Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology - PubMed (original) (raw)

Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology

D Kontoyiannis et al. EMBO J. 2001.

Abstract

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.

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Figures

Fig. 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 _Tnf_ΔARE/+ (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 _Tnf_ΔARE/+ (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.

Fig. 2. IL-10 targets TNF mRNA translation, but not TNF mRNA decay and transmembrane TNF processing. (A) Mouse TEPM and BMDM (5 × 105 adherent cells/ml) were stimulated with LPS (1 µg/ml) for 12 h in the presence of various concentrations of rmIL-10; TNF levels in cultured supernatants were determined by ELISA. Results shown as mean percentages of LPS values from at least three experiments (n = 5 mice/group/experiment). (B) Immunocytometric detection of transmembrane TNF on the surface of LPS-stimulated TEPM, in the presence or absence of IL-10. A, _Tnf_–/– control; B, non-stimulated; C, LPS (1 µg/ml) + rmIL-10 (5 ng/ml) for 2 h; D, LPS (1 µg/ml) for 2 h. (C) Northern analysis and quantitation of TNF mRNA isolated from TEPM (hatched bar) or BMDM (white bar) following stimulation with LPS (1 µg/ml) for 2 h, in the presence or absence of IL-10 (5 ng/ml). Results shown as percentages of the LPS values from three different experiments. (D) Decay analysis. TNF and β-actin mRNA from TEPM and BMDM stimulated with LPS for 1 h and then with actinomycin D, in the absence (open circles) or presence (closed circles) of IL-10. Northern analysis and semilogarithmic plots of data values obtained from densitometric analysis of the corresponding autoradiographs. (E) Sucrose gradient analysis. TEPM were activated with LPS in the presence or absence of IL-10 (5 ng/ml) for 2 h, and cell lysates were analyzed by sucrose gradient centrifugation. (i) Representative profile of the 254 nm UV absorption across the gradients, indicating the peaks corresponding to the 60S, 80S and the polysome containing fractions. Quantitation of GAPDH (ii) and TNF (iii) transcripts in individual fractions was determined by hybridization and autoradiography using a phosphoimager device. Closed circles, LPS-treated macrophages; open circles, LPS plus IL-10 treated macrophages. Representative dot-blots of fractions hybridized with a TNF probe are also presented.

Fig. 3. TNF 3′ARE absence desensitizes TNF translation to IL-10-mediated suppression. (A) TEPM and BMDM (5 × 105 adherent cells/ml) from Tnf+/+ and _Tnf_ΔARE/ΔARE mice were stimulated with LPS (1 µg/ml) for 12 h in the presence of various concentrations of IL-10; TNF levels in cultured supernatants were determined by ELISA. Results shown as mean percentages of LPS values from at least three experiments with cells derived from individual mice (n = 5 mice/group/experiment). (B) Detection of nitrite release using cultured Tnf+/+ and _Tnf_ΔARE/ΔARE TEPM stimulated for 24 h with LPS (1 µg/ml) plus IL-10 (5 ng/ml). Results shown as mean ± SD percentages from the LPS values from two experiments with TEPM derived from five individual mice/group. (C) Northern analysis (autoradiographs) and quantitation of TNF and β-actin mRNA isolated from Tnf+/+ (gray bar) and _Tnf_ΔARE/ΔARE (white bar) TEPM, activated with LPS in the presence or absence of IL-10 (5 ng/ml) for 2 h. Results shown as mean ± SD percentages of the LPS values (black bar) normalized to the GAPDH values from three different experiments. (D) mRNA decay analysis. Semi logarithmic plots of data values obtained from densitometric analysis of the corresponding autoradiographs following hybridization with TNF and β-actin probes before and after IL-10 treatment of Tnf+/+ (open circles) and _Tnf_ΔARE/ΔARE (closed circles) TEPM stimulated with LPS for 3 h, in the presence of actinomycin D. (E) Sucrose gradient analysis. _Tnf_ΔARE/ΔARE TEPM were activated with LPS in the presence or absence of IL-10 (5 ng/ml) for 2 h. Quantitation of labeled TNF transcripts in individual gradient fractions was determined using a phosphoimager device. Closed circles, LPS-treated macrophages; open circles, LPS plus IL-10 treated macrophages. Representative dot-blot of fractions hybridized with a TNF probe are presented.

Fig. 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 _Tnf_ΔARE/+ 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 _Tnf_ΔARE/+ 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).

Fig. 5. IL-10 inhibits p38 phosphorylation in LPS-induced macro phages. 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.

Fig. 6. IL-10 inhibits p38-mediated signals that activate TNF mRNA translation. (A) Impaired efficacy of IL-10 to suppress TNF production in mk2-deficient macrophages. LPS-stimulated TEPM from mk2+/+ and _mk2_–/– mice were cultured with various concentrations of rmIL-10 for 12 h, and TNF protein was detected in culture supernatants by ELISA. Results are from a representative of two experiments with cells derived from five mice/group. Data are as absolute values (upper) as well as percentages (+ SD) of the corresponding LPS values (lower). (B) Defective inhibition of polysome TNF mRNA in the absence of MK2. Sucrose gradient analysis of monosomal/polysomal-associated TNF mRNA in LPS plus IL-10-stimulated mk2+/+, _mk2_–/– TEPM as well as RAW 264.7 macrophages. Results from representative experiments shown as percentages of total cytoplasmic fractions. (C) Normal IL-10 targeting of TNF production in MKK3-deficient macrophages. TEPM from mkk3+/+ and _mkk3_–/– macrophages were stimulated with LPS (1 µg/ml) in the presence of 5 ng/ml rmIL-10. Data shown as mean (± SD) of the corresponding LPS values. Results from two experiments with macrophages from three mice/group. (D) Transfection of a constitutively active form of MKK6 inhibits IL-10-mediated targeting of TNF production. RAW 264.7 cells were transfected via electroporation with control plasmid or the expression plasmid for the mutated MKK6(Glu) (MKK6 CA). Following 18 h starvation, cells were challenged with LPS supplemented with the indicated quantities of IL-10. TNF protein quantitation was performed as before. Data shown as mean (± SEM) percentages of LPS values. Results from three independent transfected cultures for each group. *p <0.01.

Fig. 7. Proposed scheme of interactions between IL-10R and TNF modulating signals. LPS binding/receptor complex leads to the transmission of p38/MAPK signals activating TNF mRNA translation (open circles and arrows). At similar or consecutive time points, it leads to the activation of stability and translation determining factors that act in a negative fashion, apparently via p38-independent mechanisms (gray shaded circles and arrows). On the other hand, IL-10 receptor engagement may transmit STAT-3-dependent and -independent signals towards SOCS-3 activation, building up a negative regime targeting p38/MK2 activating signals towards ARE-dependent TNF mRNA translation.

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Interleukin-10 targets p38 MAPK to modulate ARE-dependent TNF mRNA translation and limit intestinal pathology - PubMed (original) (raw)