Therapeutic enhancement of protective immunity during experimental leishmaniasis

Abstract

Background: Leishmaniasis remains a significant cause of morbidity and mortality in the tropics. Available therapies are problematic due to toxicity, treatment duration and emerging drug resistance. Mouse models of leishmaniasis have demonstrated that disease outcome depends critically on the balance between effector and regulatory CD4(+) T cell responses, something mirrored in descriptive studies of human disease. Recombinant IL-2/diphtheria toxin fusion protein (rIL-2/DTx), a drug that is FDA-approved for the treatment of cutaneous T cell lymphoma, has been reported to deplete regulatory CD4(+) T cells.

Methodology/principal findings: We investigated the potential efficacy of rIL-2/DTx as adjunctive therapy for experimental infection with Leishmania major. Treatment with rIL-2/DTx suppressed lesional regulatory T cell numbers and was associated with significantly increased antigen-specific IFN-γ production, enhanced lesion resolution and decreased parasite burden. Combined administration of rIL-2/DTx and sodium stibogluconate had additive biological and therapeutic effects, allowing for reduced duration or dose of sodium stibogluconate therapy.

Conclusions/significance: These data suggest that pharmacological suppression of immune counterregulation using a commercially available drug originally developed for cancer therapy may have practical therapeutic utility in leishmaniasis. Rational reinvestigation of the efficacy of drugs approved for other indications in experimental models of neglected tropical diseases has promise in providing new candidates to the drug discovery pipeline.

Figure 1. Short-term treatment with rIL-2/DTx leads to transient T_reg depletion.

(A) Uninfected C57BL/6 mice were treated intraperitoneally with a single dose of normal saline (open bars) or rIL-12/DTx (12 µg/kg, gray bars; 50 µg/kg, black bars) and splenic T_reg (TCRβ⁺CD4⁺CD25⁺Foxp3⁺ cells) were quantified by flow cytometry at the time indicated. (B) Uninfected C57BL/6 mice were treated intraperitoneally with 4 or 8 weekly doses of normal saline (open bars) or rIL-12/DTx 50 µg/kg (filled bars) and splenic T_reg (TCRβ⁺CD4⁺CD25⁺Foxp3⁺ cells) were quantified by flow cytometry 7 d after the final dose. (C and D) C57BL/6 mice were given weekly intraperitoneal doses of normal saline (open bars) or rIL-2/DTx (50 µg/kg; filled bars), starting 1 week after intradermal infection in both ears with 3×10³ metacyclic L. major promastigotes. Lesional T_reg (TCRβ⁺CD4⁺CD25⁺Foxp3⁺ cells) were quantified by flow cytometry (C), and IgG₁ antibodies to diphtheria toxin were measured by ELISA in serially diluted serum samples (D), 7 d after the last indicated dose of rIL-2/DTx. Data represent means +/− SE in a single experiment; n = 3 (A), n = 4–6 (B) and n = 5–6 (C and D). (A) ANOVA P<0.01; Tukey's correction; *P<0.05; (B and C) Student's t test *P<0.05, **P<0.01.

Figure 2. Treatment with rIL-2/DTx enhances resolution of experimental L. major infection.

C57BL/6 mice were infected intradermally in both ears with 3×10³ metacyclic L. major promastigotes. (A, B) Beginning 30 d after infection, mice were treated three times, at 5 d intervals, with normal saline (open symbols) or rIL-2/DTx (12 µg/kg; filled symbols). (A) Lesion size; (B) Lesional parasite burden, quantified 45 d after infection. (C, D) Beginning 7 d after infection, mice were treated at weekly intervals with normal saline (open symbols) or rIL-2/DTx (12 µg/kg; filled symbols), and lesion size (C) and parasite burden (D) was quantified 7 d after administration of the last indicated dose. Data represent means +/− SE of 8 mice/group (with individual data points shown for parasite burden). (A and C) MANOVA P<0.05; (A–D) Student's t test; *P<0.01, **P<0.005, ***P<0.001.

Figure 3. Combined rIL-2/DTx and sodium stibogluconate treatment provides additive efficacy in experimental L. major infection.

C57BL/6 mice were infected as in Figure 2. Beginning 30 d after infection, mice were treated: (1) daily for 10 d with SSG (250 mg/kg; filled triangles); (2) three times at 5 d intervals with rIL-2/DTx (12 µg/kg; open squares); (3) three times at 5 d intervals with rIL-2/DTx (50 µg/kg; open circles); (4) daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (12 µg/kg; filled squares); or (5) daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 µg/kg; filled circles); or (6) with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; crossed symbols). (A) Lesion size, (B) lesional parasite burden, and (C) lesional T_reg percentage were quantified in the indicated groups, 45 d after infection. Data represent means +/− SE of 8 mice/group in a single experiment (with individual data points shown for parasite burden); n = 8 (A and B), n = 4 (C). (A) MANOVA P<0.05; (A–C) ANOVA P<0.02; Tukey's correction; *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Enhanced resolution of infection with combined therapy correlates with amplification of the effector immune response.

C57BL/6 mice were infected as in Figure 2; treatment was begun 30 d after infection. (A) Lesion size. Mice were treated: (1) daily for 10 d with SSG (250 mg/kg; open circles); (2) daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 µg/kg; filled circles); or (3) with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; crossed symbols); harvested 45 d after infection. (B) Lesional parasite burden; (C) Lesional T_reg percentage; (D) T_reg percentage in draining lymph nodes; Antigen-specific (E) IFN-γ and (F) IL-10 secretion by leukocytes isolated from lymph nodes draining lesional sites and cultured in presence of soluble Leishmania antigen (quantified by ELISA); Data represent means +/− SE of 6–8 animals/group in a single experiment (with individual data points shown for parasite burden); (A) MANOVA P<0.05; (A–F) statistical analysis on data obtained 45 d after infection; ANOVA P<0.05; Tukey's correction; *P<0.05, **P<0.001.

Figure 5. Addition of rIL-2/DTx to antibiotic therapy allows for treatment duration reduction in experimental cutaneous leishmaniasis.

C57BL/6 mice were infected as in Figure 2. (A and B) lesion size. Beginning 30 d after infection, mice were treated as follows: (1) SSG 250 mg/kg daily for 10 d [black circles]; (2) SSG 25 mg/kg daily for 10 d [blue circles]; (3) SSG 250 mg/kg daily for 5 d [red circles]; (4) SSG 250 mg/kg daily for 10 d+rIL-2/DTx 50 µg/kg 3 times at 5 d intervals [black squares]; (5) SSG 25 mg/kg daily for 10 d+rIL-2/DTx 50 µg/kg 3 times at 5 d intervals [blue squares]; (6) SSG 250 mg/kg daily for 5 d+rIL-2/DTx 50 µg/kg 3 times at 5 d intervals [red squares]; (7) normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx) [open circles]. Note, while regimens are separated into 2 panels for ease of visibility, the control (saline) group in both panels is one and the same. (A and B) MANOVA P<0.0004; ANOVA P<0.001; Tukey's correction; *P<0.05, **P<0.01, ***P<0.001. Linear random effects modeling, done in a second order of analysis, rejected the null hypothesis of all treatments having equal effects (P<0.001), and sorted the therapeutic efficacy of the regimens as follows, from best to worst: groups 4 and 6; groups 1, 3 and 5; groups 2 and 7. (C) Lesional parasite burden quantified in the indicated groups, 45 d after infection. ANOVA P<0.001; Tukey's correction; *P<0.05. Data represent means +/− SE of 5–6 mice/group in a single experiment (with individual data points shown for parasite burden).

Figure 6. Combined therapy enhances resolution of experimental L. major infection in BALB/c mice.

BALB/c mice were infected as in Figure 2. (A) Lesion size. Beginning 30 d after infection, mice were treated: daily for 10 d with SSG (250 mg/kg; blue circles); 3 times at 5 d intervals with rIL-2/DTx (50 µg/kg; red circles); daily for 10 d with SSG (250 mg/kg) plus three times at 5 d intervals with rIL-2/DTx (50 µg/kg; black circles); or with normal saline (per route and schedule for 10 d SSG plus 3 doses of rIL-2/DTx; open circles). (B) Lesional parasite burden; (C) Parasite burden in draining lymph nodes; (D) Parasite burden in liver (dotted line represents the limit of detection in the assay); (E) Parasite burden in spleen; (F) Splenic T_reg (TCRβ⁺CD4⁺CD25⁺Foxp3⁺) percentage; (G) Draining lymph node T_reg percentage; and antigen-specific (H) IFN-γ, (I) IL-10 and (J) IL-4 secretion (by leukocytes isolated from lesional lymph nodes and cultured in presence of soluble Leishmania antigen) were quantified 45 d after infection. Data represent means +/− SE of 6–7 mice/group in a single experiment (with individual data points shown for parasite burden); (A) MANOVA P<0.0003; ANOVA P<0.0001; Tukey's correction; *P<0.05, **P<0.01, ***P<0.001. (B–E) Non-parametric ANOVA (Kruskal-Wallis test for over-all comparison of treatment to no treatment); (B) P<0.02; (C) P<0.006; (D) not statistically significant; (E) P<0.005. Wilcoxon test (for pairwise comparisons), (B–E) *P<0.05, **P<0.01. (F–H) ANOVA P<0.0001; Tukey's correction; *P<0.05, **P<0.01, ***P<0.001.