p38γ and p38δ kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation

Abstract

On the basis mainly of pharmacological experiments, the p38α MAP kinase isoform has been established as an important regulator of immune and inflammatory responses. However, the role of the related p38γ and p38δ kinases has remained unclear. Here, we show that deletion of p38γ and p38δ impaired the innate immune response to lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) ligand, by blocking the extracellular signal-regulated kinase 1/2 (ERK1/2) activation in macrophages and dendritic cells. p38γ and p38δ were necessary to maintain steady-state levels of tumor progression locus 2 (TPL2), the MKK kinase that mediates ERK1/2 activation after TLR4 stimulation. TNFα, IL-1β, and IL-10 production were reduced in LPS-stimulated macrophages from p38γ/δ-null mice, whereas IL-12 and IFNβ production increased, in accordance with the known effects of TPL2/ERK1/2 signaling on the induction of these cytokines. Furthermore, p38γ/δ-deficient mice were less sensitive than controls to LPS-induced septic shock, showing lower TNFα and IL-1β levels after challenge. Together, our results establish p38γ and p38δ as key components in innate immune responses.

Fig. 1.

Bone marrow-derived macrophages from p38γ/δ-deficient mice show altered cytokine production in response to LPS. (A) BMDM from WT (black bars) or p38γ/δ^−/− (white) mice were exposed to LPS (100 ng/mL) for the indicated times, and culture supernatants harvested for luminex cytokine analysis of TNFα, IL-1β, IL-10, IL-6, and IL-12(p70). Values show mean ± SD for one representative experiment of three performed in duplicate. (B) qPCR of TNFα, IL-1β, IL-10, IL-6, IL-12(p35), and IL-12(p40) mRNA in total RNA from WT (black bars) or p38γ/δ^−/− (white) BMDM stimulated with LPS (100 ng/mL). Results were normalized to 18S RNA expression and x-fold induction was calculated relative to WT expression at 0 h. Data show mean ± SD from one representative experiment of two in triplicate, with similar results. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 relative to WT BMDM exposed to LPS at each time. (C) WT and p38γ/δ^−/− BMDM were exposed to LPS (100 ng/mL). Cell lysates (50 μg) were immunoblotted with a murine anti-TNFα antibody.

Fig. 2.

Role of IL-10 in LPS-induced cytokine production. (A and B) BMDM from WT (black bars) or p38γ/δ^−/− (white) mice were exposed to LPS (100 ng/mL) alone or in the presence of neutralizing antibody to IL-10 (anti-IL10) or an isotype control antibody (IgG) (both at 1 μg/mL, 12 h); culture supernatants were harvested for luminex cytokine analysis of (A) IL-12(p70) and IL-12(p40) and (B) IFNβ. Data are mean ± SD of one representative experiment of two in triplicate with similar results. Real-time qPCR analysis of (A) IL-12(p35), IL-12(p40), and (B) IFNβ in total RNA from WT (black bars) or p38γ/δ^−/− (white) BMDM stimulated as before for the times indicated. Results show mean ± SD of triplicate wells normalized to GAPDH mRNA. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 relative to WT BMDM in each experimental condition.

Fig. 3.

p38γ and p38δ are necessary for LPS activation of ERK1/2 but not of other signaling pathways. (A) Scheme of various signaling pathways activated in macrophages after LPS stimulation. (B–D) BMDM from WT, p38γ^−/−, p38δ^−/−, or p38γ/δ^−/− mice were stimulated with 100 ng/mL LPS for the times indicated (B and C) or for 15 min (D). Cell lysates (50 μg) were immunoblotted with antibodies to (B) active phosphorylated p38α (P-p38α) and active phosphorylated JNK1/2 (P-JNK1/2) or IκBα and (C) active phosphorylated ERK1/2 (P-ERK1/2) and MKK1 (P-MKK1). (B and C) Total protein levels of tubulin, p38MAPK, JNK1/2, ERK1/2, and MKK1 were also measured in the same lysates as loading controls. Duplicate lanes are shown; similar results were obtained in at least three independent experiments. (D) Cell lysates immunoblotted with anti–TPL-2, anti–ABIN-2, anti-phospho p105 (P-p105), anti-p105, anti–P-ERK1/2, anti–ERK1/2, anti–P-MKK1, or anti–MKK1.

Fig. 4.

Deletion of p38γ and p38δ decrease TPL-2 and ABIN-2 protein levels in cells. (A) Quantitative PCR of Tpl-2 mRNA or Abin-2 mRNA in total RNA from WT or p38γ/δ^−/− BMDM. Results were normalized to 18S RNA expression. Data show mean ± SD from one representative experiment of two with similar results. (B) BMDM from WT or p38γ/δ^−/− mice were incubated with the proteasome inhibitor PSI (60 μM) for 0 or 8 h before lysis with SDS-containing buffer A. Immunoblotting was carried with anti-total TPL-2, anti–ABIN-2, or anti-total p38α antibody as protein loading control. (C) WT and p38γ/δ^−/− MEF were transiently transfected with plasmids encoding p38γ, p38δ, or with no insert (EV). (D) p38γ/δ^−/− MEF were transiently transfected with plasmids encoding ABIN-2, TPL2 (as positive control), or with no insert (EV). (C and D) Lysates were immunoblotted with the antibodies indicated, and the band intensity of blots in D and E were quantified and the relative amount of TPL-2 or ABIN-2 in the lysates was calculated. Histogram show mean ± SD of two to three independent experiments. BMDM from WT or p38γ/δ^−/− mice were stimulated with (E) 100 ng/mL TNFα or (F) 100 ng/mL PMA. Immunoblotting was carried out with antibodies to active phosphorylated-ERK1/2 (P-ERK1/2) or total protein (ERK1/2) as loading control. E shows duplicate lanes. Results were similar in two independent experiments. (G and H) Uninfected WT BMDM or p38γ/δ^−/− BMDM infected with recombinant retroviruses encoding TPL-2 or with no insert (EV). Cells were stimulated with LPS at various times (G), and immunoblots were carried out as in E and F. (H) BMDM were stimulated with LPS for 6 h, and qPCR of TNFα was performed as in Fig. 1. Data show mean ± SD from one representative experiment of two in triplicate, with similar results. ***P ≤ 0.001. (I) Schematic representation of the signaling pathways involved in cytokine production, which are controlled by p38γ and p38δ in LPS-stimulated macrophages.

Fig. 5.

Deletion of p38γ and p38δ decreases LPS/d-Gal–induced lethality. (A–C) Mice received injections of LPS (50 μg/kg) and d-Gal (1 g/kg), and death was monitored for up to 36 h. Wild type (n = 14) and (A) p38γ^−/− (n = 15), (B) p38δ^−/− (n = 11), or (C) p38γ/δ^−/− (n = 12) are shown. (D) Serum from mice in A–C was collected 2 h after LPS and d-Gal challenge, and TNFα and IL-1β were measured in a luminex cytokine assay. Data show mean ± SD (n = 4–6 mice/group). *P ≤ 0.05 and **P ≤ 0.01 relative to WT mouse serum.