A2EN cells or CECs were treated with stated cytokines at 1 or 10 ng/ml for 4 or 24 h before RNA extraction. Gene expression of IFNα, IFNβ, or IFNε was determined by RT-PCR analysis. As expected, the baseline of IFNα or IFNβ was low in A2EN and CECs. The IFNε mRNA level was 315- and 23-fold more abundant than IFNα and IFNβ mRNAs in A2EN cells, respectively, and 97-fold and 26-fold more abundant than IFNα and IFNβ mRNA levels in CECs, respectively (data not shown). Pro-inflammatory cytokines such as TNFα (1 ng/ml) and IL-1β at 1 and 10 ng/ml significantly induced IFNε by 7.7 and 4.6-fold, respectively. IL-1β had a moderate effect on IFNα expression (twofold induction) in A2EN cells (Fig. 1a). Gene induction of IFNα and IFNε by TNFα was also found in CECs (Fig. 1b). Interestingly, TNFα suppressed IFNβ expression by 40–50% in A2EN cells. IFNβ suppression by TNFα at 1 ng/ml for 4 h incubation in CECs was observed; however, treatment of TNFα for 24 h induced IFNβ in CECs.
IL-6 or IL-8, which are often elevated during inflammation at the mucosa, was noted to induce IFNε in A2EN cells after 24 h incubation, but the induction was not significant when various experiments were compared (Fig. 1c). The effects of IL-6 and IL-8 on IFNε induction were not prominent in CECs. Interestingly, IL-8 significantly suppressed IFNβ in A2EN cells (25–77% suppression) and CECs (61% suppression). IL-8 treatment for 4 h also suppressed IFNα in A2EN cells (53–71% suppression) but the significant suppression of IFNα was observed in CECs after the 1 ng/ml for 24 h incubation (64% suppression).
IL-17 and IL-22 play a crucial role in mucosal immune homeostasis [11]. In A2EN cells, IL-17 slightly elevated the IFNα mRNA level by 1.8-fold but had a trend to suppress expression of IFNβ (p=0.057) (Fig. 1c). In CECs, IL-17 significantly suppressed IFNα and β after 4 h incubation. The suppression of IFNε by IL-17 was observed but not significant (p=0.07). IL-22 has similar but distinct functions compared to IL-17 [11]. In A2EN cells, IL-22 significantly suppressed IFNα and β at both concentrations after the 4 h incubation. IL-22 continued to suppress IFNβ after incubation for 24 h. The suppressive effect of IL-22 on IFNε was limited at 10 ng/ml for 4 h incubation. IL-22 did not have a significant impact on IFNα expression but slightly induced IFNβ or IFNε (1 ng/ml for 24 h) in CECs. IL-22 at 10 ng/ml significantly suppressed IFNβ after 4 h incubation, indicating that IL-22-mediated IFNβ gene regulation depended on the cytokine concentration and the incubation time.
IFNα, induced in response to infection or Toll-like receptor activation, is known to induce type I IFNs [12]. Because type I IFN signaling can be cell-type specific [13], we determined the effect of IFNα on expression of IFNs in A2EN cells and CECs. IFNα moderately induced all three IFNs by 2–3 fold in A2EN cells after 24 h treatment although only induction of IFNα was statistically significant (Fig. 1c). IFNα slightly induced IFNα but had no effect on IFNε in CECs. Regulation of IFNβ by IFNα appeared to be time and concentration dependent. IFNα at 10 ng/ml suppressed IFNβ expression by 60% after 4 h incubation, but there was a trend of IFNβ induction after 24 h incubation.
TNFα is known to activate the transcription factor NFκB [14] although the previous report showed that it did not induce the IFNε promoter activity in HeLa cells [9]. To determine whether the NF-κB pathway was involved in TNFα-mediated IFNε gene expression, primary cervical cells were treated with the IκB kinase (IKK) inhibitor, BAY 11-0782, to block NF-κB activation. BAY 11-0782 blocked TNFα-mediated IFNε induction, suggesting the role for the NFκB pathway in IFNε gene regulation (Fig. 2a). Note that the suppression of IFNε induction by BAY 11-0782 was not due to cytotoxicity as determined by using the CytoTox-Glo Cytotoxicity assay (data not shown) (Promega). We confirmed that the IKK inhibitor blocked NFκB p65 serine-536 phosphorylation in response to TNFα stimulation (Fig. 2b). We then generated a DNA construct with a luciferase reporter gene under the control of IFNε promoter (nt − 1000 to + 20). The pGL3 with IFNε promoter construct had a low basal luciferase activity compared to the promoterless pGL3 basic vector, and TNFα significantly induced the IFNε promoter activity (Fig. 2c). Importantly, BAY 11-0782 blocked TNFα-mediated activation of the IFNε promoter, suggesting the involvement of the NFκB pathway in IFNε gene regulation (Fig. 2d).
Our results showed that TNFα induced IFNε gene expression in both A2EN and CECs through NF-κB signaling. The degree of induction in these cells was more pronounced than that previously reported in HeLa cells [9]. Additionally, we observed TNFα-mediated IFNα induction, which is absent in HeLa cells [9]. The discrepancy may be due to the limited passage of A2EN and CECs, which could preserve a more primary cell-like immune response. Activation of the IFNε promoter by TNFα was not observed in the study by Matsumiya et al. [9]. The discrepancy may be due to the different promoter region (Matumiya, nt − 497 to + 296 vs nt − 1000 to + 20 in this study) or different cell types. NF-κB binding sites have been identified at the − 53 and − 854 nt position in the proximal promoter of IFNε [2], which were included in our promoter construct. Our study suggests the role for NFκB in IFNε gene regulation. Because both TNF-α and IL-1β activate NF-κB, p38 MAPK, and JNK [14, 15], further analysis of the IFNε promoter function and of the role of transcriptional factors and kinases involved in IFNε gene expression will produce a better understanding of IFNε regulation by cytokines at mucosa.
Although TNFα-mediated induction of IFNα and IFNε was consistent between A2EN and CECs, induction of IFNε in response to various cytokines was noted to be stronger in A2EN cells than CECs. Similarly, suppression of IFNs by IL-22 was apparent in A2EN cells. Differential profiles of IFN regulation between A2EN and CECs may be due to the tissue origin (e.g. ectocervix or endocervix), the presence of HPV E6/E7 in A2EN cells, or cells at different differentiation states. A2EN is an endocervical cell line, representing a uniform cell population, whereas CECs were composed of heterogeneous epithelial cells from cervical tissues, mostly ectocervix. It is possible that some cell populations in CECs may not be responsive to stimulation.
In summary, our study demonstrated that TNFα and IL-1β were potent inducers of IFNε and that the NF-κB pathway was involved in TNFα-mediated IFNε gene regulation. Expression of IFNα, IFNβ and IFNε was regulated differentially in response to specific cytokines, and gene regulation of IFNs by specific cytokines can be concentration-, time-, and cell type-dependent.