Cysteinyl leukotriene receptor-1 as a potential target for host-directed therapy during chronic schistosomiasis in murine model

Abstract

Schistosomiasis remains the most devastating neglected tropical disease, affecting over 240 million people world-wide. The disease is caused by the eggs laid by mature female worms that are trapped in host's tissues, resulting in chronic Th2 driven fibrogranulmatous pathology. Although the disease can be treated with a relatively inexpensive drug, praziquantel (PZQ), re-infections remain a major problem in endemic areas. There is a need for new therapeutic drugs and alternative drug treatments for schistosomiasis. The current study hypothesized that cysteinyl leukotrienes (cysLTs) could mediate fibroproliferative pathology during schistosomiasis. Cysteinyl leukotrienes (cysLTs) are potent lipid mediators that are known to be key players in inflammatory diseases, such as asthma and allergic rhinitis. The present study aimed to investigate the role of cysLTR1 during experimental acute and chronic schistosomiasis using cysLTR1^-/- mice, as well as the use of cysLTR1 inhibitor (Montelukast) to assess immune responses during chronic Schistosoma mansoni infection. Mice deficient of cysLTR1 and littermate control mice were infected with either high or low dose of Schistosoma mansoni to achieve chronic or acute schistosomiasis, respectively. Hepatic granulomatous inflammation, hepatic fibrosis and IL-4 production in the liver was significantly reduced in mice lacking cysLTR1 during chronic schistosomiasis, while reduced liver pathology was observed during acute schistosomiasis. Pharmacological blockade of cysLTR1 using montelukast in combination with PZQ reduced hepatic inflammation and parasite egg burden in chronically infected mice. Combination therapy led to the expansion of Tregs in chronically infected mice. We show that the disruption of cysLTR1 is dispensable for host survival during schistosomiasis, suggesting an important role cysLTR1 may play during early immunity against schistosomiasis. Our findings revealed that the combination of montelukast and PZQ could be a potential prophylactic treatment for chronic schistosomiasis by reducing fibrogranulomatous pathology in mice. In conclusion, the present study demonstrated that cysLTR1 is a potential target for host-directed therapy to ameliorate fibrogranulomatous pathology in the liver during chronic and acute schistosomiasis in mice.

Keywords: Th2 immune responses; cysteinyl leukotriene 1 (CYSLTR1); host-directed therapy; montelukast; praziquantel; schistosomiasis.

Figure 1

Disruption of cysLTR1 does not alter immune cell composition but there is expansion of CD4 T cm and CD8 Tcm in secondary lymphoid tissues. (A) Total number of splenic immune cell populations (CD4, CD8, CD19 B cells). (B) Total number of CD4 T central memory (Tcm) and CD8 Tcm in the spleen. (C) Total number of MLN immune cell populations (CD4, CD8, CD19 B cells). (D) Total number of CD4 Tcm and CD8 Tcm in the MLN. Data are representative of two independent experiments. n=6 - 8 mice. *p<0.05 by unpaired Student’s t-test. ns, statistically not significant.

Figure 2

Deletion of cysLTR1 leads to amelioration of granulomatous inflammation during chronic schistosomiasis. CysLTR1 deficient mice and littermate control mice were infected with 35 live S. mansoni cercariae, killed at 16 weeks post infection respectively and the liver was harvested. (A) Experimental layout. (B) Survival curve of S. mansoni infected cysLTR1 and control mice. Survival curves were compared using log-rank test. cysLTR1 mice were infected with S. mansoni cercariae and analyzed 16 weeks post infection. (C) Histological examination of H&E and CAB-stained liver sections. (D) Granuloma area was measured using computerized morphometric analysis (NIS Elements, Nikon) by measuring 20 - 25 granulomas per mouse. (E) Liver fibrosis determined by assaying hydroxyproline concentration normalized to tissue eggs. (F) Hepatocellular damage indicated by serum aspartate transaminase (AST) concentration. (G) Egg burden in S-mansoni infected liver. (H) Body weight. (I) Spleen weight index (as a ratio of total body weights). (J) Liver weight index. Data are representative of two independent experiments. n= 6 - 10 mice. *p<0.05 and ***p<0.001 vs wild type mice using unpaired Student’s t-test.

Figure 3

Deletion of cysLTR1 leads to reduced Th2 immune response during chronic schistosomiasis. CysLTR1 deficient mice and littermate control mice were infected with 35 live S. mansoni cercariae, killed at 16 weeks post infection and the serum, hLN and liver were harvested. (A) Cytokine production in the serum. Livers from infected mice were homogenized and levels of the indicated (B) cytokines were detected by ELISA and normalized to mg of liver tissue at week 16 post infection. Total hLN cells were stimulated with (C) SEA and (D) αCD3. (E) Representative total number of CD4⁺ intra-epithelial lymphocytes (IEL), CD8⁺ IEL, CD4⁺ CD8⁺ IEL, CD8⁺ dendritic cells, neutrophils (CD11b⁺Ly6G⁺), macrophages (CD11b⁺F4/80⁺), and eosinophils (CD11b⁺SiglecF⁺). (F) Total number of IFN-γ, IL-4, IL-9, and IL-17-expressing CD4⁺ T cells. (G) Total number of IL-4 expressing ILC2 cells in the liver. (H) SEA-specific IgG1 antibody titre. (I) Total IgE antibody titre. Data are representative of two independent experiments. n=6 - 10 mice. *p<0.05, **p<0.01 and ***p<0.001 by unpaired Student’s t-test.

Figure 4

Pharmacological blockade of cysLTR1 led to reduced liver pathology during chronic schistosomiasis. Four groups of wild type mice were infected with 35 live S. mansoni cercariae and at 11 weeks pi, one group was treated with Praziquantel only once daily for 7 days, Montelukast only once every other day for 3 weeks, a combination of Praziquantel and Montelukast therapy and one group was given a mock treatment and kept as a control. Animals were killed at week 16 pi and the liver and serum were harvested. (A) Experimental layout. (B) Histological examination of H&E-stained liver sections. (C) Granuloma area was measured using computerized morphometric analysis (NIS Elements, Nikon) by measuring 20 - 25 granulomas per mouse. (D) Liver fibrosis determined by assaying hydroxyproline concentration normalized to tissue eggs. (E) Hepatocellular damage indicated by serum aspartate transaminase (AST)/alanine transaminase (ALT) ratio. (F) Parasite egg burden in liver. (G) Liver weight index. (H) Spleen weight index. (I) Body weight. Data are representative of two independent experiments. n= 4 - 7 mice. *p<0.05 and ***p<0.001 by unpaired Student’s t-test.

Figure 5

Inhibition of cysLTR1 in combination PZQ results in expansion of CD4⁺Foxp3⁺ T cells during chronic schistosomiasis. (A–F) Cytokine production in the serum and liver homogenates (Livers from infected mice were homogenized and levels of the indicated cytokines were detected by ELISA and normalized to mg of liver tissue at week 16 post infection). Representative total number of CD3⁺ lymphocytes (G), CD4⁺ lymphocytes (H) and CD8⁺ lymphocytes (I). Total number of T-bet (J), Gata3 (K) and Foxp3 (L) expressing CD4⁺ T cells. Data are representative of two independent experiments. n= 4-7 mice. *p<0.05, **p<0.01 and ***p<0.001 by unpaired Student’s t-test.

Figure 6

Deletion of cysLTR1 leads to amelioration of granulomatous inflammation during acute schistosomiasis. CysLTR1 deficient mice and littermate control mice were infected with 80 live S. mansoni cercariae, killed at 8-weeks post infection and the liver was harvested. (A) Experimental layout. (B) Histological examination of H&E and CAB-stained liver sections. (C) Granuloma area was measured using computerized morphometric analysis (NIS Elements, Nikon) by measuring 20 - 25 granulomas per mouse. (D) Liver fibrosis determined by assaying hydroxyproline concentration normalized to tissue eggs. (E) Egg burden in S-mansoni infected liver. (F) Hepatocellular damage indicated by serum aspartate transaminase (AST) concentration. (G) Liver weight. (H) Spleen weight index. (I) Body weight. Data are representative of two independent experiments. n= 6 - 7 mice. ***p<0.001 vs wild type mice using unpaired Student’s t-test.