Murine beta-defensin 2 promotes TLR-4/MyD88-mediated and NF-kappaB-dependent atypical death of APCs via activation of TNFR2

Abstract

Mammalian antimicrobial peptides, including beta-defensins, represent an ancient arm of innate immunity designed to directly neutralize invading microbes. Previously, we demonstrated that murine beta-defensin 2 (mDF2beta) also acted as an endogenous ligand for TLR-4-activating maturation of dendritic cells (DCs). Herein, we report that this TLR-4 -dependent activation leads to induction of an atypical cell death that is unexpectedly exaggerated by the inhibition of caspases. Experiments using APCs with nonfunctional TNF-alpha or its receptors suggest that this is a NF-kappaB- and TNF-alpha-dependent process that does not require TNFR1. We demonstrate that mDF2beta triggers a TNFR2-mediated signaling cascade of "self-destruction" through up-regulation of membrane-bound TNF-alpha and TNFR2. This appears not to be an isolated phenomenon, as human synthetic beta-defenisn 3 was also able to activate and kill DCs. We propose that beta-defenins may play an important immunoregulatory role as controllers of the natural process of elimination of activated APCs.

Fig. 1

MDF2β activates APCs. (A) Maturation of XS52 cells was judged by up-regulation of CD11c⁺/CD40⁺/B7.2 cells after overnight incubation with indicated amounts (μg/ml) of mDF2β fusion protein (mDF2bsFv315). Control cells were treated with 5 μg/ml sFv315 in the presence or absence of indicated amounts of LPS (ng/ml). Comparable results were reproduced at least five times, and the mean of the representative triplicate experiment is shown ± sem. P < 0.03 is for comparison between treatments with mDF2β and sFv315. (B) Surface expression of H2K^d and CD40 on overnight-treated macrophage cell RAW264.7. Cells were treated with mDF2β-sFv315 (mDF2b; 5 μg/ml), sFv315 (5 μg/ml), or LPS (10 ng/ml) in the presence or absence of polymixin B (PxB; 5 μg/ml). Shown are representative data from four independent experiments with similar results ± sem. CM, Conditioned media. (C) ELISA results of cytokine production (pg/ml) in culture media of XS52 cells overnight treated with 5 μg/ml proteins or 10 ng/ml LPS in the presence or absence of 5 μg/ml polymixin B. The mean of the single representative triplicate experiment is shown.

Fig. 2

mDF2β kills XS52 cells, as detected by light microscope (A-F) and scattered electron microscopy (G and K). Cells were treated for 3 days with 5 μg/ml mDF2β-sFv315 (A) or 10 ng/ml LPS (B) alone and with 5 μg/ml polymixin B (D and E, respectively). Control treatments were 5 μg/ml murine MIP-3α-sFv315 (mMIP3a; C) and mproDF2β-sFv38 (Pro-mDF2β; F). Overnight treatment with mDF2β did not show signs of abnormalities and death (G), and numerous large blebs (arrows) and loss of intact mitochondria could only be detected after 3 days of treatment (K). The experiment was independently reproduced twice.

Fig. 3

Treatment with mDF2β reduces survival of XS52 cells (A and D) and bone marrow-derived DCs (BMDCs; B and C). Cells, treated for the indicated days with 5 μg/ml mDF2β-sFv38 (mDF2b), sFv315, or 10 ng/ml LPS, with or without 5 μg/ml polymixin B. To assess cell survival, cells were stained with Annexin V/propidium iodide (PI; A, B, and D). In parallel, cells were stained with antibodies for CD11c⁺/CD40⁺/CD86⁺ to assess cell activation (C). Inhibition of pan-caspases using ZVAD exaggerates the mDF2β-induced killing, leading to the cell death after only overnight incubation (D). XS52 cells were incubated overnight with 5 μg/ml mDF2β or sFv315 in the presence or absence of ZVAD-fmk or control ZFA-fmk. All results shown (A-D) were independently reproduced at least three times, and the mean of the representative triplicate experiment is shown ± sem. P values are for comparisons of pooled mDF2β data with LPS, mDF2β + LPS, or mDF2β + polymixin B (A) or between groups indicated by solid lines (D).

Fig. 4

mDF2β induces death of APCs via activation of signaling cascades and secretion of cytotoxic factors. (A) Conditioned media from XS52 cells treated for 3 days with mDF2β (mDF2b CM d3) but not sFv315 are cytotoxic for naïve XS52 cells. In contrast, overnight conditioned media from overnight, mDF2β-treated XS52 cells are not toxic (mDF2b CM d1). Conditioned media are used at 1:5 and 1:10 dilutions. Cell survival (left y-axis) and activation (right y-axis) were measured as in Figure 3. The data were reproduced three times in triplicate experiments. (B and C) Experiments with macrophage cells with nonfunctional NF-κB (NFκB KO) and TNF-α (TNFα KO). mDF2β cannot kill (B) or activate (C) when cells do not express NF-κB. Moreover, cytotoxicity of mDF2β (B) is significantly abrogated when cells do not express TNF-α. The ability to be activated with mDf2β is not affected (C). Used, immortalized macrophage cells from B6/129 mice with KO p50 NF-κB or TNF-α genes, respectively. Controls were immortalized macrophage cells from B6/129 mice. Cell were treated for 3 days and stained in parallel to assess cell survival (B) or cell activation (C), as described in Figure 3. (D) Survival of naïve DCs incubated overnight with conditioned media (at dilutions 1:10) from wt DCs (mDF2b-DC-CM) or with nonfunctional genes TNFR1 (mDF2b-TNFR1-KO-CM) and TNF-α (mDF2b-TNFα-KO-CM) treated with 5 μg/ml mDF2β for 3 days. Control DCs were treated with 20 ng/ml TNF-α, or conditioned media were from sFv315-treated wt DCs (sFv315-DC-CM). Data were compared with effects of CM from XS52 cells treated with mDF2β (mDF2β-XS52-CM). (E) mDF2β (solid line) but not mproDF2β (dotted line) induces expression of cell-anchored TNF-α on the surface of the B6/129 macrophage. The black line is for untreated cells. Cells were stained with FITC-labeled anti-mouse TNF-α mAb after overnight incubation with proteins. No specific staining was detected with FITC-labeled, isotype-matched control antibody (data not shown). All results shown (A-E) were reproduced at least three times, and the mean of the representative triplicate experiment is shown ± sem. P values are for comparisons of between Day 1 and Day 3 conditioned media (A); wt and TNF-α KO or NF-κB KO macrophages treated with mDF2β or LPS (B and C); or between conditioned media of TNFR1 KO and TNF-α KO (D).

Fig. 5

The mDF2β-induced cell death is mediated through TNF-α and its receptor TNFR2 but not TNFR1. BMDCs from nonfunctional TNFR1 (A) or TNFR1 and TNFR2 mice (B) were treated with mDF2β, mDF3β, and sFv315 or mock-treated for 3 days to assess cell survival (solid bars, left y-axis) or DC maturation (shaded bars, right y-axis), as described in Figure 3. (C) The mDF2β-induced death of B6/129 macrophage cells is abrogated in the presence of anti-mouse TNFR2-neutralizing mAb (10 μg/ml) but not isotype-matched IgG (10 μg/ml). The y-axis is in arbitrary proportion of Annexin V/PI-positive cells (dead cells). (D) TNFR2 is up-regulated on the surface of immortalized B6/129 macrophages treated overnight with mDF2β or LPS but not with mproDF2β. The cells were stained for TNFR2 and CD40 (solid bars, left y-axis) and Annexin/PI (shaded bars, right y-axis). All results shown (A-D) were reproduced at least two times, and the mean of the representative triplicate experiment is shown ± sem. P values are for comparisons of viability of cells treated with mDF2β and control protein sFv315 (A) or mproDF2β (D); between two groups as depicted by horizontal lines (C); or between mDF2β or LPS with mproDF2β (D). (B) The survival difference between mDF2β- and mproDF2β-treated groups was not insignificant.

Fig. 6

Human β-defensin 3 (HBD3) also induces both maturation death of human DCs. Human PBL-derived iDCs were treated for 1 day (A) and 3 days (B) with human β-defensin 3 or human β-defensin 1 (20 μg/ml) or with 10 μg/ml mDF2β or saline (conditioned media). Cell death (solid bars) and DC maturation (shaded bars) were assessed as described in Figure 3. Shown are data of a triplicate experiment using individual human donor PBL ± sem. The results were reproduced using DCs from two donors. P values are for comparisons of maturation (A) and survival (B) between two groups as depicted by horizontal lines.