Blaž Rozman

Mechanism of Endosomal TLR Inhibition by Antimalarial Drugs and Imidazoquinolines

Endosomal TLRs play an important role in innate immune response as well as in autoimmune processes. In the therapy of systemic lupus erythematosus, antimalarial drugs chloroquine, hydroxychloroquine, and quinacrine have been used for a long time. Their suppression of endosomal TLR activation has been attributed to the inhibition of endosomal acidification, which is a prerequisite for the activation of these receptors. We discovered that chloroquine inhibits only activation of endosomal TLRs by nucleic acids, whereas it augments activation of TLR8 by a small synthetic compound, R848. We detected direct binding of antimalarials to nucleic acids by spectroscopic experiments and determined their cellular colocalization. Further analysis revealed that other nucleic acid-binding compounds, such as propidium iodide, also inhibited activation of endosomal TLRs and colocalized with nucleic acids to endosomes. We found that imidazoquinolines, which are TLR7/8 agonists, inhibit TLR9 and TLR3 even in the absence of TLR7 or TLR8, and their mechanism of inhibition is similar to the antimalarials. In contrast to bafilomycin, none of the tested antimalarials and imidazoquinolines inhibited endosomal proteolysis or increased the endosomal pH, confirming that inhibition of pH acidification is not the underlying cause of inhibition. We conclude that the direct binding of inhibitors to nucleic acids mask their TLR-binding epitope and may explain the efficiency of those compounds in the treatment of autoimmune diseases.

Autoantibodies in Nonautoimmune Individuals during Infections

Abstract:u2002 Infections can act as environmental triggers inducing or promoting autoimmune disease in genetically predisposed individuals. Identification of microbial peptides similar to self‐tissues may by molecular mimicry, provide the inducing mechanism for an immune response. The aim of this study was to identify autoantibodies (autoAbs) in nonautoimmune individuals during acute bacterial, viral, or parasitic infections. Specific Abs or specific infections with an increased autoAb load may shed insight into the mechanisms of autoimmune disease. Sera from 88 patients with acute infections (41 bacterial, 23 viral, 17 parasitic, and 7 rickettsial) were tested by the ELISA method for antinuclear antibodies (ANA) 8 Pro, and Abs to thyroid peroxidase (TPO), thyroglobulin, phospholipids, annexin‐V, laminin, anti–Saccharomyces cervisiae (ASCA), and prothrombin, along with 80 normal controls. Elevated titers of Abs to annexin‐V and prothrombin were the most prevalent in viral, parasitic, and rickettsial infections and to laminin in viral and parasitic infections. Elevated titers of ASCA and ANA were found in viral and bacterial infections. Antiphospholipid Abs were found in parasitic and Q‐fever infections. Thirty‐four individuals harbored elevated titers of at least two Abs. An autoAb burden was detected in individuals with hepatitis A, hepatitis B, toxoplasma or Q‐fever infections. In nonautoimmune individuals with various (bacterial, viral, parasitic, and rickettsial) infections, elevated titers of Abs to annexin‐V, prothrombin, laminin, ASCA, ANA, and phospholipids were most frequently detected.

Nanoparticles isolated from blood: a reflection of vesiculability of blood cells during the isolation process

Background Shedding of nanoparticles from the cell membrane is a common process in all cells. These nanoparticles are present in body fluids and can be harvested by isolation. To collect circulating nanoparticles from blood, a standard procedure consisting of repeated centrifugation and washing is applied to the blood samples. Nanoparticles can also be shed from blood cells during the isolation process, so it is unclear whether nanoparticles found in the isolated material are present in blood at sampling or if are they created from the blood cells during the isolation process. We addressed this question by determination of the morphology and identity of nanoparticles harvested from blood. Methods The isolates were visualized by scanning electron microscopy, analyzed by flow cytometry, and nanoparticle shapes were determined theoretically. Results The average size of nanoparticles was about 300 nm, and numerous residual blood cells were found in the isolates. The shapes of nanoparticles corresponded to the theoretical shapes obtained by minimization of the membrane free energy, indicating that these nanoparticles can be identified as vesicles. The concentration and size of nanoparticles in blood isolates was sensitive to the temperature during isolation. We demonstrated that at lower temperatures, the nanoparticle concentration was higher, while the nanoparticles were on average smaller. Conclusion These results indicate that a large pool of nanoparticles is produced after blood sampling. The shapes of deformed blood cells found in the isolates indicate how fragmentation of blood cells may take place. The results show that the contents of isolates reflect the properties of blood cells and their interaction with the surrounding solution (rather than representing only nanoparticles present in blood at sampling) which differ in different diseases and may therefore present a relevant clinical parameter.

Toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles.

Extracellular vesicles from patients with rheumatoid arthritis stimulate inflammation in a TLR4-dependent manner. Stressing out TLR4 In addition to bacterial products like lipopolysaccharide (LPS), endogenous molecules released in response to sterile inflammation activate Toll-like receptor 4 (TLR4). Sterile inflammation contributes to chronic diseases, such as rheumatoid arthritis (RA). Indeed, mice with defective TLR4 signaling are protected from RA. Manček-Keber et al. found that extracellular vesicles (membrane-encapsulated particles) from the serum of RA patients, but not healthy controls, contained oxidized phospholipids and stimulated the TLR4-dependent expression of genes encoding inflammatory cytokines in cells. Cells expressing TLR4 mutants that cannot be activated by LPS also failed to respond to vesicles from RA patients, suggesting that these vesicles signal conditions of oxidative stress to stimulate inflammation. Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids that stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress–induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.

Prevention of microvesiculation by adhesion of buds to the mother cell membrane--a possible anticoagulant effect of healthy donor plasma.

Microvesicles (MVs) found in peripheral blood are derived from the budding of cell membranes and are associated with a higher risk of thrombosis. Recently, a hypothesis has been suggested that certain plasma proteins could suppress microvesiculation by mediating adhesion of the buds to the mother cell membrane. In a pilot study, we have tested this hypothesis by considering the relation between the amount of MVs in peripheral blood and the ability of plasma to induce adhesion between giant phospholipid vesicles (GPVs). MVs were isolated from human plasma and counted by flow cytometry. The adhesion between GPVs was measured by assessing the average angle of contact between the adhered vesicles. It was found that greater ability of plasma to induce adhesion relates to smaller concentration of MVs in plasma. The ratio between the concentration of MVs and the concentration of platelets proved the most efficient parameter to predict the propensity of the membrane to shed vesicles. Our results indicate that a stronger attractive interaction between GPVs mediated by plasma is associated with a smaller amount of MVs per platelets. Plasma that mediates stronger attractive interaction between GPVs might potentially be associated with a smaller risk of thrombosis.

Autoimmune and proinflammatory activity of oxidized immunoglobulins

AIMSnOxidation reactions can modify protein activity or specificity. Recently, a novel redox-reactive family of autoantibodies was described, which indicated involvement of altered antibodies (beside altered antigens) into autoimmune reactions. The aim of our study was to determine the binding capacity alterations of electro-oxidized blood donors IgGs, and to evaluate their effects on released proinflammatory interleukin 6 in HUVEC.nnnRESULTSnWe found out that 1.) Isolated blood donor IgGs bound after electro-oxidation to beta2-glycoprotein I, cardiolipin, citrullinated cyclic peptide and protein 3 by enzyme-linked immunosorbent assay, extractable nuclear antigens by counterimmuno-electrophoresis, and cell antigens by indirect immunofluorescence; 2.) Alterations in immunoreactivity of IgGs due to oxidation highly depend on electric current, time of exposure and the presence of antioxidants, 3.) Treatment of HUVEC with oxidized IgGs resulted in changed cell morphology, accompanied by an increase in released interleukin-6.nnnCONCLUSIONSnOur data suggest repeatable transformation of antibodies present in the blood of healthy persons and patients. Inter-individual differences in chemical stability of antibodies, patients antioxidant status, and the microenvironmental changes at the cellular level may influence the range of antibody alterations and their involvement in pathophysiological autoimmune processes.

Avidity of Anti‐β2‐Glycoprotein I and Thrombosis or Pregnancy Loss in Patients with Antiphospholipid Syndrome

Abstract: We aimed to evaluate avidity of anti‐β2‐glycoprotein I antibodies (anti‐β2‐GPIs) in patients with antiphospholipid syndrome (APS) at the time of acute thrombotic events or pregnancy loss as compared with clinical event‐free periods. To do so, 69 sera samples from 16 patients (6 with primary APS and 10 with APS secondary to systemic lupus erythematosus) were selected on the basis of anti‐β2‐GPI positivity. Avidity of IgG anti‐β2‐GPIs was determined by chaotropic enzyme‐linked immunosorbent assay (ELISA), using increased NaCl concentration during antibody binding. High, heterogeneous, and low‐avidity anti‐β2‐GPIs were measured in APS patients, with no clear pattern regarding the time of thrombotic events or pregnancy failure. In general, anti‐β2‐GPI avidity did not change substantially during disease course. We concluded that avidity of anti‐β2‐GPIs appears to be a rather stable parameter in an individual APS patient. Considering the previously shown association of high‐avidity anti‐β2‐GPIs with venous thrombosis, avidity of anti‐β2‐GPIs may be a better predictor of predisposition to thrombosis and unsuccessful pregnancy than levels of antiphospholipid antibodies, which may fluctuate over time owing to several factors.

Agglutination of like-charged red blood cells induced by binding of β2-glycoprotein I to outer cell surface

Plasma protein-mediated attractive interaction between membranes of red blood cells (RBCs) and phospholipid vesicles was studied. It is shown that beta(2)-glycoprotein I (beta(2)-GPI) may induce RBC discocyte-echinocyte-spherocyte shape transformation and subsequent agglutination of RBCs. Based on the observed beta(2)-GPI-induced RBC cell shape transformation it is proposed that the hydrophobic portion of beta(2)-GPI molecule protrudes into the outer lipid layer of the RBC membrane and increases the area of this layer. It is also suggested that the observed agglutination of RBCs is at least partially driven by an attractive force which is of electrostatic origin and depends on the specific molecular shape and internal charge distribution of membrane-bound beta(2)-GPI molecules. The suggested beta(2)-GPI-induced attractive electrostatic interaction between like-charged RBC membrane surfaces is qualitatively explained by using a simple mathematical model within the functional density theory of the electric double layer, where the electrostatic attraction between the positively charged part of the first domains of bound beta(2)-GPI molecules and negatively charged glycocalyx of the adjacent RBC membrane is taken into account.

Influence of ionic strength and beta2-glycoprotein I concentration on agglutination of like-charged phospholipid membranes

The effect of ionic strength on adhesion between negatively charged giant unilamellar vesicles induced by beta2-glycoprotein I (β2-GPI) was studied experimentally and theoretically. Measuring the effective angle of contact between adhering vesicles indicated that the strength of adhesion between vesicles decreases with increasing ionic strength, and increases with concentration of β2-GPI. In the theoretical part we focused on the study of the average orientation of β2-GPI near the charged membrane and its role in mediating the attractive interactions between the vesicles. β2-GPI proteins were modelled as rods with internal distribution of electric charge. The predictions of Monte Carlo simulations show orthogonal orientation of some of the membrane attached β2-GPI in narrow gap between two vesicles. On the contrary, at larger distances between vesicles the proteins are parallelly attached to the membrane surface. A local minimum of the free energy corresponding to β2-GPI-mediated adhesion of two neighbouring vesicles was predicted. The strength of adhesion was confirmed to decrease at high ionic strength.

The Effect of in vitro Anticoagulant Disodium Citrate on Beta-2-glycoprotein I - Induced Coalescence of Giant Phospholipid Vesicles

In order to elucidate the mechanisms of blood coagulation the complex interactions between phospholipid membranes, serum protein beta-2-glycoprotein I (β2GPI), antiphospholipid antibodies (aPL) and disodium citrate were studied by observing collective interactions between giant phospholipid vesicles (GPVs) in the sugar solution. GPVs composed of palmitoyl-oleoyl-sn-glycero-3-phosphocholine (POPC), tetraoleoyl cardiolipin and cholesterol were obtained by the electroformation method and observed under the phase contrast microscope. β2GPI or aPL acted as mediators inducing the coalescence of the vesicles. The strength of the adhesion between the coalesced vesicles was dependent on the content of cardiolipin and the species of the mediator. The addition of disodium citrate to the coalesced GPVs solution caused disintegration of the complexes of coalesced vesicles. The extend of the disintegration between coalesced vesicles was interpreted to be connected to the strength of the adhesion between GPVs. It was found that the disintegration of the GPV complexes was more pronounced in the system where the vesicles coalesced due to the presence of antiphospholipid antibodies compared to the system where the vesicles coalesced due to the presence of β2GPI. The effect of the disintegration of the coalesced GPVs was more pronounced for smaller vesicles which originated in the budding of the the membrane of larger GPVs.