Acidic environment could modulate the interferon-γ expression: Implication on modulation of cancer and immune cells’ interactions

Exponentially growing HeLa cells (human cervical cancer), Jurkat cells (human T cell lymphoma), and THP-1 cells (monocytes) were procured from the National Centre for Cell Science (NCCS), India. THP-1 (monocytes) were differentiated to macrophages by using PMA (5 ng/mL) for 48 h [26]. HeLa cells were allowed to adhere for 48 h before being exposed to experimental conditions. The cells were cultured at 37°C under humidified 5% CO₂ and a 95% air atmosphere. The cell density was maintained at a level lesser than 3 × 10⁵ cells/mL in 25 cm² plastic tissue culture flasks with 10 mL of RPMI-1640. The culture medium was supplemented with 10% fetal bovine serum (Hi Media). LPS-free reagents were used during the experiments. The pH of the culture media was adjusted using 1N HCl. The experiments were carried out in the exosome-free fetal bovine serum [27].

To ascertain the effect of acidic pH on immune cells, Jurkat (T cells) and THP-1 (macrophages) cells were cultured in pH 7.4, pH 6.9, and pH 6.5 media (Figure 1A). Further, to ascertain how cancer cells in an acidic environment can influence immune response, HeLa cells were plated at a density of 0.5 × 10⁶/mL in media having pH values of 6.5, 6.9, and 7.4. A low cell density was maintained, avoiding the effect of media starvation. After 24 h of incubation, the supernatant from HeLa cells was centrifuged at 2000 rpm for 5 min followed by filtration through a 0.22 μm membrane for further experimentation. This was referred to as HCM (Figure 1B). Further, Jurkat and THP-1 cells were plated at the density of 0.5 × 10⁶/mL in HCM of pH 7.4, pH 6.9, and pH 6.5 for 24 h (Figure 1B). The experiment was not extended beyond 24 h and at a pH lower than 6.5 since both these conditions have been shown to induce apoptosis in cells [28].

Figure 1.

The cytotoxic effect of acidic pH was studied on Jurkat, HeLa, and macrophages cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [29]. Briefly, the cells were cultured in 96 well plates at a density of 1 × 10⁴ cells per well at different pH values (7.4, 6.9, and 6.5). After incubation for 24 h, MTT (5 mg/mL in PBS) was added to each well and further incubation was carried out at 37°C for 2 h. Dark blue formazan crystals formed were dissolved in DMSO and optical density was measured by a microplate reader (Bio-Rad) at a wavelength of 570 nm.

Isolation of total RNA from cells after exposing them to experimental conditions was done using TRIzol reagent (Sigma). First strand cDNA (complementary DNA) synthesis was carried out using the Verso cDNA kit (Thermo Scientific). DNA contamination in the isolated RNA was eliminated using the RT enhancer available with the kit. Semi-quantitative PCR was carried out and amplified PCR products were resolved by 2% agarose gel electrophoresis. Quantitative real-time PCR analysis was performed in a real-plex system (Eppendorf) using the SYBR Green PCR Master mix (Thermo Scientific). Real-time PCR was carried out for IFN-γ, IL-10, IL-18, and β-actin using gene specific primers, as mentioned in Table 1. β-actin served as an internal control. Melting curve analysis was performed to confirm the specificity of amplified products, and delta CT method was used to quantify the alteration in gene expression.

Both Jurkat and THP-1 cells (0.5 × 10⁶ cells/mL) were washed in PBS, then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Bioscience) for 20 min on ice. Cells were pelleted, washed with perm wash buffer (BD Bioscience), and stained for 1 h at 4°C with a fluorochrome-conjugated anti–IFN-γ monoclonal antibody (BD Bioscience). Cells were washed twice with a perm wash buffer and finally re-suspended in a perm wash buffer for flow cytometry analysis. Flow cytometric analysis was performed on BD FACS Canto II (BD Biosciences) for maximum cell counts of 10,000 and analyzed using BD FACSDiva software.

After culturing of HeLa cells for 24 h at different pH, the supernatant was collected and subjected to the centrifugation steps of 400 × g (10 min) to remove the intact cells, 3000 × g (20 min) to remove the cell debris, and 10,000 × g (30 min) to remove micro vesicles. The exosomes were then pelleted at 64,000 × g (110 min) and 100,000 × g (sucrose gradient) for 1 h. Total amount and concentration of exosomal proteins in the pooled samples were measured by the BCA method using protein estimation kit (Bangalore Genei) and subjected to western blot analysis (IL-18, CD 63).

Cells were harvested, washed with cold PBS, and lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM ethylene glycol-bis (b-aminoethyl ether)-tetraacetic acid, 1 mM EDTA, 50 mM NaF, 1 mM b-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM orthovanadate, protease inhibitor cocktail, and 1 mM PMSF). Protein estimation was determined by the BCA method using a protein estimation kit (Bangalore Genei). An equal amount of cell lysate from different test samples was resolved on 10% SDS-PAGE followed by transfer onto a PVDF membrane. The blots were probed with (1:1000) anti NF-κB (Sigma), IL-18, p38 MAPK, and CD63. Horseradish peroxidase-conjugated secondary antibodies were used to detect immune reactive bands using 3,3′-diaminobenzidine as a substrate.

After treatment, Jurkat and THP-1 cells were suspended in hypotonic buffer (250 mM sucrose, 20 mM Hepes [pH 7.5], 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 100 μM PMSF) on ice and incubated for 30 min, followed by lysing the cells by vigorous pipetting for 30 min. Cell lysate from the previous step was centrifuged at 3000 rpm for 5 min at 4°C to remove nuclei. The supernatant was saved as cytoplasmic fraction. The pellet was washed again and saved as nuclear fraction and further subjected to western blotting as described above.

To determine the role of acidic environment (as an intermediate medium between cancer cells and immune cells) in the modulation of immune and cancer cells’ interactions, Jurkat or THP-1 cells were cultured in HCM of pH 6.5, pH 6.9, and pH 7.4 for 20 h and the obtained cell-free medium (CM1) was saved for cytotoxicity assessment (Figure 2). Further, the cells thus harvested were then cultured in fresh medium at pH 7.4 for 4 h and cell-free culture medium (CM2) was again saved for cytotoxicity assessment (reversal of acidic pH 6.9, pH 6.5 to physiological pH 7.4). HeLa cells were then cultured in CM1 and CM2 separately for each pH condition and viability was assessed by MTT assay to examine the cytotoxic effect of biological factors secreted by Jurkat cells, macrophages and modulated by different pH conditions and after reversal of pH to 7.4 (Figure 2).

Figure 2.

The needed statistical procedures were applied using SPSS 13.0, and data are presented as mean ± standard error of mean (SEM). One-way ANOVA (Analysis of variance) was used to analyze the results and to determine the significance of the mean between groups. P values <0.05 were considered significant.

Before initiating the experimental study, the effect of acidic pH on the viability of cells was examined. The viability of HeLa and THP-1 cells did not differ significantly (P = 0.3281), whereas Jurkat cells showed a nearly 10% decrease in viability at pH 6.5 when compared with cells cultured at pH 7.4 (P = 0.04).

Upon exposure of Jurkat cells to acidic conditions (pH 6.5), the expression of IFN-γ mRNA declined in comparison with Jurkat cells exposed to pH 7.4 (Figures 3A and 3C). On the other hand, Jurkat cells cultured in pH 6.9 medium demonstrated upregulation of IFN-γ mRNA by 2.3 fold when compared with control (pH 7.4) (Figures 3A and 3C) (P = 0.22). These results were further supported by flow cytometric analysis (Figure 3E). Jurkat cells cultured at physiological pH 7.4 and stained with fluorochrome-conjugated anti–IFN-γ antibody had a mean fluorescence of 2227 whereas cells cultured at pH 6.9 and pH 6.5 had mean fluorescence of 2477 and 2043, respectively (Figure 3E). Culturing of differentiated THP-1 cells in the acidic microenvironment could have resulted in a decrease in IFN-γ expression at both pH 6.5 (P = 0.022) and pH 6.9 (P = 0.41) (Figures 3B and 3D). Flow cytometric analysis of fluorochrome-conjugated anti–IFN-γ antibody stained THP-1 cells cultured at physiological pH 7.4 revealed a mean fluorescence of 1465, while cells cultured at pH 6.9 and pH 6.5 revealed mean fluorescence of 971 and 965, respectively (Figure 3F). Collectively, this indicated a restriction in IFN-γ expression in both macrophages and T cells in the acidic microenvironment, which may play a role in thwarting the immune response.

Figure 3.

To explore the role of factors secreted by cancer cells that help them to evade the immune response, Jurkat cells and macrophages were cultured in HCM. Jurkat cells cultured in HCM at pH 6.9 and pH 6.5 demonstrated 1.5 fold (P = 0.24) and 3.7 fold (P = 0.07) upregulation in levels of IFN-γ mRNA, respectively, than Jurkat cells cultured in HCM at pH 7.4 (Figures 4A and 4C). Flow cytometric analysis of fluorochrome-conjugated anti–IFN-γ antibody stained Jurkat cells cultured in HCM at pH 7.4 revealed a mean fluorescence of 1931, whereas cells cultured in HCM at pH 6.9 and pH 6.5 revealed mean fluorescence of 2133 and 2695, respectively (Figure 4D and E). These results suggest that the acidic microenvironment may be beneficial to IFN-γ expression. Following that, the level of IFN-γ mRNA in Jurkat cells cultured at pH 7.4 (without HCM) was significantly downregulated (8 fold) than in Jurkat cells cultured in HCM at pH 7.4 (Figure 5A) (P = 0.02). When Jurkat cells were cultured at pH 6.9 without and with HCM, there was a further downregulation (4 fold) in IFN-γ expression (Figure 5A) (P = 0.48). In contrast, when similar studies were performed at pH 6.5, IFN-γ mRNA was found to be upregulated (2 fold) (Figure 5A) (P = 0.29). Furthermore, when THP-1 cells were cultured in HCM, the level of IFN-γ was downregulated at pH 6.9 (P = 0.58) and pH 6.5 (P = 0.35) compared with pH 7.4 (Figure 4B). Similarly, flow cytometric analysis of THP-1 cells cultured in HCM at pH 7.4 revealed a mean fluorescence of 1264, while cells cultured in HCM at pH 6.9 and pH 6.5 revealed mean fluorescence of 995 and 1067 (Figure 4F). When THP-1 cells were cultured in media with pH 7.4, pH 6.9, and pH 6.5 and THP-1 cells were cultured in HCM with pH 7.4, pH 6.9, and pH 6.5 compared, upregulation of IFN-γ (4.5 fold) was observed at HCM pH 6.5 as compared to pH 6.5 (Figure 5B) (P = 0.065). RT PCR was used to examine IFN-γ expression in HeLa cells under different pH conditions (pH 7.4, pH 6.9, and pH 6.5). The results showed that IFN-γ was not expressed in HeLa cells at any pH.

Figure 4.

Figure 5.

The upregulation of IFN-γ in Jurkat and THP-1 cells cultured in HCM at acidic pH suggests that cancer cells may secrete biological factors to stimulate these cells. As a result, we examined the expression of IL-10 and IL-18, which have been shown to alter IFN-γ expression in various cells [34]. Total mRNA from HeLa cells (pH 7.4, pH 6.9, pH 6.5) was isolated and analyzed for the expression of IL-10 and IL-18 to investigate their role in IFN-γ expression. No expression of IL-10 was observed. However, enhanced expression of IL-18 was observed in HeLa cells cultured in a medium of acidic pH (Figure 6A). As a result, efforts were made to investigate the role of IL-18 in the acidic microenvironment and its relationship to IFN-γ expression. The acidic microenvironment around cancer cells stimulated the expression of IL-18, which in turn might be correlated with the IFN-γ mediated immune response. Further, western blot analysis of exosomes prepared from the cell-free supernatant of HeLa cells showed an association of IL-18 with the exosomes (Figure 6B). IL-18 was reported to modulate IFN-γ expression via NF-κB and MAPK kinase mediated pathways [35]. Therefore, the role of NF-κB and MAPK molecules in the modulation of IFN-γ were further investigated. At lower pH, HCM treated Jurkat and THP-1 cells showed a decrease in p38 MAPK expression (Figure 7). An increase in the level of NF-κB in the nuclear fraction was evident both in Jurkat cells and THP-1 cells cultured in HCM at lower pH. These results suggest the involvement of NF-κB in the modulation of IFN-γ. These findings suggest that under acidic conditions, HeLa cells secreted exosome-anchored IL-18, which might be correlated with IFN-γ expression.

Figure 6.

Figure 7.

As previously stated, the acidic environment induced IL-18 correlated with IFN-γ upregulation in Jurkat and THP-1 cells. Therefore, attempts were made to decipher the behavior of HeLa cells in the culture medium taken from Jurkat and THP-1 cells grown in a medium of different pH. To investigate this, HeLa cells were cultured for 24 h in CM1 and CM2 (pH 7.4, pH 6.9, and pH 6.5) obtained from Jurkat cells to mimic the natural tumor conditions as described in the Methods section. HeLa cells showed 88% viability at pH 6.5 (Figure 8B) in CM1, whereas the viability was decreased to 48% in cells cultured in CM2 obtained from the growth of Jurkat cells (reversion of pH to 7.4) (P = 0.32). The viability of HeLa cells was determined similarly to the CM1 and CM2 obtained from THP-1 cells. At pH 6.5, only 55% of the HeLa cells were viable in CM1, while viability was reduced to 17% in cells cultured in CM2 (Figure 8A) (P = 0.04).

Figure 8.