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-gamma 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.

Figure 1. Mortality reduction by aPC requires EPCR and PAR1 expression in BM-derived cells.

BM chimeras were generated by reciprocal BM transfers between CD45 isotype–mismatched animals of the indicated genotypes (EPCR^lo: reduced EPCR expression; Par1^–/–: PAR1-knockout mice). Eight to 10 weeks after documented hematopoietic reconstitution, animals received a dose of LPS causing 50% mortality in wild-type mice, followed by intravenous infusion of a 10-μg bolus of murine recombinant 5A-aPC (aPC) in PBS or carrier alone. (A) BM transfer between wild-type mice does not alter the sensitivity to LPS and response to aPC therapy. Full efficacy of mortality reduction by 5A-aPC requires normal expression of EPCR and PAR1 in hematopoietic cells (B and D), as well as in non-hematopoietic cells (C and E). Significance of the aPC effect on 7-day survival was determined by Kaplan-Meier log-rank test.

Figure 2. EPCR is expressed in HSCs and spleen DCs.

(A) Whole BM from 10 wild-type mice was pooled and fractionated by FACS into HSCs (Lin^–Sca-1^hic-kit^hi; Lin: CD3ε, CD4, CD8a, CD19, Ly6G, CD45R), granulocyte-macrophage progenitors (GMP: Lin^–Sca-1^–c-kit⁺CD34⁺FcRγII/III^hi), common myeloid progenitors (CMP: Lin^–Sca-1^–c-kit⁺CD34⁺FcRγII/III^lo), megakaryocytic erythroid progenitors (MEP: Lin^–Sca-1^–c-kit⁺CD34^–FcRγII/III^lo), or a progenitor mix (CMP, GMP, MEP, CLP: Lin^–Sca-1^–c-kit^lo/–). Epcr and Gapdh mRNA were amplified by 35- and 30-cycle RT-PCR, respectively, from RNA isolated from sorted cell pools. (B) Detection of EPCR expression in wild-type splenocytes. Back-gating of EPCR-positive cells (gate P1, gray line indicates signal obtained with isotype control antibody) shows EPCR expression in CD11c^hiPDCA-1^– DCs (red). (C) Abundance of EPCR-expressing CD11c^hiPDCA-1^– DCs is diminished in EPCR^lo mice. Spleen DCs were enriched by capture on CD11c/PDCA-1 magnetic beads and analyzed for EPCR expression as in B. (D) Detection of EPCR surface expression captures the majority of DCs expressing Epcr mRNA. Spleen DCs were enriched on magnetic beads as in C, and EPCR⁺ cells were isolated by FACS via gating on CD11c^hiPDCA-1^– DCs, followed by sorting into EPCR⁺ and EPCR^– CD11c^hiPDCA-1^– cells (left panel, solid gray line: isotype control on post-sort EPCR⁺ cells; dotted red line: FITC intensity of post-sort EPCR-depleted CD11c^hiPDCA-1^– cells; solid red line: FITC intensity of post-sort EPCR⁺ CD11c^hiPDCA-1^– cells). Quantitative RT-PCR analysis of Epcr mRNA on sorted cells shows depletion of Epcr mRNA in cells lacking EPCR surface expression as detected by flow cytometry (right panel; bars indicate the average ± SD of the detection threshold expressed as the ΔC_T value for Epcr mRNA determined in 2 independent sorting experiments, with 3 measurements/sample).

Figure 3. EPCR is expressed in the CD8⁺DEC205⁺ subset of spleen DCs.

Spleen DCs were enriched by capture on CD11c/PDCA-1 magnetic beads, and EPCR⁺ cells in gate P1 were back-gated onto the CD11^hi population defined by gate P2 (gray events) to visualize EPCR expression (black events) in relation to the indicated markers.

Figure 4. EPCR⁺ DCs are required for the therapeutic efficacy of 5A-aPC.

(A) Spleen DCs were enriched by selection on CD11c/PDCA-1 magnetic beads from unchallenged wild-type or EPCR^lo mice, and the CD11c^hiPDCA-1^– population was isolated by preparative FACS. (B) Sorted DCs (10⁶) were infused intravenously into mice with hematopoietic EPCR deficiency, treated 24 hours later with LPS/5A-aPC, and monitored for 7-day survival. A control group (ctrl) received wild-type DCs and LPS/S360A-aPC. (C) In an independent experiment, mice with hematopoietic EPCR deficiency received either wild-type DCs or wild-type DCs depleted of EPCR⁺ cells, followed by treatment with LPS/5A-aPC. Efficacy of 5A-aPC was measured by Kaplan-Meier log-rank analysis of survival.

Figure 5. APC effects on LPS-induced gene expression in DCs.

(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 CD11c^hi 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 CD11c^hi DCs in isolated from Par1^–/– and EPCR^lo 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 EPCR^lo 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.

Figure 6. APC alters the function of IFN-producing NK-DCs.

Spleen DCs were isolated from unchallenged (no LPS), LPS/S360A-aPC–treated, and LPS/5A-aPC–treated mice (14–16 hours after LPS challenge) by selection on CD11c/PDCA-1 magnetic beads, followed by FACS gating on cells expressing high levels of CD11c, as in Figure 4A. (A) Expression of the surface markers NK1.1 and CD3ε live cells. (B) Detection of intracellular IFN-γ in permeabilized CD11c⁺PDCA-1^–CD3ε^–NK1.1⁺ cells (gray line indicates isotype control staining for IFN-γ). Data shown are derived from one individual mouse each and are representative of 3 independent experiments.