Fig 2: Neutralizing maternal TNFα is protective against embryonic malformation induced by the combination of high maternal iron and Western diet.a Clustering analysis of 32-plex cytokine panel in maternal serum from iron-adequate (gray circles), dietary iron-loaded (Fe diet, light blue circles), and genetically iron-loaded hepcidin KO (HKO, dark blue circles) dams fed standard or Western diet (WD). Color key indicates Z-score for each cytokine. b–e E18.5 TNFα measurements in b maternal serum, c embryo serum pooled from each litter, d pooled amniotic fluid, and e placental Tnf gene expression. Placentas were randomly selected for analysis. f Hepcidin KO females were fed Western diet (100 ppm iron) starting at 3 weeks of age and were mated after 9 weeks. Pregnant dams received intravenous injections of neutralizing TNFα antibody (white circles) or isotype IgG (dark blue circles) targeting trinitrophenol as a control (250 µg/injection) on E5.5, E8.5, E12.5, and E15.5, and embryo outcome was evaluated at E18.5. g Embryo gross morphology. h–j Incidence of eye malformations. b–e, h, j Error bars represent mean ± s.e.m. Statistical differences were determined by one-way ANOVA on ranks followed by Dunn’s method for multiple comparisons (denoted by #) or two-tailed Fisher’s exact test (indicated by ‡). P-values are indicated in each figure panel. Source data are provided as a Source data file.

Fig 3: Age-dependent differences in transcriptional profiles of ICOS+CD38+ cTfh(A) t-Distributed stochastic neighbor embedding (t-SNE) analysis was performed for all CD4 T cell subsets. ICOS+CD38+ cTfh shown before and after vaccination for young (n = 6) and elderly (n = 8) adults.(B) Aggregated GSEA results for Hallmark gene sets comparing day 7 versus day 0 for ICOS+CD38+ cTfh in young (orange) and elderly (purple) adults.(C–F) GSEA shown for day 7 versus day 0 for ICOS+CD38+ cTfh from young (orange) and elderly (purple) adults for TGF-β signaling (C), E2F targets (D), IL-2 signaling (E), and TNF-NF-κB signaling (F) gene sets.

Fig 4: TNF-NF-κB pathway is enriched with aging in ICOS+CD38+ cTfh(A) Schematic for RNA-seq analyses.(B and C) Weighted gene correlation network analysis on ICOS+CD38+ cTfh at day 7 from young (B) and elderly (C) adults.(D) Module preservation analysis was performed using Fisher’s exact test. Heatmap color and value indicate the −log10(p value) for the overlap in genes in modules.(E) Genes ranked based on module membership for elderly module EM4. Then, GSEA was performed using the MSigDB HALLMARK collection. Positive enrichment scores indicate enrichment for genes in EM4.(F) Transcription factors with module membership >0.80 in EM4 displayed as a multiple association network (GeneMANIA without gene prediction).(G) Ingenuity Pathway Analysis for predicted upstream regulators for ICOS+CD38+ cTfh at day 7 for young and elderly adults.(H) Differentially expressed NF-κB target genes comparing ICOS+CD38+ cTfh at day 7 from elderly (purple) and young (orange) adults.(I) GSEA for NF-κB target genes to compare ICOS+CD38+ cTfh from elderly and young adults.(J) Example plot from 1 young adult and 1 elderly adult for total NF-κB p50 protein from an independent cohort of young and elderly adults. Geometric mean fluorescence intensity (MFI) shown.(K) Total NF-κB p50 for ICOS+CD38+ cTfh at baseline in young (orange) and elderly (purple) adults (n = 16 in each group), as taken from an independent cohort of young and elderly adults.(L and M) Gene expression for TNFRSF1A (L) (n = 6 for young; n = 8 for elderly) and TNFRSF1B (M) (n = 8 for elderly) shown at day 7 after vaccine from log2-transformed counts data for young (orange) and elderly (purple) adults.

Fig 5: TAT–RHIM induces necrotic cell death that is independent of endogenous RHIM proteins and cannot be inhibited.(A) Murine NIH3T3 cells were incubated with 40 µM TAT–M45RHIM for 8 h in the presence of either 25 µM of the pan-caspase inhibitor zVAD, 20 µM of the RIPK1 inhibitor Nec-1s, 1 mM of the RIPK1 inhibitor primidone, or 10 µg/ml of the TNF antagonist etanercept. Cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. The graphs present the means ± standard deviations of three independent experiments. None of the investigated inhibitors of RCD could ameliorate TAT–M45RHIM-induced cell death. (B) Murine non-edited, Ripk1-, Ripk3- or Ripk1/Ripk3/Mlkl-edited NIH3T3 cells (designated as parental, Ripk1-ko, Ripk3-ko and Ripk1/3/Mlkl-tko, respectively) were incubated for 8 h at 37°C as indicated with 100 ng/ml TNFα + 25 µM zVAD, 20 µM Nec-1s, or 40 µM TAT–M45RHIM. Cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. FACS dot plots of a representative experiment are shown (n = 3 independent repeats). While TNFα/zVAD induces necroptosis in parental NIH3T3 cells that can be rescued by Nec-1s, the respective Ripk1, Ripk3 single- or Ripk1/3/Mlkl triple knockout cells are completely protected against necroptosis. Nevertheless, like non-edited cells, these ko-cells are as susceptible to TAT–M45RHIM-induced cell death. (C) Primary murine BMDMs isolated from wild-type (wt) or Nlrp3-knockout (Nlrp3-ko) mice were incubated at 37°C with 40 µM TAT–M45RHIM for 8 h as indicated. Cell death was quantified by FACS analysis using 7-AAD and phosphatidylserine accessibility (Annexin V staining) as markers. FACS dot plots of a representative experiment are shown (n = 3 independent repeats). Note that knockout of Nlrp3 has no protective effect against TAT–M45RHIM-induced cell death.