Elevated IL-17 levels in semi-immune anaemic mice infected with Plasmodium berghei ANKA

Previous studies [21, 22, 30] indicated destruction of relatively high uninfected red blood cells (uRBC) in Balb/c semi-immune mice at low parasite density, while relatively low uRBC were destroyed in CBA. Uninfected RBC destruction was implicated as no parasite sequestration was observed in any organs (spleen, liver, brain, kidney, lung, heart and muscle) [30]. Based on this observation Balb/c (described as the more destructive) and CBA (less destructive type) were chosen for the study. And to understand if any gene is involved in this phenomenon, the F1 cross between Balb/c and CBA was made. The procedure for this method has been reported elsewhere [23, 24]. Briefly, 8-week-old female Balb/c, CBA and F1 (Balb/c × CBA) mice supplied by SLC laboratories, Fukuoka, Japan, were injected intraperitoneally (i.p.) with 10⁴ P. berghei (ANKA strain) infected red blood cells. This line of P. berghei ANKA was generously donated by Dr. Tetsuo Yanagi, of National Bio-Resource Center (NBRC), NEKKEN, Nagasaki University, Nagasaki, Japan. For every 2 days, levels of parasitaemia and reticulocyte were monitored by Giemsa-stained thin blood film. These were expressed as a percentage of more than 500 RBCs. Even though a recent study [31] has shown that estimates of polychromatophilic cells as done after Giemsa staining in this study are typically less than percentages of reticulocytes, Giemsa has been successfully used previously for reticulocyte staining [23, 24, 32]. Method used in estimating the level of blood haemoglobin (Hb) is as previously described [23]. By day 6 after infection, chloroquine (10 mg/kg i.p.) and pyrimethamine (10 mg/kg i.p.) were used to treat mice daily for 6 days. This infection and treatment of the mice were done for 5 cycles. This was to ensure mice attain semi-immune status. A mouse is considered semi-immune due to the several cycles of infection and treatment. An additional parameter is the observation of lower parasitaemia growth during other cycles of infection in relation to the first cycle (Fig. 1). Between each round of infection (after the first cycle), mice were rested for 2 weeks before being re-challenged with 10⁴ P. berghei. The mice were then monitored and drug-cured before parasitaemia attains 10%. A graphic outline of the infections, chemotherapy and time point of analysis are indicated in Fig. 1. Parasitaemia and haemoglobin profile in the semi-immune mice strains was similar to that described previously [24]. For baseline value, blood was collected from the tail after the 5th cycle of infection and treatment, after parasitaemia was checked to be negative (zero). Serum was harvested from the tail blood before the final cycle of infection without treatment. Mice were monitored every other day during the final cycle of infection (without treatment). Blood harvesting was done on days in which minimum Hb (i.e. maximum Hb reduction) was observed, while the procedure for serum preparation and storage is as described previously [23].

Immunoglobulin G and its subtype antibody responses were assessed by ELISA from the sera collected after the fifth cycle. Briefly, 96-well plates were coated with 100 μL of 0.5 μg/mL of P. berghei crude antigen in coating buffer and kept overnight at 4 °C. Plates were washed three times with 400 μL/well of 0.05% Tween-PBS (phosphate-buffered saline) and then blocked for nonspecific binding using 340 μL/well of 0.1% blocking reagent (Roche Diagnostics, Mannheim, Germany) for 1 h at 37 °C. Plates were washed three times with 400 μL/well of 0.05% Tween-PBS and 100 μL of serially diluted pooled sera (1:80) was added and incubated at 37 °C for 3 h. Plates were then washed five times with 400 μL/well of 0.05% Tween-PBS, and 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and subclasses IgG1, IgG2a and IgG3 (Southern Biotechnology, Birmingham, AL) diluted with blocking buffer (1:2500) was added and incubated for 1 h at room temperature. Plates were washed 5 times with 400 μL/well of 0.05% Tween-PBS and antigen–antibody reaction was visualized by the addition of 50 μL/well of 3, 3′, 5, 5′-tetramethylbenzidine (TMB) (Vector Laboratories, CA, USA). The colour development reaction was stopped after 30 min by adding 50 μL of 1 N of H₂SO₄, and the absorbance was measured in an automated plate reader (Bio-Rad, Hercules, CA) at 450 nm.

Spleens were excised from individual mouse during the sixth cycle of infection without treatment (days zero for the mice strains used in the experiment; days 12 for CBA; days 16 and 28 for F1 and Balb/c). The frequency of CD4(+) CD25(+) Treg cell was measured by FACS. Analysis of cell surface markers (CD3, CD4, and CD25) from BD Pharmingen (BD Biosciences San Jose, California, USA), intracellular cytokine (Foxp3) expression recognition were done. Fluorescent-conjugated monoclonal antibodies, and isotype control were purchased from Biolegend (San Diego, California, USA). Purified Rat Anti Mouse CD16/CD32 antibody was purchased from BD Biosciences Pharmingen (San Jose, California, USA) to block non-antigen-specific binding of immunoglobulins to the FcγIII and FcγII. Cells were stained with CD3, CD4, CD25 surface markers. Cells were then fixed for 20 min at 4 °C in 4% paraformaldehyde, washed and permeabilized in the presence of 0.1% saponin. Finally, re-suspended cells were incubated for 30 min at 4 °C in the presence of optimal concentrations of Foxp3 and conjugated rat IgG2a isotype controls were used for the intracellular staining. The Tregs frequency was evaluated by flow cytometry using Becton Coulter Gallios, Flow Cytometer (Beckman Coulter, Inc.), and data were analysed using Kaluza software.

This method as described previously [23] was done according to the manufacturer’s instructions. Cytokines measurement was done using Procarta Mouse Cytokine Assay Kit plex according to manufacturer’s instructions (Luminex, Affymetrix). Briefly, after the reading buffer was used to wet the plate, it (the plate with the reading buffer) was incubated at room temperature for 5 min, and later filtered. To each well was added antibody beads (50 μL), then filtered and washed once with washing buffer (150 μL). This procedure was followed by the addition of 25 μL of serum standard buffer to all the sample wells, followed with the addition of equal volume (25 μL) of serum to each well, and then incubated for 60 min at room temperature. Washing was done three times, later followed with the addition of 25 μL premixed detection, and then incubated for 30 min on the shaker at room temperature. Washing and filtration was done three times afterwards. One hundred and fifty (150) μL of washing buffer was used during each wash. Washing and filtration was done three times after incubation was done for 30 min with Streptavidin-PE (50 μL).The plate was prepared for analysis after the third wash, with the addition of 120 μL reading buffer.

GraphPad Prism Version5.00 for Windows, GraphPad Software, San Diego California, USA, [Graph pad prism version 5.0 for Windows; http://​www.​graphpad.​com] was used for the data analysis. Data are expressed as the mean with standard deviation (SD) unless otherwise stated. To ensure normal distribution, data were log transformed before one-way analysis of variance (ANOVA, with Tukey’s post-test, two tailed), were performed. The differences of means were considered statistically significant if p < 0.05.

The Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan (Notice no. 71) was strictly followed during the study. All efforts were made to humanely minimize animal suffering. The Nagasaki University, Board of Animal Research approved all animal experiments, according to Japanese Guideline for use of Experimental animals (Permit Number 0811130716).

Balb/c, CBA and the cross between them called F1 were taken through five cycles of infection with 10⁴ P. berghei followed by pyrimethamine and chloroquine treatment to generate the semi-immune status. After the fifth cycle, these semi-immune mice strains were challenged with 10⁴ P. berghei without treatment to observe how the semi-immune status of the mice can be measured. Figure 2 revealed that, while all the CBA mice were dead by day 28 (probably due to high parasitaemia, Fig. 3c), most of F1 and Balb/c mice survived, Fig. 2.

Figure 3 shows the parasitaemia in the semi-immune mice strains in another experiment after the mice strains were challenged with 10⁴ P. berghei. It can be seen that prepatent period was 4 days in the mice strains (Fig. 3a–c). Another interesting observation made is that while high parasitaemia (50% for F1 and 30% for Balb/c) were also recorded by some mice in the F1 and Balb/c mice, some recovered and a few died (Fig. 3a [M6 mouse], b [M4 and M5 mice]). Much higher parasitaemia was, however, observed in the individual mice strains of CBA (Fig. 3c). It was also observed that the semi-immune mice strains of CBA died, even though some mice appeared to resolve their parasitaemia (Fig. 3c), they could not fully recover. The mean blood haemoglobin, reticulocytes, and parasitaemia kinetic profiles of F1, Balb/c and CBA strains were also recorded as shown in Fig. 4. A clear observation made was that an Hb decrease and an increase of reticulocyte count was associated with peak parasitaemia. Hb improved gradually within F1 and Balb/c semi-immune mice while parasites were cleared as well within these semi-immune mice strains (F1 and Balb/c) (Fig. 4a, b). On the other hand CBA could not clear the parasites resulting in continued fall in Hb and eventual death (Fig. 4c).

Haemoglobin levels fell to 48.6, 54.0 and 62.7% of normal levels in F1, Balb/c and CBA, respectively, on days with minimum Hb (Hbm), Table 1. These Hb reductions were not significantly different within the three mice strains (p = 0.088). However, the mean parasitaemia at which this Hb reductions occurred within F1, Balb/c (< 4.0%) and CBA (27.5%) were significantly different, p = 0.012. Moreover, this minimum percentage Hb drop occurred at about the same period for each semi-immune mouse strain (p = 0.52, Table 1). Reticulocyte count was estimated in the semi-immune mice to assess extent of erythropoietic response. Similar reticulocyte count (p = 0.427) was recorded in the three semi-immune mice strains. The extent of reticulocyte production to Hb loss in the three semi-immune mice strains in this study was shown to be similar, p = 0.240. During the repeated cycles of infection and treatment, it was observed that relatively higher RBC destruction occurred at later cycles (semi-immune status of mice), but at low parasitaemia in comparison with the first cycle of infection, implying iRBC are not solely responsible in addition to inadequate reticulocyte response. With this observation, the extent to which destruction occurred in the semi-immune mice strains at the final cycle and the contribution of parasitaemia was evaluated. Figure 5 revealed that, the mean percentage Hb loss per parasitaemia was significantly different statistically in the semi-immune mice strains (p = 0.012).

To further explore the role of other immune mechanism and at what point the immune system becomes more aggressive, in relation to the recovery by F1 and Balb/c and death in the CBA, the kinetics of IgG subtypes and T cell populations were assessed. IgG subtypes were generally high in the semi-immune mice strains when compared to the uninfected negative control suggesting the antibody production was initiated by the parasite infection (Fig. 6). Furthermore, IgG subtypes were highest at the point when minimum Hb (Hm) was observed in the three mice strains. In all the three points that antibody type was measured, IgG1 was highest in F1 and Balb/c than CBA. Finally, IgG subtypes were more than two times higher in the F1, Balb/c than the CBA semi-immune mice strains, at D0 and Hm (Additional file 1).

Kinetics of T cell populations in the three semi-immune mice strains revealed that while CD4 T cells were very low in Balb/c semi-immune mice on day zero, CD4⁺CD25⁺ and CD3 were much higher on day zero (D0) (Figs. 7, 8). These T cell types were very low at Hm (minimum Hb), with only CD4⁺CD25⁺ increasing sharply again at recovery in the Balb/c semi-immune mice (Additional file 1). F1 on other hand had very high CD4 T cell, with very low CD4⁺CD25⁺ and CD3 levels recorded on D0. Levels of CD4 T cell was drastically reduced with an increase in CD4⁺CD25⁺ and CD3 at minimum Hb (Hm) for F1. Interestingly, there was a sharp increase in the CD4⁺CD25⁺ with low CD4 and CD3 T cell types in the F1 semi-immune mice at recovery (Rec), Figs. 7, 8. Since CBA semi-immune mice did not recover and died due to high parasitaemia, T cell type assessment was done at two-time points, Figs. 7iii and 8. While T cells expressing CD4 in CBA was high and similar at D0 and Hm, CD4⁺CD25⁺ was low on both days (D0 and Hm in the CBA semi-immune mice). CD3 T cells was, however, much higher at Hm. Another interesting finding is that T cells expressing CD4 and CD3 were highest in the CBA semi-immune mice strains than the F1 and Balb/c semi-immune mice strains at Hm.

As IgG subtypes were elevated at Hm with relatively low T cell population observed at the Hm (F1 and Balb/c in particular), the role of anti- and pro-inflammatory cytokines levels were evaluated at the Hm (Table 2). Hm was the point where low Hb was observed in the last cycle of infection without treatment (Fig. 1). In F1 and Balb/c semi-immune mice, this was the time the mice start clearing the parasite by themselves (without treatment, Fig. 1) suggestive of protection, thus IL-17 was measured together with the other anti- and pro-inflammatory cytokines that are thought to be involved in suppressing erythropoiesis, and others contributing to anaemia in children with falciparum malaria [33, 34]. It was observed that significantly high levels of IL-4, IL-12, IL-17 and IFN-γ were recorded in the F1 and Balb/c mice strains, p < 0.05 (Table 2). The other cytokines and the TNF: IL-10 ratio was comparable in the three semi-immune mice strains studied in this current paper (p > 0.05), Table 2.