A novel role for programmed cell death receptor ligand 2 in sepsis-induced hepatic dysfunction

Abstract

The liver is an organ that, when dysfunctional in a septic patient, is strongly associated with morbidity and mortality. Understanding the pathophysiology of liver failure during sepsis may lead to improved diagnostics and potential therapeutic targets. Historically, programmed cell death receptor (PD) ligand 1 (PD-L1) has been considered the primary ligand for its checkpoint molecule counterpart, PD-1, with PD-L2 rarely in the immunopathological spotlight. PD-1 and PD-L1 contribute to liver dysfunction in a murine cecal ligation and puncture (CLP) model of sepsis, but virtually nothing is known about PD-L2's role in sepsis. Therefore, our central hypothesis was that sepsis-induced changes in hepatic PD-L2 expression contributed to worsened liver function and, subsequently, more pronounced morbidity and mortality. We found that although PD-L1 gene deficiency attenuated the hepatic dysfunction seen in wild-type mice after CLP, the loss of PD-L2 appeared to actually worsen indices of liver function along with a trend toward higher liver tissue vascular permeability. Conversely, some protective effects of PD-L2 gene deletion were noted, such as reduced liver/peritoneal bacterial load and reduced IL-6, IL-10, and macrophage inflammatory protein 2 levels following CLP. These diverse actions, as well as the unique expression pattern of PD-L2, may explain why no overt survival advantage could be witnessed in the septic PD-L2^-/- mice. Taken together, these data suggest that although PD-L2 has some selective effects on the hepatic response seen in the septic mouse, these factors are not sufficient to alter septic mortality in this adult murine model. NEW & NOTEWORTHY Our study shows not only that ligands of the checkpoint protein PD-1 respond inversely to a stressor such as septic challenge (PD-L2 declines, whereas PD-L1 rises) but also that aspects of liver dysfunction increase in septic mice lacking the PD-L2 gene. Furthermore, these differences in PD-L2 gene-deficient animals culminated in the abrogation of the survival advantage seen in the septic PD-L1-knockout mice, suggesting that PD-L2 may have roles beyond a simple immune tolerogen.

Keywords: inflammation; posttranslational modification; programmed cell death receptor ligand 1; programmed cell death receptor-1; sepsis.

Fig. 1.

Programmed cell death receptor ligand 2 (PD-L2) is expressed on a variety of organs and leukocytes present in the blood, peritoneum, or spleen, and with the exception of a consistent decline in the gene expression of the liver, is not altered in mice 24 h post-cecal ligation and puncture (CLP). PD-L2 gene expression (normalized to housekeeping gene GAPDH) in blood leukocytes, splenocytes, and peritoneal exudate cells (A) (n = 4/group) or lung, liver, large intestine, and small intestinal tissue lysates (B) (n = 7–8/group) obtained from mice 24 h after CLP was extracted for mRNA and analyzed using quantitative real-time PCR. PD-L2 protein expression in various whole tissue lysates or serum was determined by Western immunoblot analyses, in which the bands detected were quantitated by densitometry. Summary of the fold expression change (ratio of densitometry) for PD-L2/GAPDH (C) (n = 4/group). The number of PD-L2⁺ liver cells by hepatic subpopulations [liver sinusoidal endothelial cells (LSEC), Kupffer cells, or hepatocytes] at 24 h after CLP was determined by flow cytometry of the various isolated/enriched cell subpopulations (D) (n = 6/group). Data are expressed as mean ± SE; *P < 0.05 by nonparametric Mann-Whitney U-test to evaluate P values when testing two subgroups.

Fig. 2.

Indices of dysfunction and damage as a measure of septic mouse liver injury. Vascular permeability measured in nanograms of Evans blue dye over milligrams of liver tissue in wild-type (WT), programmed cell death receptor ligand 2 (PD-L2)^−/−, and PD-L1^−/− mice 24 h after cecal ligation and puncture (CLP) (n = 4/group) (A). Total blood bilirubin measured in mg/dL in WT, PD-L2^−/−, and PD-L1^−/− mice 24 h after CLP (n = 5/group) (B). Blood alanine aminotransferase (ALT) and aspartate aminotransferase (AST) measured in IU/L in WT, PD-L2^−/−, and PD-L1^−/− mice 24 h after CLP (n = 5/group) (C and D, respectively). Data are expressed as mean ± SE; *P < 0.05 by nonparametric Mann-Whitney U-test to evaluate P values when testing two subgroups. For multiple groups analysis, intergroup comparisons were performed by the Holm-Sidak test. ND, not detected.

Fig. 3.

TNF-α, IL-6, and IL-10 as markers of pro-/anti-inflammation in the liver, locally in the peritoneum (relative to the septic insult), and systemically in blood. TNF-α (A–C), IL-6 (D–F), and IL-10 (G–I) levels 24 h after CLP were determined for the liver tissue lysates (A, D, and G), clarified peritoneal fluid (B, E, and H), and plasma (C, F, and I) by commercial ELISA and reported in pg/mg protein (n = 6/group). Data are expressed as mean ± SE; *P < 0.05 by nonparametric Mann-Whitney U-test to evaluate P values when testing two subgroups. For multiple groups analysis, intergroup comparisons were performed by the Holm-Sidak test. CLP, cecal ligation and puncture; PD-L2, programmed cell death receptor ligand 2; WT, wild-type.

Fig. 4.

Survival is enhanced in programmed cell death receptor ligand 1 (PD-L1)^−/− mice compared with wild-type (WT) post-cecal ligation and puncture (CLP) but not in PD-L2^−/− mice. Survival of PD-L1^−/−, PD-L2^−/−, and WT mice after CLP measured at 12 h time points for the first 3 days and then at 24 h time points for a total of 360 h (14 days) (n = 15/group). *P < 0.05 by a Kaplan-Meier analysis, and comparisons were performed by the log-rank test.

Fig. 5.

Bacterial burden in blood, peritoneal fluid, and liver post-cecal ligation and puncture (CLP). Bacterial load in liver tissue (A), peritoneal fluid (B), or the blood (C) of mice harvested 24 h after CLP or sham protocol, as measured in colony-forming unit (CFU)/ml or CFU/mg tissue (n = 6). Data are expressed as mean ± SE; *P < 0.05 by nonparametric Mann-Whitney U-test to evaluate P values when testing two subgroups. For multiple groups analysis, intergroup comparisons were performed by the Holm-Sidak test. PD-L, programmed cell death receptor ligand; WT, wild-type; ND, not detected.

Fig. 6.

Monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 2 (MIP-2) as markers of leukocyte chemotactic environment in the liver, locally in the peritoneum (relative to the septic insult), and systemically in blood. MCP-1 (A–C) and MIP-2 (D–F) levels 24 h after cecal ligation and puncture (CLP) were determined for the liver tissue lysates (A, and D), clarified peritoneal fluid (B and E), and plasma (C and F) by commercial ELISA and reported in pg/mg protein (n = 6/group). Data are expressed as mean ± SE; *P < 0.05 by nonparametric Mann-Whitney U-test to evaluate P values when testing two subgroups. For multiple groups analysis, intergroup comparisons were performed by the Holm-Sidak test. PD-L2, programmed cell death receptor ligand 2; WT, wild-type.