Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology
2001; Springer Nature; Volume: 20; Issue: 14 Linguagem: Inglês
10.1093/emboj/20.14.3760
ISSN1460-2075
AutoresDimitris L. Kontoyiannis, Alexey Kotlyarov, Ester Carballo, Lena Alexopoulou, Perry J. Blackshear, Matthias Gaestel, Roger J. Davis, Richard A. Flavell, George Kollias,
Tópico(s)Immune Response and Inflammation
ResumoArticle16 July 2001free access Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology Dimitris Kontoyiannis Dimitris Kontoyiannis Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Search for more papers by this author Alexey Kotlyarov Alexey Kotlyarov Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, D-06120 Halle, Germany Search for more papers by this author Ester Carballo Ester Carballo Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709 USA Search for more papers by this author Lena Alexopoulou Lena Alexopoulou Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT, 06520-8011 USA Search for more papers by this author Perry J. Blackshear Perry J. Blackshear Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709 USA Search for more papers by this author Matthias Gaestel Matthias Gaestel Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, D-06120 Halle, Germany Search for more papers by this author Roger Davis Roger Davis Howard Hughes Medical Institute, Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA, 01605 USA Search for more papers by this author Richard Flavell Richard Flavell Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT, 06520-8011 USA Search for more papers by this author George Kollias Corresponding Author George Kollias Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Search for more papers by this author Dimitris Kontoyiannis Dimitris Kontoyiannis Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Search for more papers by this author Alexey Kotlyarov Alexey Kotlyarov Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, D-06120 Halle, Germany Search for more papers by this author Ester Carballo Ester Carballo Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709 USA Search for more papers by this author Lena Alexopoulou Lena Alexopoulou Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT, 06520-8011 USA Search for more papers by this author Perry J. Blackshear Perry J. Blackshear Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709 USA Search for more papers by this author Matthias Gaestel Matthias Gaestel Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, D-06120 Halle, Germany Search for more papers by this author Roger Davis Roger Davis Howard Hughes Medical Institute, Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA, 01605 USA Search for more papers by this author Richard Flavell Richard Flavell Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT, 06520-8011 USA Search for more papers by this author George Kollias Corresponding Author George Kollias Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece Search for more papers by this author Author Information Dimitris Kontoyiannis1, Alexey Kotlyarov2, Ester Carballo3, Lena Alexopoulou1,4, Perry J. Blackshear3, Matthias Gaestel2, Roger Davis5, Richard Flavell4 and George Kollias 1 1Institute of Immunology, BSRC 'Alexander Fleming', 166 72 Vari, Greece 2Innovationskolleg Zellspezialisierung, Martin-Luther-Universität Halle Wittenberg, D-06120 Halle, Germany 3Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709 USA 4Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT, 06520-8011 USA 5Howard Hughes Medical Institute, Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, MA, 01605 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3760-3770https://doi.org/10.1093/emboj/20.14.3760 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Interleukin-10 (IL-10) is a key inhibitory signal of inflammatory responses that regulates the production of potentially pathogenic cytokines like tumor necrosis factor (TNF). We show here that the development of chronic intestinal inflammation in IL-10-deficient mice requires the function of TNF, indicating that the IL-10/TNF axis regulates mucosal immunity. We further show that IL-10 targets the 3′ AU-rich elements (ARE) of TNF mRNA to inhibit its translation. Moreover, IL-10 does not alter TNF mRNA stability, and its action does not require the presence of the stability-regulating ARE binding factor tristetraprolin, indicating a differential assembly of stability and translation determinants on the TNF ARE. Inhibition of TNF translation by IL-10 is exerted mainly by inhibition of the activating p38/MAPK-activated protein kinase-2 pathway. These results demonstrate a physiologically significant cross-talk between the IL-10 receptor and the stress-activated protein kinase modules targeting TNF mRNA translation. This cross-talk is necessary for optimal TNF production and for the maintenance of immune homeostasis in the gut. Introduction Tumor necrosis factor (TNF) plays a central role in diverse immune and inflammatory processes (Kollias et al., 1999). Although, physiologically, TNF actions are beneficial, it is clear that aberrations in TNF production in vivo may be pathological. The potential of TNF to induce chronic inflammatory disease has been exemplified in animal models, where deregulation of its production leads to the development of various pathologies (Kollias et al., 1999). Conversely, interleukin-10 (IL-10) has emerged as a macrophage deactivator competent to suppress the expression of inflammatory mediators as well as the macrophages' ability to support accessory functions to adaptive immunity (for review see Moore et al., 1993). The anti-inflammatory potential of IL-10 has been repeatedly demonstrated in vivo, in preventing endotoxemia (Gerard et al., 1993; Marchant et al., 1994) and suppressing the development of intestinal inflammation (Kuhn et al., 1993). In particular, an indication of a homeostatic TNF/IL-10 axis against inflammatory bowel disease (IBD) is provided by the fact that mice deficient in IL-10, or in macrophage signaling molecules that mediate IL-10 receptor signals, overproduce TNF and develop IBD (Kuhn et al., 1993; Takeda et al., 1999). Activation of macrophages by lipopolysaccharide (LPS) results in the rapid production of both TNF and IL-10, with similar kinetics (Marchant et al., 1994). However, the mechanism by which IL-10 suppresses TNF expression remains elusive. It is possible that IL-10 may affect the same pathways that are required for LPS-induced TNF production (Geng et al., 1994; Wang et al., 1995). However, there is a great degree of contradictory data as to what level(s) IL-10 exerts its inhibitory action (Bogdan et al., 1991, 1992; de Waal et al., 1991; Wang et al., 1995; Brown et al., 1996; Kishore et al., 1999). Homeostatic control of TNF biosynthesis is exerted at multiple levels and proceeds through a multitude of signals. For example, binding of LPS to its Toll-receptor complex on macrophages transmits signals through tyrosine kinases, protein kinase C, NFκB, and the mitogen- and stress-activated protein kinases (MAPK/SAPK) ERK, JNK and p38 (reviewed by Beutler, 2000). These signals may affect TNF gene expression directly, at the level of transcription (Collart et al., 1990), splicing (Osman et al., 1999), mRNA stability and translation (Carballo et al., 1998; Kontoyiannis et al., 1999), and protein processing (Peschon et al., 1998). We have recently demonstrated that the AU-rich elements (ARE) residing in the 3′UTR of TNF mRNA are important in controlling message stability and translational activation (Kontoyiannis et al., 1999). The latter appears to rely on the activity of the MAPK/SAPK pathways (Kontoyiannis et al., 1999). Although data on TNF ARE binding factors remain scarce, recent evidence indicated that the prototype member of a zinc finger family of RNA binding proteins, tristetraprolin (TTP), has the capacity to modulate TNF expression in vivo by destabilizing TNF mRNA in an ARE-dependent fashion (Carballo et al., 1998). Whether TTP exclusively modulates TNF mRNA stability, and with what signaling requirements, remains to be demonstrated. In this report we demonstrate that, in mouse macrophages, IL-10 exerts its anti-inflammatory and immunosuppressive action mainly by targeting ARE-mediated translation of the TNF message through modulation of activating p38/SAPK signals. Aberrations in TNF production due to the absence of IL-10 function in mutant mice are shown here to be causal for intestinal inflammation, indicating that correct regulation of the IL-10/TNF axis is of physiological significance to intestinal immune homeostasis. Results Absence of TNF attenuates development of IBD in IL-10-deficient mice Examination of TNF production in LPS-induced macrophages from normal or IL-10-deficient mice demonstrated that TNF was overexpressed in the latter (Figure 1D). To substantiate whether a defective TNF/IL-10 axis is a general criterion for the development of intestinal inflammation, we assessed the role of TNF in the development of enterocolitis in IL-10-deficient mice by generating double-mutant (Il-10−/− Tnf−/−) mice. As shown in Figure 1B, the macroscopic disease developing in Il-10−/− and Il-10−/− Tnf+/−Tnf+/− mice was similar, and resulted in lethality for ∼40–50% (n = 27 and 24 mice, respectively) of the animals by the age of 10 weeks. Clinical symptoms included gradual weight loss (Figure 1A) and an increased incidence of colorectal prolapses (in ∼70–80% of mice examined). In sharp contrast, 80% (18/21) of Il-10−/− Tnf−/− mice showed no symptoms of macroscopic disease and appeared normal throughout the period examined (20 weeks). However, three out of the 21 double-homozygous mice developed clinical symptoms similar to those presented by control groups. Histological evaluation of the large intestine of the control groups revealed moderate to severe inflammatory occurrences (Table I). In sharp contrast, colitis was completely prevented in 77% of the double-homozygous mice (Figure 1C; Table I). However, in the three diseased as well as in two asymptomatic Il-10−/− Tnf−/− mice, the histological hallmarks of colitis developed with differential onset. These results demonstrate that TNF is dominantly involved in the development of colitis in IL-10-deficient mice. Figure 1.Evidence for a defective TNF/IL-10 axis in IL-10 knockout and TNFΔARE mutant mice. (A) Weight distribution and (B) cumulative percent survival of Il-10−/− Tnf−/− and control mice. (C) Representative photomicrographs (×200) of the proximal colon of Il-10−/− Tnf−/− from: (i) 8-week-old Il-10−/− Tnf+/− control mice showing prominent transmural inflammation (arrows), epithelial cell hyperplasia and goblet cell loss; (ii) 14-week-old Il-10−/− Tnf−/− colon showing normal epithelial integrity; (iii) 12-week-old Il-10−/− Tnf−/− asymptomatic mouse showing mild signs of submucosal inflammation (asterisk); (iv) 12-week-old Il-10−/− Tnf−/− diseased mouse showing severe inflammation. (D) Kinetics of TNF protein accumulation (upper) and production per hour (lower) in Il-10+/+ and Il-10−/− TEPM following LPS stimulation in vitro. TNF and IL-10 protein levels in collected supernatants were determined by ELISA. Results shown as mean ± SD values from five cultures/group. (E) Kinetics of TNF and IL-10 protein production in Tnf+/+ (closed circles) and TnfΔARE/+ (open circles) mice following LPS challenge in vivo. Mice were challenged with 100 μg/ml LPS intraperitoneally and subsequently exsanguinated via cardiac puncture at the indicated time points. TNF and IL-10 protein levels in sera were determined by ELISA. Results shown as mean ± SD values from four mice/time point. (F) Kinetics of TNF and IL-10 in Tnf+/+ (closed circles) and TnfΔARE/+ (open circles) TEPM and following LPS stimulation, in vitro. TEPM were cultured as before and subsequently stimulated with LPS (1 μg/ml) for 12 h. Download figure Download PowerPoint Table 1. Effects of TNF absence on the incidence and severity of colitis in IL-10−/− mice Genotype No. of mice affected Disease score distributiona Mean disease score (0–20)a 0 >0–5 >5–10 >10–15 >15–20 Il-10−/− 14/14 (100%)† 0 0 4 7 3 13.1 ± 4.1* Tnf−/− 0/5 (0%) 5 0 0 0 0 nil Il-10−/− Tnf+/− 13/15 (87%)†† 2 2 3 6 3 10.6 ± 5.9** Il-10−/− Tnf−/− 5/21 (23%) 16 2 0 1 2 3.47 ± 5.71 a Mean severity of colonic lesions in affected mice; assessment as in Materials and methods. Data are represented as mean ± SD. * Denotes a significant difference to the corresponding test group score (assessed by using unpaired Student's t-test): p = 0.002; p = 0.00005. † Denotes a significant difference to the corresponding test group score (assessed by using two-tailed Fisher's exact test): †p = 0.00018; †† p = 0.00000324. We next tested in the TnfΔARE mouse whether defective IL-10 production could be associated with the development of IBD in this system. Interestingly, TNF overexpression in TnfΔARE mice is accompanied by exacerbated production of IL-10 both in vivo and in vitro (Figure 1E and F). Thus, IL-10 production is enhanced in TnfΔARE mice, yet its action appears ineffective in suppressing the high TNF load. IL-10 inhibits TNF production by macrophages at multiple levels We examined in detail the effect of IL-10 on LPS-stimulated TNF production by murine macrophages. Since IL-10 production coincides with TNF production (Figure 1E and F), we hypothesized that, physiologically, IL-10 targets TNF expression concurrently with the LPS signals. IL-10 inhibited, in a dose-dependent manner, the secretion of TNF by thioglycolate-elicited peritoneal macrophages (TEPM) and bone marrow-derived (BMDM) macrophage cultures, reaching a maximal value of >80% inhibition at 5 ng (Figure 2A). At the same dose, IL-10 also reduced the levels of LPS-induced transmembrane TNF protein accumulation by 80–90%, as indicated by flow cytometry (Figure 2B). In contrast, IL-10 reduced the levels of steady-state TNF mRNA by 9 h. Comparison of the corresponding mRNA values showed a similar increase in TNF mRNA accumulation in LPS-stimulated TTP−/− and TnfΔARE BMDM relative to wild-type values, indicating a comparable effect of both mutations on TNF mRNA stability (data not shown). Thus, the difference in TNF protein production between TTP- and TNF ARE-deficient macrophages lies in the added modulation of TNF mRNA translation by the ARE. This was confirmed by the use of SB203580, which is known to inhibit the translation of TNF mRNA in a TNF ARE-dependent manner. SB203580 was as efficient in blocking TNF production by TTP−/− BMDM as it was in the case of TTP+/+ macrophages (Figure 4B). Similarly, IL-10-mediated suppression of TNF production, although minimal in the absence of TNF ARE, occurred normally in TTP−/− as well as control macrophages (Figure 4B). Taken together, these results show that TTP is not required for the ARE-dependent modulation of TNF mRNA translation in macrophages, indicating differential organization of TNF ARE-mediated stability and translational controls. Figure 4.Functional uncoupling of ARE-mediated functions: TTP does not interfere with ARE-dependent modulation of TNF mRNA translation. (A) Kinetics of TNF protein production/h. Supernatants from wild-type (wt), TTP−/− and TnfΔARE/+ BMDM (5 × 105 adherent cells/ml) were collected at hourly intervals after LPS stimulation. Following each sample collection, cells were washed and incubated with fresh medium. TNF protein levels were quantitated as before. Data shown as actual mean ± SD values (left) or as mean + SD percentages to each of the corresponding maximal values (right). (B) BMDM (5 × 105 adherent cells/ml) from wild-type (wt), TTP−/− and TnfΔARE/+ BMDM were stimulated with LPS (1 μg/ml) for 12 h in the presence of various concentrations of IL-10 or SB203580; TNF levels in cultured supernatants were as before. Results shown as mean percentages of LPS values; data from at least three experiments with cells derived from individual mice (n = 5 mice/group/experiment). Download figure Download PowerPoint Reduced p38/SAPK activation in the presence of IL-10 in LPS-stimulated macrophages The p38, JNK and ERK MAPK/SAPK pathways have previously been demonstrated to modulate TNF biosynthesis (Lee et al., 1994; Swantek et al., 1997; Kontoyiannis et al., 1999; Srivastava et al., 1999; Dumitru et al., 2000). A paradigm of cross-talk between signals activated by the IL-10 receptor and MAPK/SAPK has also been demonstrated previously (Kallunki et al., 1994; Niiro et al., 1998; Turkson et al., 1999). However, other reports (reviewed by Donelly et al., 1999) have not been able to reproduce an effect of IL-10 on these signaling modules. We examined the effect of IL-10 on the LPS-induced activation of ERK, JNK and p38 MAPK in TEPM, and the mouse macrophage cell line RAW 264.7, by detecting the corresponding native and phosphorylated forms via western blotting. As can be readily seen in Figure 5A, IL-10 inhibited the LPS-induced phosphorylation of p38 MAPK by 90% in TEPM and by 50% in RAW 264.7 macrophages, while the levels of the native form of this protein remained unaltered. In contrast, no reduction was observed in the levels of the phosphorylated forms of JNK1/2 (Figure 5B) and ERK1/2 (not shown). These results suggest that IL-10 interferes with p38 signaling at the level of its phosphorylation by upstream kinases, but not at the level of p38 abundance. Figure 5.IL-10 inhibits p38 phosphorylation in LPS-induced macrophages. TEPM and RAW 264.7 macrophages were challenged with LPS (1 μg/ml) and IL-10 (5ng/ml) for 15 min. Total cell lysates were immunoblotted with antibody probes for the phosphorylated forms of p38/SAPK (A) and JNK/SAPK (B). Membranes were stripped and reprobed for the detection of the total protein content of each kinase. Representative blots are shown. Quantitation of phospho-p38 kinase levels, normalized to the total p38 content, is also shown (A). Results from one out of three experiments, shown as mean densitometric units (+ SD) from independent cultures. Download figure Download PowerPoint IL-10 targets the p38/MAPKAP-2 pathway to modulate ARE-dependent TNF mRNA translation The p38/MAPK cascade (reviewed by Ono and Han, 2000) activates many downstream protein kinases, including the serine/threonine kinase MK2 [MAPK-activated protein (MAPKAP)-kinase 2]. MK2-deficient macrophages show reduced levels of TNF protein production, but not of the corresponding mRNA, following LPS challenge (Kotlyarov et al., 1999), indicating that the p38/MK2 pathway is required for activation of TNF translation by LPS. To examine whether MK2 modulates TNF translation in macrophages in an ARE-dependent manner, we have recently generated TNFΔARE/+ mk2−/− mice (A.Kotlyarov, D.Kontoyiannis, A.Neininger, R.Winzen, R.Eckert, H.D.Volk, H.Holtmann, G.Kollias and M.Gaestel, in preparation). Macrophages from these mice show a pattern of TNF production similar to that observed in TNFΔARE macrophages, indicating that MK2 targets TNF ARE. We further examined whether the absence of MK2 signals interferes with IL-10 targeting of TNF production. Exposure of LPS-stimulated mk2−/− TEPM to IL-10 reduced TNF protein production by 30–40% instead of 80% in mk2+/+ macrophages (Figure 6A). In addition, the LPS-induced, polysome-associated TNF mRNA profile in MK2-deficient TEPM, although it appears lower than in LPS-induced wild-type TEPM, is not significantly altered in the presence of IL-10 (−IL-10: 3.0 + 0.1; +IL-10: 2.5 + 0.70; n = 3) (Figure 6B). These observations demonstrate that MK2 is required for the ARE-dependent, IL-10-mediated inhibition of TNF production. Figure 6.IL-10 inhibits p38-mediated signals that activate TNF mRNA translation. (
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