Paula Laranjeira

Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells

IntroductionThe ability to self-renew, be easily expanded in vitro and differentiate into different mesenchymal tissues, render mesenchymal stem cells (MSCs) an attractive therapeutic method for degenerative diseases. The subsequent discovery of their immunosuppressive ability encouraged clinical trials in graft-versus-host disease and auto-immune diseases. Despite sharing several immunophenotypic characteristics and functional capabilities, the differences between MSCs arising from different tissues are still unclear and the published data are conflicting.MethodsHere, we evaluate the influence of human MSCs derived from umbilical cord matrix (UCM), bone marrow (BM) and adipose tissue (AT), co-cultured with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNC), on T, B and natural killer (NK) cell activation; T and B cells’ ability to acquire lymphoblast characteristics; mRNA expression of interleukin-2 (IL-2), forkhead box P3 (FoxP3), T-bet and GATA binding protein 3 (GATA3), on purified T cells, and tumor necrosis factor-alpha (TNF-α), perforin and granzyme B on purified NK cells.ResultsMSCs derived from all three tissues were able to prevent CD4+ and CD8+ T cell activation and acquisition of lymphoblast characteristics and CD56dim NK cell activation, wherein AT-MSCs showed a stronger inhibitory effect. Moreover, AT-MSCs blocked the T cell activation process in an earlier phase than BM- or UCM-MSCs, yielding a greater proportion of T cells in the non-activated state. Concerning B cells and CD56bright NK cells, UCM-MSCs did not influence either their activation kinetics or PHA-induced lymphoblast characteristics, conversely to BM- and AT-MSCs which displayed an inhibitory effect. Besides, when co-cultured with PHA-stimulated MNC, MSCs seem to promote Treg and Th1 polarization, estimated by the increased expression of FoxP3 and T-bet mRNA within purified activated T cells, and to reduce TNF-α and perforin production by activated NK cells.ConclusionsOverall, UCM-, BM- and AT-derived MSCs hamper T cell, B cell and NK cell-mediated immune response by preventing their acquisition of lymphoblast characteristics, activation and changing the expression profile of proteins with an important role in immune function, except UCM-MSCs showed no inhibitory effect on B cells under these experimental conditions. Despite the similarities between the three types of MSCs evaluated, we detect important differences that should be taken into account when choosing the MSC source for research or therapeutic purposes.

Frequency and functional activity of Th17, Tc17 and other T-cell subsets in Systemic Lupus Erythematosus

To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n=15) and inactive disease (n=19) and healthy age- and gender-matched controls (n=15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-beta and FoxP3 mRNA. Iotan active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.

Bone marrow cells from myelodysplastic syndromes show altered immunophenotypic profiles that may contribute to the diagnosis and prognostic stratification of the disease: A pilot study on a series of 56 patients

A heterogeneous spectrum of immunophenotypic abnormalities have been reported in myelodysplastic syndromes (MDS). However, most studies are restricted to the analysis of CD34+ cells and/or other major subsets of CD34− cells, frequently not exploring the diagnostic and prognostic impact of immunophenotyping.

Functional characterization of peripheral blood dendritic cells and monocytes in systemic lupus erythematosus

With the purpose of contributing to a better knowledge of the APCs functional activity in SLE, we evaluated the distribution and functional ability to produce pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12) of peripheral blood (PB) monocytes and DC (tDC), particularly myeloid (mDC) and CD14−/lowCD16+ DC subpopulations comparing them with those obtained from healthy individuals. The study was performed in 34 SLE patients with diverse disease activity scores (SLEDAI) and 13 healthy age- and sex-matched controls (NC). Our results show an overall decrease in absolute number and relative frequency of tDC in SLE patients with active disease when compared to those with inactive disease and NC, although this decrease did not seem to have an effect on the distribution of PB DC subsets. The monocytes number in SLE patients was similar to those found in NC, whereas a higher frequency of monocytes producing cytokines as well as the amount of each cytokine per cell found without stimulation was particularly observed in those patients with active disease. After stimulation, we observed a higher frequency of IL-12-producing monocytes in active SLE patients. On the other hand, we found among DCs higher frequencies of cytokine-producing CD14−/lowCD16+ DCs and a higher amount of cytokines produced per cell, particularly in active disease. These findings support an increased production of inflammatory cytokines by APCs in active SLE, mostly associated with alterations in CD14−/lowCD16+ DC subset homeostasis that might contribute to explain the dynamic role of these cells in disease pathogenesis.

Effect of human bone marrow mesenchymal stromal cells on cytokine production by peripheral blood naive, memory, and effector T cells

IntroductionThe different distribution of T cells among activation/differentiation stages in immune disorders may condition the outcome of mesenchymal stromal cell (MSC)-based therapies. Indeed, the effect of MSCs in the different functional compartments of T cells is not completely elucidated.MethodsWe investigated the effect of human bone marrow MSCs on naturally occurring peripheral blood functional compartments of CD4+ and CD8+ T cells: naive, central memory, effector memory, and effector compartments. For that, mononuclear cells (MNCs) stimulated with phorbol myristate acetate (PMA) plus ionomycin were cultured in the absence/presence of MSCs. The percentage of cells expressing tumor necrosis factor-alpha (TNF-α), interferon gamma (IFNγ), and interleukin-2 (IL-2), IL-17, IL-9, and IL-6 and the amount of cytokine produced were assessed by flow cytometry. mRNA levels of IL-4, IL-10, transforming growth factor-beta (TGF-β), and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in purified CD4+ and CD8+ T cells, and phenotypic and mRNA expression changes induced by PMA + ionomycin stimulation in MSCs, were also evaluated.ResultsMSCs induced the reduction of the percentage of CD4+ and CD8+ T cells producing TNF-α, IFNγ, and IL-2 in all functional compartments, except for naive IFNγ+CD4+ T cells. This inhibitory effect differentially affected CD4+ and CD8+ T cells as well as the T-cell functional compartments; remarkably, different cytokines showed distinct patterns of inhibition regarding both the percentage of producing cells and the amount of cytokine produced. Likewise, the percentages of IL-17+, IL-17+TNF-α+, and IL-9+ within CD4+ and CD8+ T cells and of IL-6+CD4+ T cells were decreased in MNC-MSC co-cultures. MSCs decreased IL-10 and increased IL-4 mRNA expression in stimulated CD4+ and CD8+ T cells, whereas TGF-β was reduced in CD8+ and augmented in CD4+ T cells, with no changes for CTLA4. Finally, PMA + ionomycin stimulation did not induce significant alterations on MSCs phenotype but did increase indoleamine-2,3-dioxygenase (IDO), inducible costimulatory ligand (ICOSL), IL-1β, IL-8, and TNF-α mRNA expression.ConclusionsOverall, our study showed that MSCs differentially regulate the functional compartments of CD4+ and CD8+ T cells, which may differentially impact their therapeutic effect in immune disorders. Furthermore, the influence of MSCs on IL-9 expression can open new possibilities for MSC-based therapy in allergic diseases.

Human Bone Marrow-Derived Mesenchymal Stromal Cells Differentially Inhibit Cytokine Production by Peripheral Blood Monocytes Subpopulations and Myeloid Dendritic Cells

The immunosuppressive properties of mesenchymal stromal/stem cells (MSC) rendered them an attractive therapeutic approach for immune disorders and an increasing body of evidence demonstrated their clinical value. However, the influence of MSC on the function of specific immune cell populations, namely, monocyte subpopulations, is not well elucidated. Here, we investigated the influence of human bone marrow MSC on the cytokine and chemokine expression by peripheral blood classical, intermediate and nonclassical monocytes, and myeloid dendritic cells (mDC), stimulated with lipopolysaccharide plus interferon (IFN)γ. We found that MSC effectively inhibit tumor necrosis factor- (TNF-) α and macrophage inflammatory protein- (MIP-) 1β protein expression in monocytes and mDC, without suppressing CCR7 and CD83 protein expression. Interestingly, mDC exhibited the highest degree of inhibition, for both TNF-α and MIP-1β, whereas the reduction of TNF-α expression was less marked for nonclassical monocytes. Similarly, MSC decreased mRNA levels of interleukin- (IL-) 1β and IL-6 in classical monocytes, CCL3, CCL5, CXCL9, and CXCL10 in classical and nonclassical monocytes, and IL-1β and CXCL10 in mDC. MSC do not impair the expression of maturation markers in monocytes and mDC under our experimental conditions; nevertheless, they hamper the proinflammatory function of monocytes and mDC, which may impede the development of inflammatory immune responses.

Modified phosphatidylethanolamines induce different levels of cytokine expression in monocytes and dendritic cells

Glycation of phosphatidylethanolamine (PE) is a reaction that is known to occur under the chronic hyperglycemic conditions found in diabetes. Glycated phosphatidylethanolamines were found in plasma and atherosclerotic plaques of diabetic patients, and its presence was correlated with increased oxidative stress. Moreover, upregulation of cytokines and other inflammatory mediators can be observed not only in diabetes, but also under oxidized phosphatidylcholine stimulation. In this study, we evaluate the effect of dipalmitoyl-phosphatidylethanolamine (DPPE) and linoleoyl-palmitoyl-phosphatidylethanolamine (PLPE) structural oxidation, glycation and glycoxidation, on monocyte and myeloid dendritic cell stimulation. Expression of cytokines, IL-1β, IL-6, IL-8, MIP-1β and TNF-α, were determined using flow cytometry after cell stimulations with native PEs, oxidized, glycated and glycoxidized PEs. Native PE, PLPE and DPPE, and all modified PEs were able to increase the stimulation levels of monocytes and mDCs. Generally, in monocytes and mDCs stimulation, GluOxPLPE and GluDPPE were the PLPE/DPPE modifications that induced the most pronounced rise in cytokine production. However, GluOxDPPE was the DPPE modification that produced the lowest stimulation levels of mDCs and monocytes. Our results indicate that glycated PE and glycoxidized PE may have an important contribution to the low-grade systemic inflammation associated with diabetes and to the development of diabetic complications.

Phosphatidylethanolamines Glycation, Oxidation, and Glycoxidation: Effects on Monocyte and Dendritic Cell Stimulation

Lipid glycation is a non-enzymatic reaction between glucose and the free amino group of aminophospholipids, particularly in chronic hyperglycemia. Glycated phosphatidylethanolamine have been found in plasma and atherosclerotic plaques of diabetic patients and was correlated with increased oxidative and inflammatory stress in diabetes. However, the biological roles of glycated lipids are not fully understood. In this study, we evaluated the effect of palmitoyl-oleoyl-phosphatidylethanolamine (POPE) oxidation, glycation, and glycoxidation products on monocyte and myeloid dendritic cell stimulation. Flow cytometry analysis was used to evaluate the capability of each modified PE to induce the expression of different cytokines (IL-1β, IL-6, IL-8, MIP-1β, and TNF-α) in monocytes or myeloid dendritic cells (mDC). Our results showed that PE modifications induced different effect on the stimulation of cells producing cytokines. All PE modifications induced higher frequencies of cytokine-producing cells than basal state. Higher stimulation levels were obtained with glycated POPE, followed by glycoxidized POPE. In contrast, oxidized POPE negatively regulated the frequency of monocytes and mDC producing cytokines, when compared with non-modified POPE. In conclusion, we verified that PE glycation, compared with oxidation and glycation plus oxidation, had higher ability to stimulate monocytes and mDC. Thus detection of increased levels of PE glycation in diabetes could be considered a predictor of a inflammatory state.

The Proliferation Index of Specific Bone Marrow Cell Compartments from Myelodysplastic Syndromes Is Associated with the Diagnostic and Patient Outcome

Myelodysplastic syndromes (MDS) are clonal stem cell disorders which frequently show a hypercellular dysplastic bone marrow (BM) associated with inefficient hematopoiesis and peripheral cytopenias due to increased apoptosis and maturation blockades. Currently, little is known about the role of cell proliferation in compensating for the BM failure syndrome and in determining patient outcome. Here, we analyzed the proliferation index (PI) of different compartments of BM hematopoietic cells in 106 MDS patients compared to both normal/reactive BM (n = 94) and acute myeloid leukemia (AML; n = 30 cases) using multiparameter flow cytometry. Our results show abnormally increased overall BM proliferation profiles in MDS which significantly differ between early/low-risk and advanced/high-risk cases. Early/low-risk patients showed increased proliferation of non-lymphoid CD34+ precursors, maturing neutrophils and nucleated red blood cells (NRBC), while the PI of these compartments of BM precursors progressively fell below normal values towards AML levels in advanced/high-risk MDS. Decreased proliferation of non-lymphoid CD34+ and NRBC precursors was significantly associated with adverse disease features, shorter overall survival (OS) and transformation to AML, both in the whole series and when low- and high-risk MDS patients were separately considered, the PI of NRBC emerging as the most powerful independent predictor for OS and progression to AML. In conclusion, assessment of the PI of NRBC, and potentially also of other compartments of BM precursors (e.g.: myeloid CD34+ HPC), could significantly contribute to a better management of MDS.

Expression of CD44 and CD35 during normal and myelodysplastic erythropoiesis.

Erythroid dysplasia is a common feature of myelodysplastic syndromes (MDS). Currently available information about the immunophenotypic features of normal and dysplastic erythropoiesis is scarce and restricted to relatively few markers. Here we studied the expression of CD117, CD35 and CD44 throughout the normal (n=16) and dysplastic (n=48) bone marrow erythroid maturation. CD35 emerged as an early marker of CD34(+) erythroid-committed precursors, which is expressed before CD105 and remains positive thereafter. MDS patients (with and without morphologic dyserythropoiesis) displayed overall increased expression of CD44, associated with slight alterations on CD35 expression, suggesting that phenotypic alterations in MDS may precede morphologic dysplasia. In turn, MDS patients with anemia showed increased expression of CD117.