To analyze co-infection and experimental cerebral malaria (ECM), 5-week-old female C57BL/6J mice were bought from CLEA, Japan. To avoid any discrepancies that may arise due to the effect of testosterone, female mice were used (Djokic et al., 2019).

Cryostabilates of B. microti Peabody mjr (ATCC® PRA-99™) were obtained from the cell bank. Plasmodium berghei ANKA was provided by Nagasaki University with support in part by National BioResource Project (NBRP), MEXT, Japan. After thawing the frozen stabilates, mice were intraperitoneally inoculated to passage and were maintained for experiments. The research was performed to investigate the influence of a non-lethal B. microti primary infection on a consequent lethal P. berghei infection. One group of mice (n=6) was infected by intraperitoneal injection with 10⁸ B. microti-infected red blood cells (iRBCs). Then, the same group was challenge infected with 10³ P. berghei-infected RBCs at day 7 (henceforth mentioned as Bm/PbA7; n=6). Likewise, one more group (n=6) was intraperitoneally inoculated with 10³ P. berghei-infected RBCs only (henceforth mentioned as PbA) (n=6) and an additional group with B. microti-infected RBCs only (henceforth mentioned as Bm) (n=6). A series of experiments was undertaken to probe the effects of co-infection. Preliminary experiments were conducted to optimize the infection dose. Out of the four doses tested (10²,10³, 10⁴, 10⁵ iRBCs), the minimum infection dose of 10³ iRBCs was selected for P. berghei infection. Similarly, for B. microti infection, two doses were evaluated during the preliminary trial, and out of those, 10⁸ iRBCs was selected as the primary infection dose (Mordue and Wormser, 2019) ( Supplementary Figure 3 ). After preliminary experiments, the initial study was executed with four groups, namely B. microti-infected group (Bm), co-infected group (Bm/PbA7), a control group (PbA), and a Naïve mouse group (injected with PBS), to investigate the progression of parasitemia, ECM signs, and other parameters. The subsequent trial was conducted including co-infected (Bm/PbA7), control (PbA), and Naïve groups (n=5) wherein the extent of blood-brain barrier (BBB) disruption and the integrity of the vascular endothelial barrier in the lungs were assessed using the Evans blue assay. Another trial was conducted with Bm, PbA, Bm/PbA7, and Naïve mouse groups (n=5) to determine the parasite burden in tissues and the host immune response against infections. At the end, mice were again infected according to the aforementioned experimental design for performing the histopathological analysis.

Regular monitoring of mice was done, and parameters including parasitemia and body weight were recorded. RBCs and hematocrit (HCT) values were measured by Celltac Alpha MEK-6550K (Nihon Kohden) (Li et al., 2012). To determine the parasitemia percentage, thin blood smears were made after staining with Giemsa; the percent parasitemia was assessed from 10³ iRBCs after examining under 100 × oil immersion Eclipse E200 microscope (Nikon, Tokyo, Japan). As we assessed the parasitemia using blood smears, it was challenging to differentiate the levels of parasitemia specifically for Babesia and Plasmodium parasites in Bm/PbA7 mice. Consequently, we presented the combined parasitemia levels as an overall representation.

Mice were monitored daily for survival rates, and symptoms were recorded for the development of ECM by the use of the rapid murine coma and behavior scale (RMCBS) score, as explained by (Carroll et al., 2010). When the RMCBS score was ≤5/20, mice were categorized as exhibiting ECM symptoms (Sanches-Vaz et al., 2019).

In a new trial, brain, liver, lungs, kidney, and spleen were harvested aseptically, weighed, and photographed at day 8 post-challenge infection from Bm/PbA7 mice (day 15 post-primary infection) and at day 8 from PbA, Bm, and Naïve mice (n=5) following P. berghei, B. microti and PBS inoculation respectively. For single cell suspension, PBS was used for spleen and digest solution was used for rest of the organs (Liu et al., 2020). Then, organs were cut and triturated to make single cell suspension. Ground spleen tissues were strained a in 50 mL tube through a 70 µm nylon sterile cell strainer, whereas other organ tissues were incubated in digest solution for 1 h at 37°C. After digestion, the rest of the organ tissues were strained as described earlier. Single-cell suspensions of liver, lungs, kidney, and spleen were washed with PBS by centrifugation at 375 × g for 5 min at 4°C and RBCs were lysed with 1× ACK lysis buffer (Gibco, Massachusetts, USA) for 5 min at 25°C. After lysis was stopped by adding cold PBS, centrifugation was performed at 375 × g for 5 min at 4°C; 2 mL cell staining buffer (CSB, BioLegend, California, USA) was used to resuspend the cell pellet and maintained at 4°C until the next step. Cell suspension of brain tissues was processed differently. The strained brain tissue suspension was washed with PBS and the pellet was resuspended with 8 mL of 40% Percoll (Cytiva, Tokyo, Japan). Then, 5 mL of 80% Percoll was taken into a round bottom tube and 8 mL of 40% Percoll was layered on top of it. After centrifugation at 1,578 × g for 20 min at 25°C with low acceleration (acceleration 1) and no break, the middle interface layer was collected and transferred into a new tube containing 10 mL PBS. Later, centrifugation was carried out at 375 × g for 5 min at 4°C, and 2 mL CSB was used to resuspend the pellet. Two antibody panels were used for each sample and approximately one million cells were reconstituted in CSB ( Supplementary Tables 1.1 - 1.2 ). After, Fc block was performed by resuspending the reconstituted cells with 70 µL CSB containing CD16/CD32 monoclonal antibody (Invitrogen, Massachusetts, USA) at 4°C for 25 min. Later, staining of samples was carried out, wherein the cells were labeled with fluorophore-tagged, marker antibodies for each type of immune cell and incubated at 4°C for 30 min in the dark. Then, samples were fixed with 4% paraformaldehyde (PFA) solution for 15 mins and washed two times with CSB and centrifuged at 375 × g for 5 min at 4°C. Finally, 200 µL CSB was used to resuspend the cells. The above mentioned protocol was described by (Rico et al., 2021). Sorting of stained and fixed cells was conducted by employing the CytoFLEX flow cytometer (Beckman Coulter, California, USA) and data were carefully evaluated by CytExpert 2.4 software (Beckman Coulter, California, USA) ( Supplementary Figure 1 ). A representation of the cell sorting strategy is shown in Supplementary Figure 2 , followed according to (Bayne and Vonderheide, 2013) (Zafar et al., 2022).

After harvesting the organs from experimental groups (n=1), the fixation of tissues was done in 4% paraformaldehyde, followed by dehydration in graded alcohol and embedding in paraffin. Next, 5 μm thick slices were made. Subsequently, the sections were deparaffinized and stained using hematoxylin & eosin (H & E). Eventually, the sections were mounted on MGK-S with coverslips. Changes in tissue histology were examined by the histopathologist in a blinded manner, by utilizing a Microphot-FX from Nikon, and images were captured using a Digital Sight DS-5M camera also from Nikon.

All groups of mice (Bm, PbA, Bm/PbA7, and Naïve n=5) were exsanguinated by withdrawing the blood from the heart at day 8 post-primary infection or day 8 pci with P. berghei. Later, the serum was collected by centrifugation at 1,500 × g for 15 mins at 4°C. To detect and quantify serum cytokines, commercial enzyme-linked immunosorbent assay (ELISA) kits (Thermo Fisher Scientific, Massachusetts, USA) were used, and the assay was performed according to the manufacturers` guidelines and methods. To detect the cytokine levels in serum samples, 1:100 dilution was made in PBS. The MULTISKAN SkyHigh plate reader (Thermo Fisher Scientific) was employed to determine the optical density. The serum cytokines (IFN-γ, TNF-α, IL-2, IL-6, IL-10, and IL-12p70) were quantified by extrapolating the standard curves.

Previously established protocol by (Kim et al., 2022) was used to prepare P. berghei ANKA crude antigen. Concisely, P. berghei infected-blood was withdrawn intracardially when the parasitemia was more than 30%. Collected blood was centrifuged at 3,000 × g for 5 min and lysed chemically. For lysing, 0.15% saponin in PBS was mixed with equal volume of pelleted RBCs at room temperature for 5 mins. Followed by centrifugation, the pelleted parasites were washed 3 times with 1× PBS and re-suspended in 1× PBS. Released parasites were subjected to mechanical disruption by 2 cycles of sonication for 30 seconds, at 40% amplitude. Protein quantity of the lysate was measured by Pierce™ BCA Protein assay kit (Thermo Fisher Scientific, USA). The lysate was kept at −20°C and later used as a coating agent for ELISA plates.

To assess the humoral immune response, antibodies against B. microti and P. berghei were measured by ELISA. We had sera from Bm, PbA, Bm/PbA7, and Naïve mice (n=5) collected at day 8 pci. Co-infected mouse serum was detected for antibodies against each parasite. A previously described protocol (Zafar et al., 2022) was followed to measure the antibody response. Either 50 μL P. berghei lysate or GST-fused rBmP32 antigen was used to coat microtiter plates (Nunc, Roskilde, Denmark) and kept at 4°C overnight. Washing was performed with 0.05% Tween 20-PBS (PBST) and blocking with 3% skim milk in PBS for 1 h at 37°C. After washing, the plates were incubated with 50 µL of 1:100 diluted serum samples in a blocking solution for 1 h at 37°C. While the samples were put in incubation, goat anti-mouse-Immunoglobulin HRP-conjugated secondary antibody (IgG, IgG1, Ig2a, IgG3, and IgM) were prepared. Secondary antibodies were diluted in a blocking solution to a concentration of 1:4,000. After incubation with diluted mouse serum, plates were washed six times. Then, incubated for 1 h at 37°C with secondary antibody and again washed six times. Finally, plates were incubated with tetramethylbenzidine (TMB) substrate for 1 h at room temperature (RT) to detect bound antibodies. Absorbance values were read at 415 nm using the MULTISKAN SkyHigh plate reader (Thermo Fisher Scientific). To ensure the consistency of results and avoid any discrepancy, triplicates of each sample were used.

At day 8 pci, organs were harvested and blood was collected from mouse groups (PbA and Bm/PbA7; n=5). DNA was extracted from tissues and blood using NucleoSpin^® Tissue Kit (Takara, Japan), and QIAamp^® DNA Blood Mini Kit (Qiagen, Hilden, Germany), respectively, adhering to the directions specified by the manufacturer. The DNA was eluted to a final volume of 100 μL. To quantify the parasites, a pair of specific primers (F: 5`-AAGCATTAAATAAAGCGAATACATCCTTAC-3`; R: 5`GGAGATTGGTTTTGACGTTTATGTG-) was used to amplify a part of the 18S rRNA gene (134 bp) of P. berghei (Baptista et al., 2010). A final volume reaction of 10 μL was run in duplicates, composed of 5 μL 1 × PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Massachusetts, USA), 0.8 μM primers, 1 uL DNA, and 2.4 uL UltraPure™ DNase/RNase-free water (Thermo Fisher Scientific). The cycling regimen employed was 50°C for 2 min, 95°C for 2 min, 40 cycles of 95°C for 15 sec, and 60°C for 45 sec, with a dissociation stage. The reactions were run in the QuantStudio™ 5 Real-time PCR System (Thermo Fisher Scientific). Serially diluted standards prepared from plasmids served as reference samples. Gene copy values were derived from the average quantified cycle values of the replicates relative to the obtained values from the standards. Values were converted to their corresponding log values followed by statistical analysis.

To measure the disruption of BBB in C57BL/6 mouse groups (n=5), either single infected or challenge-infected with P. berghei ANKA and in Naïve, we followed the previously stated protocol by (Baptista et al., 2010). Concisely, 200 μL of 2% Evans blue (EB) in PBS was injected intravenously (iv) in the mouse tail after the onset of clinical symptoms of ECM. One group of Naïve mice was injected with PBS as the negative control to distinguish morphology. One hour later, mice were euthanized and perfused with PBS. Harvested brains and lungs were weighed, photographed, and kept in 2 mL formamide (Merck, New Jersey, USA) for 48 h at 37°C to recover the Evans blue dye from the tissue. The extent of BBB and lung damage was assessed by measuring the absorbance at 620 nm. Evans blue concentration was quantified by calculations using a standard curve.

GraphPad Prism 8 software (GraphPad Prism, California, USA) was used for statistical analyses of all data. To make a comparison among groups, ordinary one-way analysis of variance (ANOVA) followed by post hoc Tukey’s multiple-comparisons test and two-tailed unpaired student t-test was used. A P-value less than 0.05 was considered significant at a 95% confidence interval.

In the current study, Bm/PbA7 mice survived longer than PbA mice. All the PbA mice succumbed to infection within day 12, as evident in Figure 1A , whereas Bm/PbA7 mice survived until day 21 and died at day 22 post-challenge infection (pci). Interestingly, PbA mice showed signs of piloerection, shivering, convulsions, seizures, and coma, while co-infected mice survived without any overt signs of ECM ( Figure 1B ). These results demonstrated that, although complete survival was not observed, primary infection with B. microti allowed a longer survival than the PbA mice.

Throughout the experiment, levels of parasitemia were monitored by Giemsa-stained blood smears. The curves represented in “ Figure 1C ” were used to compare the parasitemia among the three groups. The comparison revealed that at day 0 of post-challenge infection, the Bm/PbA7 mice had a parasitemia level of 32.5% resulting from a previous B. microti infection. Subsequently, the parasitemia declined by day 6 post-challenge infection (pci) (which means day 13 post-primary infection), but it later peaked again. This subsequent increase in parasitemia was attributed to the P. berghei challenge-infection. It is important to note that the observed parasitemia represents combined parasitemia caused by both Babesia and Plasmodium parasites. Surprisingly, co-infected mice showed the highest parasitemia (38%) while the peak parasitemia in PbA mice was 32.47%. In the case of Bm mice, the peak parasitemia level reached 36.39% and was observed on day 8 post-infection (pi), ( Figure 1C ). The body weights of the Bm mice showed transient loss that was recovered after the parasitemia was resolved. In PbA mice, drastic weight loss was seen from initial weight (20.058 g) to final weight (14.887 g) before they expired, whereas gradual decline in weight was seen in co-infected mice from (16.98 g) to (14 g) ( Figure 1D ). Moreover, all groups developed anemia, especially the Bm and Bm/PbA7 mice, as evidenced by lower-than-normal RBCs and hematocrit indices. Before the day of death, Bm/PbA7 mice exhibited intense anemia, whereas hematologic parameters were rehabilitated and sustained until day 28 pci in Bm mice ( Figures 1E, F ) (Zafar et al., 2022). Our results confirmed that escalation in blood parasites expedited the breakdown of iRBCs and reduced hematologic indices, resulting in anemia.

We probed the above-mentioned crucial organs by flow cytometry to shed light on the immune cell dynamics. The aim was to study the impact of co-infection on immune cell population during early stage infection, and hence determine the role of innate immunity in our study. Spleen, brain, liver, lungs, and kidneys were aseptically removed and, after processing the samples, data was collected ( Figure 2 ). Although the brain of PbA mice showed the lowest population of NK cells, DCs, and macrophages, the population of CD4+ and CD8+ T cells significantly increased compared to the other groups ( Figure 2B ). Co-infected mice had significantly higher number of B cells than PbA mice ( Figure 2A ). There were marked differences in the macrophage population between PbA and Bm/PbA7 mice. Macrophages were more predominant immune cells of all the probed organs in co-infected mice compared to PbA mice. Notably, macrophages were significantly higher in co-infected (5.36%) spleen compared to PbA (0.13%) mouse spleen ( Figure 2A ). Among all organs, the liver had the greatest percentage of macrophages in all the groups: 2.56%, 7.41%, 6.67%, and 13.27% in PbA, Bm, Bm/PbA7, and Naïve mice, respectively ( Figure 2C ). PbA mice had a comparatively lower immune cell population in kidneys and lungs than Bm/PbA7 mice ( Figures 2D, E ). Percentages of B cells, CD4+ and CD8+ T cells, macrophages, NK cells, and DCs varied among all groups, with spleen of PbA mice being the lowest in immune cell population (except for CD8+ cells) and Bm/PbA7 being comparatively higher ( Supplementary Table 2 ).

In an attempt to comprehend the cytokine crosstalk in mice, we measured the serum cytokine levels (IFN-γ, TNF-α, IL-6, IL-12p70, IL-2, and IL-10) ( Figure 3G ). Co-infected mice exhibited decreased levels of pro-inflammatory cytokines (i.e., IFN-γ, TNF-α, and IL-12p70), whereas the level of anti-inflammatory cytokine (IL-10) was elevated in the acute phase of co-infection ( Figure 3 ). Although the decrease in the level of IL-12p70 was not statistically significant, it is worth noting that PbA mice exhibited a slightly higher level compared to co-infected mice: 58.7 pg/mL in PbA mice and 57.5 pg/mL in Bm/PbA7 mice. Our findings suggested that IL-10 plays a protective role against ECM as the coinfected mice had significantly higher levels of IL-10 compared to PbA mice ( Figure 3F ). Furthermore, the absence of early secretion of IFN-γ resulted in deadly infection as significantly higher levels of IFN-γ were seen in PbA mice at day 8 pi ( Figure 3A ) compared with Bm/PbA7 mice. Relatively higher levels of TNF-α and IL-12p70 were seen in the PbA mice, however, a significant difference was not seen in the levels of IL-12p70 ( Figures 3B, D ). In PbA mice, the level of TNF-α in serum was enhanced in comparison to other groups ( Figure 3B ). The raised levels of IL-6 ( Figure 3C ) and IL-2 ( Figure 3E ) were also noticed in mice with P. berghei infection. Altogether, our findings suggest that IL-10, IFN-γ, and TNF-α are key cytokines as high levels of IFN-γ and TNF-α contributed to the lethality of disease and IL-10 acted as a protector in the course of B. microti and P. berghei co-infection.

Later, we aimed to validate if the spike in B and T cells corroborated the ( Figure 2 ) species-specific antibody response. Therefore, antibodies against B. microti and P. berghei ( Figure 4 ) were evaluated at day 8 pci. For single-infected mice, day 8 pci was day 8 post-infection while for co-infected mice it was day 14 post-B. microti primary infection and day 8 pci at the time of serum collection. Therefore, the co-infected mice had pronounced IgG-specific antibody responses against B. microti in mice ( Figure 4A ). Although the levels of IgM in single-infected Bm and PbA mice were higher compared to co-infected mice, the difference was not found to be statistically significant ( Figures 4E, J ). The specific antibody production against P. berghei was comparable between co-infected and PbA mice. Our results suggested that there was not much difference between serum antibody production in PbA and co-infected mice. Overall, our results showed that mice during the acute stage infection had lowered humoral immune response.

In H & E-stained sections of the spleen, Naïve mice had typical, normal histology with distinct red and white pulps ( Figure 5C ). In the spleens infected with P. berghei ANKA, the boundary between red and white pulps became blurred because of repletion of immune cells in the red pulp, whereas, in the co-infected spleen, the development of the marginal zone distinguished the white pulp more clearly ( Figure 5C ). In the liver of Naïve mice, normal structure, which consists of liver lobules with an orderly arrangement of hepatocyte plates, was observed ( Figure 5A ). This arrangement and morphology of hepatocytes were severely damaged in the livers infected with P. berghei ANKA, whereas in the liver of co-infected mice these were normal ( Figure 5A ). The lung of Naïve mice had intact and typical structure and normal histology, which contains pulmonary alveoli separated by thin septa ( Figure 5B ). The lungs infected with P. berghei ANKA showed severe and moderate interstitial pneumonia, with the thickening of the interstitium. This abnormality became mild in the co-infected lung ( Figure 5B ). Overall, these findings implied that co-infection lessened the disease manifestations in the spleen, liver, and the lungs of mice.

The results of flow cytometry suggested the possible role of innate immune cells in protective immunity ( Figure 2 ), likewise, our qPCR results pointed out the fact that innate immune cells played a defensive role against P. berghei infiltration in organs as evidenced by the low P. berghei burden in tissues including liver, spleen, kidneys, lungs, and brain. Thus, to corroborate whether severe disease manifestation in PbA mice is exacerbated due to the accumulation of P. berghei iRBCs in organs, qPCR was performed on DNA extracted from the brain, liver, lungs, spleen, kidneys, and blood of PbA and co-infected mice at day 8 pci ( Figure 6 ). We found the extent of tissue injury ( Figure 5 ) was directly proportional with P. berghei sequestration in the tissues, with significantly lower P. berghei burden in tissues including liver, spleen, kidneys, lungs, and brain ( Figures 6A-E ). Surprisingly, mouse blood showed the opposite result, with higher parasite load in co-infected mice compared to PbA mice ( Figure 6F ). P. berghei load was increased in organs of PbA mice as compared to co-infected mice. These results clearly established that the protective immune response led to the evasion of sequestration of high load of iRBCs in co-infected mice. Low parasite burden in liver, spleen, kidney, lung, and brain of co-infected mice is concomitant with the prevention of tissue injury ( Figures 6A–E ). These results were statistically significant.

To ascertain that co-infected mice died later without ECM, evaluation of the integrity of the BBB and changes in lung vascular endothelial barrier function was assessed by measuring Evans blue infiltration in respective tissues. As anticipated, the integrity of BBB and lung vascular endothelial barrier was present in co-infected mice, whereas in PbA mice, the BBB became more permeable with onset of ECM ( Figures 7A, B ). Comparatively higher extravasation of Evans blue was noted in PbA mice in contrast to co-infected mice ( Figure 7C ). Overall, our results suggest that the severity of CM characterized by the compromised BBB caused by P. berghei infection dramatically decreased in co-infected mice. In addition, function of the lung vascular endothelial barrier was more impaired in PbA mice in comparison to co-infected mice as indicated by higher infiltration of Evans blue dye ( Figure 7D ).