A soluble form of the triggering receptor expressed on myeloid cells-1 modulates the inflammatory response in murine sepsis

Abstract

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently discovered receptor expressed on the surface of neutrophils and a subset of monocytes. Engagement of TREM-1 has been reported to trigger the synthesis of proinflammatory cytokines in the presence of microbial products. Previously, we have identified a soluble form of TREM-1 (sTREM-1) and observed significant levels in serum samples from septic shock patients but not controls. Here, we investigated its putative role in the modulation of inflammation during sepsis. We observed that sTREM-1 was secreted by monocytes activated in vitro by LPS and in the serum of animals involved in an experimental model of septic shock. Both in vitro and in vivo, a synthetic peptide mimicking a short highly conserved domain of sTREM-1 appeared to attenuate cytokine production by human monocytes and protect septic animals from hyper-responsiveness and death. This peptide seemed to be efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines. These data suggest that in vivo modulation of TREM-1 by sTREM peptide might be a suitable therapeutic tool for the treatment of sepsis.

Figure 1.

(A) Release of sTREM-1 from cultured monocytes after stimulation with LPS with and without proteases inhibitor. LPS stimulation induced the appearance of a 27-kD protein that was specifically recognized by an anti–TREM-1 mAb (inset). sTREM-1 levels in the conditioned culture medium were measured by reflectance of immunodots. Data are shown as mean ± SD (n = 3). (B) Expression of TREM-1 mRNA in monocytes. Cultured monocytes were stimulated with LPS (1 μg/ml) for 0, 1, and 16 h as indicated. LPS induced TREM-1 mRNA production within 1 h.

Figure 2.

(A) Release of cytokines and sTREM-1 from cultured monocytes. For cell activation, primary monocytes were cultured in 24-well flat-bottom tissue culture plates in the presence of LPS (1 μg/ml). In some experiments, this stimulus was provided in combination with LP17 (10–100 ng/ml), control peptide (10–100 ng/ml), or rIL-10 (500 U/ml). To activate monocytes through TREM-1, an agonist anti–TREM-1 mAb (10 μg/ml) was added as indicated. Cell-free supernatants were analyzed for production of TNF-α, IL-1β, and sTREM-1 by ELISA or immunodot. All experiments were performed in triplicate and data are expressed as means (SEM). (a) Media; (b) LP17 10 ng/ml; (c) anti–TREM-1; (d) LPS; (e) LPS + anti–TREM-1; (f) LPS + LP17 10 ng/ml; (g) LPS + LP17 50 ng/ml; (h) LPS + LP17 100 ng/ml; (i) LPS + IL10. (B) Effect of LP17 on NF-κB activation. Monocytes were cultured for 24 h in the presence of E. coli LPS (O111:B4; 1 μg/ml), anti–TREM-1 mAb (10 μg/ml), and/or LP17 (100 ng/ml) as indicated, and the levels of NF-κB p50 and p65 were determined using an ELISA-based assay. Experiments were performed in triplicate, and data are expressed as means of optical densities (SEM).

Figure 3.

Accumulation of sTREM-1 in serum of LPS-treated mice. Male Balb/c mice (20–23 g) were treated with LPS (LD₅₀, intraperitoneally). Serum was assayed for sTREM-1 by immunodot. Serum sTREM-1 was readily detectable 1 h after LPS administration and was maintained at a plateau level from 4 to 6 h.

Figure 4.

Endotoxinic shock in mice. (A) LP17 pretreatment protects against LPS lethality in mice. Male Balb/c mice (20–23 g) were randomly grouped (10 mice per group) and treated with an LD₁₀₀ of LPS. LP17 (50 or 100 μg) or control vector was administered 60 min before LPS. (B) Delayed administration of LP17 protects LPS lethality in mice. Male Balb/c mice (20–23 g) were randomly grouped (8 mice per group) and treated with an LD₁₀₀ of LPS. LP17 (75 μg) or control vector was administered 4 or 6 h after LPS as indicated. (C) Administration of agonist TREM-1 mAb is lethal to mice. Male Balb/c mice (20–23 g) were randomly grouped (eight mice per group) and treated with a combination of an LD₅₀ of LPS + control vector, LD₅₀ of LPS + anti-TREM-1 mAb (5 μg), or LD₁₀₀ of LPS + control vector as indicated. Control vector and anti–TREM-1 mAb were administered 1 h after LPS injection.

Figure 5.

CLP polymicrobial sepsis model. (A) LP17 partially protects mice from CLP-induced lethality. Male Balb/c mice (20–23 g) were randomly grouped and treated with normal saline (n = 14) or the control peptide (n = 14; 100 μg) or with LP17 (100 μg) in a single infection at H0 (n = 18), H+4 (n = 18), or H+24 (n = 18). The last group of mice (n = 18) was treated with repeated injections of LP17 (100 μg) at H+4, H+8, and H+24. (B) Dose effect of LP17 on survival. Mice (n = 15 per group) were treated with a single injection of normal saline or 10, 20, 50, 100, or 200 μg of LP17 at H0 after the CLP and monitored for survival.

Figure 6.

LP17 has no effect on bacterial counts during CLP. Mice (5 per group) were killed under anesthesia at 24 h after CLP. Bacterial counts in peritoneal lavage fluid and blood were determined and results are expressed as CFU per ml of blood and CFU per mouse for the peritoneal lavage.