Michele Maria Luchetti

Oxidative stress in scleroderma: Maintenance of scleroderma fibroblast phenotype by the constitutive up‐regulation of reactive oxygen species generation through the NADPH oxidase complex pathway

OBJECTIVE To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). METHODS Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. RESULTS Unstimulated skin fibroblasts from SSc patients released more O2- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of alpha1(I) and alpha2(I) collagen messenger RNA. CONCLUSION The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2-, H2O2, IL-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

5-Methylcytosine content of DNA in blood, synovial mononuclear cells and synovial tissue from patients affected by autoimmune rheumatic diseases

The percentage of 5-methylcytosine (m5Cyt) has been determined in peripheral blood, synovial mononuclear cells and synovial tissue from patients affected by various rheumatic autoimmune diseases. The determination was performed by reversed-phase high-performance liquid chromatography. Fifteen controls were compared to twenty-one patients affected by rheumatoid arthritis and to nine patients affected by systemic lupus erythematosus. The mean percentage of m5Cyt in normal individuals was significantly higher than in the rheumatoid arthritis and systemic lupus erythematosus patients. In addition, patients with active disease showed lower values than patients in remission. This finding is in agreement with the hypothesis that DNA hypomethylation may play a role in the pathogenesis of the autoimmune diseases, resulting in altered oncogene expression. Therapy with cyclosporin A led to a decrease in the percentage of m5Cyt in three rheumatoid arthritis patients, but a rebound was observed when the cyclosporin A was suspended. The percentage of m5Cyt in the DNA of synovial tissue from four rheumatoid arthritis patients and five patients with osteoarthritis was similar; this observation confirms that, in addition to disease-specific and disease activity-specific variations, the percentage of m5Cyt may also show tissue-specific variations.

Microcirculatory effects of the transfusion of leukodepleted or non-leukodepleted red blood cells in patients with sepsis: a pilot study

IntroductionMicrovascular alterations impair tissue oxygenation during sepsis. A red blood cell (RBC) transfusion increases oxygen (O2) delivery but rarely improves tissue O2 uptake in patients with sepsis. Possible causes include RBC alterations due to prolonged storage or residual leukocyte-derived inflammatory mediators. The aim of this study was to compare the effects of two types of transfused RBCs on microcirculation in patients with sepsis.MethodsIn a prospective randomized trial, 20 patients with sepsis were divided into two separate groups and received either non-leukodepleted (n = 10) or leukodepleted (n = 10) RBC transfusions. Microvascular density and perfusion were assessed with sidestream dark field (SDF) imaging sublingually, before and 1 hour after transfusions. Thenar tissue O2 saturation (StO2) and tissue hemoglobin index (THI) were determined with near-infrared spectroscopy, and a vascular occlusion test was performed. The microcirculatory perfused boundary region was assessed in SDF images as an index of glycocalyx damage, and glycocalyx compounds (syndecan-1, hyaluronan, and heparan sulfate) were measured in the serum.ResultsNo differences were observed in microvascular parameters at baseline and after transfusion between the groups, except for the proportion of perfused vessels (PPV) and blood flow velocity, which were higher after transfusion in the leukodepleted group. Microvascular flow index in small vessels (MFI) and blood flow velocity exhibited different responses to transfusion between the two groups (P = 0.03 and P = 0.04, respectively), with a positive effect of leukodepleted RBCs. When within-group changes were examined, microcirculatory improvement was observed only in patients who received leukodepleted RBC transfusion as suggested by the increase in De Backer score (P = 0.02), perfused vessel density (P = 0.04), PPV (P = 0.01), and MFI (P = 0.04). Blood flow velocity decreased in the non-leukodepleted group (P = 0.03). THI and StO2 upslope increased in both groups. StO2 and StO2 downslope increased in patients who received non-leukodepleted RBC transfusions. Syndecan-1 increased after the transfusion of non-leukodepleted RBCs (P = 0.03).ConclusionsThis study does not show a clear superiority of leukodepleted over non-leukodepleted RBC transfusions on microvascular perfusion in patients with sepsis, although it suggests a more favorable effect of leukodepleted RBCs on microcirculatory convective flow. Further studies are needed to confirm these findings.Trial registrationClinicalTrials.gov, NCT01584999

Plasma free hemoglobin and microcirculatory response to fresh or old blood transfusions in sepsis.

Background Free hemoglobin (fHb) may induce vasoconstriction by scavenging nitric oxide. It may increase in older blood units due to storage lesions. This study evaluated whether old red blood cell transfusion increases plasma fHb in sepsis and how the microvascular response may be affected. Methods This is a secondary analysis of a randomized study. Twenty adult septic patients received either fresh or old (<10 or >15 days storage, respectively) RBC transfusions. fHb was measured in RBC units and in the plasma before and 1 hour after transfusion. Simultaneously, the sublingual microcirculation was assessed with sidestream-dark field imaging. The perfused boundary region was calculated as an index of glycocalyx damage. Tissue oxygen saturation (StO2) and Hb index (THI) were measured with near-infrared spectroscopy and a vascular occlusion test was performed. Results Similar fHb levels were found in the supernatant of fresh and old RBC units. Despite this, plasma fHb increased in the old RBC group after transfusion (from 0.125 [0.098–0.219] mg/mL to 0.238 [0.163–0.369] mg/mL, p = 0.006). The sublingual microcirculation was unaltered in both groups, while THI increased. The change in plasma fHb was inversely correlated with the changes in total vessel density (r = -0.57 [95% confidence interval -0.82, -0.16], p = 0.008), De Backer score (r = -0.63 [95% confidence interval -0.84, -0.25], p = 0.003) and THI (r = -0.72 [95% confidence interval -0.88, -0.39], p = 0.0003). Conclusions Old RBC transfusion was associated with an increase in plasma fHb in septic patients. Increasing plasma fHb levels were associated with decreased microvascular density. Trial Registration ClinicalTrials.gov NCT01584999

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Background Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Methods Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Results Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Conclusions Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.

Induction of Scleroderma Fibrosis in Skin‐Humanized Mice by Administration of Anti−Platelet‐Derived Growth Factor Receptor Agonistic Autoantibodies

To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors.

Induction of scleroderma fibrosis in skin‐humanized mice by anti‐Platelet‐Derived Growth Factor receptor agonistic autoantibodies

To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from scleroderma patients or from healthy donors.

Diet-Induced Unresolved ER Stress Hinders KRAS-Driven Lung Tumorigenesis

Summary Dietary effects on tumor biology can be exploited to unravel cancer vulnerabilities. Here, we present surprising evidence for anti-proliferative action of high-calorie-diet (HCD) feeding on KRAS-driven lung tumors. Tumors of mice that commenced HCD feeding before tumor onset displayed defective unfolded protein response (UPR) and unresolved endoplasmic reticulum (ER) stress. Unresolved ER stress and reduced proliferation are reversed by chemical chaperone treatment. Whole-genome transcriptional analyses revealed FKBP10 as one of the most downregulated chaperones in tumors of the HCD-pre-tumor-onset group. FKBP10 downregulation dampens tumor growth in vitro and in vivo. Providing translational value to these results, we report that FKBP10 is expressed in human KRAS-positive and -negative lung cancers, but not in healthy parenchyma. Collectively, our data shed light on an unexpected anti-tumor action of HCD imposed before tumor onset and identify FKBP10 as a putative therapeutic target to selectively hinder lung cancer.

Biologics in Inflammatory and Immunomediated Arthritis

BACKGROUND Biologic drugs, introduced in clinical practice almost twenty years ago, represent nowadays a prominent treatment option in patients with chronic inflammatory arthritis, such as Rheumatoid Arthritis, Psoriatic Arthritis and Spondyloarthritis, that include ankylosing spondylitis and non-radiographic axial spondyloarthritis. METHODS Several compounds targeting different pathways have been marketed and approved for the treatment of inflammatory arthritis, with a significant impact on the clinical outcomes and the natural history of the diseases. RESULTS There are currently seven classes of biologics that are available for the treatment of inflammatory arthritis, each inhibiting a different aspect of the immune-driven inflammatory pathway. They include: • Tumor Necrosis Factor (TNF) inhibitors (infliximab, adalimumab, etanercept, golimumab and certolizumab pegol); • Interleukin-1 (IL-1) receptor antagonists (anakinra); • Interleukin-6 (IL-6) inhibition (tocilizumab); • Interleukin-12/23 (IL23) inhibition (ustekinumab); • Interleukin-17 (IL-17) inhibition (secukinumab); • B-cell inhibition (anti-CD20, rituximab); • T-cell costimulation inhibition (anti-CTLA-4, abatacept). CONCLUSION In this review, we will focus on the role of biologic drugs in the treatment strategies for inflammatory arthritis.

OP0205 Gut Dysbiosis in Patients with HLA-B27+ Ankylosing Spondylitis is Associated with Ileitis, Down-Regulation of Tight Junction Proteins, Increased Serum Levels of LPS and Monocytes Anergy

Background Intestinal dysbiosis has been recently demonstrated in the inflamed ileum of AS patients. Objectives To study the ileal localization of bacteria in AS patients and their relationship with local and systemic immune responses. Methods Consecutive gut biopsies obtained from 30 HLA-B27+ AS patients and 20 normal subjects were histologically classified in normal histology, acute inflammation and chronic inflammation. Giemsa and Silver stains were used to visualize bacteria and characterize their morphology. Intestinal bacteria were scored on the basis of the numbers of bacteria and their aggregation in clusters. The ileal expression and tissue distribution of claudin-2 and 4, Zonulin 1 and occludin were investigated by rt-PCR and immunohistochemistry. Serum levels of LPS and intestinal fatty acid binding protein (i-FABP) were also assayed by ELISA. The expression of HLA-DR, CD71 and CD14 on circulating monocytes and the monocyte immunologic behavior were studied by flow-cytometry. Results Cocci-like bacteria, but not segmented filamentous bacteria, were observed in the inflamed ileum of AS sometimes aggregated in clusters. Bacterial scores were significantly correlated with the percentages of infiltrating inflammatory cells (r2=0.56, p<0.0001). The presence of invadent bacteria in AS was invariably associated with histologic changes characterized by i) the detachment of epithelial cell layer from the basement membrane; ii) an edematous lamina propria with extravasated red blood cells and iii) the down-regulation of occludin, claudin 2 and 4 and Zonulin 1 (∼10, ∼12, ∼8 and ∼ 6 fold decrease, respectively; p<0.05) assessed by rt-PCR and confirmed by IHC. Higher serum levels of LPS and i-FABP significantly associated with the higher score of bacteria invasion and tissue inflammation were observed in AS patients. Circulating monocytes in AS displayed a reduced expression of CD14 (p>0.0001), HLA-DR (p<0.05) and CD71 (p<0.001). Ex vivo stimulation of monocytes with LPS increased the expression of IL-23 in CD14+ cells in both AS patients and controls that was, however significantly higher in AS patients. Production of IL-23 by CD14- monocytes in AS patients was however undetectable and not modified by LPS alone. Stimulation of monocytes with LPS+sCD14 increased IL-23 production in CD14+ cells only in controls, strongly inducing the production of IL-23+ in CD14- monocytes in AS. Neither LPS or LPS+CD14 modify the expression of UPR genes both in patients and controls. Conversely, LPS and more intensely, LPS+sCD14 stimulation significantly abrogated the expression of the autophagy genes ATG16L1 (∼5 fold decrease, p<0.05); IRGM (∼ 3 fold decrease, p<0.05) and MAP1LC3A (∼7 fold decrease, p<0.05) only in AS monocytes. Conclusions Bacterial ileitis, accompanied by damaged intestinal mucosal barrier, characterizes AS patients. i-FABP and LPS are increased in the peripheral blood of AS patients and associated with the down-regulation of LPS co-receptor CD14 and with an anergic monocyte phenotype. Ex vivo stimulation with sCD14 may confer LPS-responsiveness to the cells not expressing CD14. Dysregulated autophagy response is present in the peripheral blood of AS patients. Disclosure of Interest None declared