Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-beta induction via X-box binding protein 1

Abstract

Type I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-beta induction. This synergy is not dependent on autocrine type I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein 1 (XBP-1). Synergistic IFN-beta induction also occurs in HLA-B27/human beta(2)m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous 'danger' that disrupts ER homeostasis is coupled to IFN-beta induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR.

Figure 1

Synergistic induction of type I IFN in macrophages undergoing an unfolded protein response. RAW267.4 macrophages were pretreated with Tm for 8 h (A) or Tpg for 1 h (B) then stimulated with LPS (10 ng/ml) for 2 h prior to RNA isolation and quantitative PCR (qPCR) analysis for IFN-β transcripts. (C) RAW264.7 macrophages were treated with Tpg for 1 h, then LPS was added for an additional 24 h prior to collection of supernatants for IFN-β ELISA. (D) RAW264.7 cells were pretreated with Tpg for 1 h then dilutions of LPS for 3 h. (E, F) Rat BM derived macrophages were pre-treated with Tpg for 1 h then LPS for the times indicated. In (C, E, F), ν represents LPS only, and indicates Tpg plus LPS. IFN transcript levels were measured with qPCR and are shown normalized to GAPDH. Results shown are means ±SD (A–C) and are representative of 4 (Tm) or 5 (Tpg) experiments. Asterisks (*) indicate p<0.05 for average fold induction for LPS plus Tpg (or Tm) vs. LPS alone, from combined experiments. Concentration curve and time course (D–F) are representative of two experiments.

Figure 2

Specificity of ER stress effects on cytokine induction. BM-derived mouse macrophages were pre-treated with Tpg for 1 h then LPS for an additional 3 h. Tpg alone was present for 4 h, and LPS 3 h. Relative expression of cytokine mRNAs was determined by qPCR and is shown normalized to GAPDH. Results shown are mean ±SD and are representative of 3 independent experiments. Asterisks (*) indicate p<0.05 for fold induction with LPS plus Tpg vs. LPS alone from combined experiments.

Figure 3

Specificity of synergistic IFN-β induction. (A) RAW267.4 macrophages were pretreated with Tpg for 1 h, then LPS (TLR4), CpG (TLR9), exogenous poly(I:C) (TLR3), or Pam3CysSK4 (TLR2), and RNA analyzed by qPCR for IFN-β transcripts. (B) HEK293 cells were pre-treated with Tpg for 1 h, then transfected with dsRNA (poly(I:C)) as indicated. Cells were harvested at 7 h. Relative expression of IFN-β mRNA is shown normalized to GAPDH. Results shown are mean ±SD and are representative of 2 independent experiments. Asterisks (*) indicate p<0.05 for fold induction with TLR agonist plus Tpg vs. TLR agonist alone from combined experiments.

Figure 4

Synergy does not require type I IFN receptor or IRF7 induction. BM-derived macrophages from WT control mice (IFNAR+/+) or ifnar-deficient mice (IFNAR^−/−) were treated with Tpg for 1 h then LPS for 3 h. IFN-β (top) and IRF7 (bottom) transcript levels were assessed by qPCR. Relative mRNA expression normalized to GAPDH is shown. Results shown are mean ±SD and are representative of 3 independent experiments. Asterisks (*) indicate p<0.05 for fold induction with LPS plus Tpg vs. LPS alone from combined experiments.

Figure 5

XBP-1 is required for synergistic IFN-β induction. (A) WT (PERK^+/+) and PERK-deficient (PERK^−/−) MEFs were stimulated with Tpg for 1 h then LPS for 2 h. IFN-β mRNA expression was assessed by qPCR. (B) WT (XBP-1^+/+) or XBP-1-deficient (XBP-1^−/−) MEFs were incubated with Tpg for 1 h, then poly(I:C) for 6h. IFN-β mRNA expression is shown normalized to GAPDH. Results shown are mean ±SD and are representative of 2 independent experiments. Asterisks (*) indicate p<0.05 for combined experiments comparing fold induction with LPS or PolyI:C plus Tpg vs. TLR agonist alone, and in B PolyI:C plus Tpg in XBP-1+/+ vs. XBP-1−/−.

Figure 6

Knockdown of XBP-1 expression prevents synergistic IFN-β induction. (A) HEK293 cells expressing TLR4 and CD14 were transiently transfected with MD2 and control (■) or XBP-1 (ν) RNAi. At 24 h post transfection, cells were pretreated with Tpg for 1 h then stimulated with LPS for 3 h. Relative expression of XBP-1 mRNA by qPCR is shown normalized to GAPDH. (B) Semiquantitative PCR for XBP-1 and actin were performed on samples from (A), showing knockdown of both XBP-1u and XBP-1s. A representative gel used for quantitation is displayed with samples in the same order as in (A). Relative expression of IFN-β is shown normalized to GAPDH. Results shown are mean ±SD and are representative of 2 independent experiments. Asterisk (*) indicates p<0.05 for combined experiments comparing control vs. XBP-1 RNAi for XBP-1 expression (A) and IFN-β expression (B).

Figure 7

Spliced XBP-1 enhances IFN-β induction. XBP-1^−/− MEFs (A) and RAW264.7 macrophages (B) were transfected with XBP-1s (ν) or vector control (■). At 24 h post transfection, MEFs were treated as in Figure 6. RAW264.7 cells were pretreated with 1 μM Tpg for 1 h then LPS for 3 h. IFN-β mRNA expression is shown normalized to GAPDH. Results shown are mean ±SD and are representative of 3 independent experiments. Asterisks (*) indicate p<0.05 for combined experiments comparing control vs. XBP-1s for the conditions indicated.

Figure 8

Synergistic IFN-β induction occurs in B27-Tg macrophages and correlates with increased XBP-1 splicing. BM-derived macrophages from F344 B27-Tg (ν) or WT control rats () were activated with IFN-γ for 20 h, then stimulated with 10 ng/ml LPS for 1–4 h. (A) Relative IFN-β transcripts were measured by qPCR. In (B), XBP-1 splicing in WT () and B27-Tg (ν) macrophages was determined as described in the legend to Figure 6. IFN-γ activated macrophages from Lewis WT (), B7-Tg(■), or B27-Tg (ν) rats, were stimulated with LPS for 2 h. IFN-β message levels were determined by qPCR (C) and XBP-1 splicing by PCR (D) as in (B). Results in A are the average of duplicates and are representative of 3 experiments. Results shown in B-D are mean ±SD and are representative of at least 2 experiments. In (B) p<0.05 for B27-Tg vs. WT, where it is barely detected. In (C and D) asterisks (*) indicate p<0.05 for B27-Tg vs. B7-Tg and WT. Representative XBP-1 splicing gel lanes in B and D are in the same sequence as the quantitative data.