Diana Castaño

Multimolecular Signaling Complexes Enable Syk-Mediated Signaling of CD36 Internalization

CD36 is a versatile receptor known to play a central role in the development of atherosclerosis, the pathogenesis of malaria, and the removal of apoptotic cells. Remarkably, the short cytosolically exposed regions of CD36 lack identifiable motifs, which has hampered elucidation of its mode of signaling. Using a combination of phosphoprotein isolation, mass spectrometry, superresolution imaging, and gene silencing, we have determined that the receptor induces ligand internalization through a heteromeric complex consisting of CD36, β1 and/or β2 integrins, and the tetraspanins CD9 and/or CD81. This receptor complex serves to link CD36 to the adaptor FcRγ, which bears an immunoreceptor tyrosine activation motif. By coupling to FcRγ, CD36 is able to engage Src-family kinases and Syk, which in turn drives the internalization of CD36 and its bound ligands.

Microparticles That Form Immune Complexes as Modulatory Structures in Autoimmune Responses

Microparticles (MPs) are induced during apoptosis, cell activation, and even “spontaneous” release. Initially MPs were considered to be inert cellular products with no biological function. However, an extensive research and functional characterization have shown that the molecular composition and the effects of MPs depend upon the cellular background and the mechanism inducing them. They possess a wide spectrum of biological effects on intercellular communication by transferring different molecules able to modulate other cells. MPs interact with their target cells through different mechanisms: membrane fusion, macropinocytosis, and receptor-mediated endocytosis. However, when MPs remain in the extracellular milieu, they undergo modifications such as citrullination, glycosylation, and partial proteolysis, among others, becoming a source of neoantigens. In rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), reports indicated elevated levels of MPs with different composition, content, and effects compared with those isolated from healthy individuals. MPs can also form immune complexes amplifying the proinflammatory response and tissue damage. Their early detection and characterization could facilitate an appropriate diagnosis optimizing the pharmacological strategies, in different diseases including cancer, infection, and autoimmunity. This review focuses on the current knowledge about MPs and their involvement in the immunopathogenesis of SLE and RA.

Dyslipidemia-associated alterations in B cell subpopulation frequency and phenotype during experimental atherosclerosis

Lymphocytes, the cellular effectors of adaptive immunity, are involved in the chronic inflammatory process known as atherosclerosis. Proatherogenic and atheroprotective properties have been ascribed to B cells. However, information regarding the role of B cells during atherosclerosis is scarce. Both the frequency and the phenotype of B cell subpopulations were studied by flow cytometry in wild type and apolipoprotein-E-deficient (apoE(-/-)) mice fed a high-fat (HFD) or control diet. Whereas the proportion of follicular cells was decreased, transitional 1-like cells were increased in mice with advanced atherosclerotic lesions (apoE(-/-) HFD). B cells in atherosclerotic mice were more activated, indicated by their higher surface expression of CD80, CD86, CD40 and CD95 and increased serum IgG1 levels. In the aorta, a decreased frequency of B cells was observed in mice with advanced atherosclerosis. Low expression of CD19 was observed on B cells from the spleen, aorta and lymph nodes of apoE(-/-) HFD mice. This alteration correlated with serum levels of IgG1 and cholesterol. A reduction in CD19 expression was induced in splenic cells from young apoE(-/-) mice cultured with lipemic serum. These results show that mice with advanced atherosclerosis display a variety of alterations in the frequency and phenotype of B lymphocytes, most of which are associated with dyslipidemia.

Endothelial Alterations in Systemic Lupus Erythematosus and Rheumatoid Arthritis: Potential Effect of Monocyte Interaction

Patients with systemic autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are prone to develop atherosclerosis and cardiovascular diseases five times more often than the general population; this increase in frequency could be partially explained by an increase in the macrovasculature endothelial damage. In these autoimmune diseases, a microvascular endothelial injury has also been reported in different organs and tissues, especially in sites where ultrafiltration processes occur. Different components that are characteristic to the immunopathology of RA and SLE could be involved in the endothelial cell activation, permeability increase, functional alteration, and vascular injury. Circulating immune complexes (IC) detected in SLE and RA have been proposed to participate in the endothelial injury. In the vascular environment, IC can generate different responses that could be mediated by monocytes, because these cells have patrolling and monitoring functions on the endothelium. However, with certain stimuli such as TLR ligands, the monocytes are retained in the lumen, releasing proinflammatory mediators that participate in the endothelial damage. This paper aims to review some aspects about the endothelial activation and dysfunction in the context of SLE and RA, as well as the potential role that monocytes apparently play in this process.

Potential Involvement of Platelet-Derived Microparticles and Microparticles Forming Immune Complexes during Monocyte Activation in Patients with Systemic Lupus Erythematosus

Microparticles (MPs) are vesicles derived from the plasma membrane of different cells, are considered a source of circulating autoantigens, and can form immune complexes (MPs-ICs). The number of MPs and MPs-ICs increases in patients with systemic lupus erythematosus (SLE). MPs activate myeloid cells by inducing IL-6 and TNF-α in both SLE and other diseases. Therefore, we propose that the recognition of MPs-ICs by monocytes rather that MPs may define their phenotype and contribute to the inflammatory process in patients with SLE. Thus, the aims of this study were to evaluate the association among circulating MPs-ICs from different cell sources, alterations observed in monocyte subsets, and disease activity in patients with SLE and to establish whether monocytes bind and respond to MPs-ICs in vitro. Circulating MPs and monocyte subsets were characterized in 60 patients with SLE and 60 healthy controls (HCs) using multiparametric flow cytometry. Patients had higher MP counts and frequencies of MPs-CD41a + (platelet-derived) compared with HCs, regardless of disease activity. MPs from patients with SLE were C1q + and formed ICs with IgM and IgG. MPs-IgG + were positively correlated with active SLE (aSLE), whereas MPs-IgM + were negatively correlated. Most of the circulating total ICs-IgG + were located on MPs. The proportion and number of non-classical monocytes were significantly decreased in patients with SLE compared with HCs and in patients with aSLE compared with patients with the inactive disease. Non-classical monocytes obtained from patients with SLE exhibited increased levels of CD64 associated with MPs-IgG +, MPs-C1q +, total circulating ICs-IgG +, and disease activity. The direct effects of MPs and MPs-IgG + on monocytes were evaluated in cell culture. Monocytes from both HCs and patients bound to and internalized MPs and MPs-IgG + independent of CD64. These vesicles derived from platelets (PMPs), mainly PMPs-IgG +, activated monocytes in vitro and increased the expression of CD69, CD64, and pro-inflammatory cytokines such as IL-1β, TNF-α, and IFN-α. Therefore, MPs are one of the most representative sources of the total amount of circulating ICs-IgG + in patients with SLE. MPs-IgG + are associated with SLE activity, and PMPs-IgG + stimulate monocytes, changing their phenotype and promoting pro-inflammatory responses related to disease activity.

Data in support of dyslipidemia-associated alterations in B cell subpopulations frequency and phenotype during experimental atherosclerosis.

Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE−/−) mice fed or not with high-fat diet (HFD), by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis (DOI: 10.1016/j.atherosclerosis.2015.12.022, article reference: ATH14410) [1]. The data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia.

Different phenotypes of non-classical monocytes associated with systemic inflammation, endothelial alteration and hepatic compromise in patients with dengue

Although dengue can progress to severe stages, the exact causes of this phenomenon are unknown; however, the possibility of monocyte participation is acknowledged. It has been suggested that monocyte subsets (classical, intermediate and non‐classical) play differential roles in dengue immunopathology. Therefore, we determined the count of monocyte subsets and obtained the clinical information of patients with dengue. We noted a significant decrease in the count of non‐classical monocytes in patients compared with controls. With this finding, we focused on studying the phenotype of non‐classical monocytes in the present study. An increase in activation and differentiation markers, such as CD64, CD86, the percentage of tumor necrosis factor‐α+ cells and exposure of phosphatidylserine, were recorded in the non‐classical monocytes of patients compared with controls. Moreover, a significant decrease in the expression of CX3CR1 with a corresponding increase in the expressions of CCR2, CCR5, CD11b and CD54 was detected in the non‐classical monocytes of patients in comparison with that of the controls. Significant increases in the frequency of microparticles from endothelium and in the concentrations of interleukin‐6 (IL‐6), IL‐8 and IL‐10 were noted in the plasma of patients. These findings demonstrate that in patients with dengue, non‐classical monocytes are activated, exhibiting a phenotype associated with more differentiation, produces tumor necrosis factor‐α and has a profile of less endothelial surveillance closer to the cellular migration. These changes were associated with hepatic compromise, endothelial alteration and high concentration of circulating cytokines. Hence, alterations of non‐classical monocytes seem to be associated with the immunopathology of dengue infection.

Role of Monocytes in the Pathogenesis of Dengue

Diseases caused by dengue virus (DENV) are a major public health problem worldwide, considered one of the infections with more prevalence in tropical and subtropical zones of the world. Despite the intense research in the pathogenesis of DENV, this feature is not well understood. One of the main target cells for DENV infection is monocytes; these phagocytes can play a dual role, since they are essential to control viremia, but they also participate in the induction of tissue damage during DENV infection. Monocytes produce different pro-inflammatory cytokines and chemokines in response to infection, and also mediate endothelial damage. In peripheral blood, monocytes can be divided into three different subpopulations, namely classical, intermediate and non-classical, which differ in frequency, cytokine production, among others. Studies in the last years suggest that non-classical monocytes have higher affinity for microvasculature endothelium compared to other type of monocytes, which implies that they could be more involved in the increase of endothelial permeability observed during DENV infection. This review provides a general view of the role of monocytes and their subpopulations in DENV pathogenesis and its effect in viral replication. Finally, the potential contribution of these phagocytes in the alterations of endothelial permeability is discussed.

Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic apoE −/− mice

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10+ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10+ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10+ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5+ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10+ B cells with T1-like and MZ, and decreased IL-10+ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4+CD25− T cells. CD5+, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)+ and tumor necrosis factor alpha (TNF-α)+ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ+ and TNF-α+ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.

AB0129 HMGB1+ microparticles in systemic lupus erythematosus patients with lupus nephritis

Background High mobility group box protein 1 (HMGB1) is a nuclear DNA-binding protein that can function as an alarmin when is released from activated and dying cells. In association with nucleosomes, HMGB1 may contribute to the pathogenesis of systemic lupus erythematosus (SLE). Some previous reports have associated HMGB1 with the pathogenesis of cutaneous lupus and lupus nephritis (LN). HMGB1 may also be contained in microparticles (MPs). These vesicles have a wide spectrum of biological activities in intercellular communication, and they compete with apoptotic cells to bind mononuclear phagocytes. Objectives To evaluate the association of MP-HMGB1+ circulating with LN and to correlate them with LN activity. Methods Blood samples from 60 SLE patients were used to isolated MPs from platelet-poor plasma by centrifugation and their count, cell source and phenotype were characterized by flow cytometry. Renal pathology was reported using the standardized International Society of Nephrology/Renal Pathological Society classification. Inactive lupus nephritis (LN) was defined by the presence of one or more of the following criteria: 24 hrs proteinuria 500 mg/dl or inactive urine sediments (<5 red cells/HPF) and no red cell casts and no leucocyturia (<5 white cells/HPF) and stable serum creatinine. Results Mean age of SLE patients was 31.9±10.8 years, and mean disease duration was 7.8±6.2 years. 73% patients had LN and 89% were female. Patients with LN had significantly higher frequency of MP-HMGB1+, no significant differences were found among patients with active versus inactive LN or among patients with proliferative vs non-proliferative LN; MP-HMGB1+ had a moderate positive correlation with disease activity (SLEDAI, r=0.367, p=0.020), anti-C1q antibodies titers (r=0.42, p=001) and 24 hours proteinuria (r=0,33, p=,032), but no correlation was found with activity or chronicity indexes on renal biopsies. A ROC curve for MP-HMGB1+ and renal involvement showed a good discriminative ability (AUC 0.706). A cutoff of 15.7% of MP-HMGB1+ showed the best discrimination threshold with a sensitivity of 63.3% and specificity of 83.3%. Conclusions In our cohort of patients with SLE, MP-HMGB1+ was significantly higher in patients with LN and in patients with active disease. Given the multiple implication of HMGB1 in SLE, including the active kidney recruitment of mononuclear phagocytes, we consider that MP-HMGB1+ could be considered as a potential biomarker for LN in SLE patients. References Harris, H. E. et al. HMGB1: A multifunctional alarmin driving autoimmune and inflammatory disease. Nat Rev Rheumatol. 8, 195–202 (2012). Zickert, A. et al. Renal expression and serum levels of high mobility group box 1 protein in lupus nephritis. Arthritis Res Ther. 20;14(1):R36 (2012). Acknowledgements C. Burbano is recipient of a doctoral scholarship from COLCIENCIAS (call 617–2013). The authors are grateful to the grants “Sostenibilidad and Sistema Universitario de Investigaciones, CODI (2013–05–42869836) from Universidad de Antioquia, and to COLCIENCIAS (111565740575)”. Disclosure of Interest None declared