In this study,we show that protein kinases p38γ/δ mediate the development of LPS-induced acute hepatitis by acting within a protein kinase signaling network that regulates the production of TNF-α by hematopoietic cells. MKK3 and MKK6 activate p38γ/δ (14
), which in turn phosphorylate and inactivate eEF2 kinase (41
). Once eEF2K is inactivated, eEF2 is dephosphorylated and activated, allowing the translational elongation of nascent TNF-α. To our knowledge, this is the first report that eEF2 or p38γ/δ control cytokine production in myeloid cells. Our findings raise 3 key areas of interest: the specific role of myeloid cells, the involvement of p38γ/δ compared with that of the other kinases of the same family, and how acute inflammatory responses can be controlled through tight regulation of translational elongation.
Myeloid cells such as Kupffer cells and other macrophages are well known to be a critical source of TNF-α in LPS-induced hepatitis (14
). This specific role of hematopoietic cells is consistent with our finding that the protective effects of MKK3/6 deficiency are also present in irradiated WT mice reconstituted with ΔMKK3/6 BM (Figure ). Furthermore, this protection was also observed in mice specifically lacking p38γ/δ in the myeloid compartment (Figure ). Two cell types of special relevance in acute hepatitis are neutrophils and monocytes/macrophages (including Kupffer cells), and both of these BM-derived cell populations are essential for the innate immune response (42
). Monocytes/macrophages bind to microbial constituents (such as LPS and cell wall constituents of Gram-positive bacteria), producing large amounts of pro- and antiinflammatory cytokines (3
). Neutrophil recruitment and activation occur through TNF-α–mediated chemokine production (3
). This is evidenced by our finding that low expression of TNF-α in ΔMKK3/6 or p38γ/δLyz-KO
mice results in decreased chemokine production and neutrophil migration, an effect reversed by injection with TNF-α. We did not find, however, any evidence for a role of p38γ/δ in neutrophil migration, and we found only a minor effect on cytokine expression. This contrasts with the recent finding that p38α can control cytokine production by neutrophils (29
), illustrating the different roles and clinical potential of p38 kinase isoforms as targets for preventing LPS-induced damage.
Previous reports have identified p38α as a key kinase involved in TNF-α production (43
). p38α MAPK deficiency in macrophages results in decreased TNF-α production, with a modest effect on IL-6 expression (44
). However, specific inhibition of this kinase has been shown to be hepatotoxic, hindering its clinical use. For example, the p38α inhibitor AMG 548 showed more than 85% inhibition of ex vivo LPS-induced TNF-α in healthy males, but its production and clinical use were suspended due to random liver enzyme elevations that were not dose or exposure dependent (45
). Our results show that specific inhibition of p38α/β with SB203580 intensifies D-gal+LPS–induced liver damage, whereas BIRB796, which inhibits all 4 p38 isoforms (α/β/γ/δ) improves liver condition, including a reduction in apoptosis and necrosis. The toxicity associated with SB203580 might be caused by inhibition of p38α in hepatocytes, since mice with specific p38α deficiency in hepatocytes have increased JNK activity and increased susceptibility to liver damage (17
). In clinical use, BIRB796 also produces some liver enzyme elevations (46
), most likely because of inhibition of p38. Our findings indicate that the generation of inhibitors that specifically target p38γ/δ kinases might avoid the adverse effects found with p38α inhibitors.
Our results further show that p38γ/δ control macrophage production of TNF-α by promoting protein elongation during translation by eEF2. Protein synthesis is tightly regulated at transcriptional and posttranscriptional levels. Owing to the relatively long life time of mRNA transcripts, transcriptional regulation is commonly involved in slow, long-term cell responses, whereas immediate cell responses require posttranscriptional regulation of mRNA stability or translation. Two physiological situations requiring rapid regulation of protein synthesis are starvation and inflammatory conditions. In starvation conditions, mTOR stops protein synthesis at the elongation step — the step that consumes most metabolic energy — by inhibiting eEF2, thereby preventing cell energy depletion. Less is known about the mechanisms involved in the termination of the inflammatory response once it has achieved its goal. This process needs to be rapidly regulated in order to avoid more tissue damage. Our results suggest that control of protein elongation might be an important mechanism by which cells rapidly shut down production of proinflammatory proteins that could extend tissue damage in the body, such as TNF-α during an inflammatory response. This might be an important mechanism by which cells tightly regulate cytokine production induced by stress kinases activated during inflammation.
In summary, we conclude that p38γ/δ in myeloid cells promote TNF-α production by activating its translation without changes in mRNA levels. This is achieved by phosphorylation of eEF2K, releasing the inhibitory action of this kinase on eEF2. This posttranscriptional regulation might be an important mechanism regulating cytokine secretion during the innate immune response and provides potential targets for the treatment of liver diseases.