Mariana H. García-Hernández

Dysregulated miR-155 expression in peripheral blood mononuclear cells from patients with type 2 diabetes

MicroRNAs (miRNAs) are involved in gene regulation of several physiological processes. Alterations in the concentrations of miRNAs may result in cancer and autoimmune diseases. In cells of the immune system, miRNA expression is regulated by several cytokines and this expression is related to the inflammatory process. In the present work we evaluated miR-155 and miR-146a levels in peripheral blood mononuclear cells (PBMC) from patients with type 2 diabetes (T2D).We analysed the expression of miRNAs in PBMC from T2D patients (n=20) and control subjects (n=20) using real-time PCR. The quantity of IL-1β and IL-6 in culture supernatants was measured by ELISA.The basal expression of miR-155 and miR-146a in patients with T2D was decreased compared to control subjects and associated with age, gender and metabolic control but not with the therapeutic treatment used. We found significant correlations between the basal expression of miR-155 and miR-146a with HbA1c, Glucose and BMI, as well as of miR-155 expression stimulated by LPS with the values of TG, HbA1c, Glucose and BMI. Additionally, we detected an altered distribution of miR-155 and miR-146a expression related with HbA1c, glucose and BMI using the analysis of a three dimensional association of variables in the group of T2D patients.Downregulated levels of miR-155 could play an important role in the pathogenesis of T2D due to their relationship with metabolic control.

The His155Tyr (489C>T) single nucleotide polymorphism of P2RX7 gene confers an enhanced function of P2X7 receptor in immune cells from patients with rheumatoid arthritis

We assessed the possible association between several single nucleotide polymorphisms (SNP) of P2RX7 gene with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). We determined the function of P2X7 receptor and the frequency of the 489C>T, 1096C>G, and 1513A>C SNP of P2RX7 gene in 111 and 122 patients with SLE and RA, and 98 healthy subjects. We found no significant association between the SNPs studied and SLE or RA. We also detected that lymphocytes from SLE and RA patients with the 489C>T SNP showed a higher ethidium bromide uptake in response to ATP than wild type or 1096C>G/1513A>C subjects. In addition, cells from RA patients and the 489C>T genotype, showed higher [Ca(2+)]i responses to ATP. Our data indicate that the 489C>T SNP of P2RX7 gene confers an enhanced function of this receptor in patients with RA, which may contribute to the pathogenesis of this condition.

In vitro assessment of agave fructans (Agave salmiana) as prebiotics and immune system activators.

The prebiotic effect of agave fructans (Agave salmiana) was evaluated through the growth of two lactic acid bacterial (LAB) strains (Lactobacillus casei and Bifidobacterium lactis). The immune system was activated through the stimulation of peripheral blood mononuclear cells (PBMC) of healthy subjects testing fructans, LAB or a mixture of these compounds at different concentrations. Immune responses, such as early cell activation (CD69), cell cycle progression, nitric oxide (NO) production and the expression of transcription factors for lymphocyte differentiation, were analyzed. Compared with other fructans, the extracted agave fructans showed the highest prebiotic activity and increased levels of CD69 expression, proliferative activity and NO production when administered with the probiotic L. casei. The Th1 lymphocyte differentiation produced through LAB stimulation was greatly diminished after the incorporation of agave fructans. In conclusion, these types of fructans (A. salmiana) are involved in the activation and selective differentiation of cells of the immune system through interactions with probiotics. Thus, agave fructans represent a novel immunomodulator that might benefit the functional food industry.

Expression and function of P2X7 receptor and CD39/Entpd1 in patients with type 2 diabetes and their association with biochemical parameters

Chronic inflammation is an important contributor to the insulin resistance observed in type 2 diabetes (T2D). We evaluated the expression and function of the P2X(7) receptor and CD39/Entpd1, molecules involved in the cellular regulation of inflammation, in peripheral blood mononuclear cells from T2D patients, and their correlation with the concentration of HbA1c in blood. T2D patients with deficient metabolic control (DC) showed increased proportion of P2X(7)(+) cells compared with healthy individuals; T2D-DC subjects also displayed higher proportion of CD14(+), CD4(+) and CD19(+) subpopulations of P2X(7)(+) cells when compared with T2D patients with acceptable metabolic control. A significant association was observed between the proportion of P2X(7)(+)CD14(+) cells and blood concentration of LDL-c. In addition, the percentages of CD39(+) cells and CD39(+)CD19(+) cells were significantly associated with HbA1c and fasting plasma glucose levels. No changes were observed in the function of P2X(7)(+) cells from T2D patients; however, enhanced CD39/Entpd1 enzyme activity and low serum levels of IL-17 were detected. Therefore, CD39(+) cells could have a balancing regulatory role in the inflammatory process observed in patients with T2D.

LL-37 Immunomodulatory Activity during Mycobacterium tuberculosis Infection in Macrophages

ABSTRACT Tuberculosis is one of the most important infectious diseases worldwide. The susceptibility to this disease depends to a great extent on the innate immune response against mycobacteria. Host defense peptides (HDP) are one of the first barriers to counteract infection. Cathelicidin (LL-37) is an HDP that has many immunomodulatory effects besides its weak antimicrobial activity. Despite advances in the study of the innate immune response in tuberculosis, the immunological role of LL-37 during M. tuberculosis infection has not been clarified. Monocyte-derived macrophages were infected with M. tuberculosis strain H37Rv and then treated with 1, 5, or 15 μg/ml of exogenous LL-37 for 4, 8, and 24 h. Exogenous LL-37 decreased tumor necrosis factor alpha (TNF-α) and interleukin-17 (IL-17) while inducing anti-inflammatory IL-10 and transforming growth factor β (TGF-β) production. Interestingly, the decreased production of anti-inflammatory cytokines did not reduce antimycobacterial activity. These results are consistent with the concept that LL-37 can modulate the expression of cytokines during mycobacterial infection and this activity was independent of the P2X7 receptor. Thus, LL-37 modulates the response of macrophages during infection, controlling the expression of proinflammatory and anti-inflammatory cytokines.

Specific therapy to regulate inflammation in rheumatoid arthritis: molecular aspects.

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which persistent inflammation of synovial tissue results in a progressive functional decline of the joint and premature mortality. TNF inhibitors were the first biological disease-modifying antirheumatic drugs (DMARDs) used to treat RA. Since then, new biological drugs have emerged, such as inhibitors of IL-1, IL-6 and others, with different mechanisms of action that include the depletion of B cells and the inhibition of T-cell costimulation. Recently, RA treatments have incorporated the use of synthetic DMARDs. This review describes the molecular aspects of the mechanisms of action of biological and synthetic DMARDs, discusses the adverse effects and limitations of established therapies and analyses the alternative approaches to RA treatment.

Abnormal expression and function of Dectin-1 receptor in type 2 diabetes mellitus patients with poor glycemic control (HbA1c>8%)

Dectin-1 is a key innate receptor involved in various cellular responses and may have a direct role in chronic inflammatory conditions such as type 2 diabetes mellitus. The aim of this work was to evaluate the expression and function of Dectin-1 in peripheral blood mononuclear cells from T2D patients. Dectin-1 expression was analyzed by flow cytometry and RT-PCR in monocytes and lymphocyte subpopulations from T2D patients (n=34) and healthy subjects (n=29). Functional assays were used to assess cytokine synthesis, ROS levels and oxidative stress ratio. We found increased expression (MFI) of Dectin-1 in monocytes from T2D patients. Significantly higher Dectin-1 expression was also detected in CD4(+) T, CD8(+) T, B cells and NK cells from T2D patients compared to controls. In contrast, monocytes from T2D patients with poor glycemic control (HbA1c>8%) showed a diminished percentage of Dectin-1(+)/TLR2(+) cells. Negative correlations between the percent of Dectin-1(+)/TLR2(+) cells and fasting plasma glucose levels (FPG) and HbA1c levels were found. A significant reduction in basal levels of IL-10 was observed in patients with poor glycemic control (HbA1c>8%) compared to patients with appropriate glycemic control (HbA1c≤6.5%) and healthy controls, an effect that was not observed in monocytes stimulated with zymosan. Higher ROS levels in zymosan-stimulated cells from patients with poor glycemic control positively correlated with FPG levels, and the oxidative stress ratio was higher in T2D cells compared with controls. Our data indicate that Dectin-1 may be involved in the abnormal immune responses that are observed in patients with T2D.

Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets.

Regulatory T cells that express CD39 (CD39+ Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39+ Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39+ Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25+ CD39+ T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39+ Treg cells and higher levels of ART1 expression in CD4+ CD39+ T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39+ Treg or CD39- Treg cells was observed by 1mM ATP or 60μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39+ Treg or CD39- Treg cells could participate in the resistance against cell death induced by ATP or NAD.

Regulatory T-cell subsets in response to specific Mycobacterium tuberculosis antigens in vitro distinguish among individuals with different QTF and TST reactivity.

Regulatory T cells (Tregs), a subset of CD4+ T cells related with immune regulation, have been associated with active and latent tuberculosis infection (LTBI). Treg frequencies were evaluated by multicolor flow cytometry (FC) in peripheral blood mononuclear cells (PBMCs) stimulated with mycobacterial antigens ESAT-6, CFP-10, and TB7.7 to assess their capacity to distinguish subjects with different reactivity to the QuantiFERON-TB® Gold In-Tube (QFT-IT) test and the tuberculin skin test (TST). Increased frequencies of CD4+CD25highCD39+ cells were found for the [TST+, QTF+] compared with the [TST+, QTF-] group. Also, higher frequencies were observed for the [TST+, QTF+] compared with the [TST+, QTF-] and [TST-, QTF-] groups in CD4+CD25highFoxp3+ and CD4+CD25highCD39+Foxp3+ populations. Receiver operating characteristics (ROC curve) analysis confirmed these discriminating results. QFT-IT and TST quantitative values correlated with several Treg population frequencies.

Type-2 diabetes alters the basal phenotype of human macrophages and diminishes their capacity to respond, internalise, and control Mycobacterium tuberculosis

BACKGROUND Type 2 diabetes (T2D) is a risk factor for the development of tuberculosis (TB), although the associated mechanisms are not known. OBJECTIVES To study the association between T2D and the basal phenotype of macrophages, and their immune response to Mycobacterium tuberculosis (Mtb) infection. METHODS We evaluated the influence of T2D on the response of monocyte-derived macrophages (MDM) to Mtb in patients with T2D (n = 10) compared to healthy subjects (n = 9), before and after infection with Mtb clinical isolates bearing different degrees of virulence. The levels of cell surface markers for activation secreted cytokines and chemokines, bacterial association, and intracellular bacterial growth were evaluated. FINDINGS The expression levels of HLA-DR, CD80, and CD86 were low while those of of PD-L1 were high in uninfected MDMs derived from patients with diabetes; as a result of Mtb infection, changes were only observed in the expression levels of PD-L1. The levels of cytokines (e.g., IL-6, IL-1β, IL-10, and IL-12) and chemokines (e.g., MCP-1, MIG, and RANTES) are perturbed in MDMs derived from patients with diabetes, both before infection and in response to Mtb infection. In response to the more virulent Mtb strains, the levels of association and bacterial clearance were diminished in MDMs derived from patients with diabetes. CONCLUSIONS T2D affects the basal activation state of the macrophages and its capacity to respond and control Mtb infection.