Regulation of dendritic cell function improves survival in experimental sepsis through immune chaperone

Abstract

Dendritic cells (DCs) are professional Ag-presenting cells that play a critical role in both innate and adaptive immune responses. DCs recognize and respond to bacteria through multiple PRRs, including TLRs. Heat shock protein gp96/grp94 is a master essential chaperone for TLRs in the endoplasmic reticulum. We generated DC-specific gp96-knockout (KO) mice and showed that gp96 KO DCs were unable to respond to multiple TLR ligands. TLR-mediated hyperinflammatory response can lead to sepsis. However, the roles of neither DCs nor the DC-intrinsic gp96 in the process are completely understood. In a LPS-induced sepsis model, we hereby found that deletion of gp96 in DCs significantly reduced serum TNF-α levels and improved survival. Furthermore, using the well-defined polymicrobial sepsis model of cecal ligation and puncture, we found that DC-specific ablation of gp96 improved survival with significantly attenuated liver and renal injuries, decreased circulating inflammatory cytokines, altered DC maturation and activation, and increased serum Ig. Collectively, we demonstrate that deletion of gp96 in DCs is beneficial in protecting mice against sepsis induced by both endotoxemia and polymicrobial infections. We conclude that targeting gp96 in DCs may provide a potential novel approach for reducing the morbidity and mortality of sepsis.

Keywords: Dendritic cell; TLR; chaperone; gp96/grp94; sepsis.

Figure 1.

Attenuation of LPS-induced endotoxemia in DC-specific gp96-deficient mice. (a) 8–12-wk-old KO mice (n = 7) and control littermates (n = 6) were administered i.p. with 25 mg/kg body mass LPS, and mouse survival was monitored for 72 h. (b) Mice were bled at 1.5 h after LPS injection. Serum TNF-α was measured by ELISA. ***P < 0.001, ****P < 0.0001.

Figure 2.

Deletion of gp96 in DCs protects mice against CLP-Induced polymicrobial sepsis. (a) KO mice (n = 21) and WT littermates (n = 21) were subjected to CLP and monitored for survival. (b, c) Sera were collected at 24 h after CLP from KO and WT littermates (n = 5), and cytokine IL-12p40 (b) and IL-10 (c) were measured by ELISA. (d–g) Sera were collected at 24 h from KO and WT littermates after a sham operation (n = 3) or CLP (n ≥ 4), and ALT (d), AST (e), creatinine (f), and BUN (g) levels were measured by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3.

Deletion of gp96 in DCs impairs DC maturation in CLP-induced septic mice. (a) Flow cytometry analysis of splenic DCs by negative gating strategy to define DCs (B220⁻MHCII⁺, left panel), followed by examining cell surface maturation markers CD83 and CD86 (right panel, open histogram with solid line). Gray-shaded histograms represent isotype controls. (b, c) Quantification of CD83⁺ (b) and CD86⁺ (c) splenic DCs as defined in (a) in WT and KO mice after Sham treatment (n = 3) or CLP (n = 7). ***P < 0.001.

Figure 4.

Deletion of gp96 in DCs results in altered the systemic Ig production. (a–f) KO mice and WT littermates were subjected to Sham (n = 3–4) or CLP (n = 8). Serum was collected at 24 h after the surgery, and then the Ig levels of IgA (a), IgG1 (b), IgG2b (c), IgG2c (d), IgG3 (e), and IgM (f) in the sera were measured by ELISA. Error bars indicate SEM. *P < 0.05; **P < 0.01.

Figure 5.

Microarray analysis of mRNAs levels in WT and gp96 KO BMDCs in response to LPS. WT or KO BMDCs were stimulated with LPS (200 ng/mL) for 6 h followed by mRNA extraction, reverse transcription, and cDNA microarray analysis using the whole mouse cDNA array. The heat map shows the genes involved in the inflammatory pathway using the KEGG Pathway Database analysis. Comparing with WT BMDCs, KO cells expressed significantly less inflammatory cytokines in response to LPS.