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Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

Lee WB, Kang JS, Choi WY, Zhang Q, Kim CH, Choi UY, Kim-Ha J, Kim YJ - Nat Commun (2016)

Bottom Line: Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production.In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution.Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.

ABSTRACT
In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

No MeSH data available.


Related in: MedlinePlus

Mincle signalling results in efficient iNOS mRNA translation.(a) WT and Mincle−/− BMDMs were stimulated with Pam3 or co-stimulated with Pam3 and TDM for the indicated times. Left: qRT-PCR analysis of iNOS mRNA levels from each type of stimulated macrophage; right: immunoblot analysis of iNOS protein expression. *P<0.05 (Student's t-test). (b) The half-life of the iNOS protein was measured from Supplementary Fig. 7. The ratio of iNOS signals from the actinomycin D- and cycloheximide-treated samples to those from samples treated only with LPS or co-treated with LPS and TDM for 6 h were calculated and plotted as percentages (initial value set at 100%). (c) Immunoblot analysis of 4EBP-1 and specific phosphor-4EBP-1 (Thr 37/46) from WT and Mincle−/− BMDMs treated with Torin1, Pam3 (P), TDB (T) or co-treated with Pam3 and TDB (PT) for 24 h. UT, untreated. (d) Polysome profiles and immunoblot analysis of the S6 (small) and L7a (large) ribosomal subunit components and β-actin (nonribosomal protein) in lysates of WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM for 12 h. qRT-PCR analysis of iNOS (left) and β-actin (right) mRNA in polysome fractions from WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM, presented for each fraction relative to the sum of all 14 fractions. Data are representative of at least three independent experiments (a,b,d: mean and s.d.).
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f3: Mincle signalling results in efficient iNOS mRNA translation.(a) WT and Mincle−/− BMDMs were stimulated with Pam3 or co-stimulated with Pam3 and TDM for the indicated times. Left: qRT-PCR analysis of iNOS mRNA levels from each type of stimulated macrophage; right: immunoblot analysis of iNOS protein expression. *P<0.05 (Student's t-test). (b) The half-life of the iNOS protein was measured from Supplementary Fig. 7. The ratio of iNOS signals from the actinomycin D- and cycloheximide-treated samples to those from samples treated only with LPS or co-treated with LPS and TDM for 6 h were calculated and plotted as percentages (initial value set at 100%). (c) Immunoblot analysis of 4EBP-1 and specific phosphor-4EBP-1 (Thr 37/46) from WT and Mincle−/− BMDMs treated with Torin1, Pam3 (P), TDB (T) or co-treated with Pam3 and TDB (PT) for 24 h. UT, untreated. (d) Polysome profiles and immunoblot analysis of the S6 (small) and L7a (large) ribosomal subunit components and β-actin (nonribosomal protein) in lysates of WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM for 12 h. qRT-PCR analysis of iNOS (left) and β-actin (right) mRNA in polysome fractions from WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM, presented for each fraction relative to the sum of all 14 fractions. Data are representative of at least three independent experiments (a,b,d: mean and s.d.).

Mentions: To understand how Mincle stimulates iNOS expression, we examined iNOS mRNA and protein levels in WT and Mincle mutant macrophages following stimulation. Pam3 treatment increased iNOS mRNA, which peaked at 6 h in both WT and Mincle−/− macrophages (Fig. 3a). However, iNOS protein was not detectable, hinting that these mRNA may not be actively translated. TDM co-treatment dramatically enhanced iNOS mRNA and protein in a Mincle-dependent manner, and iNOS protein was gradually increased even 48 h after stimulation, temporally distinct from the mRNA peak. Although moderate levels of iNOS mRNA were induced by the treatment, only slight amount of iNOS protein was detected in Mincle−/− macrophages, demonstrating the requirement of Mincle for iNOS protein expression. Because LPS activates diverse immune signalling pathways21, LPS treatment elicited strong iNOS mRNA production independent of Mincle, and triggered iNOS protein synthesis (Supplementary Fig. 6). Nevertheless, TDM co-stimulation further extended iNOS protein synthesis beyond 24 h, only in WT macrophages. These results suggest possible post-transcriptional regulation of iNOS by Mincle.


Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

Lee WB, Kang JS, Choi WY, Zhang Q, Kim CH, Choi UY, Kim-Ha J, Kim YJ - Nat Commun (2016)

Mincle signalling results in efficient iNOS mRNA translation.(a) WT and Mincle−/− BMDMs were stimulated with Pam3 or co-stimulated with Pam3 and TDM for the indicated times. Left: qRT-PCR analysis of iNOS mRNA levels from each type of stimulated macrophage; right: immunoblot analysis of iNOS protein expression. *P<0.05 (Student's t-test). (b) The half-life of the iNOS protein was measured from Supplementary Fig. 7. The ratio of iNOS signals from the actinomycin D- and cycloheximide-treated samples to those from samples treated only with LPS or co-treated with LPS and TDM for 6 h were calculated and plotted as percentages (initial value set at 100%). (c) Immunoblot analysis of 4EBP-1 and specific phosphor-4EBP-1 (Thr 37/46) from WT and Mincle−/− BMDMs treated with Torin1, Pam3 (P), TDB (T) or co-treated with Pam3 and TDB (PT) for 24 h. UT, untreated. (d) Polysome profiles and immunoblot analysis of the S6 (small) and L7a (large) ribosomal subunit components and β-actin (nonribosomal protein) in lysates of WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM for 12 h. qRT-PCR analysis of iNOS (left) and β-actin (right) mRNA in polysome fractions from WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM, presented for each fraction relative to the sum of all 14 fractions. Data are representative of at least three independent experiments (a,b,d: mean and s.d.).
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Related In: Results  -  Collection

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f3: Mincle signalling results in efficient iNOS mRNA translation.(a) WT and Mincle−/− BMDMs were stimulated with Pam3 or co-stimulated with Pam3 and TDM for the indicated times. Left: qRT-PCR analysis of iNOS mRNA levels from each type of stimulated macrophage; right: immunoblot analysis of iNOS protein expression. *P<0.05 (Student's t-test). (b) The half-life of the iNOS protein was measured from Supplementary Fig. 7. The ratio of iNOS signals from the actinomycin D- and cycloheximide-treated samples to those from samples treated only with LPS or co-treated with LPS and TDM for 6 h were calculated and plotted as percentages (initial value set at 100%). (c) Immunoblot analysis of 4EBP-1 and specific phosphor-4EBP-1 (Thr 37/46) from WT and Mincle−/− BMDMs treated with Torin1, Pam3 (P), TDB (T) or co-treated with Pam3 and TDB (PT) for 24 h. UT, untreated. (d) Polysome profiles and immunoblot analysis of the S6 (small) and L7a (large) ribosomal subunit components and β-actin (nonribosomal protein) in lysates of WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM for 12 h. qRT-PCR analysis of iNOS (left) and β-actin (right) mRNA in polysome fractions from WT macrophages stimulated with Pam3 or co-stimulated with Pam3 and TDM, presented for each fraction relative to the sum of all 14 fractions. Data are representative of at least three independent experiments (a,b,d: mean and s.d.).
Mentions: To understand how Mincle stimulates iNOS expression, we examined iNOS mRNA and protein levels in WT and Mincle mutant macrophages following stimulation. Pam3 treatment increased iNOS mRNA, which peaked at 6 h in both WT and Mincle−/− macrophages (Fig. 3a). However, iNOS protein was not detectable, hinting that these mRNA may not be actively translated. TDM co-treatment dramatically enhanced iNOS mRNA and protein in a Mincle-dependent manner, and iNOS protein was gradually increased even 48 h after stimulation, temporally distinct from the mRNA peak. Although moderate levels of iNOS mRNA were induced by the treatment, only slight amount of iNOS protein was detected in Mincle−/− macrophages, demonstrating the requirement of Mincle for iNOS protein expression. Because LPS activates diverse immune signalling pathways21, LPS treatment elicited strong iNOS mRNA production independent of Mincle, and triggered iNOS protein synthesis (Supplementary Fig. 6). Nevertheless, TDM co-stimulation further extended iNOS protein synthesis beyond 24 h, only in WT macrophages. These results suggest possible post-transcriptional regulation of iNOS by Mincle.

Bottom Line: Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production.In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution.Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Republic of Korea.

ABSTRACT
In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

No MeSH data available.


Related in: MedlinePlus