Distinct activation profiles of cells treated with IFN-α2a or IFN-γ
To determine any differences between the cellular response of IFN-α2a and IFN-γ, we first examined the activation of STAT1 after each IFN treatment. Both IFN-α2a and IFN-γ treatment increased production of total STAT1 protein with similar kinetics, starting at 6 h and continuing through 48 h (). However, they activate STAT1 differently; treatment with IFN-α2a leads to immediate phosphorylation of STAT1 that begins to decrease at 2 h and remains at low levels for up to 48 h, whereas IFN-γ treatment results in the same initial phosphorylation profile as IFN-α2a, with an additional increase in pSTAT1 at 15–24 h ().
FIGURE 1 STAT1 signaling response in A549 cells treated with IFN-α2a and IFN-γ. Western blot of A549 cells treated with IFN-α2a and IFN-γ showing kinetics of STAT1 (via phosphorylation at Y701) and STAT2 (via phosphorylation at (more ...)
Upregulation of ISRE-containing genes by IFN-γ in A549 cells
Because the profile of STAT activation between IFN-α2a and IFN-γ showed a major difference in STAT1 phosphorylation at 24 h, we used gene expression microarrays to examine differences in gene expression at this time point. includes the data comparing statistically significant differences in gene expression (of at least 1.8-fold), as determined by Student t test, of A549 cells treated with 1 ng/ml IFN-α2a (200 IU/ml) or 1 ng/ml IFN-γ (10 IU/ml) for 24 h. Type I IFN (IFN-α2a) treatment resulted in distinct upregulation of several genes, including PLSCR1, TRIM6–TRIM34 (IFP1), LY6E (RIG-E), PNPT1, and HERC5 (). Similarly, IFN-γ treatment resulted in upregulation of a specific subset of genes, including IRF1, GBP2, GBP3, WARS, and genes involved in Ag presentation such as CD74 (). Interestingly, IFN-α2a and IFN-γ both upregulated a subset of genes that are traditionally associated with a type I IFN antiviral response and contain ISRE promoter motifs, including MxA (Mx1), PKR (EIF2AK2), and OAS1 (). We used quantitative RT-PCR (qRT-PCR) to validate the microarray data for a small subset of genes involved in the antiviral response. As expected, IFN-α2a treatment resulted in upregulation of MxA, OAS1, PKR, and IFIT3. However, these genes were also upregulated after treatment with IFN-γ (). Addition of a neutralizing Ab to IFNAR1 (which blocks type I IFN signaling) had no effect on induction of these genes by IFN-γ (data not shown).
Gene expression of select ISGs differentially regulated by either type I, type II, or both type I and type II IFNs
FIGURE 2 Gene expression of select antiviral ISGs after IFN-α2a and IFN-γ treatment. Cells were treated with IFN for 24 h, after which total RNA was collected and assayed for relative mRNA transcript amounts of the ISGs MxA (A), OAS1 (B), PKR ( (more ...)
We also examined expression of MxA, PKR, and IFIT3 (which is also regulated by an ISRE-containing promoter element) at the protein level. PKR is both constitutively expressed and IFN inducible. There was a significant increase in PKR after treatment with IFN-α2a beginning at 6 h. We observed the same pattern of expression in IFN-γ–treated cells, albeit to a much lesser extent (). Additionally, IFN-γ treatment resulted in a delay of PKR expression compared with IFN-α2a: induction started at 6 h for IFN-α2a and 15 h for IFN-γ. MxA and IFIT3 had the same pattern of protein expression as PKR after treatment with both IFN-α2a and IFN-γ; however, in both cases, IFN-α2a induction of these proteins was greater than for IFN-γ.
FIGURE 3 Expression of antiviral ISGs in A549 cells treated with IFN-α and IFN-γ. Cell lysates (from ) were evaluated for expression of the ISGs MxA, PKR, and IFIT3. Cells were treated with 200 IU/ml IFN-α2a or 10 IU/ml IFN-γ, (more ...)
IFN-γ induces formation of ISGF3II
To determine how these ISRE-containing genes were being upregulated, we examined the possibility that IFN-γ induces ISGF3. We were able to isolate the three components of the ISGF3 complex by coimmunoprecipitation following treatment with IFN-α2a or IFN-γ for 24 h (, lanes 7 and 8, respectively). The amount of the ISGF3 proteins precipitated did not decrease in the presence of a neutralizing Ab to IFNAR2 during the course of IFN-γ treatment (, lane 9). However, the amount of STAT1 and IRF9 that precipitated with STAT2 at 24 h decreased when an Ab that neutralized IFN-γ signaling was added to the media after 15 h of IFN-γ treatment (, lane 10). The complex was not present in untreated cells (, lane 6) or in samples precipitated with nonspecific immune serum (, lane 11). One percentage of the total cell lysate used for each sample was included to compare relative amounts of ISGF3/ISGF3II formation to the total amount of STAT1, STAT2, and IRF9 in each treatment group (, lanes 1–5). STAT1 and IRF9 were upregulated with each treatment, but STAT2 was not. We also used coimmunoprecipitation to determine whether STAT1 and STAT2 were phosphorylated after IFN-α and IFN-γ treatment. As expected, STAT1 was phosphorylated after each treatment (). Although IFN-γ treatment results in more STAT1 phosphorylation at 24 h, there is less pSTAT1 in the precipitated complex in response to IFN-γ compared with IFN-α2a. It is also interesting to note that whereas STAT2 was phosphorylated after IFN-α treatment, we were not able to detect phosphorylation of STAT2 in response to IFN-γ treatment (). Therefore, to avoid confusion with the classical ISGF3 complex, the complex containing unphosphorylated STAT2 will be referred to as ISGF3II.
FIGURE 4 Neutralization of STAT1 activation in IFN-γ–treated cells and accumulation of ISGF3 lacking STAT2 phosphorylation. A, STAT2 Abs were used to coimmunoprecipitate the ISGF3 complex in A549 cells. Cells were left untreated (lane 6) or treated (more ...)
To exclude the possibility that autocrine type I IFN led to the formation of ISGF3II, we then used qRT-PCR to determine whether a 24-h IFN-γ treatment induced other IFNs. IFN-γ treatment did not upregulate IFN-α1, IFN-α2, IFN-β, IFN-λ1, IFN-λ2 or IFN-λ3, and IFN-ω at 24 h (). Although IFN-α2a treatment led to statistically significant upregulation of IFN-λ1 at 24 h, none of the other IFNs were upregulated.
FIGURE 5 Gene expression of IFN genes after IFN treatment. Cells were left untreated or treated with 10 IU/ml IFN-α2a or IFN-γ for 24 h, after which total RNA was collected and assayed for relative mRNA transcript amounts of IFN-α1, IFN-α2, (more ...)
Next, we added a neutralizing Ab for IFNAR2 prior to treating cells with IFN-γ, then used pSTAT1 as a marker for IFN activation. There was no difference in the activation of pSTAT1 in IFN-γ–treated cells with neutralized IFNAR2 compared with those treated with IFN-γ alone (, lanes 4 and 2, respectively). The Ab was capable of neutralizing the activity of any type I IFN present over the course of the treatment (, lane 6). An Ab that neutralizes IFN-γ signaling was added to IFN-γ–treated cells at 15 h to allow for any possible cytokine production that might have corresponded to the induction of the second signaling peak from . The presence of this Ab completely abrogated pSTAT1 activity at later time points (, lane 5). We also used a neutralizing Ab for IL-10Rβ to inhibit any possible type III IFN signaling. This also had no effect on pSTAT1 activity after treatment with IFN-γ (data not shown).
FIGURE 6 Gene expression of select ISGs in IFN-γ–treated cells after neutralization with IFNGR1 Ab. A, IFN-treated cells were examined by Western blot for phosphorylation of STAT1 (at Y701) as an indicator of autocrine or paracrine IFN action. (more ...)
To further demonstrate that no other cytokines were responsible for the second pSTAT1 signaling peak, we added an Ab to neutralize IFN-γ signaling at 15 h, then harvested the cells at 24 h. The upregulation of PKR, IFIT3, MxA, and OAS1 gene expression was also reversed upon neutralization of IFN-γ signaling after treatment for 15 h; each of these ISRE-inducible genes showed significant downregulation to steady state or near steady state levels at 24 h (, respectively). In contrast, HLA-A, which is regulated by IRF1-containing transcription factors rather than ISGF3, showed no significant downregulation upon addition of the neutralizing Ab (), demonstrating that IRF1-mediated transcription continued after neutralization of IFN-γ signaling, and PKR, IFIT3, MxA, and OAS1 transcription was independent of IRF1.
STAT2 binds to ISRE-containing antiviral gene promoters after IFN-γ treatment
To show that ISGF3II is involved in transcribing IFN-γ–inducible genes, we used ChIP to demonstrate that STAT1, STAT2, and IRF9 physically bind to the promoters of the ISRE-containing genes following IFN-γ treatment. As expected, IFN-α2a induces recruitment of STAT1- and STAT2-containing complexes to the PKR promoter at 24 h (). However, STAT1-, STAT2-, and IRF9-containing transcription factor complexes are also recruited to the PKR promoter after treatment with IFN-γ (). Interestingly, IFN-α2a treatment did not result in statistically significant recruitment of IRF9 to the PKR promoter compared with untreated cells in this assay ().
FIGURE 7 Occupancy of the PKR promoter by (A) STAT1, (B) STAT2, and (C) IRF9 after IFN-γ and IFN-α2a treatment. Cross-linked, sheared chromatin ~1 kb in length from cells left untreated or treated with 10 IU/ml IFN-γ or 10 IU/ml IFN-α2a (more ...)
In addition, we analyzed the expression of ISRE-driven antiviral genes in IFN-γ–treated cells lacking STAT2 and IRF9. Both STAT2 and IRF9 siRNA treatment in A549 cells resulted in efficient inhibition of protein expression to nearly undetectable amounts (). Knocking down one protein had no effect on expression of the other, and neither siRNA treatment resulted in decreased expression of STAT1. We used qRT-PCR to examine how absence of these proteins affected the gene expression of the ISGF3-inducible proteins PKR and IFIT3. Loss of both STAT2 and IRF9 resulted in significant downregulation of PKR mRNA expression when treated with IFN-α and IFN-γ (). Similar results were obtained for IFN-mediated mRNA expression of IFIT3 following STAT2 and IRF9 siRNA treatment ().
FIGURE 8 Gene and protein expression of ISGs in STAT2- and IRF9-deficient cells after treatment with IFN-α2a or IFN-γ. A, STAT2 and IRF9 protein expression was knocked down by siRNA. Cells were either mock treated (lipofectamine only), treated (more ...)
Biological role for ISGF3II in the antiviral activity of IFN-γ
Although ISGF3II is present and classical antiviral genes driven through the ISRE promoter are expressed in A549 cells treated with IFN-γ, it is unclear whether this phenomenon has any biological function in these cells. Therefore, to disrupt ISGF3/ISGF3II formation after IFN treatment, we used siRNA to knock down STAT2 and IRF9. STAT2 and IRF9 siRNA reduced the ability of A549 cells to mount an antiviral response against ECMV after treatment with either IFN-α2a () or IFN-γ (). In IFN-α2a–treated samples, antiviral activity was inhibited up to a concentration of 25 IU/ml in the STAT2 knockdowns, and activity was partially abrogated even at 100 IU/ml in the IRF9 knockdown cells. Either STAT2 or IRF9 knockdown inhibited the antiviral activity of up to 10 IU/ml IFN-γ.
FIGURE 9 The absence of IRF9 or STAT2 abrogates the antiviral activity of IFN-γ in A549 cells. Specific siRNAs targeting IRF9, STAT2, or nonspecific (NS) control siRNAs were transfected at a concentration of 20 nM for 24 h before addition of the indicated (more ...)
We also added neutralizing Abs for IFNAR2 and IL-10Rβ both during transfection and IFN treatment as well as upon addition of EMCV to block any possible production and signaling by type I or type III IFNs. These treatments did not result in increased abrogation of antiviral activity, demonstrating that these IFNs do not play a role in the antiviral activity of IFN-γ (data not shown).