Lipopolysaccharide shock reveals the immune function of indoleamine 2,3-dioxygenase 2 through the regulation of IL-6/stat3 signalling

Abstract

Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified catalytic enzyme in the tryptophan-kynurenine pathway that is expressed primarily in monocytes and dendritic cells. To elucidate the biological role of Ido2 in immune function, we introduced lipopolysaccharide (LPS) endotoxin shock to Ido2 knockout (Ido2 KO) mice, which led to higher mortality than that in the wild type (WT) mice. LPS-treated Ido2 KO mice had increased production of inflammatory cytokines (including interleukin-6; IL-6) in serum and signal transducer and activator of transcription 3 (stat3) phosphorylation in the spleen. Moreover, the peritoneal macrophages of LPS-treated Ido2 KO mice produced more cytokines than did the WT mice. By contrast, the overexpression of Ido2 in the murine macrophage cell line (RAW) suppressed cytokine production and decreased stat3 expression. Finally, RAW cells overexpressing Ido2 did not alter nuclear factor κB (NF-κB) or stat1 expression, but IL-6 and stat3 expression decreased relative to the control cell line. These results reveal that Ido2 modulates IL-6/stat3 signalling and is induced by LPS, providing novel options for the treatment of immune disorders.

Figure 1

Ido2 KO mice are highly sensitive to LPS-induced endotoxin shock. (a) Kaplan–Meier plot of age-matched WT (n = 13) and Ido2 KO mice (n = 12) treated with LPS (15 mg/kg). Mortality was assessed for 7 days. Statistical analyses were performed using the log-rank test: **p < 0.001 between WT and Ido2 KO mice. (b) Serum concentration of Trp and KYN in WT and Ido2 KO mice 24 h after LPS administration (n = 5 each). (c) Representative images from H&E staining of lung and spleen sections from WT and Ido2 KO mice 24 h after LPS administration. Scale bar, 200 μm. (d) Cytokine concentrations in serum were determined with Bioplex. Samples were taken at 1 and 4 h after LPS administration in WT and Ido2 KO mice (n = 5 each). (e) Protein expression of phospho-stat3 was examined in the spleen of WT and Ido2 KO mice 4 h after LPS administration (n = 5 each). Data are presented as the mean ± SD. *p < 0.05.

Figure 2

The peritoneal macrophages of Ido2 KO mice upregulate cytokine production following LPS stimulus. The inflammatory cytokine and chemokine levels of peritoneal macrophages in WT and Ido2 KO mice 24 h after LPS treatment (100 ng/mL) were examined with Bioplex. The Ido2 KO accelerated the production of various cytokines and chemokines. Data are presented as the mean ± SD (n = 5–7). *p < 0.05.

Figure 3

The overexpression of Ido2 in RAW cells blocks cytokine signalling. (a) RAW cells were transfected with mouse full-length Ido2 (RAW-Ido2) or empty vector (RAW-MOC). A representative microscopic image of GFP in RAW-MOC or RAW-Ido2 cells is shown. (b) The expression of Ido2 mRNA in RAW-MOC or RAW-Ido2 cells was determined by RT-PCR. (c) The expression of Ido2 protein in RAW-MOC or RAW-Ido2 cells was determined by western blot. (d) Functional Ido2 activity level was determined by measuring the concentrations of Trp and its metabolite, KYN in RAW- Ido2 cells using HPLC (n = 4 each). (e) The concentration of cytokines was examined in the conditioned medium of RAW-MOC and RAW-Ido2 cells (n = 4 each). Data are presented as the means ± SD. *p < 0.05 by one-way ANOVA.

Figure 4

Ido2 inhibits the cytokine signalling. (a) Heat maps representing the relative expression of the signalling pathways in RAW-MOC and RAW-Ido2 cells. (b) The protein expression of stat3 in RAW-MOC and RAW-Ido2 cells. (c) qRT-PCR analysis of LPS induced and stat3-relative gene expression in RAW-MOC and RAW- Ido2 cells. Data are presented as the mean ± SD (n = 3 each). *p < 0.05.

Figure 5

Schematic overview of Ido2 functional role. First, LPS binds to TLR4 receptor and stimulates early cytokine expression via NF-κB. These cytokines, when released, induce stat1 and stat3 activation. Ido2 gene affects IL-6/stat3 signalling after LPS treatments.