Impact of co-infection with Plasmodium berghei ANKA in Leishmania major-parasitized mice on immune modulation and cutaneous leishmaniasis

Abstract

Background: Malaria and leishmaniasis are vector-borne diseases responsible for a significant number of deaths worldwide. Despite the co-endemicity of these diseases in regions with tropical and subtropical climates, our understanding of the complex interplay between Plasmodium spp. and Leishmania spp. co-infections on host immune response and resultant disease outcomes remains limited.

Methodology/principal findings: This study employs C57BL/6 mice co-infected with Leishmania major and Plasmodium berghei ANKA, well-established models of cutaneous leishmaniasis and experimental cerebral malaria, respectively. Our findings demonstrate that an acute infection with P. berghei ANKA mitigates the progression of ongoing cutaneous leishmaniasis, as evidenced by a reduction in lesion size and parasite burden in the dermis of L. major-infected mice. Co-infection also led to elevated serum levels of TNF compared to the levels observed in mice infected with L. major alone, which may contribute to a more effective control of the Leishmania parasite. Furthermore, co-infected mice exhibited reduced recruitment of activated T cells and inflammatory monocytes to the site of L. major infection. As inflammatory monocytes can be exploited by Leishmania as host cells that support parasite replication, their reduced infiltration may limit parasite growth. This diminished cellular infiltration is likely to contribute to reduced local inflammation, thereby limiting tissue damage and resulting in smaller lesion size.

Conclusions/significance: These findings elucidate the potential cross-regulation of immune responses in co-infections, underscoring the necessity to consider co-infecting pathogens in disease management and therapeutic strategies in endemic areas.

Fig 1. Co-infection with P. berghei ANKA on day 28 of L. major infection reduces dermal lesion in C57BL/6 mice with cutaneous leishmaniasis.

On the 28th day following infection with L. major, mice were either infected or not with P. berghei ANKA (A). The development of dermal lesions was monitored and measured using a digital caliper on a weekly basis (B) prior to P. berghei ANKA infection, and on a daily basis, specifically on days 6 (C), 7 (D), and 8 (E) following infection with P. berghei ANKA. The experimental groups were designated as Lm (mice infected with L. major only) and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data presented are representative of a single experiment, with n = 7-8 mice per group. Asterisks indicate statistically significant differences, analyzed by Student’s t test, considering p < 0.05. Mouse illustration obtained from Openclipart (https://openclipart.org), public domain (CC0 license).

Fig 2. Co-infection with P. berghei ANKA on day 14 of L. major infection reduces dermal lesions in C57BL/6 mice with cutaneous leishmaniasis.

On the 14th day following infection with L. major, mice were either infected or not with P. berghei ANKA (A). The development of dermal lesions was monitored and measured using a digital caliper on a weekly basis (B) prior to P. berghei ANKA infection, and on a daily basis, specifically on days 6 (C), 7 (D), and 8 (E) following infection with P. berghei ANKA. The experimental groups were designated as Lm (mice infected with L. major only) and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data presented are representative of a single experiment, with n = 6-8 mice per group. Asterisks indicate statistically significant differences, analyzed by Student’s t test, considering p < 0.05. Mouse illustration obtained from Openclipart (https://openclipart.org), public domain (CC0 license).

Fig 3. Co-infection with P. berghei ANKA reduces parasite load in the ear of L. major-infected C57BL/6 mice.

On the 14th day following infection with L. major, mice were either infected or not with P. berghei ANKA. Parasite load was assessed on day 20 of L. major infection (corresponding to day 6 of P. berghei ANKA infection) in the ear lesion (A) and the draining lymph node (dLN) (B). The experimental groups were designated as Lm (mice infected with L. major only) and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data represent a pool of two independent experiments, with n = 13 mice/group. Asterisks indicate statistically significant differences, analyzed by Student’s t test, considering p < 0.05.

Fig 4. Malaria progression analysis shows no difference between co-infected and P. berghei ANKA-only infected C57BL/6 mice.

On the 28th day (A) and 14th day (B) following infection with L. major, mice were infected with P. berghei ANKA and monitored daily for survival. Mice infected with P. berghei ANKA only or L. major only, as well as uninfected controls, were also monitored over time. (C) Parasitemia on days 4 and 6, and (D) body weight and (E) temperature on day 6 post-P. berghei ANKA infection in mice co-infected on day 14 of L. major infection. The experimental groups were designated as Naïve (uninfected animals), Lm (mice infected with L. major only), and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). Asterisks indicate statistically significant differences, as analyzed by One-Way ANOVA with p < 0.05.

Fig 5. Recruitment of T cells and myeloid cells to the site of L. major infection is diminished during co-infection.

Flow cytometry was employed to assess the total number of CD4 T cells (CD3⁺CD4⁺CD62L^-) (A), CD8 T cells (CD3⁺CD8⁺CD62L^-) (C), the percentage of IFN-γ⁺ CD4 T cells (B) and IFN-γ⁺ CD8 T cells (D), as well as the total number of myeloid cells (CD11b⁺) (E), inflammatory monocytes (CD11b⁺Ly6G^-Ly6C^hi) (F), and neutrophils (CD11b⁺Ly6G⁺Ly6C^int) (G) recruited to the ear of the different experimental groups on day 20 of L. major infection (corresponding to day 6 of P. berghei ANKA infection). The experimental groups were designated as Naïve (uninfected animals), Lm (mice infected with L. major only), and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data are representative of two independent experiments with n = 4-8 mice/group. Statistical analyses were performed by One-way ANOVA with Tukey’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.

Fig 6. Co-infection with P. berghei ANKA reduces the number of T cells in the draining lymph node associated with the L. major infection site.

Flow cytometry was employed to assess the total number of CD4 T cells (CD3⁺CD4⁺) (A), activated CD4 T cells (CD3⁺CD4⁺CD62L^-) (B), CD8 T cells (CD3⁺CD8⁺) (C), and activated CD8 T cells (CD3⁺CD8⁺CD62L^-) (D) present in the draining lymph node of the experimental groups on day 20 of L. major infection (corresponding to day 6 of P. berghei ANKA infection). The experimental groups were designated as Naïve (uninfected animals), Lm (mice infected with L. major only), and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data are representative of two independent experiments with n = 4-8 mice/group. Statistical analyses were performed by One-way ANOVA with Tukey’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.

Fig 7. Analysis of T cell and myeloid cell populations in the spleen during L. major and P. berghei ANKA infections.

Flow cytometry was employed to assess the total number of CD4 T cells (CD3⁺CD4⁺) (A), activated CD4 T cells (CD3⁺CD4⁺CD62L^-) (B), CD8 T cells (CD3⁺CD8⁺) (C), activated CD8 T cells (CD3⁺CD8⁺CD62L^-) (D), myeloid cells (CD11b⁺) (E), inflammatory monocytes (CD11b⁺Ly6G^-Ly6C^hi) (F) and neutrophils (CD11b⁺Ly6G⁺Ly6C^int) (G) present in the spleen of the experimental groups on day 20 of L. major infection (corresponding to day 6 of P. berghei ANKA infection). The experimental groups were designated as Naïve (uninfected animals), Lm (mice infected with L. major only), PbA (mice infected with P. berghei ANKA only), and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data are representative of two independent experiments with n = 5-6 mice/group. Statistical analyses were performed by One-way ANOVA with Tukey’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.

Fig 8. Modulation of serum cytokine levels following P. berghei ANKA co-infection in L. major-infected mice.

Serum cytokine levels of the experimental groups on day 20 of L. major infection (corresponding to day 6 of P. berghei ANKA infection) were analyzed using the Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 kit. TNF (A), IFN-γ (B), IL-6 (C), IL-4 (D) and IL-10 (E). The experimental groups were designated as Naïve (uninfected animals), Lm (mice infected with L. major only), PbA (mice infected with P. berghei ANKA only), and Lm + PbA (mice co-infected with both L. major and P. berghei ANKA). The data represent a pool of three independent experiments, with n = 10-15 mice/group. Statistical analyses were performed by One-way ANOVA with Tukey’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.