins were negatively correlated to serum complement C3 and C4 and the highest relative levels was detected in platelets of normal size. Conclusion Platelets from SLE patients shared a specific protein profile, including immunoglobulins, complement proteins and autoantigens, largely independent of platelet size and in agreement with an integrated role for platelets in SLE.
spite of immune activation. Hierarchical clustering is presented in figure 1. Cluster analysis demonstrates that the group with the highest mean SLEDAI-2K (10.7) had lower Hb, transferrin and LCN2 in addition to elevated IL1b, IL6 and hepcidin compared with those with lower SLEDAI-2K. Elevated haptoglobin levels were seen in those in the higher disease activity groups, suggesting that haemolysis was an unlikely cause for anaemia.Conclusions The findings of this study suggest increased lupus disease activity results in abnormal iron homeostasis through impaired cellular iron import (via reduced transferrin), a lack of stored iron release (under the actions of elevated hepcidin) and reduced iron sequestration by LCN2, which may represent a novel cause of non-haemolytic anaemia.
BackgroundSystemic Lupus Erythematosus (SLE) is characterized by autoimmunity towards apoptotic/necrotic cells, complement activation and excessive amounts of circulating immune complexes. Platelets are recognized as immune cells that interacts with innate and adaptive immune functions. They are activated in SLE patients and contribute to an increased susceptibility to thrombosis [1]. Decreased platelet size has been observed in patients with SLE [2], but the mechanism(s) remains unclear. In this study, we have analyzed the complete proteome of platelets with normal and decreased size from SLE patients and from healthy controls (HC).ObjectivesOur aim was to find clues that could explain the morphological differences observed in platelets from SLE patients and to better characterize the role of platelets in SLE.MethodsWe included 23 consecutive patients with SLE, median SLEDAI-2K score was 2, and 10 HC. Blood count, serum complement levels and the presence of antiphospholipid or dsDNA antibodies were analyzed in all patients. Platelet size (forward scatter) and activation status (CD154, PAC1, CD32, PAR1, CD62P and Annexin V) was determined using flow cytometry. The proteome of 10 platelet isolates from SLE (five with smallest and the five with largest average size) and five HC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Data were analyzed using ANOVA, t-test, hierarchical cluster analysis, protein interactions using the STRING software and correlation analysis using spearman correlation.ResultsWe identified a total of 2572 proteins from the platelet isolates. Out of the identified proteins, 396 had significantly different levels, meeting an ANOVA q-value ≤ 0.01. Pairwise t-test analysis, using a fold difference (FD) of ≥ 1.5 and a p-value of ≤ 0.05 as cut off reveled significant differences in the distribution of proteins between groups. Platelets of both SLE groups (small and normal sized) shared higher levels of forty proteins and twenty proteins were reduced, compared to HC. Cytoskeletal functions were overrepresentation in the group of reduced proteins, while proteins with higher levels in platelets from SLE patients included proteins associated with complement and autoantibody targets such as Beta-2-glycoprotein 1, Annexin A5, and Prothrombin. Platelets from SLE patients also shared an abundance in immunoglobulin proteins, with even greater accumulation in the normal sized platelets. SLE platelet heavy constant alpha 1 (r -0.85, p=0.003), heavy constant mu (r -0.64, p=0.05) and heavy constant gamma 3 (r -0.80, p=0.008) was inversely correlated with complement C4 in serum and heavy constant gamma 2 (r -0.648, p=0.049) with complement C3.ConclusionThis study revealed an accumulation of complement proteins, immunoglobulins and known autoantigens in platelets from SLE patients compared to HC. The signature was largely independent of platelet size, but the enrichment of proteins involved in SLE pathogenesis indicates that the composition is influenced by SLE disease mechanisms. This was supported by the inverse correlation between platelet immunoglobulin and serum levels of complement protein C3 and C4. Platelets are known to interact with complement and express the low-affinity immunoglobulin gamma Fc region receptor IIA (CD32), suggesting a role in the clearance of immune complexes [3]. Future studies will have to determine if platelets play a role in the turnover of complement and immune complexes and the potential role of platelets as a source of autoantigens.References[1]Linge, P., et al., The non-haemostatic role of platelets in systemic lupus erythematosus. Nat Rev Rheumatol, 2018. 14(4): p. 195-213.[2]Lood, C., et al., Decreased platelet size is associated with platelet activation and anti-phospholipid syndrome in systemic lupus erythematosus. Rheumatology (Oxford), 2017. 56(3): p. 408-416.[3]Huang, Z.Y., et al., Human platelet FcgammaRIIA and phagocytes in immune-complex clearance. Mol Immunol, 2011. 48(4): p. 691-6.Disclosure of InterestsPetrus Linge: None declared, Andreas Jern: None declared, Helena Tydén: None declared, Birgitta Gullstrand: None declared, Hong Yan: None declared, Charlotte Welinder: None declared, Robin Kahn: None declared, Andreas Jonsen Consultant of: Astra Zeneca and glaxosmithkline, John Semple: None declared, Anders Bengtsson: None declared.
Background Neuropsychiatric (NP) involvement and fatigue are major problems in systemic lupus erythematosus (SLE). S100A8/A9 is a marker of inflammation and responds to therapy in SLE patients. S100A8/A9 has an immunopathogenic role in various neurological diseases. We investigated S100A8/A9 in relation to NP-involvement and fatigue in SLE. Methods 72 consecutive SLE outpatients at a tertiary centre and 26 healthy controls were included in this cross-sectional study. NPSLE was determined by specialists in rheumatology and neurology and defined according to three attribution models: “ACR”, “SLICC A” and “SLICC B”. Cerebral MRI was assessed by a neuroradiologist and neurocognitive testing by a neuropsychologist. The individuals were assessed by scores of pain (VAS), fatigue (VAS and FSS), depression (MADRS-S), and health-related life quality (EQ5D). Concentrations of S100A8/A9 in serum and cerebrospinal fluid were measured with ELISA. Statistical calculations were performed using non-parametric methods. Results Serum concentrations of S100A8/A9 were higher in NPSLE patients compared with non-NPSLE patients when applying the ACR and SLICC A models, but not significant when applying the SLICC B model (medians 1400 ng/ml; 920 ng/ml, p=0.011; 1560 ng/ml; 1090 ng/ml, p=0.050; 1460 ng/ml; 1090 ng/ml, p=0.083, respectively). The concentrations were higher in SLE patients compared with controls (medians 1230 ng/ml; 790 ng/ml, p=0.023). No differences of CSF S100A8/A9 concentrations were observed between NPSLE and non-NPSLE patients. SLE patients with depression or cognitive dysfunction as an ACR NPSLE manifestation had higher serum S100A8/A9 concentrations than non-NPSLE patients (median 1460 ng/ml, p=0.007 and 1380 ng/ml, p=0.013, respectively). Higher serum S100A8/A9 correlated with higher VAS fatigue (r=0.31; p=0.008), VAS pain (r=0.27, p=0.021), and lower EQ5D (r=-0.29, p=0.014) in SLE patients. NPSLE was associated with higher plasma CRP concentrations, higher scores of fatigue and pain, lower EQ5D, and fewer weekly work hours. Serum S100A8/A9 was not independently associated with NPSLE when adjusting for plasma CRP, and scores of fatigue (FSS) and pain (VAS), (OR 1.70, 95% CI 0.82-3.53, p=0.15). Conclusions Serum S100A8/A9 concentrations may be associated with NPSLE and fatigue. S100A8/A9 may be of interest in evaluating NPSLE, although further investigations are needed.
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