PDIA3 Expression in Glioblastoma Modulates Macrophage/Microglia Pro-Tumor Activation

Abstract

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia-glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

Keywords: IL6; PDIA3; STAT3; glioma; microglia; punicalagin.

Figure 1

Correlation between protein disulfide-isomerase A3 (PDIA3) expression in glioma tissues and patients’ survival: data from The Cancer Genome Atlas (TCGA) lower grade glioma and glioblastoma (GB) were examined for the correlation between overall survival and PDIA3 gene expression levels. A total of 690 patients (male and female combined) were divided into two groups with low (blue lines, n = 338) and high (red lines, n = 352) PDIA3 expression. Kaplan–Meier curves were generated to test for correlation by Log-rank (Mantel–Cox) test. p < 0.00001.

Figure 2

PDIA3 staining in human GB specimens: the photographs show a representative tumor field (A) and a representative positive parenchyma field (B) stained for PDIA3 and IBA1 (magnification: 40×). Yellow arrows point to double IBA1/PDIA3 stained cells, whereas black and green arrows point to IBA1 and PDIA3 positive cells, respectively. (C,D) The histograms represent the percentage of PDIA3 positive (C) or PDIA3/IBA1 double-positive cells (D) in human GB specimens, taking into account the tumor or the parenchyma regions (periphery). Data are expressed as mean ± SEM of samples collected from 18 patients. Tumor versus periphery, * p < 0.05; ** p < 0.01.

Figure 3

Effects of PDIA3 silencing on IL6, IL1β, and COX2 in human T98G cells: transcript detection was performed by qRT-PCR. Results indicate relative mRNA expression and are the mean ± SEM of three independent determinations. Data show a comparison between (A) IL6, (B) IL1β, and (C) COX2 gene expression of control T98G (calibrator) versus PDIA3-silenced (shPDIA3) T98G cells. **** p < 0.0001.

Figure 4

Semi-quantitative analysis of multiple cytokines, chemokines, growth factors, and other soluble proteins in cell culture media of glioma cells: (A,B) Results of protein analysis in B-Conditioned Medium (CM) collected from control (A) and PDIA3-silenced (B) T98G cells. Yellow squares and green squares indicate the downregulated factors and upregulated factors, respectively. (C) Semi-quantitative analysis of the released proteins showing a statistically significant increase or decrease with p < 0.05. (D) Proteins whose expression was induced after PDIA3 silencing. Data are the mean ± SD (4). For a complete list of proteins that are modified, see Supplementary Figure S3A.

Figure 5

Effects of PDIA3 silencing on IL6, IL1β, and COX2 in activated human GB cells: T98G cells were treated or not with TII (TNFα, IL1β, and hIFNγ) for 8 h, and detection of transcripts was performed by qRT-PCR. Results indicate relative IL6 (A), IL1β (B), and COX2 (C) mRNA expression in TII-treated cells compared to untreated cells (control or silenced cells, to which the value of 1 was attributed) and are the mean ± SEM of three independent determinations. Statistically significant differences between TII-treated control andPDIA3-silenced cells are shown. **** p < 0.0001.

Figure 6

Semi-quantitative analysis of multiple cytokines, chemokines, growth factors, and other soluble proteins in cell culture media of pre-stimulated GB cells: (A,B) Results of protein analysis in PS-CM collected from control (A) and PDIA3-silenced (B) T98G cells. Yellow squares and green squares indicate the downregulated factors and upregulated factors, respectively. (C) Semi-quantitative analysis of the released proteins showing a statistically significant increase or decrease with p < 0.05. (D) Proteins whose expression was detected only after PDIA3 silencing. Data are mean ± SD (4). For a complete list of proteins that appear to be modified, see Supplementary Figure S3B.

Figure 7

Viability of T98G cells after punicalagin (PUN) challenge: T98G cells were treated with 5 μM PUN for 24 h and then analyzed by the Annexin V-propidium iodide assay and flow cytometry. Viability was obtained from negative control cells without the Annexin V/PI conjugated fraction and expressed as percentage. Data are the mean (n = 8) ± SEM.

Figure 8

Viability of CHME-5 after punicalagin (PUN) challenge: CHME-5 cells were treated with 5 μM PUN for 24 h and then analyzed by the Annexin V-propidium iodide assay and flow cytometry. Viability was obtained from negative control cells without the Annexin V/PI conjugated fraction and expressed as percentage. *** p < 0.001. Data are the mean (n = 8) ± SEM.

Figure 9

Analysis of M2 phenotype of CHME-5 cells exposed to GB-derived CM: (A) analysis of urea production by CHME-5 cells treated with the indicated CMs for 48 h and (B) flow cytometry analysis of ARG1 expression. CHME-5 cells were treated for 24 h with CMs from T98G and PDIA3-silenced T98G cells. Data are shown as percentage of control. (C) Flow cytometry analysis of ARG1: (i) fluorescence intensity measurements for B-CMs and (ii) fluorescence intensity measurements for PS-CMs. Data are shown as mean (n = 8) ± SEM. * p < 0.05; ° p < 0.05; ** p < 0.01.

Figure 10

Effects of GB-derived CMs on the IL6-STAT3 pathway in microglial cells: (A) IL6 release, expressed as pg/mL per number of cells from CHME-5. Cells were treated with CMs for 24 h. IL6 was measurable only in cells treated with PS-CM. (B) Evaluation of p-STAT3 and STAT3 expression in microglia CHME-5 cells treated for 2 h with CMs from control and PDIA3-silenced T98G cells. Treatments: lanes 1 and 2, controls; lane 3, B-CM; lane 4, PDIA3-silenced B-CM; lane 5, PS-CM; and lane 6, PDIA3-silenced PS-CM. Data represent the results of densitometric analysis and are shown as the mean ± SEM of three independent determinations. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 11

Anti-inflammatory effects induced by punicalagin (PUN) on CHME-5 cells: (A) CHME-5 cells were treated for 48 h with proinflammatory cytokines mix (TII) alone and/or in association with punicalagin. (B) Reactive oxygen species (ROS) production after 48 h of treatment with TII alone and in association with punicalagin, in Relative Fluorescence Units (RFU). (C) IL6 release after 24 h of treatment with punicalagin. Data are expressed as mean ± SEM. **** p < 0.0001. Data are the mean (n = 6) ± SEM.

Figure 12

Evaluation of IκBα expression after punicalagin (PUN) challenge of microglial cells: CHME-5 cells were untreated (control) or treated with the different stimuli for 2 h. Treatments: lane 1, control; lane 2, TII; lane 3, TII + PUN 1 µM; lane 4, B-CM; lane 5, B-CM + PUN 1 µM; and lane 6, PUN 1 µM. TII, B-CM, and PUN are compared to the control; TII + PUN 1 µM and B-CM + PUN 1 µM are compared to the same treatments given alone. Data are shown as mean ± SD. * p < 0.05; **, °° p < 0.01; **** p < 0.0001.