Ferritin H deficiency deteriorates cellular iron handling and worsens Salmonella typhimurium infection by triggering hyperinflammation

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1β pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.


Supplementary Table S1
Supplementary Table S1. Table of  Estimateinteraction: linear regression estimate for the iron: genotype interaction. The table is available online.

Supplementary Table S2
Supplementary Table S2. Results of GO term enrichment analysis for transcripts downregulated by the iron: genotype interaction. Transcripts significantly regulated by iron: genotype interaction in course of S. tm infection were identified as described in Figure

Supplementary Table S4
Supplementary Table S4. Results of transcription factor binding site enrichment in the set of genes downregulated by the iron: genotype interaction. Transcripts significantly regulated by iron: genotype interaction in course of S. tm infection were identified as described in Figure 3, Materials and Methods and Supplementary Figure S5. Transcription factor (TF) binding site enrichment in the set of genes found downregulated by the iron: genotype interaction over the mouse whole-genome occurrence was investigated by a bootstraping procedure described in Materials and Methods. TF ID: Transfac database binding site identifier, TF symbol: Transfac database TF symbol, Enrichment: fold enrichment over binding site occurrence in mouse genome, Raw P: raw bootstrap test p value, P FDR: Benjamini-Hochberg-corrected raw bootstrap test p value. The table is available online.

Supplementary Table S5
Supplementary Table S5. Results of transcription factor binding site enrichment in the set of genes upregulated by the iron: genotype interaction. Transcripts significantly regulated by iron: genotype interaction in course of S. tm infection were identified as described in Figure      expressed as percent of STG-positive cells within the parent population (Fth +/+ ctrl: n = 11, Fth +/+ iron: n = 11, Fth Δ/Δ ctrl: n = 12, Fth Δ/Δ iron: n = 13). Each point denotes single animal, bars with whiskers represent means ± SEM. Statistical significance was assessed with two-way ANOVA with Benjamini-Hochberg-corrected two-tailed post-hoc T tests. In the plots, post-hoc test p values are indicated.

Strategy of whole-transcriptome data analysis.
Fth flox/flox (Fth +/+ ) and LysM-Cre Fth fl/fl (Fth ∆/∆ ) mice were intravenously administered PBS or iron isomaltoside (2 mg Fe) and infected three days later with 500 CFU GFP-expressing S.tm (STG). 12 hours post infection, total spleen RNA was isolated and subjected to a whole transcriptome measurement with gene microarrays (Mouse Gene 2.0 ST Array). Probe signal intensities were RMA (Robust Multiarray Average) normalized, probes allocated to gene identifiers and log2 expression calculated with the Bioconductor package oligo. For each gene, two-way ANOVA with the genotype, iron and genotype: iron interaction terms was performed, p values were calculated and adjusted with the Benjamini-Hochberg method. Genes with adjusted p < 0.05 for the genotype: iron interaction term were deemed significant. The gene set significantly regulated by the genotype: iron interaction was subjected to hierarchical clustering (average linkage algorithm, Euclidean distance, Genesis software), visualized as a heat-map and the cluster of genes significantly upregulated and downregulated in iron-loaded Fth ∆/∆ mice were identified. To identify functional relationships for the genes significantly upregulated in iron-loaded Fth ∆/∆. mice, gene ontology (GO) term enrichment analysis was performed with the DAVID 6.8 online tool.
To identify common transcription regulatory pathways for the genes significantly upregulated in ironloaded Fth ∆/∆. mice, transcription factor (TF) binding site enrichment analysis was performed with an inhouse written R script. In brief, the total number of predicted binding sites of the particular TF (D-Light) in promoters of the significantly regulated gene set was compared with numbers of TF binding sites in 10 6 random gene sets from the entire mouse genome. For details see Materials and Methods.
(A) Transcript levels of Il1b, Il6 and Tnf genes normalized to Gusb mRNA levels determined by qRT-PCR (B) Serum levels of IL1β, IL6, IL18 and TNFα measured by ELISA (C) Bacterial burden of the spleen and liver determined by plating Each point denotes single animal, bars with whiskers represent means ± SEM. Statistical significance was assessed with two-way ANOVA with Benjamini-Hochberg-corrected two-tailed post-hoc T tests (A) and Mann-Whitney U tests (B, C). In the plots, post-hoc test p values are indicated.

Supplementary Figure S7
Administration of diverse intravenous iron preparations induces cytokine storm in Fth ∆/∆ animals upon S.tm infection.
LysM-Cre Fth fl/fl (Fth ∆/∆ ) mice mice were intravenously injected with PBS (n = 7) or 2 mg elementary Fe in form of iron gluconate (FG, n = 5) or iron isomaltoside (FM, n = 7) and infected three days later with 500 CFU GFP-expressing S.tm (STG). Animals were analyzed 20 hours post infection. Serum levels of IL1β, IL6 and TNFα were measured by ELISA. Each point denotes single animal, bars represent with whiskers means ± SEM. Statistical significance was assessed with one-way ANOVA with Benjamini-Hochberg-corrected two-tailed post-hoc T tests. In the plots, post-hoc test p values are indicated.