Interleukin-27R (WSX-1/T-cell cytokine receptor) gene-deficient mice display enhanced resistance to leishmania donovani infection but develop severe liver immunopathology

Abstract

The interleukin-27 (IL-27)/T-cell cytokine receptor (TCCR) pathway plays an important role in development of protective immunity against cutaneous leishmaniasis caused by Leishmania major. In this study, we analyzed the role of IL-27/TCCR pathway in the host defense against visceral leishmaniasis (VL) by monitoring the course of L. donovani infection in TCCR-deficient C57BL/6 (TCCR-/-) mice. TCCR-/- mice mounted a robust inflammatory response, produced high levels of pro-inflammatory cytokines, and developed severe liver pathology after L. donovani infection that eventually resolved. Interestingly, L. donovani-infected TCCR-/- mice controlled the parasite growth in their organs significantly faster than similarly infected TCCR+/+ mice. Adoptive cell transfer and cell depletion studies revealed that CD4(+) T cells were involved in mediating liver immunopathology and controlling L. donovani growth in TCCR-/- mice. These results indicate that the IL-27/TCCR pathway is not essential for the induction of protective Th1 response during VL but is involved in mediating susceptibility to L. donovani. Additionally, the data demonstrate that although the IL-27/TCCR interaction limits the severity of liver inflammation during VL by controlling CD4(+) T-cell activity, it is not required for the resolution of hepatic immunopathology.

Figure 1

Analysis of TCCR gene deletion in TCCR−/− mice. A: Tail DNA isolated from TCCR+/+ and TCCR−/− mice were analyzed by PCR for the presence of TCCR gene. Cont, control with no DNA. Expression of TCCR on the T cells isolated from the spleens of TCCR+/+ (B) and TCCR−/− (C) mice was analyzed by flow cytometry. Gray line denotes the cells stained with an isotype control. Solid black area denotes the cells stained with anti-TCCR Ab.

Figure 2

TCCR−/− mice are relatively resistant to L. donovani infection and rapidly control parasite burdens. Liver and spleen parasite loads (A and B) were determined 15, 30, and 60 days after intravenous inoculation with 1 × 10⁷ L. donovani. Parasite burdens in liver (A) and spleen (B) are expressed as the mean LDU ± SEM. The data are the mean values from six to eight animals per group at each time point in two independent experiments with similar results. *Statistically significant differences between each group (P < 0.05).

Figure 3

TCCR−/− mice develop severe liver inflammation after L. donovani infection. Histopathology of livers from both TCCR+/+ and TCCR−/− mice at 15 days (A, B, C, and D), 30 days (E, F, G, and H), and 60 days (I, J, K, and L) after L. donovani infection. On days 15 and 30 after infection, livers from TCCR+/+ mice displayed formation of well-organized granulomas with occasional clusters of amastigotes within individual macrophages (A, B, E, and F). By contrast, livers from TCCR−/− mice (G and H) displayed severe liver pathology with large granulomas associated with diffuse foci of inflammation (gray arrows) and necrosis (white arrows) compared with those from TCCR+/+ mice (E and F). By day 60, both TCCR+/+ (I and J) and TCCR−/− (K and L) mice resolved liver inflammation although some small granulomas were still visible in livers from TCCR+/+ mice (I and J).

Figure 4

Immunohistochemical analysis of cell populations in livers from TCCR+/+ and TCCR−/− mice on day 30 after L. donovani infection. Cryostat cut sections from livers were stained using rat anti-mouse CD11b (A and B) or anti-mouse CD4 antibodies (C and D), and bound primary Ab was detected by appropriate secondary biotinylated Ab and streptavidin AlexaFluor 488 (green) or AlexaFluor 594 (red). Livers from TCCR+/+ mice showed well-organized granulomas composed of CD11b+ macrophages (A) and CD4⁺ T cells (C), whereas those from TCCR−/− mice displayed diffuse parenchymal infiltration by these cell populations (B and D).

Figure 5

Analysis of collagen and reticulin fiber deposition in livers from TCCR+/+ and TCCR−/− mice on days 15 (A, B, C, and D), 30 (E, F, G, and H), and 60 (I, J, K, and L) after L. donovani infection. Livers from TCCR−/− mice showed significantly more deposition of collagen (C and G) and reticulin fiber (D and H) on days 15 and 30 after infection compared with those from the wild-type mice (A and E (collagen); B and F (reticulin fibers)). At day 60 after infection, livers from TCCR−/− mice displayed less collagen and reticulin fiber deposition compared with TCCR+/+ mice (I, J, K, and L). Note that collagen is stained blue and that reticulin fibers, which stain black, are denoted by white arrows. Magnification, ×10.

Figure 6

Kinetics of serum cytokine responses in TCCR+/+ and TCCR−/− mice after L. donovani infection. Mice were bled on days 15, 30, and 60 after infection and levels of IL-12p70 (A), TNF-α (B), IFN-γ (C), and IL-10 (D) were measured in serum by ELISA. Data shown are the mean ± SD of triplicate measurement from six to eight mice per group. The data are representative of two independent experiments. *Statistically significant differences between each group (P < 0.05).

Figure 7

Kinetics of in vitro IL-12p70 (A), IFN-γ (B), IL-10 (C), and NO (D) production by spleen cells from L. donovani-infected TCCR+/+ and TCCR−/− mice. Spleen cells were stimulated with 20 μg/ml of LdAg, and levels of cytokines were measured by ELISA. NO production was measured using Griess’s assay. Note that the levels of IFN-γ are shown in nanograms per milliliter. Data shown are the means ± SD of triplicate from four to five mice per group and are representative of two independent experiments. *Statistically significant differences between each group (P < 0.05).

Figure 8

Effect of CD4⁺ and CD8⁺ cell depletion on liver pathology and host resistance in TCCR−/− mice during L. donovani infection. Histopathology of livers on day 21 after infection from TCCR−/− mice treated with control Ab (A), anti-CD4 (B), and anti-CD8 (C) antibodies. Parasite burdens in liver (D) and spleen (E) are expressed as the mean LDU ± SEM. The data are the mean values from three to four animals per group.*Statistically significant differences compared with mice treated with control Ab (P < 0.05).

Figure 9

Course of L. donovani infection in RAG2−/− mice reconstituted with TCCR+/+ or TCCR−/− T cells. Histopathology of livers on day 30 after L. donovani infection from RAG2−/− mice reconstituted with (A and B) TCCR+/+ T cells or (C and D) TCCR−/− T cells. Parasite burdens in liver (E) and spleen (F) are expressed as the mean LDU ± SEM. The data are the mean values from three to four animals per group.*Statistically significant differences between each group (P < 0.05). Note: B and D are livers from RAG2−/− mice that only received T cells from TCCR+/+ and TCCR−/− mice, respectively.

Figure 10

Effect of IFN-γ and/or TNF-α blockade on liver pathology and host resistance in TCCR−/− mice during L. donovani infection. Histopathology of livers on day 30 after infection from TCCR−/− mice treated with control antibodies (A), anti-IFN-γ (B), anti-TNF-α (C), and anti-IFN-γ/anti-TNF-α (D). Magnification, ×40. Parasite burdens in liver (E) and spleen (F) expressed as the mean LDU ± SEM. The data are the mean values from three to four animals per group.*Statistically significant differences between each group (P < 0.05).