Fig 2: 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 3: 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 4: 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.