PD-L2 negatively regulates Th1-mediated immunopathology during Fasciola hepatica infection

Abstract

Macrophage plasticity is critical for controlling inflammation including those produced by helminth infections, where alternatively activated macrophages (AAM) are accumulated in tissues. AAM expressing the co-inhibitory molecule programmed death ligand 2 (PD-L2), which is capable of binding programmed death 1 (PD-1) expressed on activated T cells, have been demonstrated in different parasitic infections. However, the role of PD-L2 during F. hepatica infection has not yet been explored. We observed that F. hepatica infection or a F. hepatica total extract (TE) injection increased the expression of PD-L2 on peritoneal macrophages. In addition, the absence of PD-L2 expression correlated with an increase in susceptibility to F. hepatica infection, as evidenced by the shorter survival and increased liver damage observed in PD-L2 deficient (KO) mice. We assessed the contribution of the PD-L2 pathway to Th2 polarization during this infection, and found that the absence of PD-L2 caused a diminished Th2 type cytokine production by TE stimulated splenocytes from PD-L2 KO infected compared with WT mice. Besides, splenocytes and intrahepatic leukocytes from infected PD-L2 KO mice showed higher levels of IFN-γ than those from WT mice. Arginase expression and activity and IL-10 production were reduced in macrophages from PD-L2 KO mice compared to those from WT mice, revealing a strong correlation between PD-L2 expression and AAM polarization. Taken together, our data indicate that PD-L2 expression in macrophages is critical for AAM induction and the maintenance of an optimal balance between the Th1- and Th2-type immune responses to assure host survival during F. hepatica infection.

Keywords: F. hepatica; PD-L2; Th1 response; infection; macrophage.

Figure 1. Effect of F. hepatica infection or total extract antigen injection on PD-L2 expression by peritoneal macrophages

A. BALB/c mice were infected with 8 F. hepatica metacercariae, and PC were isolated at days 1, 3 and 7 p.i. Uninfected animals were analyzed in parallel as controls. PC were processed by flow cytometry and analyzed for F4/80+PD-L2+ cells. Representative dot plots of each group of mice are shown, with bars displaying the percentage of PD-L2+ cells gated on F4/80+ cells, ** p< 0.005. B. BALB/c WT mice were injected with 80 μg of TE or PBS as control and the PC were obtained 24 h later. F4/80+ PD-L2+ cells were analyzed by flow cytometry. A representative dot plot for each group of mice is shown. Bars display percentage of F4/80+ PD-L2+ cells, **p=0.0038. Data are representative of two independent experiments and expressed as mean ± SD.

Figure 2. Increased susceptibility to F. hepatica infection in PD-L2 KO mice

WT and PD-L2 KO mice were orally infected with 8 F. hepatica metacercariae. A. Survival was followed for 50 days, *p<0.02, Gehan–Breslow Wilcoxon test, n=12 per group. B. Representative hematoxylin and eosin images of livers from each group are shown (400x). Arrow heads in fibrosis areas, inflammatory foci (IF) and the presence of worms (P) are indicated. Inset shows the extensive inflammatory infiltrate. C. Intrahepatic leukocytes were isolated by percoll gradient and quantified in a Neubauer chamber. Each dot represents the total liver leukocyte number in individual livers of each group p=0.0065. D. Intrahepatic leukocytes were stained with anti-Ly6G before being incubated with 20μM of DAF-FMDA probe for intracellular NO detection. Bars show the percentage of Ly6G+NO+ cells. Representative dot plots of each group of control or infected WT and PD-L2 mice are shown,**p= 0.021; * p=0.0149; # p=0.0354. The data are representative of two experiments with similar results, and are expressed as mean ± SD.

Figure 3. Th1 and Th2 cytokine levels in WT and PD-L2 KO infected mice

Spleen mononuclear cells were obtained from WT and PD-L2 KO mice orally infected with 8 F. hepatica metacercariae. Then, the cells were stimulated with 80 μg/ml of TE or left unstimulated, and the cytokines measured in culture supernatants by ELISA. A. IFN-γ production by splenocytes at days 3 and 7 p.i. Dots represent mean cytokine levels of individual animals, lines represent the mean value *p<0.05 B. IL-10, IL-4 and IFN-γ production from splenocytes at day 21 p.i. Bars show fold increase of TE stimulated vs unstimulated cells. Data are expressed as mean ± SD *p<0.05;**p<0.005. C. CD4+ and CD8+ splenocytes producing IFN-γ are shown, each dot represents an individual animal *p<0.05. D. CD4+ intrahepatic lymphocytes producing IFN-γ are shown, with each dot representing an individual mouse * p<0.05. One representative experiment is depicted out of two.

Figure 4. Reduced alternative activation of macrophages in total extract antigen (TE) injected or infected PD-L2 KO

A. BALB/c WT and PD-L2 KO mice were injected with 80 μg of TE or PBS as control, and 24 h later arginase I and iNOS expression mice were analyzed in PC by Western blot. Mouse liver lysate was used as a positive control for arginase I expression and LPS-stimulated J774 cells were used as a positive control for iNOS expression. ATP Citrate expression was used as control loading. Right panel shows the densitometric analysis using GelPro software, n =2 experiments **p=0.0011; *p< 0.05. B. Arginase activity was evaluated by measuring urea levels in lysated PC, ***p=0.0003. C. WT and PD-L2 KO mice were infected with 8 F. hepatica metacercariae, and the PC were isolated at days 3 and 7 pi. Arginase I expression was measured by Western blot. Mouse liver lysate was used as a positive control for Arginase I expression, and Actin expression was analyzed as control loading. Right panel shows the densitometric analysis using GelPro software, n =2 experiments **p=0.0046; *p<0.05. D. Arginase activity was evaluated through urea production *p<0.05. E. PC were stained with anti-F4/80 antibody. Then, PC were fixed and permeabilized to evaluate arginase I and IL-10 expression by using APC labeled anti-arginase I antibody and a PE-Cy7 labeled anti-IL-10 antibody. Left panel: representative dot plots for each group of mice at days 3 and 7 p.i. are shown. Right panel: Bars display the percentage of IL-10+ arginase I+ cells on F4/80+ gated populations, *p <0.05. All experiments were repeated twice with similar results being obtained and are expressed as mean ± SD.