https://orcid.org/0000-0003-0297-6341
Figures
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.
Author summary
The co-endemicity of malaria and leishmaniasis affects thousands of individuals annually, potentially complicating the diagnosis and resolution of both diseases. However, few studies have explored how the presence of a secondary infection might modulate the host immune response, either exacerbating or suppressing it. In this study, we utilize experimental models to investigate the impact of Plasmodium berghei ANKA infection on the progression of Leishmania major infection, the causative agent of cutaneous leishmaniasis in both humans and mice. Our findings demonstrate that, while the clinical course of malaria remains unaffected by co-infection, the progression of cutaneous leishmaniasis is significantly altered. Co-infection leads to reduced tissue damage and a lower Leishmania parasite load in the dermis. This phenomenon can be attributed to a decrease in the recruitment of inflammatory monocytes and activated T cells to the site of infection, leading to reduced inflammation and lower availability of host cells, particularly inflammatory monocytes, that support Leishmania replication.
Citation: Ornellas-Garcia U, Freire-Antunes L, Rangel-Ferreira M, de Sousa CHG, Ribeiro-Almeida ML, Daniel-Ribeiro CT, et al. (2025) Impact of co-infection with Plasmodium berghei ANKA in Leishmania major-parasitized mice on immune modulation and cutaneous leishmaniasis. PLoS Negl Trop Dis 19(7): e0013302. https://doi.org/10.1371/journal.pntd.0013302
Editor: Claudia Ida Brodskyn, Centro de Pesquisa Gonçalo Moniz-FIOCRUZ/BA, BRAZIL
Received: February 17, 2025; Accepted: June 30, 2025; Published: July 28, 2025
Copyright: © 2025 Ornellas-Garcia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.
Funding: This work was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro- FAPERJ (E-26/200.487/2023, E-26/010.002168/2019 and E-26/203.253/2017 to P.C., 26010.002418/2019 and 260003/001169/2020 to C.T.D.R., E-26/211.323/2019 and 26/210.420/2022 to F.L.R.G.); the Fundação Oswaldo Cruz (8121512557 to F.L.R.G.); the Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq (465489/2014-1 and 310445/2017-5 to C.T.D.R., 312573/2020-0 to P.C., 431230/2016-1 and 311128/2022-0 to F.L.R.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Vector-borne diseases, including malaria and leishmaniasis, are endemic in tropical and subtropical regions, impacting a substantial portion of the global population and responsible for a significant number of deaths annually [1]. The World Malaria Report (2024) indicates that 263 million cases of malaria caused by Plasmodium spp. were reported in 2023, resulting in 597,000 deaths [2]. The mortality rate associated with malaria is closely linked to the development of severe disease manifestations, such as cerebral malaria (CM) [3]. Although the immunopathological mechanisms are not fully understood, CM is intrinsically related to parasitized red blood cells (pRBCs) adhering to the endothelium of cerebral microvasculature. Furthermore, an exacerbated type 1 immune response is observed, which triggers heightened endothelial activation, amplified pRBCs adherence, mechanical obstruction of cerebral blood vessels, decreased blood flow and oxygen delivery, and ultimately, tissue damage and blood-brain barrier disruption [4,5].
Similarly, Leishmania parasites cause a spectrum of diseases responsible for nearly 1 million new cases annually [2], and more than 4000 deaths were reported in the last decade [6]. The pathophysiology of leishmaniasis is determined by a number of interacting factors, including parasite species, host-related characteristics (particularly immune responses), and the occurrence of co-infections [7]. The disease can range from self-limiting skin lesions to fatal visceral forms if left untreated [7]. Cutaneous leishmaniasis (CL) represents the most prevalent form of the disease. Despite the World Health Organization (WHO) reporting 205,990 cases in 2022, the actual number of cases is estimated to exceed 600,000, due to underreporting of the disease [8]. In CL caused by L. major and L. braziliensis, an inflammatory type-1 immune response is typically associated with parasite control [9,10]; however, it is not always correlated with less severe pathology [11].
Leishmaniasis and malaria are co-endemic in several countries [12–14], and co-infection between these two pathogens is a recurrent phenomenon [14–22]. Nevertheless, the study of cases of co-infection and their clinical and immune implications for the host is an overlooked issue.
To address this knowledge gap, the present study utilized well-established models in C57BL/6 mice. These mice are known for their inherent resistance to L. major (Friedlin strain) infection, developing self-healing lesions following intradermal inoculation. This resistance is associated with a robust Th1-polarized immune response, characterized by IFN-γ and TNF production and macrophage activation, which are essential for parasite elimination [23,24]. Conversely, C57BL/6 mice demonstrate a marked susceptibility to P. berghei ANKA infection, thereby serving as the principal model for experimental cerebral malaria (eCM). As indicated by the extant literature, this infection has been shown to lead to neurological disorders and a high mortality rate between six and nine days post-infection [5,25]. The pathogenesis in this model mirrors human CM, with excessive production of IFN-γ and TNF contributing to immune-mediated vascular damage [4,26]. Therefore, to explore the immunological interplay between these two protozoan parasites and their impact on disease outcomes, C57BL/6 mice were first infected with L. major and subsequently challenged with P. berghei ANKA.
Our findings demonstrate that the immune response induced by P. berghei ANKA modulates the ongoing response in C57BL/6 mice previously infected with L. major, leading to reduced recruitment of T cells and inflammatory monocytes to the dermal site of Leishmania infection, along with increased systemic levels of TNF. This immunomodulation alters the clinical course of CL, as evidenced by reduced lesion size and parasite load.
Materials and methods
Ethics statement
Female C57BL/6 mice aged 6–8 weeks were obtained from the Instituto de Ciências e Tecnologia em Biomodelos (ICTB), Fiocruz. The animals were maintained under controlled light and temperature conditions and provided with ad libitum access to food and water. All animal procedures were conducted in strict accordance with relevant regulations and guidelines. All experimental protocols were approved by the local Ethical Committee for Animal Use (Comissão de Ética no Uso de Animais, CEUA) of the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz - FIOCRUZ (license numbers L030-2017 and L-030–2017 A1).
Parasites and infection
Leishmania major Friedlin FV1 (MHOM/IL/80/FN) parasites were cultured at 26°C in Schneider medium supplemented with 100U/mL penicillin, 100μg/mL of streptomycin, 2% sterile human urine, and 10% heat-inactivated fetal bovine serum (FBS) (Gibco). These parasites were utilized up to the fourth in vitro passage. Metacyclic promastigotes of L. major were isolated through a 10% Ficoll gradient centrifugation (1690g, 10 minutes, 28ºC) from stationary in vitro cultures that had been maintained for 5–6 days [27]. Mice were infected with 2 x105 metacyclic forms via injection into the ear dermis.
P. berghei ANKA expressing a green fluorescent protein was generated as previously described [28] and supplied by the Malaria Research and Reference Reagent Resource Center-MR4, Manassas, VA (MR4 number: MRA-865). The blood samples containing pRBCs were cryopreserved in liquid nitrogen. Before infecting the experimental groups, a 100 µL aliquot of the blood was thawed and inoculated intraperitoneally into healthy mice. Four to five days later, blood samples were obtained from the animals via cardiac puncture, and parasitemia and red blood cell (RBC) counts were estimated. Subsequently, the mice comprising the experimental group were intraperitoneally injected with 106 fresh pRBCs.
The experimental design, detailing the sequence of infections, is described in S1 Fig. Mice were randomly assigned to four groups: one group infected with L. major only (Lm group), one infected with P. berghei ANKA only (PbA group), one co-infected with both L. major and P. berghei ANKA (Lm + PbA group), and one group of uninfected animals (Naïve group).
Evaluation of Plasmodium berghei ANKA parasitemia and body temperature
On the sixth day following infection with P. berghei ANKA, parasitemia and body temperature were assessed. The parasitemia was determined by flow cytometry, based on the percentage of GFP+pRBCs. Rectal temperature was determined by using a thermocouple probe (Oakton Acorn; Oakton Instruments, IL, USA). Furthermore, the occurrence of clinical signs, including lethargy, ataxia, seizures, and coma, was monitored daily.
Evaluation of L. major lesion size and parasite load
The thickness and diameter of dermal lesions resulting from L. major infection were evaluated over a period of approximately 16 weeks using a digital caliper (DIGIMESS). The parasite loads in the ear and the draining lymph node were determined through limiting dilution analysis [29,30]. Briefly, tissue suspensions were serially diluted in 96-well flat-bottom culture plates containing Schneider medium supplemented with 20% heat-inactivated FBS and 2% sterile human urine. The plates were examined microscopically for the presence of promastigotes on days 4, 7, and 14. The parasite burden in each tissue was calculated by counting the number of parasite-positive wells.
Measurement of serum cytokine levels
The cytokine levels in the serum of the experimental groups were evaluated using a BD Cytometric Bead Array Kit for Mouse Th1/Th2/Th17 Cytokines (BD Biosciences, San Jose, CA) in accordance with the manufacturer’s instructions.
Processing of tissue samples from the ear, draining lymph node, and spleen
The preparation of the ear tissue was conducted in accordance with the methodology previously described [31] with minor modifications. In brief, the ears were removed and promptly immersed in 70% ethanol for a period of 2–5 minutes, followed by air-drying. The two layers of ear dermis were separated and placed in RPMI medium containing 0.160 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp.), and incubated for 1 hour and 30 minutes at 37°C. Following digestion, the tissue was homogenized through a 70 μm-pore-size cell strainer using a syringe plunger in RPMI medium containing 0.05% DNase I (SIGMA, ALDRICH). The resulting cell suspension was then subjected to centrifugation (609g, 8 minutes, 4ºC) and resuspended in 1 mL of complete Schneider medium.
The ear-draining lymph node was excised and transferred to separate Eppendorf tubes containing 300 µL each of supplemented Schneider medium. The lymph node was macerated individually using a Pellet Pestle Motor (Kontes) until complete dissociation of the organ was achieved. Thereafter, the volume was adjusted to 700 µL using Schneider medium. The total number of cells was calculated after counting them using a Neubauer chamber.
The spleens were carefully harvested, cut into smaller pieces using scissors, and subsequently homogenized in cold phosphate-buffered saline (PBS) using a 70 μm-pore cell strainer to obtain a single cell suspension. The resulting cell suspensions were subjected to centrifugation (609g, 8 minutes, 4ºC), and the pellet was resuspended in Red Blood Cell Lysing Buffer (Sigma, Life Science) to eliminate RBCs. The splenocyte suspensions were then thoroughly washed and resuspended in PBS containing 5% FBS. The total count of splenocytes was determined by diluting the cell suspension in Trypan blue and counting them using a Neubauer chamber.
Immunophenotyping of myeloid and lymphoid cell populations
Following the extraction and processing of spleen, draining lymph nodes, and ear tissues from both infected and uninfected animals, immunolabeling was performed. Specifically, 1 x106 cells from the spleen and draining lymph nodes, as well as half of the homogenized ear tissue, were washed and labeled with various combinations of anti-mouse antibodies. To identify lymphoid populations, cells were incubated on ice for 20 minutes with a pool of antibodies, including anti-Fc-γ III/II (CD16/32) receptor antibody (2.4G2), anti-TCRβ PE (H57-597) or anti-CD3 PercpCy7 (145-2C11), anti-CD4 APC-H7 (GK1.5), anti-CD8 PeCy7 (53-6.7), and anti-CD62L FITC (MEL-14). To identify myeloid populations, cells were incubated with the following antibodies: anti-Fc-γ III/II (CD16/32) receptor antibody (2.4G2), anti-CD11b PeCy7 (M1/70), anti-Ly6C APC-Cy7 (AL-21), and anti-Ly6G APC (1A8). In some experiments, to assess IFN-γ production, cells were fixed, permeabilized, and incubated with anti-IFN-γ APC (XMG1.2) antibodies following surface staining. All antibodies utilized in this study were from BD Biosciences. Following the incubation period, the cells were washed twice prior to acquisition. Samples were acquired using either FACSCanto II (BD Biosciences) or CytoFLEX (Beckman Coulter) flow cytometer. Data were analyzed using FlowJo 10.0 software (BD Biosciences).
Statistical analysis
All graphs and statistical analyses were made using GraphPad Prism version 8.0 (GraphPad Software, USA). Comparison between the two groups was conducted using the unpaired t-test with a 95% confidence level. To compare more than two groups, a One-way ANOVA test was conducted, followed by a Tukey’s multiple comparison test. Only results with p-values less than 0.05 (p < 0.05) were considered statistically significant.
Results
Acute infection with Plasmodium berghei ANKA in Leishmania major-parasitized mice reduces dermal lesion size and parasite load in those with cutaneous leishmaniasis
To assess whether Plasmodium infection might impact the clinical course of an ongoing Leishmania infection, we used well-known experimental models of eCM [32–34] and CL [24]. P. berghei ANKA infection of C57BL/6 mice has been shown to induce eCM, resulting in death 6–9 days post-infection [32,33]. In contrast, L. major infection of C57BL/6 mice has been observed to result in a cutaneous lesion that self-resolves within 12 weeks after inoculation [23,35]. In this study, mice were injected into the ear dermis with metacyclic promastigote forms of L. major, and at the peak (day 28th) or beginning (day 14th) of the dermal lesion development, the animals were co-infected or not with P. berghei ANKA (Figs 1A and 2A). Co-infection with P. berghei ANKA reduced the cutaneous lesion size of mice previously parasitized with L. major, independently of the time (28- or 14-days post-L. major infection) (Figs 1B and 2B). Lesion thickness between co-infected (Lm + PbA group) and L. major-only infected mice (Lm group) was statistically different at days 7 (Fig 1D) and 8 post-P. berghei ANKA infection (Figs 1E and 2E) exhibiting a reduction in the Lm + PbA group. Note that although the reduction in lesion thickness appeared statistically different earlier (on day 7 post-P. berghei ANKA infection) in the group co-infected at day 28, no such difference was observed in the group co-infected at day 14. However, any direct comparisons between the two timepoints should be interpreted with caution, as these experiments were conducted independently. Taken together, both datasets support the notion that P. berghei ANKA co-infection mitigates the progression of CL.
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).
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).
Given the similarity of the results obtained from our co-infection analyses with P. berghei ANKA at 28- and 14-days post-infection with L. major regarding the lesion progression, we opted to proceed with our analysis by maintaining the co-infection at day 14 post-L. major infection. This timepoint represents a critical phase of active lesion development, marked by intense parasite replication and immune cell recruitment, thereby providing a valuable window to investigate the immediate impact of a secondary infection. While this earlier stage may not fully capture the extent of P. berghei ANKA-mediated modulation, it presents a technically feasible and biologically informative opportunity to dissect the initial host-pathogen interactions during co-infection.
The co-infection of L. major-parasitized mice at day 14 with P. berghei ANKA led to a reduction in the load of Leishmania parasite in the ear dermis when compared to L. major-only infected mice (Fig 3A). No difference was observed in the parasite load between Lm + PbA and Lm groups in the skin-draining lymph nodes (Fig 3B).
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.
Of note, the Lm + PbA and PbA groups exhibit comparable survival rates, with mice from both groups succumbing to eCM between 6 and 9 days post-P. berghei ANKA infection, regardless of whether the co-infection was established on day 28 (Fig 4A) or day 14 (Fig 4B) following L. major infection. Furthermore, parasitemia, body weight, and temperature were also assessed to provide a more comprehensive characterization of the disease course. Parasitemia (Fig 4C) measured on days 4 and 6 post-P. berghei ANKA infection showed similar kinetics in both co-infected and P. berghei ANKA-only infected mice. Body weight (Fig 4D) and temperature (Fig 4E) measurements on day 6 post-P. berghei ANKA infection revealed similar patterns of weight loss and hypothermia (used to define late stage of eCM in various studies [36,37] in both the Lm + PbA and PbA groups, in contrast to the Naive and Lm groups. Together, these results indicate that a pre-existing L. major infection does not significantly alter the clinical progression of P. berghei ANKA infection in this co-infection model.
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.
Co-infection with Plasmodium berghei ANKA results in reduced T cell and myeloid cell counts at the dermal site of Leishmania major infection and in the skin-draining lymph node
In order to determine the biological processes initiated by P. berghei ANKA infection that could be influencing L. major pathology, we evaluated the recruitment of CD4 and CD8 T cells and myeloid cell subpopulations to the site of L. major infection and the T cell numbers in the draining lymph nodes 6 days post-P. berghei ANKA infection. To this end, we established gate strategies for the selective identification of the cells of interest in both tissues (S2 and S3 Figs). Flow cytometry analysis of the dermal tissue revealed a robust recruitment of CD4 (CD3+CD4+CD62L-) and CD8 (CD3+CD8+CD62L-) T cells in the ear dermis of L. major-only infected mice, as compared to the Naïve group (Fig 5A and 5C). However, acute infection with P. berghei ANKA in L. major-parasitized mice resulted in decreased number of these cells in the ear dermis (Fig 5A and 5C). Among the CD4 T cells in the ear dermis, it was possible to identify an increase in the percentage of IFN-γ+ cells in L. major-only infected mice, which was reduced following co-infection with P. berghei ANKA (Fig 5B). In contrast, co-infection resulted in an increase in the percentage of IFN-γ+ CD8 T cells (Fig 5D). However, it is noteworthy that the total number of CD8 T cells was significantly reduced in co-infected mice (Fig 5C). Moreover, the absolute number of IFN-γ+ CD8 T cells did not differ between co-infected mice and those infected with L. major alone (S5 Fig), suggesting that the observed increase in the percentage of IFN-γ+ CD8 T cells does not correspond to a biologically significant change in cell numbers. As anticipated, all CD4 and CD8 T cells present at the site of infection exhibited the CD62L- phenotype, indicative of activation.
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-Ly6Chi) (F), and neutrophils (CD11b+Ly6G+Ly6Cint) (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.
The recruitment of myeloid cells (CD11b+) was also evaluated across all experimental groups, including inflammatory monocytes (CD11b+Ly6G-Ly6Chi) and neutrophils (CD11b+Ly6G+Ly6Cint). Notably, L. major infection induced a significant increase in the total number of CD11b+ cells and inflammatory monocytes. However, in L. major-parasitized mice co-infected with P. berghei ANKA, the overall count of myeloid cells and inflammatory monocytes at the site of L. major infection was reduced compared to animals infected with L. major only (Fig 5E and 5F). No difference was observed in the total number of neutrophils between the groups in the dermis (Fig 5G).
A comparable phenomenon was observed in the draining lymph node. The total number of CD4 T cells (CD3+CD4+), activated CD4 T cells (CD3+CD4+CD62L-), CD8 (CD3+CD8+) and activated CD8 T cells (CD3+CD8+CD62L-) significantly increased following L. major infection, but declined in co-infected animals, except for activated CD8 T cells, which remained unchanged compared to animals infected only with L. major (Fig 6A–6D).
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.
In summary, our analysis of immune cell populations in different tissues from L. major-only infected mice and co-infected mice underscored that the introduction of P. berghei ANKA co-infection during ongoing CL results in a reduction of T cells at both the site of L. major infection and the draining lymph node. Furthermore, there was a marked decrease in inflammatory monocytes at the site of infection.
Co-infection of Leishmania major-parasitized mice with Plasmodium berghei ANKA induces alterations in the cell counts recovered from the spleen
We next evaluated the number of T cells and myeloid cells in the spleen, a pivotal organ in the immune response during the erythrocytic phase of Plasmodium spp. infection [38]. Using flow cytometry and gating strategies to delineate the cells of interest (S4 Fig), an increase in the numbers of total (Fig 7A and 7C) and activated (Fig 7B and 7D) CD4 and CD8 T cells in the spleens of mice infected with P. berghei ANKA alone or co-infected (PbA and Lm + PbA groups) was observed in comparison to uninfected animals (Fig 7A–7D). Interestingly, the number of CD4 (activated) and CD8 (total and activated) T cells in co-infected mice consistently exceeded the values observed in mice infected only with L. major, although their numbers remained lower than in mice infected exclusively with P. berghei ANKA (Fig 7A–7D).
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-Ly6Chi) (F) and neutrophils (CD11b+Ly6G+Ly6Cint) (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.
Turning our attention to myeloid cell subpopulations (Fig 7E–7G), both L. major and P. berghei ANKA infections heightened the numbers of total splenic CD11b+ cells (Fig 7E). It is notable that P. berghei ANKA infection resulted in an increase in the number of inflammatory monocytes (CD11b+Ly6G-Ly6Chi) even in the presence of an ongoing L. major infection (Fig 7F). Conversely, an augmented presence of splenic neutrophils (CD11b+Ly6G+Ly6Cint) was observed exclusively in mice infected with L. major only (Fig 7G). Additionally, it appears that the co-infection with P. berghei ANKA resulted in a reduction in the number of neutrophils recovered from the spleen (Fig 7G).
Plasmodium berghei ANKA co-infection enhances systemic TNF levels in L. major-parasitized mice, compared to mice infected with L. major alone
Plasmodium spp. infection is known to induce substantial levels of systemic pro-inflammatory cytokines [39–42]. Pro-inflammatory cytokines, such as IFN-γ and TNF, play crucial roles in regulating CL [43]. To further investigate this interplay, we assessed serum levels of pro- and anti-inflammatory cytokines across our experimental groups.
Mice infected solely with P. berghei ANKA exhibited elevated levels of TNF, IFN-γ, and IL-6 in comparison to uninfected mice (Fig 8A–8C). Interestingly, mice co-infected with both pathogens (Lm + PbA group) exhibited reduced levels of these inflammatory cytokines (TNF, IFN-γ, and IL-6) compared to the PbA group (Fig 8A–8C). However, it is worth noting that P. berghei ANKA co-infection resulted in a significant increase in TNF levels in mice with an ongoing L. major infection compared to mice infected with L. major alone (Fig 8A), which may contribute to improving L. major parasite control. The levels of other cytokines, including IL-4 (Fig 8D) and IL-10 (Fig 8E), demonstrated no statistically significant differences between the groups.
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.
Discussion
The co-infection of hosts with multiple parasites has been identified as a critical factor influencing the susceptibility and severity of numerous diseases. Simultaneous Plasmodium spp. infections, whether occurring naturally or in experimental settings, can disrupt the immune responses and affect the progression of other diseases, including those caused by viruses [44–46], bacteria [47,48], and parasites [49–51]. The clinical outcome of Plasmodium spp. - Leishmania spp. co-infection is influenced by a number of factors such as the parasite species, the order of infection, and host genetics. In natural infections, it is challenging to accurately measure and evaluate some of these variables. However, experimental models afford us the opportunity to elucidate the intricate details involved in co-infections with greater robustness and precision.
Infection of C57BL/6 mice with L. major (MHOM/IL/80/Friedlin) typically results in the development of a single lesion that heals spontaneously within a few weeks without the need for therapeutic intervention [23]. The present study demonstrated that co-infection with P. berghei ANKA in L. major-parasitized mice accelerated the healing process, significantly reducing lesion size and parasite load in the dermis just a few days following co-infection, in comparison to animals only infected with L. major. In a different murine experimental model, Pinna et al. (2016) demonstrated a comparable outcome, whereby the lesion and ulcerations were diminished in BALB/c animals co-infected with L. braziliensis and P. yoelii 17XNL (non-lethal strain) [52]. Moreover, a reduction in the parasite burden was observed in the lymph nodes of co-infected animals when compared to those infected only with L. braziliensis [52]. It is noteworthy that in another co-infection model, infection of C57BL/6 mice with T. brucei brucei was demonstrated to regulate the pathology of CL by L. major, resulting in a reduction of the lesion size and parasite load, the same parameters evaluated in our study [53]. Our findings align with these studies involving P. yoelii 17XNL and T. brucei brucei, indicating that the presence of a second parasite simultaneously in the same host can influence the clinical course of CL. However, studies employing L. amazonensis infection of BALB/c and C57BL/6 mice, followed by co-infection with P. yoelii 17XNL a few days later, demonstrated a worsening of skin lesions [52,54–56]. In this experimental context of L. amazonensis infection, concomitant plasmodial infection has the potential to exacerbate the severity of CL.
In addition, our study suggests that a previous infection with L. major did not prevent the development of eCM in animals co-infected with P. berghei ANKA. Moreover, no differences were observed in parasitemia, body weight, or temperature between the P. berghei ANKA-infected and co-infected animals. However, more subtle effects of co-infection with L. major on brain pathology cannot be excluded and warrant further investigation. The findings in the literature regarding the clinical course of a plasmodial infection in co-infected animals exhibit considerable variability, depending on the Leishmania spp. utilized in the study, as well as the mouse model employed. In brief, BALB/c mice co-infected with L. braziliensis and P. yoelii 17XNL exhibited a reduction in plasmodial parasitemia compared to mice infected only with P. yoelii 17XNL [52]. In contrast, BALB/c and C57BL/6 mice infected with L. amazonensis followed by P. yoelii 17XNL a few days later demonstrated a notable increase in parasitemia compared to those mice that had only been infected with P. yoelii 17XNL [52,54–56].
In infections caused by Plasmodium spp. and Leishmania spp., cell recruitment is a critical process that supports an effective or harmful immune response, which directly influences the clinical outcome of these diseases [57,58]. The analysis of the animals’ ears revealed that infection with L. major resulted in the expected recruitment of lymphoid and myeloid cells to the site of infection, as would be anticipated. However, in animals co-infected with both parasites, this recruitment was found to be reduced for both activated T cells, including IFN-γ+ CD4 T cells, and inflammatory monocytes. Notably, although we observed an increase in the percentage of IFN-γ ⁺ CD8 T cells in co-infected animals, this did not translate into an increase in their absolute number. This discrepancy probably reflects the overall decrease in total CD8 T cells in the tissue and suggests that the higher frequency does not represent a biologically significant increase in effector CD8 T cells. In the initial stages of Leishmania infection of the skin, IFN-γ plays a pivotal role in recruiting permissive monocytes, which are essential for the early establishment and proliferation of the parasite [59]. This finding suggests that co-infection with P. berghei ANKA limits cell migration to the site, thereby contributing to the reduction of the inflammatory process in the dermis.
The findings of this study are consistent with those of other experimental models, which have demonstrated the impact of co-infection with Plasmodium spp. on the recruitment of immune cells and the modulation of inflammation [51,60,61]. Teo et al. (2018) demonstrated that animals co-infected with chikungunya virus (CHIKV) and P. berghei ANKA exhibited a reduction in the infiltration of various immune cells in the joints, including CD4 T cells and monocytes, in comparison to those infected with the chikungunya virus alone [60]. This reduction in CD4 T cells was correlated with a decrease in joint inflammation, as the pathology is linked to IFN-γ-producing CD4 T cells [60]. Similarly, Vieira-Santos et al. (2021) observed that mice co-infected with Ascaris suum and P. berghei NK65-NY exhibited decreased cellularity in bronchoalveolar lavage, including reductions in lymphocytes, macrophages, and neutrophils [51]. In a separate model, Edwards et al. (2015) reported that co-infection of P. chabaudi chabaudi with murine pneumovirus significantly suppressed the recruitment of lymphocytes, neutrophils, and eosinophils to the lungs, in contrast to the recruitment observed in animals infected with the virus alone [61]. These studies indicate that co-infection with Plasmodium spp. plays a significant role in modulating the recruitment of different cell populations, which may contribute to the control of the inflammatory process or, in some cases, compromise the effectiveness of the immune response.
The restricted recruitment of cells to the site of infection can be attributed, at least in part, to mechanisms originating in secondary lymphoid organs, such as the lymph nodes and spleen. The evaluation of T cell populations in the lesion’s draining lymph node of co-infected mice also revealed a reduction in the number of T cells compared to those infected with L. major only. The reason for these decreases was not assessed in the present study; however, it may be associated with T cell apoptosis, as has been reported in the CHIKV + P. berghei ANKA and P. yoelii 17XNL + Listeria monocytogenes co-infection [60,62]. Moreover, co-infection can impact the migration of these cells. In the CHIKV + P. berghei ANKA model, co-infection not only induces apoptosis of T cells in lymph nodes but also suppresses the production of chemoattractant factors such as MIP-1α and MIP-1β, thereby limiting the migration of CD4 T cells to joints [60]. In a recent study, Foo et al. (2024) demonstrated that during co-infection with the neurotropic arbovirus SFV (Semliki Forest virus) and a non-neurotropic strain of influenza virus (IAV), there was an increase in influenza-specific CD8 T lymphocyte traffic to the brain, accompanied by augmented blood-brain barrier permeability [63]. Another potential mechanism for the observed reduction in T cells is the process of cell exhaustion, which has been demonstrated to impair the proliferation of these cells. Plasmodial infection has been shown to induce the expression of exhaustion molecules, including PD-1 and CTLA-4, in T cells [64–66]. In advanced stages of exhaustion, cells may enter apoptosis, thereby generating a state of immune hyporesponsiveness. This phenomenon has been described not only in Plasmodium spp. infections, but also in Leishmania spp. infections [67,68]. It is possible that co-infection may exacerbate this process.
Splenomegaly is a commonly observed phenomenon in the scientific literature concerning the infection of Plasmodium spp. [69–71]. The enlargement of the organ is attributed to the accumulation of RBCs and pRBCs, along with cell recruitment and clonal expansion [32]. This cellular expansion comprises the activation of T and B cells, which are essential for the control of the parasite [72,73]. The results of our study confirm that infection with P. berghei ANKA results in the expansion and activation of splenic CD4 and CD8 T cells. Although the overall numbers of these cells in animals co-infected with both parasites were lower than in those infected only with P. berghei ANKA, they were higher than in animals infected only with L. major. The data indicate that a prior infection with L. major had a partial inhibitory effect on the expansion of T cells following co-infection with P. berghei ANKA.
A type 1 immune response is strongly induced in C57BL/6 mice during malaria and leishmaniasis, resulting in the production of pro-inflammatory cytokines such as IFN-γ and TNF [57,74,75]. Nevertheless, research has demonstrated that co-infections involving Plasmodium spp., whether with viral, bacterial, or parasitic pathogens, result in a distinct alteration of the serum cytokine profile in comparison to single-pathogen infections [52,76,77]. Our findings align with these observations, as evidenced by the data, which indicate that co-infection with P. berghei ANKA in previously L. major-infected animals does not result in an elevation of type 1 cytokines (IFN-γ and IL-6) observed in animals infected with P. berghei ANKA alone. However, animals co-infected with L. major and P. berghei ANKA exhibited elevated serum TNF levels in comparison to those infected with L. major alone. This may contribute to the control of L. major parasite load in the lesion.
In brief, our study elucidated the immunological and clinical consequences of Plasmodium spp. – Leishmania spp. co-infection demonstrating that infection with P. berghei ANKA modulates the progression of ongoing L. major infection by a reduction in lesion size and parasite burden in the dermis, and reduction of activated T cells and inflammatory monocytes recruitment to the site of infection. Although co-infection with P. berghei ANKA resulted in an increase in the pro-inflammatory cytokine TNF that may be related to the control of the L. major parasite load, co-infection exerted an immunomodulatory effect that attenuated local inflammation, minimizing tissue damage and accelerating the healing process. However, the precise mechanisms underlying this cross-regulation, including the signaling pathways and molecular interactions, remain to be fully elucidated.
Supporting information
S1 Fig. Experimental design.
On day 0, the Lm and the Lm + PbA groups received 2 x105 metacyclic forms of L. major intradermally (ID) in the ears. Subsequently, on either the 14th or 28th day, depending on the experiment and detailed in the legends of the subsequent figures, the Lm + PbA and PbA groups received an intraperitoneal inoculation (IP) of 106 pRBCs infected with P. berghei ANKA. The uninfected animals (Naive group) received intradermal and intraperitoneal inoculations of RPMI and PBS, respectively, at the corresponding time points. On the 20th or 34th day following L. major infection (which corresponds to a period of 6 days after P. berghei ANKA infection of the PbA and Lm + PbA groups), all groups were euthanized for analyses. In some experiments, the size of the lesion and the survival rate were monitored over time. Mouse illustration obtained from Openclipart (https://openclipart.org), public domain (CC0 license).
https://doi.org/10.1371/journal.pntd.0013302.s001
(TIF)
S2 Fig. Representative plots illustrating the gating strategy applied to identify the T cells and myeloid cells in the ear.
(A) After gating on “all cells” (i), singlets (ii), and CD3+ cells (iii), the CD4 and CD8 T cells were defined (iv). Activated CD4 (CD3+CD4+CD62L-) (v) and CD8 (CD3+CD8+CD62L-) (vi) T cells were defined based on the lack of CD62L expression, and among them, the T cells expressing IFN-γ were further characterized (vii and viii). Identification of positive populations was facilitated by employing the fluorescence minus one (FMO) control as a negative reference. (B) After gating on “all cells” (i), singlets (ii), and CD11b+ cells (iii), the neutrophils (CD11b+Ly6G+Ly6Cint) and monocytes (CD11b+Ly6G-Ly6Chi) were defined.
https://doi.org/10.1371/journal.pntd.0013302.s002
(TIF)
S3 Fig. Representative plots illustrating the gating strategy applied to identify the T cells in the draining lymph node.
After gating on “all cells” (i), singlets (ii), and CD3+ cells (iii), the CD4 and CD8 T cells were defined (iv). Activated CD4 (CD3+CD4+CD62L-) (v) and CD8 (CD3+CD8+CD62L-) (vi) T cells were defined based on the lack of CD62L expression. Identification of positive populations was facilitated by employing the fluorescence minus one (FMO) control as a negative reference.
https://doi.org/10.1371/journal.pntd.0013302.s003
(TIF)
S4 Fig. Representative plots illustrating the gating strategy applied to identify the T cells and myeloid cells in the spleen.
(A) After gating on “all cells” (i), singlets (ii), and CD3+ cells (iii), the CD4 and CD8 T cells were defined (iv). Activated CD4 (CD3+CD4+CD62L-) (v) and CD8 (CD3+CD8+CD62L-) (vi) T cells were defined based on the lack of CD62L expression. Identification of positive populations was facilitated by employing the fluorescence minus one (FMO) control as a negative reference. (B) After gating on “all cells” (i), singlets (ii), and CD11b+ cells (iii), the neutrophils (CD11b+Ly6G+Ly6Cint) and monocytes (CD11b+Ly6G-Ly6Chi) were defined.
https://doi.org/10.1371/journal.pntd.0013302.s004
(TIF)
S5 Fig. Total number of IFN-γ+ T cells is not altered during coinfection.
Flow cytometry was employed to assess the total number of IFN-γ+ CD4 T cells (A) and IFN-γ+ CD8 T cells (B) present in ear 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.
https://doi.org/10.1371/journal.pntd.0013302.s005
(TIF)
Acknowledgments
The authors thank the Multi-user Research Facility of Flow Cytometry—Multiparametric Analysis, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. The authors thank DVM Mônica Nogueira and her team of the Centro de Experimentação Animal (IOC, Fiocruz) for caring for the animals and supporting the experiments.
References
- 1.
World Health Organization. Global vector control response 2017–2030 [Internet]. Geneva: World Heath Organization. Licence: CC BY-NC-SA 3.0 IGO; 2017. Available from: https://www.who.int/publications/i/item/9789241512978
- 2.
World Health Organization. World Malaria Report (2023) [Internet]. Geneva: World Heath Organization. Licence: CC BY-NC-SA 3.0 IGO. [cited 2023 Dec 28. ]. Available from: https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023
- 3. Ghazanfari N, Gregory JL, Devi S, Fernandez-Ruiz D, Beattie L, Mueller SN. CD8 and CD4 T cells infiltrate into the brain during Plasmodium berghei ANKA infection and form long-term resident memory. J Immunol [Internet]. 2021;207(6):1578–90. Available from: https://journals.aai.org/jimmunol/article/207/6/1578/234132/CD8-and-CD4-T-Cells-Infiltrate-into-the-Brain
- 4. Dunst J, Kamena F, Matuschewski K. Cytokines and chemokines in cerebral malaria pathogenesis. Front Cell Infect Microbiol [Internet]. 2017;7. Available from: http://journal.frontiersin.org/article/10.3389/fcimb.2017.00324/full
- 5. Ghazanfari N, Mueller SN, Heath WR. Cerebral malaria in mouse and man. Front Immunol [Internet]. 2018;9(SEP):1–11. Available from: https://www.frontiersin.org/article/10.3389/fimmu.2018.02016/full
- 6.
Jain S, Madjou S, Virrey F, Jain S, Madjou S, Virrey F. Global leishmaniasis surveillance updates 2023: 3 years of the NTD road map Surveillance mondiale de la leishmaniose, 2023: la feuille de route pour les MTN a 3 ans [Internet]; 2023. Available from: https://www.who.int/publications/i/item/who-wer-9945-653-669
- 7. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet [Internet]. 2018;392(10151):951–70. Available from: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(18)31204-2/abstract
- 8.
World Health Organization. Global report on neglected tropical diseases 2024: executive summary [Internet]; 2024. Available from: https://www.who.int/teams/control-of-neglected-tropical-diseases/global-report-on-neglected-tropical-diseases-2024
- 9. Ajdary S, Alimohammadian MH, Eslami MB, Kemp K, Kharazmi A. Comparison of the immune profile of nonhealing cutaneous Leishmaniasis patients with those with active lesions and those who have recovered from infection. Infect Immun. 2000;68(4):1760–4. pmid:10722561
- 10. Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Rev Immunol [Internet]. 2016;16(9):581–92. Available from: https://www.nature.com/articles/nri.2016.72
- 11. Charmoy M, Hurrell BP, Romano A, Lee SH, Ribeiro-Gomes F, Riteau N, et al. The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice. Eur J Immunol. 2016;46(4):897–911. pmid:26689285
- 12.
WHO. Malaria [Internet]; 2022 [cited 2022 Aug 10]. Available from: https://www.who.int/news-room/fact-sheets/detail/malaria
- 13.
WHO. Leishmaniasis [Internet]; 2022 [cited 2022 Jul 20]. Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis
- 14. Ornellas-Garcia U, Cuervo P, Ribeiro-Gomes FL. Malaria and leishmaniasis: updates on co-infection. Front Immunol. 2023;14:1122411. pmid:36895563
- 15. Henderson LH. Clinical observations on kala-azar in the province of the Sudan. Trans R Soc Trop Med Hyg [Internet]. 1937;31(2):179–90. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0035920337900361
- 16. Yoeli M. Non-pigmented malaria parasites in the bone marrow from a mixed infection of leishmania and plasmodium vivax. Trans R Soc Trop Med Hyg. 1948;42(1):99-IN5. pmid:18878521
- 17. Nandy A, Addy M, Guha SK, Maji AK, Chaudhuri D, Chatterjee P. Co-existent kala-azar and malaria in India. Trans R Soc Trop Med Hyg. 1995;89(5):516. pmid:8560528
- 18. Ab Rahman AK, Abdullah FH. Visceral leishmaniasis (kala-azar) and malaria coinfection in an immigrant in the state of Terengganu, Malaysia: a case report. J Microbiol Immunol Infect. 2011;44(1):72–6. pmid:21531357
- 19. van den Bogaart E, Berkhout MMZ, Adams ER, Mens PF, Sentongo E, Mbulamberi DB, et al. Prevalence, features and risk factors for malaria co-infections amongst visceral leishmaniasis patients from Amudat Hospital, Uganda. PLoS Negl Trop Dis. 2012;6(4):e1617. pmid:22506087
- 20. van den Bogaart E, Talha A-BA, Straetemans M, Mens PF, Adams ER, Grobusch MP, et al. Cytokine profiles amongst Sudanese patients with visceral leishmaniasis and malaria co-infections. BMC Immunol. 2014;15:16. pmid:24886212
- 21. Aschale Y, Ayehu A, Worku L, Tesfa H, Birhanie M, Lemma W. Malaria-visceral leishmaniasis co-infection and associated factors among migrant laborers in West Armachiho district, North West Ethiopia: community based cross-sectional study. BMC Infect Dis. 2019;19(1):239. pmid:30849958
- 22. Tekalign S, Adera C, Den Boer M, Miecha H, Zewde A, Mulugeta D, et al. Clinical features and treatment outcomes of visceral leishmaniasis patients admitted to three centers in Oromia, Ethiopia. J Infect Dev Ctries. 2020;14(6.1):42S-47S. pmid:32614795
- 23. Belkaid Y, Mendez S, Lira R, Kadambi N, Milon G, Sacks D. A natural model of Leishmania major infection reveals a prolonged “silent” phase of parasite amplification in the skin before the onset of lesion formation and immunity. J Immunol [Internet]. 2000;165(2):969–77. Available from: https://journals.aai.org/jimmunol/article/165/2/969/32962/A-Natural-Model-of-Leishmania-major-Infection
- 24. Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol. 2002;2(11):845–58. pmid:12415308
- 25. Schmidt KE, Kuepper JM, Schumak B, Alferink J, Hofmann A, Howland SW, et al. Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators. PLoS One. 2018;13(2):e0192717. pmid:29438386
- 26. de Souza JB, Riley EM. Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis. Microbes Infect. 2002;4(3):291–300. pmid:11909739
- 27. Späth GF, Beverley SM. A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol. 2001;99(2):97–103. pmid:11748963
- 28. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, van der Linden R, et al. A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol. 2004;137(1):23–33. pmid:15279948
- 29. Titus RG, Marchand M, Boon T, Louis JA. A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol. 1985;7(5):545–55. pmid:3877902
- 30. Sacks DL, Melby PC. Animal models for the analysis of immune responses to leishmaniasis. Curr Protoc Immunol. 2015;108:19.2.1-19.2.24. pmid:25640990
- 31. Ribeiro-Gomes FL, Peters NC, Debrabant A, Sacks DL. Efficient capture of infected neutrophils by dendritic cells in the skin inhibits the early anti-leishmania response. PLoS Pathog. 2012;8(2):e1002536. pmid:22359507
- 32.
Engwerda C, Belnoue E, Grüner AC, Rénia L. Experimental models of cerebral malaria. In: Current topics in microbiology and immunology [Internet]; 2005. p. 103–43. Available from: https://link.springer.com/10.1007/3-540-29967-X_4
- 33. Amante FH, Haque A, Stanley AC, Rivera F de L, Randall LM, Wilson YA, et al. Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria. J Immunol. 2010;185(6):3632–42. pmid:20720206
- 34. De Souza B, Hafalla JCR, Riley EM, Couper KN. Cerebral malaria: why experimental murine models are required to understand the pathogenesis of disease. Parasitology [Internet]. 2010;137(5):755–72. Available from: https://www.cambridge.org/core/journals/parasitology/article/abs/cerebral-malaria-why-experimental-murine-models-are-required-to-understand-the-pathogenesis-of-disease/DDC424D1002232B179CD9B08FAA70BD6#
- 35. Cangussú SD, Souza CC de, Campos CF, Vieira LQ, Afonso LCC, Arantes RME. Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model. Mem Inst Oswaldo Cruz. 2009;104(6):918–22. pmid:19876567
- 36. Clemmer L, Martins YC, Zanini GM, Frangos JA, Carvalho LJM. Artemether and artesunate show the highest efficacies in rescuing mice with late-stage cerebral malaria and rapidly decrease leukocyte accumulation in the brain. Antimicrob Agents Chemother. 2011;55(4):1383–90. pmid:21220531
- 37. Gul S, Ribeiro-Gomes FL, Moreira AS, Sanches GS, Conceição FG, Daniel-Ribeiro CT, et al. Whole blood transfusion improves vascular integrity and increases survival in artemether-treated experimental cerebral malaria. Sci Rep. 2021;11(1):12077. pmid:34103601
- 38. Engwerda CR, Beattie L, Amante FH. The importance of the spleen in malaria. Trends Parasitol. 2005;21(2):75–80. pmid:15664530
- 39. Artavanis-Tsakonas K, Tongren JE, Riley EM. The war between the malaria parasite and the immune system: immunity, immunoregulation and immunopathology. Clin Exp Immunol. 2003;133(2):145–52. pmid:12869017
- 40. Schofield L, Grau GE. Immunological processes in malaria pathogenesis. Nat Rev Immunol [Internet]. 2005;5(9):722–35. Available from: https://www.nature.com/articles/nri1686
- 41. Gomes AP, Vitorino RR, Costa A de P, Mendonça EG de, Oliveira MG de A, Siqueira-Batista R. Severe Plasmodium falciparum malaria. Rev Bras Ter Intensiva. 2011;23(3):358–69. pmid:23949409
- 42. Clark IA, Budd AC, Alleva LM, Cowden WB. Human malarial disease: a consequence of inflammatory cytokine release. Malar J. 2006;5:85. pmid:17029647
- 43. de Moura TR, Novais FO, Oliveira F, Clarêncio J, Noronha A, Barral A, et al. Toward a novel experimental model of infection to study American cutaneous leishmaniasis caused by Leishmania braziliensis. Infect Immun. 2005;73(9):5827–34. pmid:16113301
- 44. Patnaik P, Jere CS, Miller WC, Hoffman IF, Wirima J, Pendame R, et al. Effects of HIV-1 serostatus, HIV-1 RNA concentration, and CD4 cell count on the incidence of malaria infection in a cohort of adults in rural Malawi. J Infect Dis. 2005;192(6):984–91. pmid:16107950
- 45. Guerra CV, da Silva BM, Müller P, Baia-da-Silva DC, Moura MAS, Araújo JDA, et al. HIV infection increases the risk of acquiring Plasmodium vivax malaria: a 4-year cohort study in the Brazilian Amazon HIV and risk of vivax malaria. Sci Rep [Internet]. 2022;12(1):9076. Available from: https://www.nature.com/articles/s41598-022-13256-4
- 46. Hussein R, Guedes M, Ibraheim N, Ali MM, El-Tahir A, Allam N, et al. Impact of COVID-19 and malaria coinfection on clinical outcomes: a retrospective cohort study. Clin Microbiol Infect. 2022;28(8):1152.e1-1152.e6. pmid:35367364
- 47. Hawkes M, Li X, Crockett M, Diassiti A, Liles WC, Liu J, et al. Malaria exacerbates experimental mycobacterial infection in vitro and in vivo. Microbes Infect. 2010;12(11):864–74. pmid:20542132
- 48. Mueller A-K, Behrends J, Hagens K, Mahlo J, Schaible UE, Schneider BE. Natural transmission of Plasmodium berghei exacerbates chronic tuberculosis in an experimental co-infection model. PLoS One. 2012;7(10):e48110. pmid:23110184
- 49. Legesse M, Erko B, Balcha F. Increased parasitaemia and delayed parasite clearance in Schistosoma mansoni and Plasmodium berghei co-infected mice. Acta Trop. 2004;91(2):161–6. pmid:15234665
- 50. Metenou S, Dembele B, Konate S, Dolo H, Coulibaly YI, Diallo AA, et al. Filarial infection suppresses malaria-specific multifunctional Th1 and Th17 responses in malaria and filarial coinfections. J Immunol. 2011;186(8):4725–33. pmid:21411732
- 51. Vieira-Santos F, Leal-Silva T, de Lima Silva Padrão L, Ruas ACL, Nogueira DS, Kraemer L, et al. Concomitant experimental coinfection by Plasmodium berghei NK65-NY and Ascaris suum downregulates the Ascaris-specific immune response and potentiates Ascaris-associated lung pathology. Malar J. 2021;20(1):296. pmid:34210332
- 52. Pinna RA, Silva-Dos-Santos D, Perce-da-Silva DS, Oliveira-Ferreira J, Villa-Verde DMS, De Luca PM, et al. Malaria-cutaneous leishmaniasis co-infection: influence on disease outcomes and immune response. Front Microbiol. 2016;7:982. pmid:27446022
- 53. Pereira L, Oliveira F, Townsend S, Metangmo S, Meneses C, Moore IN. Coinfection with Trypanosoma brucei confers protection against cutaneous leishmaniasis. Front Immunol. 2018;9(December).
- 54. Coleman RE, Edman JD, Semprevivo LH. Leishmania mexicana: effect of concomitant malaria on cutaneous leishmaniasis. Development of lesions in a Leishmania-susceptible (BALB/c) strain of mouse. Exp Parasitol. 1988;65(2):269–76. pmid:3350106
- 55. Coleman RE, Edman JD, Semprevivo LH. Interactions between Plasmodium yoelii and Leishmania mexicana amazonensis in leishmania resistant C57B1/6 mice. Am J Trop Med Hyg. 1988;39(6):540–4. pmid:3207174
- 56. Coleman RE, Edman JD, Semprevivo LH. Interactions between malaria (Plasmodium yoelii) and leishmaniasis (Leishmania mexicana amazonensis): effect of concomitant infection on host activity, host body temperature, and vector engorgement success. J Med Entomol. 1988;25(6):467–71. pmid:3204626
- 57. Scott P, Novais FO. Cutaneous leishmaniasis: immune responses in protection and pathogenesis. Nat Publ Gr [Internet]. 2016. Available from: https://www.nature.com/articles/nri.2016.72
- 58. Kurup SP, Butler NS, Harty JT. T cell-mediated immunity to malaria. Nat Rev Immunol. 2019;19(7):457–71.
- 59. Carneiro MB, Lopes ME, Hohman LS, Romano A, David BA, Kratofil R, et al. Th1-Th2 cross-regulation controls early leishmania infection in the skin by modulating the size of the permissive monocytic host cell reservoir. Cell Host Microbe. 2020;27(5):752-768.e7. pmid:32298657
- 60. Teo T-H, Lum F-M, Ghaffar K, Chan Y-H, Amrun SN, Tan JJL, et al. Plasmodium co-infection protects against chikungunya virus-induced pathologies. Nat Commun. 2018;9(1):3905. pmid:30254309
- 61. Edwards CL, Zhang V, Werder RB, Best SE, Sebina I, James KR, et al. Coinfection with blood-stage plasmodium promotes systemic type I interferon production during pneumovirus infection but impairs inflammation and viral control in the lung. Clin Vaccine Immunol. 2015;22(5):477–83. pmid:25716232
- 62. White CE, Villarino NF, Sloan SS, Ganusov VV, Schmidt NW. Plasmodium suppresses expansion of T cell responses to heterologous infections. J Immunol. 2015;194(2):697–708. pmid:25505280
- 63. Foo IJ-H, Chua BY, Clemens EB, Chang SY, Jia X, McQuilten HA, et al. Prior infection with unrelated neurotropic virus exacerbates influenza disease and impairs lung T cell responses. Nat Commun. 2024;15(1):2619. pmid:38521764
- 64. Hafalla JCR, Claser C, Couper KN, Grau GE, Renia L, de Souza JB, et al. The CTLA-4 and PD-1/PD-L1 inhibitory pathways independently regulate host resistance to Plasmodium-induced acute immune pathology. PLoS Pathog. 2012;8(2):e1002504. pmid:22319445
- 65. Horne-Debets JM, Faleiro R, Karunarathne DS, Liu XQ, Lineburg KE, Poh CM, et al. PD-1 dependent exhaustion of CD8+ T cells drives chronic malaria. Cell Rep. 2013;5(5):1204–13. pmid:24316071
- 66.
Wykes MN, Horne-debets JM, Leow C, Karunarathne DS. Malaria drives T cells to exhaustion. 2014;5(May):1–5.
- 67. Gigley JP, Bhadra R, Moretto MM, Khan IA. T cell exhaustion in protozoan disease. Trends Parasitol. 2012;28(9):377–84. pmid:22832368
- 68. Garcia de Moura R, Covre LP, Fantecelle CH, Gajardo VAT, Cunha CB, Stringari LL. PD-1 blockade modulates functional activities of exhausted-like T cell in patients with cutaneous leishmaniasis. Front Immunol [Internet]. 2021;12(March):1–12. Available from: https://www.frontiersin.org/articles/10.3389/fimmu.2021.632667/full
- 69. Imbert P, Rapp C, Buffet PA. Pathological rupture of the spleen in malaria: analysis of 55 cases (1958-2008). Travel Med Infect Dis. 2009;7(3):147–59. pmid:19411041
- 70. Del Portillo HA, Ferrer M, Brugat T, Martin-Jaular L, Langhorne J, Lacerda MVG. The role of the spleen in malaria. Cell Microbiol. 2012;14(3):343–55. pmid:22188297
- 71. Ferrer M, Martin-Jaular L, De Niz M, Khan SM, Janse CJ, Calvo M, et al. Imaging of the spleen in malaria. Parasitol Int. 2014;63(1):195–205.
- 72. Marsh K, Kinyanjui S. Immune effector mechanisms in malaria. Parasite Immunol. 2006;28(1–2):51–60. pmid:16438676
- 73. Beeson JG, Osier FHA, Engwerda CR. Recent insights into humoral and cellular immune responses against malaria. Trends Parasitol. 2008;24(12):578–84. pmid:18848497
- 74. Hunt NH, Grau GE. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 2003;24(9):491–9. pmid:12967673
- 75. Novais FO, Wong AC, Villareal DO, Beiting DP, Scott P. CD8 T cells lack local signals to produce IFN-γ in the skin during leishmania infection. J Immunol [Internet]. 2018;200(5):1737–45. Available from: https://journals.aai.org/jimmunol/article/200/5/1737/106766/CD8-T-Cells-Lack-Local-Signals-To-Produce-IFN-in
- 76. Lobaloba Ingoba L, Adedoja A, Peko SM, Vairo F, Haider N, Kock R, et al. Diagnosis of chikungunya virus in febrile patients from a malaria holoendemic area. Int J Infect Dis. 2021;109:247–52. pmid:34174430
- 77. Kotepui M, Mala W, Kwankaew P, Kotepui KU, Masangkay FR, Wilairatana P. Distinct cytokine profiles in malaria coinfections: a systematic review. PLoS Negl Trop Dis. 2023;17(1):e0011061. pmid:36716305