Ferritin Light Chain Confers Protection Against Sepsis-Induced Inflammation and Organ Injury

Abstract

Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to protection against lipopolysaccharide-induced endotoxemia and cecal ligation and puncture (CLP)-induced model of sepsis as evidenced by reduced cytokine levels, multi-organ dysfunction and mortality. We identified that such protection is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin light chain restrains an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of FtL on NF-κB activation, a key signaling pathway that is implicated in the pathogenesis of sepsis. We further identified that LPS mediated activation of MAPK pathways, specifically, JNK, and ERK were also reduced with FtL pre-treatment. Taken together, our findings elucidate a crucial immunomodulatory function for circulating ferritin that challenges the traditional view of this protein as a mere marker of body iron stores. Accordingly, these findings will stimulate investigations to the adaptive nature of this protein in diverse clinical settings.

Keywords: LPS; NF-κB; cytokine; ferritin; inflammatory response; multi-organ injury; myeloid cells; sepsis.

Figure 1

Myeloid FtH deficiency prevents multi-organ failure and mortality in experimental sepsis. (A,B) Percent survival of mice deficient in myeloid-specific FtH (FtH^LysM−/−) and their floxed controls (FtH^fl/fl) after cecal ligation puncture (CLP) surgery. (A) Males, n = 11 per group. (B) Females, n = 7–12 per group. Data are from three independent experiments; ^#p < 0.05 vs. FtH^fl/fl. (C) Serum creatinine levels were measured and expressed in milligrams per deciliter (mg/dL) as mean ± SEM 24 h after CLP or sham surgeries. Sham, n = 3 per group; CLP, n = 10 per group. *p < 0.05 vs. sham. (D) Transcutaneous glomerular filtration rate (GFR) was measured using FITC-sinistrin 24 h after CLP or sham surgery and expressed in microliters per minute (μL/min) as mean ± SEM. Sham, n = 3 per group; CLP, n = 10 per group. *p < 0.05 vs. sham. (E) Serum aspartate aminotransferase (AST) was measured 24 h after CLP surgery and expressed in units per liter (U/L) as mean ± SEM. Sham, n = 5–6 per group; CLP, n = 12 per group. *p < 0.05 vs. sham (F–I). (F) Blood pressure was measured using radiotelemetry. Baseline measurements were recorded 24 h prior to CLP. Mice were monitored for 48 h. (G) Mean arterial pressure, (H) systolic, and (I) diastolic blood pressure were recorded 24 h following CLP in FtH^LysM−/− and FtH^fl/fl controls and expressed as mean ± SEM. n = 6 per group; *p < 0.05 vs. sham.

Figure 2

Myeloid FtH deficiency dampens the inflammatory response during sepsis Serum levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6), IL-12, chemokine ligand 1 (CXCL1), IL-10, IL-1β, IL-4, IL-2, and IL-5 were measured 24 h following sham or CLP surgery. Data are expressed in nanograms or picograms per milliliter (ng/mL or pg/mL) as mean ± SEM. Sham, n = 7 per group; CLP, n = 9–11 per group. Data are from two independent experiments. *p < 0.05 vs. sham, ^#p < 0.05 vs. FtH^fl/fl.

Figure 3

Myeloid FtH deficiency dampens the inflammatory response without altering the proportions of immune cell populations. (A,B) Representative flow cytometry histograms of (A) myeloid and (B) lymphoid panels from FtH^fl/fl sham control spleen demonstrating gating scheme and proportions of immune cell populations. (C,D) Quantification of number of cells in the (C) blood and (D) spleen 24 h after CLP. Data are from three independent experiments and are represented as number of cells per gram spleen (cells/g) or number of cells per ml blood as mean ± SEM. n = 4–5 per group. *p < 0.05 vs. sham.

Figure 4

Myeloid FtH deletion diminishes response to lipopolysaccharide (LPS). (A) Serum creatinine levels were measured and expressed in mg/dL as mean ± SEM 24 h after LPS (8 mg/kg) or vehicle (normal saline). n = 5 per group; ^#p < 0.05 vs. FtH^fl/fl. (B–G) Real-time polymerase chain reaction (RT-PCR) analysis of mRNA expression of (B) FtH, (C) TNF-α, (D) IL-6, (E) NACHT, LRR, and PYD domains-containing protein (NLRP3), and (F) IL-1β in bone marrow-derived macrophages (BMDMs) of FtH^fl/fl and FtH^LysM−/− mice that were treated with vehicle or LPS for 8 h. Data are analyzed and represented relative to GAPDH as mean ± SEM. n = 5–6 per group; *p < 0.05 vs. vehicle, ^#p < 0.05 vs. FtH^fl/fl. (G) Untreated BMDMs from FtH^fl/fl and FtH^LysM−/− mice were analyzed for expression of FtH and FtL. Membranes were stripped and reprobed for GAPDH expression to demonstrate equal loading. (H,I) BMDMs from FtH^fl/fl and FtH^LysM−/− mice were collected at 0, 0.25, 0.5, 1, 2, 4, and 8 h after LPS treatment and analyzed for expression of (H) phosphorylated p65 (pP65), total P65, phosphorylated extracellular signal-related kinase (pERK), FtH, and (I) phosphorylated c-Jun N-terminal kinase (pJNK), total JNK, and FtH. GAPDH was used as a loading control. Total ERK levels were examined in these lysates on a different membrane. Representative western blots from one out of three mice per genotype.

Figure 5

Serum ferritin is significantly higher in myeloid FtH deficient mice. Serum from FtH^fl/fl and FtH^LysM−/− mice 24 h after CLP and sham surgeries was collected and analyzed for (A) high mobility group box-1 (HMGB-1; ng/ml), (B) iron (μg/dL), (C) hepcidin (ng/ml), (D) neutrophil gelatinase associated lipocalin (NGAL; μg/ml), (E) hemopexin (mg/ml), (F) haptoglobin (mg/ml), and (G) ferritin (μg/ml). Data are expressed as mean ± SEM. n = 8–10 per group. *p < 0.05 vs. sham, ^#p < 0.05 vs. FtH^fl/fl.

Figure 6

Ferritin light chain (FtL) mitigates LPS-induced MAPK and NF-κB activation. (A) BMDMs from FtH^fl/fl mice were pre-treated with FtL for 16 h followed by treatment with LPS or saline control. Cell lysates were collected 8 h later and analyzed for mRNA expression levels of IL-6, monocyte chemoattractant protein-1 (MCP-1), TNF-α, inducible nitric oxide synthase (iNOS), and IL-1β, Data are normalized to GAPDH and fold change relative to saline vehicle are expressed as mean ± SEM. n = 3 from three independent experiments. *p < 0.05 vs. vehicle, ^#p < 0.05 vs. LPS+FtL. (B,C) FtH^fl/fl BMDMs were pre-treated with FtL for 16 h, then washed and treated with LPS and collected at 0, 0.25, 0.5, 1, 2, 4, and 8 h. Cell lysates were analyzed for expression of (B) pJNK, total JNK, pERK, FtL, and FtH. β-actin was used as a loading control. Total ERK levels were examined in these lysates on a different membrane. (C) pP65, P65, and IκBα. GAPDH was used as a loading control. (D) FtH^fl/fl BMDMs were pre-treated with Apoferritin (ApoF) for 16 h, then washed and treated with LPS and collected at 0, 0.5, 1, 3, and 8 h. Cell lysates were analyzed for expression of pP65, P65, IκBα, pJNK, total JNK, and FtH. GAPDH was used as a loading control. Representative western blot from one out of three mice per treatment group.

Figure 7

Alteration of gene expression profile of blood leukocytes following sepsis. RNA sequencing was performed on blood leukocytes isolated from FtH^fl/fl and FtH^LysM−/− mice following CLP or sham surgery. (A) Hierarchical clustering of genes that are significantly altered (p < 0.01) between leukocytes from the transgenic mice at 8 h following surgery. Heatmap displays z-transformed expression values. n = 3 per group. (B) NF-κB signaling pathway genes differentially expressed in FtH^fl/fl and FtH^LysM−/− CLP compared to FtH^fl/fl sham. The differentially expressed genes were identified based on absolute fold change of two or more and adjusted p value < 0.01. n = 3 per group. (C) Genes associated with “immune response” biological process [GO: 0006955] down-regulated or differentially expressed in FtH^LysM−/− CLP compared to FtH^fl/fl CLP. The differentially expressed genes were identified based on absolute fold change of two or more and adjusted p < 0.01. n = 3 per group.