Fig 2: Gut-Derived MAIT Cells Show a Broadly Similar Response Pattern toward Innate and Adaptive Stimuli Compared with Their Blood-Derived CounterpartsRepresentative plots showing the percentage of cells positive for the indicated effector molecules as a proportion of CD8+ MAIT cells.(A–C) Proportions of blood-derived (n = 32) CD8+ MAIT cells producing IFN-γ (A), TNF-α (B), or GrB (C) following overnight stimulation with combinations of suboptimal concentrations of IL-12 and IL-18, TL1A, and αCD3/CD28 beads as indicated.(D–F) Proportions of gut-derived (n = 13) CD8+ MAIT cells producing IFN-γ (D), TNF-α (E), or GrB (F) stimulated in the same way as in (A)–(C).(G and H) Expression of IFN-γ, TNF-α, and GrB by blood-derived (G, n = 7) or gut-derived (H, n = 6) CD8+ MAIT cells 20 h after coculture with THP1 cells alone or THP1 cells incubated with 25 fixed E. coli bacteria per cell.Data were acquired from multiple donors as indicated in 3–5 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests (A–F), two-way ANOVA (G), or Wilcoxon tests (H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001.See also Figure S3.

Fig 3: TCR- and Cytokine-Activated MAIT Cells Possess Distinct Transcriptional Profiles(A–C) Venn diagrams showing genes that are significantly differentially modulated (p < 0.05, fold change > 4) in TCR (T)-, cytokine (C)-, or TCR and cytokine (TC)-treated CD8+ MAIT cells compared with untreated (UT) MAIT cells of three healthy individuals. The cytokine (C) stimulation consisted of a cocktail of 4 cytokines: IL-12 (2 ng/mL), IL-18 (50 ng/mL), IL-15 (25 ng/mL), and TL1A (100 ng/mL). Genes with significantly altered expression levels (A) are divided into two sets: those are that are upregulated upon stimulation (B) and those that are downregulated upon stimulation (C).(D) Heatmap showing 1,594 significantly differentially expressed transcripts (p < 0.05, fold change > 4) between TCR/C/TC-stimulated and UT CD8+ MAIT cells among the same three healthy individuals.(E) Visualization of the CD8+ MAIT cell transcripts elicited by differential stimulations in the subspace of the first principle components (PCs). Each colored circle represents a sample and is color coded in accordance with the conditions with which cells were stimulated, as illustrated on the right-hand side of the graph.(F–K) Volcano plots to visualize differentially expressed transcriptional profiles of activated CD8+ MAIT cells stimulated in different ways. Each point represents a single gene, and genes expressed at significantly higher or lower levels between the compared conditions are depicted, respectively, in the upper-right or upper-left corner of each plot. Genes discussed in the text are highlighted in blue (tissue repair associated) or in red (inflammation associated). The gene expression of untreated MAIT cells was compared to (F) T-, (G) C-, or (H) TC-stimulated MAIT cells. Further, gene expression in those cells was also compared directly between the different stimulation conditions: (I) T- to C- stimulation, (J) T- to TC-stimulation, and finally (K) C- to TC-stimulation.Data were acquired from three donors in one experiment.See also Figure S4 and Tables S1, S2, and S3.

Fig 4: Targeting TRAF3IP2 or RECK overexpression blunts IL-18-induced ASMC proliferation and migration. (A–E) Silencing TRAF3IP2 inhibits IL-18-induced ASMC proliferation (B) and migration (C). ASMC were transduced with adenovirus-expressing shRNA targeting human TRAF3IP2 (moi 10 for 48 h), made quiescent, and then exposed to IL-18 (10 ng/mL; experimental design in (A)). ASMC proliferation was assessed after 48 h of IL-18 addition using the CyQUANT Cell proliferation assay (B), and migration after 18 h using Boyden chamber assay (C). ASMCs migrating to the lower surface of the membrane were counted in 10 different fields and summarized as mean ± SEM. (B,C) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6). Knockdown of TRAF3IP2 was confirmed by RT-qPCR using a TaqMan™ probe (D) and Western blotting (E). (D,E) * p < 0.01 vs. untreated (n = 3). (F,G) Dose-dependent effects of Ad.RECK on RECK expression (experimental design in (F)). Induction of RECK following adenoviral transduction was confirmed by Western blotting with tubulin serving as an internal control (G). (H–K) Forced expression of RECK inhibits IL-18-stimulated ASMC proliferation and migration. ASMCs were transduced with adenovirus-expressing human RECK cDNA (moi 10 for 24 h), made quiescent, and then treated with IL-18 (experimental design in (H)) and analyzed for proliferation (I) and migration (J) as in (B,C). (C,J) The insets show representative images of Matrigel™ Transwell invasion. Scale bar: 20 μM. (E,G,K) While a representative immunoblot is shown, the intensities of immunoreactive bands from three (E), four (G) and three (K) independent experiments were semiquantified by densitometry and are summarized on the right. (I) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+GFP (n = 6); (J) * p < at least 0.01 vs. Untreated; † p < 0.01 vs. IL-18 or IL-18+eGFP (n = 4); (K) * p < 0.01 vs. Untreated; † p < 0.05 vs. IL-18 or IL-18+eGFP (n = 3).

Fig 5: EF24 inhibits IL-18-induced ASMC proliferation and migration, without affecting cell viability. (A) Chemical structure of EF24, a curcumin analog. (B–F) EF24 is not cytotoxic to ASMC at the concentrations used. Quiescent ASMCs were exposed to EF24 at the indicated concentrations for 48 h (experimental design in (B)). Cell viability was analyzed by trypan blue dye exclusion (C), cleaved caspase-3 levels by ELISA (D) with H2O2 (100 μM) serving as a positive control, ELISA of mono-oligonucleosomal fragmented DNA (E) with H2O2 (100 μM) serving as a positive control, and LDH release with LDH release in response to 0.2% Triton-X100 being considered as 100% (F). DMSO (0.1%) alone served as a solvent control (depicted as “0”). Cells without any treatment served as a control (C). (D–F) * p < 0.01 vs. untreated controls or treated with DMSO alone. (G–H) EF24 inhibits IL-18-induced ASMC proliferation and migration. Quiescent ASMCs were incubated with EF24 at various concentrations ranging from 1 to 10 μM in DMSO for 1 h, followed by the addition of IL-18 at 10 ng/mL for 48 h (experimental design in (G)). Cell proliferation was analyzed by the CyQUANT Cell proliferation assay (H). Cell migration was analyzed by Boyden chamber assay after 18 h ((I) The insets show representative images of Matrigel™ transwell invasion). The combination of IL-18 and EF24 did not affect cell viability (J). (C,I) Scale bars, 20 μM. (H,I) * p < at least 0.05 vs. untreated controls, † p < 0.05 vs. IL-18 without EF24 (n = 6). (J) * p < 0.01 vs. untreated controls (n = 12).