Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs

Abstract

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αβ unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αβ is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αβ to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αβ in the absence of Stat1. These studies reveal that IFN-αβ is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

Figure 1. L. pneumophila growth in Stat1[−/−] and Stat2[−/−] BMMs

L. pneumophila (JR-32; Lp: MOI=0.25) growth was evaluated by a colony forming assay (24, 48 or 72 hrs post infection) in 129 (WT) control, (A) Stat1[−/−], (B) Stat2[−/−] and (C) Stat1/Stat2[−/−] double knockout BMMs in the 129 background (2.5 × 10⁵/well of 24-well plate), as previously described (17). Some BMMs were pretreated with a single dose of IFN-α_A/D (1000 U/ml) or IFN-γ (50 U/ml) 2 hours prior to infection. Please note that panels A and B were previously published (17) and are included solely for comparisons sake. Studies are representative of more than 3 independent experiments. Similar results were obtained in the C57Bl/6J background.

Figure 2. Delayed kinetics of IFN-α stimulated gene expression and Stat2 activation

(A) The kinetics of IFN-α_A/D (1000 U/ml) dependent Mx-1 and ISG-15 expression was evaluated by Q-PCR in C57Bl/6J (WT), Stat1[−/−], Stat2[−/−] and IRF-9[−/−] BMMs. Similar results were obtained with IFN-β treatment and BMMs from the 129 background. (B) Whole cell extracts (WCEs) were prepared from day 6 (d6) C57Bl/6J (WT), Stat1[−/−] and Stat2[−/−] BMMs afterstimulation with IFN-α_A/D (1,000 U/ml), as indicated. Extracts were fractionated and immunoblotted with antibodies specific for phospho-Stat1 (pSt1; Cell Signaling), phospho-Stat2 (pSt2; UBI) and Tubulin (Sigma-Aldrich). The same extracts were re-fractionated by SDS-PAGE and immunoblotted for total-Stat1 (tStat1; Santa Cruz) and total-Stat2 (tStat2; (11)). Studies are representative of more than 3 independent experiments. Similar results were obtained with IFN-β treatment and the 129 background.

Figure 3. IFN-Is activate a novel ISRE binding complex in Stat1[−/−] BMMs

(A) Nuclear extracts from IFN-α_A/D treated (1000 U/ml; 0.5 & 16 hrs), d6 C57Bl/6J (WT) and Stat1[−/−] BMMs were evaluated with a radiolabeled ISRE probe. In some samples, a seven fold excess of cold ISRE probe (comp) or antibodies specific for Stat2 (α-St2) or IRF-9 (α-IRF9) were added. Migration of ISGF3 and the “novel” complex are indicated. Studies are representative of more than 3 independent experiments. Similar results were obtained in the 129 background. (B) For ChIP studies, IFN-α treated C57Bl/6J (WT), Stat1[−/−] and Stat1[−/−]Stat2[−/−] BMMs, as above, were crosslinked, sonicated and immunoprecipitated with a Stat2-specific antibody and interrogated by Q-PCR. Error bars represent the SEM, and asterisks denote statistically significant differences (**, p<0.01; ***, p<0.0005).

Figure 4. IRF-9 is required for IFN-I stimulated ISG expression in Stat1[−/−] BMMs

(A) Efficiency of IRF-9 knockdown was evaluated by Q-PCR in C57Bl/6J (WT) and Stat1[−/−] iBMMs (top left). IFN-α_A/D dependent Mx-1 expression was determined by Q-PCR in C57Bl/6J (WT) iBMMs stably knocked down with either sh-IRF-9 or an shRNA control (Open Biosystems; top right). IFN-α_A/D dependent Mx-1 and ISG-15 expression was similarly evaluated in Stat1[−/−] iBMMs stably knocked down with either sh-IRF-9 or an sh-control. (B) IFN-α_A/D dependent Mx-1 and ISG-15 expression was similarly evaluated in C57Bl/6J (WT) or Stat1/Stat2 double knockout BMMs (Stat1/2^−/−). Data represents three independent replicates.

Figure 5. Jak1 is activated with delayed kinetics in IFN-I stimulated Stat1[−/−] BMMs

(A) Jak1 was immunoprecipitated from IFN-α_A/D C57Bl/6J (WT) and Stat1[−/−] BMM WCEs and immunoblotted with an antibody specific for activated Jak1, as in Figure 2. (B) IFN-α_A/D stimulated WCEs were prepared from C57Bl/6J (WT) and Stat1[−/−] BMMs and immunoblotted as in Figure 2 (left panels). P6 inhibitor (2 μM) was added for either 1 or 4 hours before IFN-I stimulated extracts were harvested, as indicated. Samples were then immunoblotted as described in Figure 2. (C) Mx1 and ISG15 expression was evaluated by Q-PCR in samples from panel B, as detailed in Figure 2. (D) Socs1 expression was evaluated by Q-PCR in C57Bl/6J (WT), Stat1[−/−], Stat2[−/−] and IRF9-[−/−] treated BMMs, as described in Figure 2. Data represents three independent replicates.