Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock

Abstract

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.

Fig 1. Irf6 is expressed in innate immune cells.

(A-D) Murine bone marrow-derived macrophages (A, D, mMΦ), neutrophils (B, PMN) and dendritic cells (C, DC) were immunostained for Irf6 (red) or nuclear DAPI (blue). (E-G) Human blood-derived leucocytes (hPMN) were immunostained for Irf6 (red) and ly-6G (green, E), CD63 (green, F), and CD11b (green, G).

Fig 2. Irf6 in innate immune cells is required for resistance to LPS-induced endotoxic shock.

(A) qPCR for Irf6 in bone marrow-derived macrophages from wild-type and cKO animals. Data are expressed as percentage of wild-type (n = 3; unpaired t-test, **** P < 0.0001). (B) Western blot analysis of protein extracts from WT (n = 2) and cKO macrophages (n = 2), and embryonic day (e) 17.5 skin (C) Survival of wild-type and cKO was assessed over a 7-day period following LPS-induced endotoxic shock (n = 37–39, Gehan-Breslow-Wilcoxon test, * P < 0.05).

Fig 3. In vivo cytokine levels and peritoneal migration of neutrophils are not significantly different between wild-type and cKO.

Low (LPS 15 mg/kg, A, B) and high (LPS 400 mg/kg, C, D) levels of LPS were injected intraperitoneally in wild-type (WT) and cKO animals. (A, C) Serum cytokine levels were measured 8 h following injection and reported as fold ratio cKO over wild-type. (B, D) Number of neutrophils (PMN) were counted following intraperitoneal lavage. Data are means ± SEM, n = 6–13 animals per group.

Fig 4. Irf6 promotes in vitro migration of neutrophils as determined by the EZ-TAXIScan assay.

Murine bone marrow-derived neutrophils were stimulated with an optimal ZAS spatial gradient generated by a 10% ZAS loading concentration using the EZ-TAXIScan chamber. (A) Net path length (μm), (B) Chemotactic index (arbitrary units), (C) Instantaneous velocity (μm/min), and (D) Percentage of motile cells in the tested cell population. Data are means of 7 experiments (150–250 cells total per group) ± SEM. ** P < 0.001, *** P < 0.0005 following t-test.

Fig 5. Irf6 inhibits cytokine production in bone marrow-derived macrophages in vitro.

Cytokines were measured in conditioned medium from unstimulated (baseline) macrophages, or macrophages stimulated with LPS (50 ng/ml), FSL (1 μg/ml) or Poly IC (10 μg/ml) from wild-type (WT) and Irf6 cKO (cKO). Data are expressed as fold change between stimulated conditioned medium over baseline. N = 9–11 per group. Each symbol represents a biological replicate, horizontal bar represent the mean. * P < 0.05.

Fig 6. Irf6 inhibits NFkB activation in bone marrow-derived macrophages in vitro.

(A) Western blot analysis (one representative of 3 experiments) for pIkBa, IkBa and Bactin of wild-type (WT) and Irf6 cKO (cKO) macrophages (MΦ) unstimulated or stimulated with LPS for 30, 60 and 180 min. (B, C) Quantitative analysis of three independent Western blot analysis as presented in (A) expressed as fold change from the unstimulated (T0) control cells.