Epidermal hepcidin is required for neutrophil response to bacterial infection

Abstract

Novel approaches for adjunctive therapy are urgently needed for complicated infections and patients with compromised immunity. Necrotizing fasciitis (NF) is a destructive skin and soft tissue infection. Despite treatment with systemic antibiotics and radical debridement of necrotic tissue, lethality remains high. The key iron regulatory hormone hepcidin was originally identified as a cationic antimicrobial peptide (AMP), but its putative expression and role in the skin, a major site of AMP production, have never been investigated. We report here that hepcidin production is induced in the skin of patients with group A Streptococcus (GAS) NF. In a GAS-induced NF model, mice lacking hepcidin in keratinocytes failed to restrict systemic spread of infection from an initial tissue focus. Unexpectedly, this effect was due to its ability to promote production of the CXCL1 chemokine by keratinocytes, resulting in neutrophil recruitment. Unlike CXCL1, hepcidin is resistant to degradation by major GAS proteases and could therefore serve as a reservoir to maintain steady-state levels of CXCL1 in infected tissue. Finally, injection of synthetic hepcidin at the site of infection can limit or completely prevent systemic spread of GAS infection, suggesting that hepcidin agonists could have a therapeutic role in NF.

Keywords: Bacterial infections; Chemokines; Infectious disease; Mouse models.

Figure 1. Keratinocyte hepcidin prevents bacterial systemic spread.

IHC with or without primary antibody detecting (A) hepcidin (in brown) on sections of cutaneous human biopsies of GAS NF patients and healthy control using PerkinElmer’s Lamina multilabel slide scanner Panoramic Viewer software. (B) Real-time reverse transcription PCR (qPCR) for hepcidin from GAS-infected human 3D organotypic skin equivalent model; n = 4 per group. (C) qPCR for hepcidin in murine GAS-infected skin; n ≥ 3 per group. (D) Hepcidin (in blue) and K14 (in brown) IHC on cutaneous biopsies of WT mice challenged or not with GAS. Scale bars: 100 μm. Leica DMI3000B microscope, Leica DFC310FX camera, 5/0.4; Leica LAS Core software. (E) Generation of Hamp1Δ^ker mice. (F) Plasma iron, ferritin, transferrin, and skin iron levels in Hamp1^lox/lox and Hamp1Δ^ker mice; n ≥ 4 per group. (G) Bacterial count in skin, blood, and spleen of Hamp1^lox/lox and Hamp1Δ^ker mice 4 days after injection with GAS; n ≥ 10 per group. (H) Weight variation of Hamp1^lox/lox and Hamp1Δ^ker mice during infection; n = 10 per group. Statistical analysis was performed using a Mann Whitney test (B, C, F, and G) or a 2-way ANOVA followed by Tukey’s test for weight kinetics (H). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Hepcidin promotes CXCL1 production by keratinocytes.

(A) GAS killing kinetics with 32 μM of LL-37 and hepcidin; n = 3 per group. Representative of 2 independent experiments. (B) GAS growth curve in the presence of penicillin G, LL-37, hepcidin, or PBS; n = 3 per group. Representative of 2 independent experiments. (C) Bacterial recovering at 1 hour and 3 hours following incubation of log-phase GAS with murine primary keratinocytes (KC) from Hamp1^lox/lox and Hamp1Δ^ker mice. Data are representative of 2 independent experiments performed in triplicate. (D) Cytokines measured with the V-PLEX Proinflammatory Panel1 kit in the culture supernatant of murine primary keratinocytes stimulated for 1 or 3 hours with hepcidin or PBS; n = 3 per group. Representative of 3 independent experiments. (E) IL-8 ELISA on the culture supernatant of HaCat or a human 3D skin equivalent model stimulated with 3.6 μM hepcidin; n ≥ 3 per group. (F) CXCL1 levels measured by ELISA in the culture supernatant of murine primary keratinocytes stimulated for 3 hours with 3.6 μM hepcidin in the presence of PBS or 100 μM FPN inhibitor (2D-014); n ≥ 3 per group. Representative of 3 independent experiments. (G) CXCL1 levels measured by ELISA on the culture supernatant of murine primary keratinocytes stimulated for 3 hours with 500 μM ferric ammonium citrate (FAC); n = 3 per group. Representative of 3 independent experiments. Statistical analysis was performed using a 2-way ANOVA followed by Tukey’s test (A, B, and D), unpaired Student’s t test (E and G), or a 1-way ANOVA followed by Tukey’s test (C and F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 3. Hepcidin is required for CXCL1 production and neutrophil recruitment.

(A) Anti-CXCL1 or (C) anti–polymorphonuclear leucocyte (PMN) immunostainings on skin of Hamp1^lox/lox and Hamp1Δ^ker mice challenged with GAS. PerkinElmer’s Lamina multilabel slide scanner Panoramic Viewer software. (B) CXCL1 ELISA on lysates from GAS-infected skin biopsies of Hamp1^lox/lox (n = 5) and Hamp1Δ^ker mice (n = 6). (D) Neutrophil count from GAS-infected skin biopsies of Hamp1^lox/lox (n = 5) and Hamp1Δ^ker mice (n = 4). (E) Area of necrotic ulcers in skin of Hamp1^lox/lox and Hamp1Δ^ker mice during GAS infection; n = 7 per group. (F) Scheme of the study protocol. (G) Bacterial count in the skin of Hamp1^lox/lox and Hamp1Δ^ker mice injected daily with CXCL1 or PBS; n ≥ 4 per group. Statistical analysis was performed using a Student’s t test (B and D), a 2-way ANOVA followed by Tukey’s test (E), or a 1-way ANOVA followed by Tukey’s test (G). *P < 0.05; **P < 0.01.

Figure 4. Hepcidin is resistant to SpyCEP cleavage and has a therapeutic role in NF.

(A) Mass spectroscopy analysis of CXCL1 or hepcidin incubated overnight with SpyCEP or PBS. Electrospray ionization generated a series of multiply charged ions (indicated as m/z; mass-to-charge ratio) from which the average molecular mass (m) of each was deduced. The blue arrows indicate uncleaved peptide peaks at 7.8 kDa (CXCL1) and 2.7 kDa (hepcidin). Red arrows show the cleavage products of CXCL1 with a small (1.3 kDa) and a big (5.9 kDa) fragment. (B) Therapeutic protocol. (C) Neutrophil count (3 measurements per individual mouse were averaged); n = 6 per group. (D) Weight variation and (E) bacterial count in spleen of WT infected mice treated with PBS or hepcidin (n = 9, red square) or PBS (n = 9, black square) during 4 days. Statistical analysis was performed using a Student’s t test (C), a 2-way ANOVA followed by Tukey’s test (D), or a Mann Whitney test (E). *P < 0.05.