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. 2017 Nov 2;7:14940. doi: 10.1038/s41598-017-14780-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Palmitic acid promotes the degradation of t-bet and gata-3 mRNA in iNKT cells via regulated IRE1α-dependent decay (RIDD), thereby suppressing IL-4 and IFN- γ production. (a and b) iNKT cells or α-GalCer/CD1d tetramer ⁺ TCRβ ⁺ iNKT cells sorted from C57BL/6 mouse liver mononuclear cells were treated with (a) tunicamycin (TM) and/or STF083010 or (b) palmitic acid and/or STF083010 in the presence of anti-CD3 and anti-CD28 mAbs. Reverse-transcription PCR and real-time PCR were performed to estimate un-spliced (us) or spliced (s) xbp-1. (c) The expression levels of Sec 61a1, Edem1, Pcyt1a and Atf4 were also measured in iNKT cells using real-time PCR. (d) To knock-down xbp-1, iNKT cells were transfected with control or xbp-1 siRNA and then treated with palmitic acid or vehicle in the presence of anti-CD3 and anti-CD28 mAbs for 24 h. The levels of IL-4 and IFN- γ were estimated in culture supernatants using ELISA. (e) The transcription levels of t-bet, gata-3, Il4, and Ifng were estimated in iNKT cells treated with vehicle, palmitic acid, or palmitic acid and STF083010 in the presence of actinomycin D at the indicated time points. (f) The transcription levels of t-betand gata-3 were measured in Ern1- or xbp-1-knockdown or control iNKT cells upon treatment with vehicle, palmitic acid, or palmitic acid and STF083010. (g) mRNA structural modeling for t-bet and gata-3. (h) DN32.D3 cells were transfected with mutant t-bet (G971C or C969G), mutant gata-3 (G1604C or C1602G), or wild type t-bet and gata-3 and then treated with palmitic acid or vehicle in the presence of anti-CD3 and anti-CD28 mAbs. mRNA measurement reflects the amount of relative degradation of t-bet or gata-3 transcript. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.005.