The aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNFα-and IFNγ cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNFα-and IFNγ-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes.Scientific and Technological Research Council of Turkey (TUBITAK 106T417

Small RNA data set that includes tRNA-derived fragments from Jurkat cells treated with camptothecin

In this article, we report a small RNA data set obtained from human T cell acute leukemia Jurkat cells, which were treated with the universal apoptotic agent camptothecin. Based on the Annexin-V labeling pattern, we sorted two Jurkat subpopulations in treated cells: one that is sensitive to the drug and the other being relatively more resistant. We report new original data that include the frequency of tRNA-derived fragments (tRF) in drug-sensitive and resistant cells. We also present partially analyzed data to show the origin of reads on tRNAs as well as the borders of the fragments. We believe that this data can benefit the science community working in the field of tRF and/or apoptosis.The Scientific and Technical Research Council of Turkey ( 107T475 to BA

Aggregatibacter actinomycetemcomitans GroEL Protein Promotes Conversion of Human CD4+ T Cells into IFNγ IL10 Producing Tbet+ Th1 Cells

One of the heat shock family protein (Hsp) expressing bacteria is the gram negative, periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa). A. actinomycetemcomitans' Hsp is a 64-kDa GroEL-protein, which has been shown to influence the host cells. In this study we used recombinant A. actinomycetemcomitans GroEL (rAaGroEL) protein as a model antigen to study GroEL-mediated T cell immune response. Human peripheral mononuclear cells (PBMCs), when stimulated with recombinant rAaGroEL, expressed early activation marker CD69 and IL-2R (CD25). CD25 and CD69 expressions were higher in CD4+ T cells compared to CD8+ T cells. rAaGroEL-responding CD4+ T cells expressed IL-10, IFNγ and TNFα cytokines. Interestingly, there were also IL-10 and IFNγ double cytokine producing CD4+ T cells. Additionally, IFNγ expressing CD4+ T cells were also T-bet positive. Altogether the results suggest that rAaGroEL protein affects CD4+ T cells to differentiate into IFNγ IL10-secreting T-bet+ Th1 cells.Scientific and Technological Research Council of Turkey (106T417

Use of Flow Cytometry for Detection of Apoptotic Cell Death in Th17 Cells

Flow cytometry (FC) is a powerful and reliable system for cell death studies. It allows to study the molecular changes in both the surface and cytoplasmic part of the cells. Depending on the laser range of flow cytometry, it is possible to collect a large number of information about a single cell by combining multiple labeling strategies. With these assets, flow cytometry allows scientists to accelerate their research. Flow cytometry is also widely used in clinical studies. Therefore, we used this powerful tool to study human T helper 17 (Th17) cell apoptosis. Newly discovered Th17 cells are important players of immune response regulation. They are also involved in different types of pathologies including autoimmune diseases and cancer. There are intensive data in the literature about molecules that are involved in Th17 differentiation signaling networks, but the apoptotic and survival molecular mechanisms of this cells are not fully understood yet. Therefore, apoptosis of Th17 cells were measured by Flow cytometry. Healthy human subjects have been invited to study with the informed consent which is approved by the ethics committee. Human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy volunteers. Phenotypically characterized and sorted naïve CD4+ T cells from PBMC were cultured under Th17 polarizing conditions. IL-17, IL-22 or CCR6 molecules were used to monitor Th17 cells. Apoptotic cell death of Th17 cells were measured by plasma membrane changes and DNA fragmentation. During differentiation stages, plasma membrane changes were monitored by Annexin V concomitantly with 7AAD. At day 7, there were Annexin V positive cells in Th17 cells. Apoptotic cell death occurs through sequential events including caspase activation and DNA fragmentation. DNA fragmentation data showed that Th17 cells were not apoptotic compared to negative control cultures. This finding suggested that survival molecules of Th17 cells interferes with this apoptotic process

IL-17, IL-21, and IL-22 Cytokines of T Helper 17 Cells in Cancer

WOS: 000463009500006PubMed: 30562123CD4(+) T helper (Th) cells are important regulators of cellular immune response. Newly discovered interleukin (IL)-17-producing CD4(+) T cells are known as T helper 17 cells (Th17). They are distinct subset from the T helper type 1 (Th1) and 2 (Th2) lineages. The differentiation of Th17 cells has been intensively studied; however, the role of Th17 cells in different diseases including cancer is still under investigation. Besides IL-17 family cytokines, Th17 cells produce IL-22, IL-21, and IL-26. The dysregulated function of Th17 cells and their cytokines could contribute to pathology of diseases, including cancer. The role of cytokines of Th17 cells such as IL-17, IL-21, and IL-22 in cancer will be discussed in this review

Flow Cytometry and FTIR Spectroscopy for Detection of Early Apoptosis in Human T Cells

Apoptosis, a programmed cell death, has a vital role in various cellular processes. Apoptotic cells exhibit morphological and biochemical changes, detected by a variety of assays (caspases, mitochondrial dyes, DNA laddering). Flow cytometry is a powerful tool for detection of apoptotic cell death and allows information about the cell size and molecules associated with cell-bound antibodies. Recently, Fourier transform infrared (FTIR) spectroscopy as rapid and low-cost tool has been extensively used for cellular studies, providing information on cellular structures. The aim of this study was to detect early apoptosis and obtain further insights into the capability of FTIR spectroscopy, comparing the results with flow cytometry. In this study, apoptotic cell death was induced in human Jurkat T cells with Camptothecin (CPT), a DNA topoisomerase I inhibitor. Cells were cultured with 4µM CPT in RPMI (with 5% FCS) for 24 h. Immunoflourescence labeling for multicolor flow cytometry was accomplished with Annexin V concomitantly with 7-AAD. The same cells were also analyzed with ATR-FTIR spectroscopy. Flow cytometry data represents that the cells are Annexin V positive but 7AAD negative. This indicates that cells are in the early apoptotic stage, only externalization of phosphatidylserine exists on the plasma membrane. FTIR data reveals that membrane phospholipids and proteins undergo changes; fatty acid acyl chains are disordered and increased in mobility after treatment, which result from the early apoptosis process after CPT-treatment, confirmed by the flow cytometry. A combined study of flow cytometry and FTIR spectroscopy for analysis of apoptosis in human T cells exhibited compatible and complementary results. Existence of biophysical and biochemical changes in T cells after treatment were also demonstrated

The Aggregatibacter actinomycetemcomitans heat shock protein GroEL interacts directly with human peripheral blood T cells

WOS: 000391393100002Heat shock family protein GroEL of Aggregatibacter actinomycetemcomitans (Aa) has antigenic properties. We previously demonstrated that A. actinomycetemcomitans GroEL-like protein affects human CD4 T cells by converting them into IL-10 and IFNg double cytokine producing Tbet+ Th1 cells. The objective of this study was to investigate whether or not AaGroEL communicates with T cells directly. To do this, sorted cells from peripheral blood mononuclear cells were stimulated with AaGroEL for 48 h. Flow cytometry was used to measure soluble and intracellular cytokine expression in the cell cultures and detect TLR2 expression on the surface of T cells. Expression of six different soluble cytokines was evaluated by CBA assay. To determine whether AaGroEL affects CD3+ T cells directly or not, purified CD3+ T cells or CD14+ cells were cultured with AaGroEL separately, and the quantity of soluble cytokine was measured. Results showed that sorted CD3+ cells produced soluble IL-6, TNF alpha, and IFN gamma cytokines. Additionally, the intracellular cytokine staining data showed that AaGroEL-stimulated CD3+ cells were also TNF alpha- and IFN gamma-positive. Moreover, AaGroEL-responsive T cells slightly increased their TLR2 expression. These findings suggest that CD3+ T cells produce cytokines in response to AaGroEL protein without requirements for other cells, such as CD14+ monocytes

IL12, IL10, IFNγ and TNFα expression in human primary monocytes stimulated with bacterial heat shock GroEL (Hsp64) protein

Actinobacillus (Aggregatibacter) actinomycetemicomitans (Aa) is a bacterium that lives in the oral cavity and plays an important role in periodontal diseases. The effect of A.actinomycetemcomitans's heat shock family protein GroEL on host or immune cells including monocytes is quite limited. In this study, the recombinant A.actinomycetemcomitans's GroEL protein (rAaGroEL) was used as an antigen and its effects on monocytes of peripheral blood mononuclear cells (PBMCs) was investigated. To do that, PBMCs were stimulated with rAaGroEL protein at different time points (16h to 96h) and the cytokines of CD14+ monocytes were detected with intracellular cytokine staining by Flow cytometry. Data showed that AaGroEL protein has an antigenic effect on human primary monocytes. AaGroEL protein responsive CD14 monocytes stimulates the expression of IL12, IL10, IFNγ and TNFα cytokines with different kinetics and expression profile. Therefore, A. actinomycetemcomitans's heat shock GroEL protein can modulate innate and adaptive immune responses and contribute to an inflammatory diseases pathology.Scientific and Technological Research Council of Turkey (106T417

Survival Proteins Encourage Human T Helper 17 Cells to Escape from Cell Death

IL-17 producing T helper 17 (Th17) cells are identified as a distinct subset of CD4+ T helper cells. They play a role in immune response. Abnormal regulation of Th17 cells can play a role in different type of pathologies such autoimmune diseases and cancer. The apoptotic and survival mechanisms of Th17 cells are not well known in different type of diseases. Therefore, the aim of the study was to investigate Bcl-2 family proteins to understand the regulation network of apoptosis in human Th17 cells. To do that, Peripheral blood (PB) were drawn from the healthy volunteers. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from blood by Ficoll gradient isolation method. The naïve CD4+ T cells were isolated from PBMC. Sorted naive CD4+ T cells were cultured under Th17 polarizing conditions. Th17 cells were characterized by Flow cytometry. Cell lysates were obtained from negative controls and Th17 cells. Bcl-2 family members (Bik, BID and Puma) in Th17 cells were detected by western blot. Data showed that naive T cells were differentiated into Th17 cells. Then, cell lysate of this cells were used for western blot experiments. In the Th17 cell lysate, BH3 family members Bik and Puma were not detectable but Mcl-1 expression was increased. Overall data indicated that the pro-apoptotic BH3-only subgroup proteins Bik and Puma was not detectable, however anti-apoptotic Mcl-1 protein expression was increased

Bacterial heat shock protein GroEL (Hsp64) exerts immunoregulatory effects on T cells by utilizing apoptosis

Aggregatibacter actinomycetemcomitans (Aa) expresses a 64-kDa GroEL protein belonging to the heat shock family of proteins. This protein has been shown to influence human host cells, but the apoptotic capacity of the GroEL protein regarding T cells is not yet known. The purpose of this study was to investigate the ability of A. actinomycetemcomitans GroEL (AaGroEL) protein to induce human peripheral blood T-cell apoptosis. Endogenous, purified AaGroEL protein was used as an antigen. In AaGroEL-treated T cells, the data indicated that phosphatidylserine exposure, an early apoptotic event, was dose- and time-dependent. The AaGroEL-treated T cells were also positive for active caspase-3 in a dose-dependent manner. The rate of AaGroEL-induced apoptosis was suppressed by the addition of the general caspase inhibitor Z-VAD-FMK. Furthermore, cleaved caspase-8 bands (40/36 kDa and 23 kDa) were identified in cells responding to AaGroEL. DNA fragmentation was also detected in the AaGroEL-treated T cells. Overall, we demonstrated that the endogenous GroEL from A. actinomycetemcomitans has the capacity to induce T-cell apoptosis. © 2016 Nalbant, Kant. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Scientific and Technological Research Council of Turkey (106T417