Background
Upon activation and differentiation, the endoplasmic reticulum (ER) of T cells is inundated with newly formed proteins that must be folded and exported to appropriate places in the cell. Failure of proteins to fold correctly leads to aggregates of misfolded proteins that induce stress in the ER. If this stress is not resolved, the cells die via apoptosis. To avoid apoptosis, cells have developed a response mechanism to this stressed state known as the unfolded protein response (UPR). The UPR is comprised of three conserved pathways that are named after the following initiating molecules: protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 alpha (IRE1α). PERK decreases general translation of proteins, while ATF6 and IRE1α increase the transcription of those that promote protein folding and degradation [
1].
The UPR plays an integral part in the development and differentiation of T cells. ER stress and activation of the UPR is associated with altered T helper differentiation and cytokine secretion in patients with inflammatory diseases [
2]. UPR inhibits IL-4/IL-13 signaling in T helper cells [
3], and Eukaryotic Translation Initiation Factor 2α (EIF2α) regulates IL-4 transcription in primed TH2 cells [
4]. Knocking out IRE1α halts the development of T cells at the CD4
−CD8
− double-negative stage [
5], and inhibition of IRE1α in primary mouse CD4 T cells undergoing activation using a commercially available drug, 4μ8c, results in decreased IL-4, IL-5, and IL-13 [
6].
IL-4, IL-5, and IL-13, while important for promoting the clearance of parasites, can promote a disease state when improperly expressed, such as with asthma and allergy, by activating immune cells involved in these pathologies. Inhibition of TH2 cells and TH2 cytokines improves asthma and allergy outcomes in humans and animal models [
7,
8]. This makes 4μ8c of potential interest for treatment of type 2 cytokine mediated diseases.
It is known that naïve T cells, cells undergoing differentiation, and T cells with an established phenotype have differences with regards to gene expression and regulation. Therefore, the results observed in naïve cells undergoing differentiation in the presence of 4μ8c are not necessarily representative of the effects of 4μ8c on established T cells. This work attempts to better understand the underlying mechanism of how inhibition of IRE1α by 4μ8c affects secretion of TH2 specific cytokines in established TH2 cells.
Conclusion
In summary, our results indicate that 4μ8c inhibits the secretion of IL-5 in established TH2 cells, but not IL-4. This is of importance because established effector cells contribute greatly to disease in chronic inflammatory disorders. This work and other recent studies suggest a role for 4μ8c as a candidate for the treatment of allergy and asthma.
Materials and methods
Aim and design of the study
The aim of this study was to determine how treatment of established TH2 cells with 4μ8c effected TH2 cytokine expression. D10.G4.1 (mouse TH2 cell line) and human TH2 cells were treated with 4μ8c and downstream applications, as explained below, were performed. All work was performed at Northeastern State University.
Human subjects
Blood was collected from human volunteers of both sexes between the ages of 18–65 by a trained phlebotomist. All volunteers self-reported as being healthy.
Ethics, consent, and permission regarding human subjects
All human subjects read and signed a consent form after being presented with an opportunity to ask questions related to the study. All subjects were informed of their right to request removal from the study. This study was conducted as approved by the Institutional Review Board at Northeastern State University (In vitro studies of ER gene expression upon T cell activation -IRB # 17–058).
Culture of established TH2 murine cell line D10.G4.1
D10.G41 (D10) cells are a TH2 T cell clone derived from AKR/J mice. Their TCR recognizes conalbumin peptide CA 134–146 in the context of IA
k [
10]. These cells were gifted to our lab by Dr. Deyu Fang (Northwestern University), but they were originally obtained from the American Type Cell Culture Collection (ATCC; Manassas, Va) and cultured based on the recommendations of ATCC. Briefly, the cells were cultured at 37 °C with 5% CO
2 in RPMI complete T cell media (RPMI-1640 + L-glutamine, 10% FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, and penicillin/ streptomycin) at a concentration of 2 × 10
5 cells/mL and suspended in fresh media every two to three days. The cells were treated with IL-2 (10 ng/mL), IL-1α (10 pg/mL), and conconavalin A (2 μg/mL) to induce growth. The media and supplements were obtained from Invitrogen, the cytokines were obtained from PeproTech Inc., and the conconavalin A was obtained from Sigma Aldrich.
CD4+ T cell purification and in vitro differentiation of TH cells
Blood was collected from seven individual volunteers in total. We sent out a request for blood three different times. Two individuals volunteered two of the times, and on one occasion, three individuals volunteered, giving us a total of 7 individual participants.
Peripheral mononuclear cells were isolated from human blood using Ficoll (Millipore) according to the manufacture’s guidelines. CD4+ T cells were positively selected using the Dynabead isolation kit (Life Technologies). Purified CD4+ cells were plated in 96 well (0.1 × 106/well) or 24 well dishes (0.5 × 106/well) that were coated with 5 μg/ml of α-CD28 (OKTϵ) and 2 μg/ml of α-CD3 (145-2c11) and cultured under TH1 (10 ng/ml IL-2, 10 ng/ml IL-12 and 1 μg/ml α-IL-4 [8D4–8]) or TH2 (10 ng/ml IL-2, 20 ng/ml IL-4, 1 μg/ml α-IL-12 [C8.6], and 1 μg/ml α-IFN-γ [NIB42]) skewing conditions in RPMI complete T cell media for three or eleven days. For cells cultured eleven days, the cells were split into new wells that were coated with antibody every two to three days. On day seven of the eleven day culture, the cells were harvested, counted, and plated with fresh media. The cells were then maintained as before. All cytokines were purchased from PeproTech Inc., and all antibodies were purchased from Biolegend.
4μ8c treatment of D10 cells
The cells were suspended in complete T cell media at a concentration of 0.5 × 106/mL or 1 × 106/mL for 24 h at 37 °C with 5% CO2 in the absence of stimulation. The cells were then harvested and transferred to culture treated plates at a concentration of 1 × 106/mL in complete T cell media under no stimulation (NS) or stimulation consisting of ionomycin (1 μM) and phorbol 12-myristate 13-acetate (PMA) (25 ng/mL) or plate-bound α-CD3 (2 μg/ml) and α-CD28 (5 μg/ml) in the presence of 4μ8c (10 μg/mL) or equal volume of dimethyl sulfoxide (DMSO). DMSO, PMA, and ionomycin were obtained from Sigma Aldrich. 4μ8c was obtained from Millipore.
4μ8c treatment of human cells
CD4+ cells were differentiated under TH2 conditions as above for three days in the presence of 4μ8c (5 μg/ml) or equal volume of DMSO. In some experiments the cells were differentiated for eleven days, rested for one day, and then stimulated with plate-bound α-CD3 (145-2C11) and α-CD28 (clone 2.43, rat IgG) or PMA (25 ng/mL) and ionomycin (1 μM) for 20–24 h in the presence of 4μ8c or equal volume of DMSO as above.
Analysis of cytokine expression by ELISA and flow cytometry
The cell supernatants were harvested from plates after stimulation and treatment with 4μ8c as stated above. ELISAs were conducted following the manufacture’s protocols for IL-2, IFNγ, IL-4, IL-5, and IL-13, and all ELISA kits were obtained from Biolegend, with the exception of the IL-13 kit. The IL-13 kit was obtained from Invitrogen. For flow cytometry experiments, D10 cells were treated as explained above with PMA and ionomycin or plate-bound α-CD3 and α-CD28 for 20 h when monensin (Biolegend) was added. The samples were incubated an additional four hours at 37 °C with 5% CO2. The cells were fixed and permeabilized using cytofix fixation buffer (Biolegend) according to the manufacturer’s instructions. Human T cells differentiated under TH2 conditions were stimulated as indicated above for three days in the presence or absence of 4μ8c, harvested, and stimulated with PMA and ionomycin in the presence of monensin for four hours as described above. The cells were suspended in fluorochrome-conjugated antibodies specific for mouse/human IL-5 (TRFK5, Biolegend) and IL-13 (abcam, AB95576) or human IL-4 (Biolegend, 8D4–8), and IFN-γ (Biolegend, 4S.B3) using the concentrations suggested by the manufacturer for 30 min at room temperature, washed, resuspended in FACS Buffer (2% BSA in 1x PBS), filtered, and analyzed via a Cytoflex flow cytometer (Beckman Coulter). IL-5 cytokine secretion assay was performed according to manufacture instructions (Miltenyi Biotec), with the exception that bovine serum was used in place of human serum and 80 μL 1x PBS was used in place of 80 μL cold buffer when adding the IL-5 Detection Antibody (PE). All flow cytometry data was analyzed using the Cytoflex or FLowJo v10 software.
Cell viability assays
D10 cells were treated as explained above with PMA and ionomycin in the presence or absence (DMSO alone) of 4μ8c as indicated in the figure legend. Annexin V and PI staining was done on cells incubated in the presence or absence of 4μ8c as above according to the manufacturer guidelines (Biolegend). The cells that were left unstimulated or stimulated in the presence of absence of 4μ8c were incubated with 0.5 mg/ml of MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reagent (Sigma-Aldrich) in a 96 well round bottom plate for two hours, washed with 1x PBS, and then lysed by incubating cells for 15 min at room temperature with 75% DMSO solution made in 1x PBS. The wells were immediately read by measuring absorbance at 595 nm wavelength on a Biomark plate reader (Biorad). In some experiments, the number of cells alive and dead were determined by counting cells with trypan blue (Sigma Aldrich) and calculating the percent alive of total cells.
RNA isolation and qRT-PCR
RNA was isolated from cells using Trizol (Invitrogen) and reverse transcribed using the SuperScript IV Reverse Transcriptase kit (Invitrogen) or QScript cDNA mastermix (Quanta BioSciences) according to the manufacture’s guidelines. cDNA reactions were carried out using a MiniCycler (MJ Research).
qPCR was carried out using Power Sybr Green (Invitrogen) on a MiniOpticon Real-Time PCR System (Bio-Rad), and the relative expression was calculated as previously described [
12]. β-actin was used to normalize all samples. Sample expression was then determined relative to the no stimulation control. The primers used in this study have been reported previously [
6,
42].
Western blot
Cells treated as above were lysed in RIPA buffer as previously described [
12]. The lysates were run on a 4–20% gradient SDS gel (Biorad), transferred to nitrocellulose, blocked in 3% (for the GATA-3 blots) or 5% milk in TBST, and blotted with the antibodies against the following: β-actin (Thermofisher, MA515739), IL-5 (My biosource, MBS821891), IL-13 (Abcam, ab106732), and GATA-3 (Santa Cruz, 1A12-D9).
Actinomycin D experiments
The cells were treated with 4μ8c or left untreated and stimulated with PMA and ionomycin as above for 24 h. The cells were then treated with actinomycin D (Sigma Aldrich) at a concentration of 3 μg/ml at 37 °C. The samples were harvested at 0, 10, 30, 60, and 90 min after exposure to actinomycin D. mRNA from all samples was isolated using Trizol, converted to cDNA, and analyzed via qRT-PCR as described above.
Statistical analysis
The data was analyzed using a 2-tailed Student’s unpaired T test, Welch’s correction. Use of the test was based on the fact that the samples to be analyzed were independently related and the variances between data sets could not be assumed to be equal. In Additional file
2: Figure S2a, a one-way ANOVA was run. The test was used to determine if cytokine gene expression differed between independent sample sets. The samples were considered to differ significantly if the
p value was less than 0.05. In all graphs the standard error is represented by error bars unless otherwise indicated. Power analysis was performed to determine the minimum number of samples to obtain for the experiments using previously published data where T cells were treated with 4μ8c and IL-4 was measured [
6]. The study group design is for two independent groups, continuous data, α = 0.05, and power 80%, with the minimum number needed equal to three samples per group. All statistical testing and analysis was performed after the predetermined sample size was obtained for each experiment.
Acknowledgements
We would like to thank Dr. Deyu Fang of Northwestern University for the generous gift of the D10 cells. We would like to thank Dr. Sallie Ruskoski of Northeastern State University for generously donating her time and performing the blood draws on the volunteers. We would like to thank Jocelyn Couch for working out the human T cell skewing protocol. We would like to thank Candace Morey and Awas Jasim for their help maintaining cell cultures, making buffers, and assisting in the laboratory. We would like to thank Dr. Janaki Iyer, Dr. Ralf Janknecht, and Mrs. Nadia Wieczorkowski for editing the paper, and Dr. Janaki Iyer for her help performing the MTT assays.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.