Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells

Abstract

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

ATF4 and GADD34 are induced during DC activation with pI:C. Experiments were performed in bmDCs stimulated with 10 μg/mL of pI:C or, when indicated, in RAW macrophages with 100 ng of LPS. mRNA levels of ATF4 (A), CHOP (C), GADD34 (E), PP1 (G), and CReP (H) were measured by qPCR. (B) ATF4 protein levels were detected by immunoblot in nuclear extracts. Histone deacetylase 1 (HDAC1) is shown as loading control. Protein levels of CHOP (D) and GADD34 (F) were detected by immunoblot in total cell lysates. pI:C was added as soluble or lipofected (pI:C-lip). When indicated, MG132 was used to inhibit proteasomal degradation of GADD34 and was added for 2 h before harvesting the cells. ER stress-inducing drugs thapsigargin (th) or tunicamycin (tun) serve as positive control treatments for the expression of ATF4, CHOP, and GADD34. Data represented in A, C, E, G, and H are mean ± SD of three independent experiments.

Fig. 2.

eIF2α dephosphorylation is controlled by GADD34 during pI:C-induced DC maturation. (A) Protein synthesis was quantified in protein extracts of pI:C-activated DCs using puromycin labeling followed by immunoblot. Cells not treated with puromycin (co) or cycloheximide (chx) were used as controls. (B) Cell extracts were blotted for P-eIF2α and total eIF2α; P-eIF2α decreases with time after pI:C stimulation. (C) Steady-state levels of P-eIF2α nonstimulated bmDC (iDC), MEF, and CD11c+ splenocytes. (D and E) Immature and pI:C-stimulated DCs were treated with GADD34 inhibitors guanabenz and salubrinal. Both drugs were added at the same time as pI:C and for a period of 8 h to nonstimulated cells. Protein extracts were blotted for P-eIF2α and eIF2α. (E) P-eIF2α levels of WT and GADD34ΔC/ΔC DCs stimulated with lipofected (lipo) or soluble pI:C (sol). Nonstimulated cells were treated with lipofectamine only. PKR blot is shown as control of DC activation. Thapsigargin (th) was used as positive control for P-eIF2α.

Fig. 3.

PKR phosphorylates eIF2α in pI:C-activated DCs. WT and PKR−/− DCs were stimulated with pI:C alone (sol) or in combination with lipofectamine (lip) for the indicated time points (A and C) or for 8 h (B). Protein extracts were blotted for PKR, P-eIF2α (A and B) and GADD34 (C). Sodium arsenite (as) was used as a positive control for P-eIF2α induction. (D) GADD34 immunoblot was performed on lysate of NOX2−/− bmDCs activated with either pI:C or LPS for 8 and 24 h. (E) WT and TRIF−/− bmDCs were stimulated with pI:C as indicated and tested for the presence of GADD34 in protein extracts. (F) WT and MDA5−/− bmDCs were stimulated with lipofected pI:C and immunoblotted for GADD34. Treatment with MG132 (2 h) was used to facilitate GADD34 visualization.

Fig. 4.

DCs are protected from translation inhibition induced by cytosolic pI:C detection. (A) Protein synthesis was monitored by immune detection of puromycin incorporation in WT and PKR−/− MEFs and DCs treated with soluble pI:C (sol) or lipofected (lip) for 8 h. Controls: no puromycin (co), chx, and lipofectamine-treated (mock) cells. (B) WT and GADD34ΔC/ΔC DCs were lipofected with pI:C for the indicated time and translation measured by antipuromycin immunoblot. (C) bmDCs from WT and GADD34ΔC/ΔC mice were challenged with thapsigargin for the indicated time. Protein translation, P-eIF2α, and total eIF2α were detected by immunoblotting.

Fig. 5.

GADD34 promotes normal secretion of IFN-β and IL-6 in response to pI:C. (A) Culture supernatants of WT and GADD34ΔC/ΔC bmDCs in which pI:C was delivered in the cytoplasm by lipofection were analyzed by ELISA for IFN-β and IL-6. (B) IFN-β and IL-6 mRNA expression levels were measured by qPCR. One of four independent experiments with similar results is shown for each panel. (C and D) GADD34 is relevant for normal IFN-β protein levels in vivo. IFN-β levels were measured in the serum of mice that were i.v. injected with 20 μg/mL of pI:C for 3 or 6 h. pI:C was delivered conjugated with DOTAP; controls were performed injecting PBS with DOTAP. (D) IFN-β dosage in tissues and serum of mice inoculated with 106 pfu of CHIKV via the ID route. Fifteen-day-old mice were analyzed 72 h after inoculation. Each data point represents the arithmetic mean ±SD for at least four mice. Average virus titers in the different organs are indicated.