Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice
Edward Kerschen, … , Francis J. Castellino, Hartmut Weiler
Edward Kerschen, … , Francis J. Castellino, Hartmut Weiler
Published August 16, 2010
Citation Information: J Clin Invest. 2010;120(9):3167-3178. https://doi.org/10.1172/JCI42629.
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Research Article

Activated protein C targets CD8+ dendritic cells to reduce the mortality of endotoxemia in mice

Abstract

Activated protein C (aPC) therapy reduces mortality in adult patients with severe sepsis. In mouse endotoxemia and sepsis models, mortality reduction requires the cell signaling function of aPC, mediated through protease-activated receptor–1 (PAR1) and endothelial protein C receptor (EPCR; also known as Procr). Candidate cellular targets of aPC include vascular endothelial cells and leukocytes. Here, we show that expression of EPCR and PAR1 on hematopoietic cells is required in mice for an aPC variant that mediates full cell signaling activity but only minimal anticoagulant function (5A-aPC) to reduce the mortality of endotoxemia. Expression of EPCR in mature murine immune cells was limited to a subset of CD8+ conventional dendritic cells. Adoptive transfer of splenic CD11chiPDCA-1– dendritic cells from wild-type mice into animals with hematopoietic EPCR deficiency restored the therapeutic efficacy of aPC, whereas transfer of EPCR-deficient CD11chi dendritic cells or wild-type CD11chi dendritic cells depleted of EPCR+ cells did not. In addition, 5A-aPC inhibited the inflammatory response of conventional dendritic cells independent of EPCR and suppressed IFN-γ production by natural killer–like dendritic cells. These data reveal an essential role for EPCR and PAR1 on hematopoietic cells, identify EPCR-expressing dendritic immune cells as a critical target of aPC therapy, and document EPCR-independent antiinflammatory effects of aPC on innate immune cells.

Authors

Edward Kerschen, Irene Hernandez, Mark Zogg, Shuang Jia, Martin J. Hessner, Jose A. Fernandez, John H. Griffin, Claudia S. Huettner, Francis J. Castellino, Hartmut Weiler

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Figure 5

APC effects on LPS-induced gene expression in DCs.

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APC effects on LPS-induced gene expression in DCs. (A–G) Heat maps depic...
(A–G) Heat maps depicting the effect of aPC treatment on the mRNA abundance of specific sets of genes, analyzed in FACS-selected EPCR+ DCs (A–E) or in sorted CD11chi spleen DCs comprising the EPCR+ and EPCR– populations (F and G). The number of probe sets represented in each heat map is indicated. (A) Lane 1: Genes exhibiting a more than 2-fold up-/downregulation (average of 2 independent experiments) in EPCR+ cells isolated 3 hours after LPS challenge/5A-aPC administration, as compared with the equivalent sample isolated from mice receiving LPS and the proteolytically inactive S360A-aPC variant. Lanes 2 and 4: Behavior of the same set of genes in cell samples isolated 3 or 16 hours, respectively, after exposure of mice to LPS/S360A-aPC, as compared with EPCR+ cells isolated from the spleen of unchallenged mice. Lane 3: aPC response of the same set of genes 16 hours after exposure to LPS/5A-aPC, as compared with LPS/S360A-aPC. (B) Subset of the genes in A regulated ≥2-fold in mice treated with LPS/S360A-aPC. (C) Lane 1: Set of probes with ≥2-fold up-/downregulation in EPCR+ cells isolated 16 hours after LPS challenge/5A-aPC administration, as compared with the equivalent sample isolated from mice receiving LPS/S360A-aPC. The set of genes in this map is largely non-overlapping with the gene set depicted in A and B. (D) Subset of the genes analyzed in C that also responds to LPS/S360A-aPC. (E) Subset of genes shown in A and C that respond to 5A-aPC treatment (≥2-fold up-/downregulated) at both time points. (F) Lane 1: Differential mRNA abundance of genes detected in CD11c+ cells isolated from LPS-challenged wild-type mice as 5A-aPC responsive (≥2-fold different, compared with treatment with S360A-aPC). Lane 2: Response of this gene set to LPS/S360A-aPC treatment, as compared with mice that were not challenged with LPS. Lanes 3 and 4: aPC response of these genes (relative change in abundance in mice receiving LPS/5A-aPC, as compared with mice receiving LPS/S360A-aPC) in CD11chi DCs in isolated from Par1–/– and EPCRlo mice. Lane 5: aPC response of this gene set in EPCR+ DCs isolated from wild-type mice. (G) Behavior of the 5A-aPC–responsive subset of the genes in F that is also regulated in mice receiving LPS/S360A-aPC. (H) Relation of 5A-aPC–responsive genes (≥2-fold up- or downregulated at 16 hours in LPS/5A-aPC–treated mice, as compared with LPS/S360A-aPC–treated animals) identified in CD11c+ cells isolated from wild-type, Par1–/–, and EPCRlo mice. Numbers denote regulated probe sets unique to or shared in animals with a given genotype. Data are based on the array hybridization intensity of a normalized pool of 6 independent samples for each genotype; control RT-PCR experiments verified that the abundance of select mRNAs in the pooled samples as detected by array hybridization accurately reflected mRNA abundance in each of the individual samples used to generate the pool.