Fig 2: The Trx1 system positively regulates the binding activity of NF-?B p65 to the DNA in BMDCs.(A–E) Txnrd1fl/fl;Rosa26-CreERT2 mice and Txnrd1fl/fl littermates were injected with TAM to delete the Txnrd1 gene, and bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) BMDCs were stimulated with LPS (400 ng/ml) for 10, 30, or 60 min and lysed for western blot. Expression of phospho-I?B-a, respective I?B-a, NF-?B p65, phospho-Erk1/2 and respective Erk1/2 was assessed with ß-actin as a loading control. (B, C) WT or Txnrd1-deficient BMDCs were fixed at the indicated time points post LPS treatment, stained for DNA (DAPI), NF-?B p65 and actin (Phalloidin), and imaged using a DeltaVision system. Approximately 10 randomly chosen imaging fields encapturing a total of 50–100 nuclei were analyzed per sample per condition. (B) Nuclear and whole-cell masks were made using the DAPI and phalloidin channels, and NF-?B signal intensity within the masks was quantified. Shown is the nuclear NF-?B signal strength plotted as percentage of whole-cell NF-?B signal. (C) Depicted are example images of the samples of indicated times points post LPS treatment. In the merged images, DAPI and anti-NF-?B channels are shown in red and green, respectively. Scale bar represents 10 µm (top-left panel). Squares indicate fields, which are magnified in Figure 5—figure supplement 2. (D) WT or Txnrd1-deficient BMDCs were stimulated with LPS (400 ng/ml) for 100 min, and the recruitment of NF-?B p65 to the Il12b (top-left), Il1b (top-right), Il6 (bottom-left) and Nfkbia (bottom-right) promoters was assessed by p65 chromatin immunoprecipitation (ChIP) analysis and quantified by RT-PCR. ‘Promoter’ indicates the utilization of a primer pairs that amplify a fragment close to the NF-?B binding sites at the promoter region of the indicated genes, whereas ‘Ctr primers’ indicate primer pairs that were used as a control to amplify a region several kilobases away from the NF-?B binding sites (n = 2). (E) The NF-?B p65 binding activity to its DNA response element in nuclear extracts from BMDCs stimulated with LPS (400 ng/ml) for 40 min was assessed by an ELISA-based method (n = 3). Bar graphs and dot plots represent mean + standard deviation. Data are representative of two (A–D) or three (E) independent experiments. For each panel, a representative experiment with technical replicates is shown (D, E). Two-way ANOVA adjusted by Bonferroni's multiple comparison test was used in B: ns, not significant. One-way ANOVA adjusted by Tukey’s multiple comparison test was used in D, E: *p=0.0332; **p=0.0021; ***p=0.0002; ****p=0.0001.

Fig 3: The Trx1 system is critical for proper M1 polarization but dispensable for promoting NF-?B binding to target DNA in BMDMs.(A–P) Bone marrows from Txnrd1fl/fl;Rosa26-CreERT2 mice and control Txnrd1fl/fl littermates treated with TAM were differentiated with M-CSF to obtain BMDMs. (A–H) BMDMs were polarized toward M1 and M2 with LPS/IFN-? and IL-4, respectively, or left untreated (M0). (A) Schematic showing the polarization assay. (B, C) Shown are the expression levels of Txnrd1 (B) and Txn1 (C) at the mRNA level determined by RT-PCR (n = 3). (D, E) Depicted is the level of the M1 marker Nos2 at the mRNA level determined by RT-PCR (D); n = 3) and at the protein level assessed by flow cytometry (E). (F, G) Shown is the level of the M2 marker Relma at the mRNA level (Fizz1) determined by RT-PCR (F); n = 3) and at the protein level assessed by flow cytometry (G). (H) Cell survival of M1 macrophages (eFluor780-Annexin-V-) polarized either for 1 or 2 days with LPS/IFN-? was assessed via flow cytometry (n = 3). (I, J) BMDMs were stimulated for 7 hr with CpG (400, 100 nM), LPS (100, 20 ng/ml), LTA (5, 1 µg/ml), or R837 (5, 1 µg/ml), and IL-12p40 (I) and IL-6 (J) were measured in supernatants by ELISA. ‘High’ and ‘Low’ indicate the concentration of the utilized stimulus (n = 3). (K) Analysis of Txnrd1 mRNA in BMDMs assessed by RT-PCR for confirmation of gene deletion (n = 3). (L, M) Shown is the mRNA expression of Il12b (L) and Il6 (M) determined via RT-PCR in BMDMs stimulated for 7 hr with CpG (100 nM), LPS (100 ng/ml), LTA (1 µg/ml) or R837 (5 µg/ml; n = 3). (N) Shown are the expression levels of the indicated Grx isoforms in WT BMDCs and BMDMs (n = 3). (O) BMDMs were treated overnight with BSO (3 µM) before stimulation with LPS (400 ng/ml) for 7 hr. The concentration of IL-12p40 in supernatants was subsequently determined by ELISA (n = 3). (P) Shown is the DNA binding activity of NF-?B p65 in nuclear extracts from Txnrd1fl/fl;Rosa26-CreERT2 and control Txnrd1fl/fl BMDMs treated overnight with 3 µM BSO (or medium as a control) before stimulation with LPS (400 ng/ml) for 90 min (n = 3). Bar graphs represent mean + standard deviation. Data are representative of two (B–H, K–P) or three (I, J) independent experiments. For each panel, a representative experiment with replicates of in vitro culture conditions is shown (B–D, F, H–P). One-way ANOVA adjusted by Tukey’s multiple comparison test was used in B-D, F, O, P: *p=0.0332; **p=0.0021; ***p=0.0002; ****p=0.0001; ns, not significant. Student's t test (two-tailed, unpaired) was used for two-group analysis in H–N: *p=0.05; **p=0.01; ***p=0.001; ****p=0.0001; ns, not significant.

Fig 4: Txnrd1-deficient BMDCs undergo defective transcription of pro-inflammatory cytokines.(A–J) Txnrd1fl/fl;Rosa26-CreERT2 mice and Txnrd1fl/fl littermates were injected with TAM to delete the Txnrd1 gene. Bone marrow cells were differentiated with GM-CSF to obtain BMDCs. (A) Analysis of Txnrd1 mRNA by RT-PCR in cultured Txnrd1fl/fl;Rosa26-CreERT2 and Txnrd1fl/fl BMDCs for confirmation of gene deletion (n = 3). (B) Viability (eFluor780-Annexin-V-) of BMDCs was assessed via flow cytometry (n = 3). (C) BMDCs were primed with CpG (100 nM), LPS (100 ng/ml), LTA (1 µg/ml), R837 (5 µg/ml) for 7 hr, and expression of Txn1 at the mRNA level was determined via RT-PCR (n = 3). (D, E) BMDCs were stimulated for 7 hr with CpG (100, 20 nM), LPS (100, 20 ng/ml), LTA (5, 1 µg/ml), R837 (5, 1 µg/ml), or zymosan (10, 2 µg/ml). ‘High’ and ‘Low’ indicate the concentration of the utilized stimulus. IL-6 (D) and IL-12p40 (E) were measured in supernatants by ELISA (n = 3). (F, G) BMDCs were stimulated for 7 hr with CpG (100 nM), LPS (100 ng/ml), LTA (1 µg/ml) or R837 (5 µg/ml), and the expression of Il6 (F) and Il12b (G) was determined by RT-PCR (n = 3). (H) BMDCs were stimulated with CpG (100 nM), LPS (400 ng/ml), LTA (5 µg/ml), R837 (5 µg/ml) or zymosan (10 µg/ml) before the addition of 2 mM ATP or 200 µg/ml alum for 1 hr or 4 hr, respectively. The concentration of IL-1ß in supernatants was determined by ELISA (n = 3). (I) BMDCs were stimulated with TLR ligands as in F,G), and expression of Il1b at the mRNA level was determined via RT-PCR (n = 3). (J) Naive, splenic, Smarta-1 transgenic CD4+ T cells were co-cultured with Txnrd1-deficient BMDCs (or Txnrd1-sufficient BMDCs as a control) and the indicated concentrations of the GP61-80 peptide. Shown are the frequencies of CD4+ T cells producing IFN-?+ after restimulation with PMA/ionomycin (n = 3). Bar graphs represent mean + standard deviation. Data are representative of two (A–C, F, G, I, J) or four (D, E, H) independent experiments. For each panel, a representative experiment with replicates of in vitro culture conditions is shown. Student's t test (two-tailed, unpaired) was used to compare Txnrd1fl/fl;Rosa26-CreERT2 and control Txnrd1fl/fl groups in (A, B, D–J): *p=0.05; **p=0.01; ***p=0.001; ****p=0.0001; ns, not significant. One-way ANOVA followed by Dunnett’s correction was used in C (comparison to the unstimulated control): *p=0.0332; **p=0.0021.

Fig 5: Commensal Bacteria Are Required for Mincle-Dependent Th17 Generation in PPs(A and B) Frequency of IL-17 production by intracellular staining in re-stimulated CD4+ T cells from PPs of the indicated genotypes after treatment with an antibiotic cocktail (ABX) containing ampicillin, neomycin, metronidazole, and vancomycin (A) or vancomycin only (B) in the drinking water over 4 weeks.(C) Frequency of IL-17 and IL-22 production by intracellular staining of re-stimulated OT-II T cells co-cultured with sorted WT and Mincle-deficient (Clec4e−/−) GM-DCs loaded with OVA323–339 peptide, as in Figures 1D and 1E; the cells were pulsed or not (medium) with mucosal-associated commensals from WT mice treated with ABX during gestation and lactation and gavaged with L. plantarum at weaning as indicated (L. plantarum-enriched mucus; 10:1 DC ratio).(D) ELISA of IL-6 and IL-23 from the co-culture of GM-DC in (C).(E and F) WT littermates and Mincle-deficient (Clec4e−/−) mice were treated with ABX during gestation and lactation and were gavaged with L. plantarum (1 × 106) at weaning (+L. plantarum gavage) or not (WT), as indicated in the figure. The frequency of IL-17- and IL-22-producing re-stimulated CD4+ T cells (E) or CCR6+ ILCs (F) from PPs by intracellular staining in the indicated genotypes and conditions is shown.Data represent one representative experiment of two performed (A and F) or were pooled from at least two independent experiments (B–E). Each symbol represents an individual mouse. The arithmetic mean for each group is indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (A–E: one-way ANOVA and Bonferroni post hoc test; F: unpaired two-tailed Student’s t test). See also Figure S5.