Curcumin-Arteether Combination Therapy of Plasmodium berghei-Infected Mice Prevents Recrudescence Through Immunomodulation

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha,beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation. But, parasites in liver and spleen were not cleared in AE or AC treatments, perhaps, serving as a reservoir for recrudescence. Parasitemia in blood reached up to 60% in AE-treated mice during the recrudescence phase, leading to death of animals. A transient increase of up to 2–3% parasitemia was observed in AC-treatment, leading to protection and reversal of splenomegaly. A striking increase in spleen mRNA levels for TLR2, IL-10 and IgG-subclass antibodies but a decrease in those for INFγ and IL-12 was observed in AC-treatment. There was a striking increase in IL-10 and IgG subclass antibody levels but a decrease in INFγ levels in sera leading to protection against recrudescence. AC-treatment failed to protect against recrudescence in TLR2−/− and IL-10−/− animals. IL-10 injection to AE-treated wild type mice and AC-treated TLR2−/− mice was able to prolong survival. Blood from the recrudescence phase in AE-treatment, but not from AC-treatment, was able to reinfect and kill naïve animals. Sera from the recrudescence phase of AC-treated animals reacted with several parasite proteins compared to that from AE-treated animals. It is proposed that activation of TLR2-mediated innate immune response leading to enhanced IL-10 production and generation of anti-parasite antibodies contribute to protective immunity in AC-treated mice. These results indicate a potential for curcumin-based combination therapy to be tested for prevention of recrudescence in falciparum and relapse in vivax malaria

Effect of AE and AC-treatments on changes in serum cytokine levels and survival response of TLR2^−/− and IL-10^−/− mice to <i>P.berghei</i>-infection.

<p>(A) Changes in the serum levels of INFγ, IL-10 and IL-12. The data represent Mean + S.D. from three sera preparations and collected during day 6 (D6) to day 23 (D23) at intervals. The day 0 values correspond to those of uninfected animals. The values (pg/ml) corresponding to infected animals, which all died on day 6, are as follows: INFγ, 274±22; IL-10, 422±29; IL-12, 510±39. The values corresponding to infected animals treated with curcumin alone and which all died between, 7–8 days are as follows: INFγ, 243±22; IL-10, 310±33; IL-12, 377±39. (B) Effect of IL-10 injection in AE-treated animals. IL-10 was injected on alternate days from day 10 in five doses ranging from 10 ng to 200 ng. Alternate i.p. and i.v. routes were used. (C) Survival response of <i>P.berghei</i>infected C57BL/6 mice to AE and AC-treatments. The data are from two experiments with five animals in each group. (D) Survival response of TLR^−/− animals with and without IL-10 injection (100 ng per animal for 5 days). (E) Survival response of IL-10^−/− animals. Four animals were used in each group for the experiments in (B), (D) and (E). The knock-out animals were in C57BL/6 background. While, the animals were injected with around 10⁴ parasites in general, the IL 10^−/− animals received around 10³ parasites. UI, unifected; I, infected; UT, untreated; C, curcumin; AE, ART alone; AC, ART+CUR.</p

Effect of AE and AC-treatments on changes in spleen mass and mRNA levels of cytokines, TLRs and IgG-sub-class antibodies in <i>P.berghei</i>-infected mice.

<p>(A) Changes in spleen weight through out the course of infection and drug treatments. Data provided represent Mean ± S.D. from five animals. *, infected animals died 5/6 day (B) Semi-quantitative RT-PCR analysis of spleen RNA on day 14. UI, unifected; I, infected; AE, ART alone; AC, ART+CUR.</p

Effect of AE and AC-treatments on hemozoin content and ROS generation in <i>P.berghei</i>infected mice.

<p>(A). Hemozoin content in parasite 8 hr and 12 hr after a single injection of ART and one oral dose of curcumin. The data represent Mean ± S.D. from three animals. (B) FACS analysis of <i>P.berghi</i>–infected red blood cells for ROS measurement 12 hr after drug treatment as per (A).</p

Effect of AE and AC-treatments on changes in serum total IgG and IgG-subclass antibody levels and Western analysis of parasite proteins with such sera in <i>P.berghei</i>-infected mice.

<p>Antibody levels were quantified in sera by ELISA using microtitre plates coated with parasite lysate from day 6 (D6) onwards. (A) Changes in the serum levels of total IgG and IgG-subclass antibodies against parasite lysate. The data represent Mean ± S.D. from three sera preparations (pooled from two animals each) and collected during day 6 (D6) to day 23 (D23) at intervals. The day 0 values correspond to those of uninfected animals. The values (A₄₀₅) corresponding to infected animals, which all died on day 6, are as follows: IgG, 0.07±0.002; IgG1, 0.03±0.005; IgG2a, not detectable; IgG2b, not detectable; IgG3, not detectable; IgM, 0.22±0.007. (B) Anamnestic response of AC-treated animals. The animals were injected with fresh parasitized blood 10 days before the day mentioned in the Figure. The antibody titre (IgG) was measured before and after challenge. The data represent an average from two sera preparations. (C) Western blot analysis of parasite proteins with the different sera preparations. 1, uninfected; 2, day 5- infected; 3, AE -day 22; 4, AC day-22; 5 and 6, AC - day 75, before and after challenge; 7 and 8; AC - day 180, before and after challenge.</p

Mean fluorescent intensity and percent positive cells in FACS analysis (Figure 2B).

<p>Data represent Mean ± SD from three preparations.</p

Effect of AE and AC- treatments on parasitemia in mice infected with <i>P.berghei</i> for 72 hr.

<p>(A) Changes in parasite load in blood at different time periods through real-time PCR analysis of parasite 18S rRNA after drug treatment. UT, untreated; C, curcumin, AE, ART alone; AC, ART+CUR. D, died; N, not detectable. Data provided represent Mean ± S.D. from three animals. The whole series was repeated thrice (total of 9 animals in each group) and number of animals surviving on day 30 in each group were as follows: UT, 0; C, 0; AE, 1; AC, 9. (B) Semiquantitative RT-PCR analysis of parasite 18S rRNA in blood, liver and spleen on day 10 and 15 after infection. GAPDH was used as a control. *, positive control. (C) Effect of injection of blood from recrudescing animals after AE and AC-treatments into naïve animals. Five animals were used in each group.</p