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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

Specific genes selected from the polysome profiling assay are regulated by Mincle-eIF5A-dependent translation.(a) Polysome fraction mRNAs from WT BMDMs co-treated with Pam3, TDM, with or without GC7 were analysed. qRT-PCR analysis of the indicated mRNAs in cells treated as indicated, presented for each fraction relative to the sum of all fourteen polysome fractions. (b) RIP analysis of the indicated gene transcripts from iBMDM cells stably expressing Flag-eIF5A, stimulated as indicated. Immunoprecipitation with anti-Flag antibody and qRT-PCR analysis of the indicated genes. (c) WT BMDMs were stimulated with Pam3, TDM or co-stimulated with Pam3 and TDM, in the presence of GC7 or control vehicle for 12 h. Immunoblot analysis of the indicated protein expression levels. Data are representative of three independent experiments (a–c: mean and s.d.).
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f6: Specific genes selected from the polysome profiling assay are regulated by Mincle-eIF5A-dependent translation.(a) Polysome fraction mRNAs from WT BMDMs co-treated with Pam3, TDM, with or without GC7 were analysed. qRT-PCR analysis of the indicated mRNAs in cells treated as indicated, presented for each fraction relative to the sum of all fourteen polysome fractions. (b) RIP analysis of the indicated gene transcripts from iBMDM cells stably expressing Flag-eIF5A, stimulated as indicated. Immunoprecipitation with anti-Flag antibody and qRT-PCR analysis of the indicated genes. (c) WT BMDMs were stimulated with Pam3, TDM or co-stimulated with Pam3 and TDM, in the presence of GC7 or control vehicle for 12 h. Immunoblot analysis of the indicated protein expression levels. Data are representative of three independent experiments (a–c: mean and s.d.).

Mentions: To identify additional transcripts regulated by Mincle-dependent eIF5A hypusination, we examined RNAs preferentially enriched in the polysome fraction under TDM and Pam3 co-stimulation, but shifted to the subpolysome fractions when GC7 was added. To this end, we sequenced total and polysome-associated RNAs from Pam3 and TDM co-treated macrophages with or without GC7 (Supplementary Fig. 17a,b). We observed 282 mRNAs located on the polysome by Pam3 and TDM co-stimulation; among these, 111 mRNAs were depleted from polysome fractions on GC7 treatment, revealing small number of transcripts highly translated in the activated macrophages, possibly with the help from hypusine (Supplementary Fig. 18a). To validate their Mincle and eIF5Ahyp-dependent polysome association, we examined the ratios of polysome-associated versus monosome-associated transcripts (P/M ratios) induced by TDM and Pam3 co-treatment with/without GC7 by qRT-PCR. Among 20 relatively abundant candidate transcripts tested, six transcripts (iNOS, Mincle, Arg2, Pfkfb3, Selk and Scd2) showed significant decrease in P/M ratios (Supplementary Fig. 19a) as well as eIF5Ahyp-dependent polysome association in GC7-treated cells (Fig. 6a and Supplementary Fig. 19b). In addition to iNOS and Mincle, these genes are involved in high NO production and cell survival: arginase 2 (Arg2) catalyzes arginine and plays important role in NO and polyamine metabolism31. The key enzyme for glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3), is the main energy source of macrophages32. Selenoprotein k (Selk) is required for Ca2+ flux from endoplasmic reticulum (ER) during immune response33. Steroyl-Coenzyme A desaturase 2 (Scd2) controls intracellular level of monounsaturated fatty acids in immune cells, which is important for inflammation34. Except for Selk, mRNA of the polysome-associated transcripts identified above (iNOS, Pfkfb3, Arg2 and Mincle) were abundantly induced by Mincle (Supplementary Fig. 18a,b).


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)

Specific genes selected from the polysome profiling assay are regulated by Mincle-eIF5A-dependent translation.(a) Polysome fraction mRNAs from WT BMDMs co-treated with Pam3, TDM, with or without GC7 were analysed. qRT-PCR analysis of the indicated mRNAs in cells treated as indicated, presented for each fraction relative to the sum of all fourteen polysome fractions. (b) RIP analysis of the indicated gene transcripts from iBMDM cells stably expressing Flag-eIF5A, stimulated as indicated. Immunoprecipitation with anti-Flag antibody and qRT-PCR analysis of the indicated genes. (c) WT BMDMs were stimulated with Pam3, TDM or co-stimulated with Pam3 and TDM, in the presence of GC7 or control vehicle for 12 h. Immunoblot analysis of the indicated protein expression levels. Data are representative of three independent experiments (a–c: mean and s.d.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4837483&req=5

f6: Specific genes selected from the polysome profiling assay are regulated by Mincle-eIF5A-dependent translation.(a) Polysome fraction mRNAs from WT BMDMs co-treated with Pam3, TDM, with or without GC7 were analysed. qRT-PCR analysis of the indicated mRNAs in cells treated as indicated, presented for each fraction relative to the sum of all fourteen polysome fractions. (b) RIP analysis of the indicated gene transcripts from iBMDM cells stably expressing Flag-eIF5A, stimulated as indicated. Immunoprecipitation with anti-Flag antibody and qRT-PCR analysis of the indicated genes. (c) WT BMDMs were stimulated with Pam3, TDM or co-stimulated with Pam3 and TDM, in the presence of GC7 or control vehicle for 12 h. Immunoblot analysis of the indicated protein expression levels. Data are representative of three independent experiments (a–c: mean and s.d.).
Mentions: To identify additional transcripts regulated by Mincle-dependent eIF5A hypusination, we examined RNAs preferentially enriched in the polysome fraction under TDM and Pam3 co-stimulation, but shifted to the subpolysome fractions when GC7 was added. To this end, we sequenced total and polysome-associated RNAs from Pam3 and TDM co-treated macrophages with or without GC7 (Supplementary Fig. 17a,b). We observed 282 mRNAs located on the polysome by Pam3 and TDM co-stimulation; among these, 111 mRNAs were depleted from polysome fractions on GC7 treatment, revealing small number of transcripts highly translated in the activated macrophages, possibly with the help from hypusine (Supplementary Fig. 18a). To validate their Mincle and eIF5Ahyp-dependent polysome association, we examined the ratios of polysome-associated versus monosome-associated transcripts (P/M ratios) induced by TDM and Pam3 co-treatment with/without GC7 by qRT-PCR. Among 20 relatively abundant candidate transcripts tested, six transcripts (iNOS, Mincle, Arg2, Pfkfb3, Selk and Scd2) showed significant decrease in P/M ratios (Supplementary Fig. 19a) as well as eIF5Ahyp-dependent polysome association in GC7-treated cells (Fig. 6a and Supplementary Fig. 19b). In addition to iNOS and Mincle, these genes are involved in high NO production and cell survival: arginase 2 (Arg2) catalyzes arginine and plays important role in NO and polyamine metabolism31. The key enzyme for glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3), is the main energy source of macrophages32. Selenoprotein k (Selk) is required for Ca2+ flux from endoplasmic reticulum (ER) during immune response33. Steroyl-Coenzyme A desaturase 2 (Scd2) controls intracellular level of monounsaturated fatty acids in immune cells, which is important for inflammation34. Except for Selk, mRNA of the polysome-associated transcripts identified above (iNOS, Pfkfb3, Arg2 and Mincle) were abundantly induced by Mincle (Supplementary Fig. 18a,b).

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