Host Response and Inflammation

Macrophages Are the Determinant of Resistance to and Outcome of Nonlethal Babesia microti Infection in Mice

Mohamad Alaa Terkawi, Shinuo Cao, Maria S. Herbas, Maki Nishimura, Yan Li, Paul Franck Adjou Moumouni, Asadullah Hamid Pyarokhil, Daisuke Kondoh, Nobuo Kitamura, Yoshifumi Nishikawa, Kentaro Kato, Naoaki Yokoyama, Jinlin Zhou, Hiroshi Suzuki, Ikuo Igarashi, Xuenan Xuan
J. H. Adams, Editor
DOI: 10.1128/IAI.02128-14

ABSTRACT

In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ−/− and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

INTRODUCTION

Babesia microti is the principal agent of human and rodent babesiosis, which has emerged as a life-threatening zoonosis in several parts of the world. Transmission to humans occurs through the bite of an infected Ixodes scapularis tick, the transmitting vector identified in the United States, or through blood transfusion (1–3). Infection usually is asymptomatic or causes mild malaria-like symptoms. However, severe infections have been documented in immunocompromised people, who may suffer from respiratory failure, organ dysfunction, or coma (3–5). The rapid emergence of B. microti infection worldwide as a potential threat to public health has spurred an urgent search for effective preventive strategies. Understanding the host immune response capable of controlling blood-stage parasite infection is essential for developing an effective vaccine (6, 7).

B. microti produces a self-limiting infection in BALB/c mice, which offers an experimental model for investigating the defense mechanisms against Babesia infection (8). Several pieces of evidence have implied that the host spleen plays an essential role in the elimination of parasites and parasitized erythrocytes (pRBCs). Splenectomized or inherently asplenic mice are highly susceptible to the infection, while resistance is associated with splenomegaly. However, the protective mechanism occurring in the spleen is not clearly understood (1, 7). In general, both cellular and humoral immunities are thought to play important roles in the defense mechanism of the host against many intracellular protozoa, including Babesia spp. (9, 10). During the acute phase of B. microti infection, an elevation of Th1 cell cytokines, including interleukin-12 (IL-12), gamma interferon (IFN-γ), and TNF-α, becomes crucial for controlling the initial burst of parasite multiplication (10, 11). On the other hand, a dramatic switch to Th2 cell cytokines, including IL-4 and IL-10, accompanied by the elevation of antigen-specific immunoglobulin G at later stages is necessary for parasite resolution (11). The protective role of T cell-mediated immunity against B. microti is evidenced by the fact that severe combined immunodeficiency (SCID) mice and T cell receptor αβ-deficient (TCRαβ−/−) mice display chronically severe and unresolved fluctuating parasitemia over a period of 2 months (12). Furthermore, a depletion of CD4+, but not CD8+, T cells diminishes the protective immunity against challenge infection of B. microti in mice (8). The CD4+ T cells are thought to play a central role in protective immunity to the blood stage of Babesia infections via secreting regulatory cytokines that mediate the production of high-affinity immunoglobulin isotypes (IgG2a) and the activation of macrophages for phagocytosis (6, 13). In contrast, CD8+ T cells seem not to be involved in control at the blood stage, since the pRBCs are incapable of presenting major histocompatibility complex molecules on their surfaces (8).

Macrophages are the primary immune cells that contribute to the host innate immune response, and they are thought to play a central role in host defense against many protozoan infections. They initiate phagocytosis and release cytokines and chemokines, which orchestrate the host cellular defense and mediate the clearance of invading organisms (14, 15). Although an increasing number of studies have investigated the influences of macrophages in the immune response to and control of Babesia infection (10, 16, 17), the precise roles of these immune cells and their time of efficacy for controlling infection are still unclear. Therefore, in the present study, we took advantage of an in vivo depletion protocol that allows rapid elimination of macrophages (mainly in the spleen and liver) at different times in the course of infection with B. microti in mice. Our results revealed that host macrophages are essential for the control of B. microti infection during both the acute and resolution stages.

MATERIALS AND METHODS

Mice.Specific-pathogen-free (SPF) 6- to 8-week-old female BALB/c mice were purchased from CLEA (Tokyo, Japan). Gamma interferon (IFN-γ)-deficient (IFN-γ−/−) female mice (8) and C.B-17/Icr-scid/scid (SCID) mice were bred and maintained under SPF conditions at Obihiro University of Agriculture and Veterinary Medicine. Animal experiments were conducted in accordance with the Standards Relating to the Care and Management of Experimental Animals promulgated by the Obihiro University of Agriculture and Veterinary Medicine (Obihiro, Japan).

Parasite and infections.The Munich strain of B. microti (8) was recovered from the frozen pRBC stock by intraperitoneal (i.p.) passage inoculations in mice. Challenge infection was performed with i.p. inoculation of 1 × 107 fresh pRBCs, and the parasitemia and survival rates were monitored daily. For parasitemia, thin blood smears were made, fixed in methanol, and then stained for 30 min with 10% Giemsa solution. Thereafter, the percentage of parasitemia was determined by examining at least 1,000 RBCs, using an oil immersion microscope.

In vivo depletion and repletion of macrophages.Macrophages were depleted by i.p. administration of 300 μl of clodronate liposomes (CLL) twice at 4-day intervals (16, 18). The CLL and empty liposomes in phosphate-buffered saline (PL) (19) were from ClodronateLiposomes (Haarlem, The Netherlands). The depletion of macrophages was confirmed by flow cytometry (16). Briefly, splenocytes of the mice were obtained 5 days after the injection with CLL or PL and then resuspended in cold phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin. Thereafter, the cells were incubated with fluorescein isothiocyanate-labeled anti-mouse monoclonal antibodies F4/80 (BD Biosciences, La Jolla, CA, USA) on ice for 30 min. After three washes with cold PBS, these cells were analyzed using an Epics XL flow cytometer (Beckman Coulter, Pasadena, CA, USA). For reconstituting the macrophage in CLL-treated mice, bone marrow cells were isolated from the hind limbs of naive mice (n = 14) and cultured at 1 × 106 cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml of penicillin, 100 μg/ml of streptomycin, and 10% L929 conditioned medium. Fresh medium was replaced twice on days 3 and 6 after culturing, and then the adherent cells were isolated using a cell scraper and collected by centrifugation. Bone marrow-derived macrophages (BMM) were washed, stained with anti-mouse monoclonal antibodies F4/80, and then examined by flow cytometry (≥80%). The BMM were transferred by i.p. injection to BALB/c mice that received either CLL or PL on days 7 and 11 postinfection (p.i.) (acute phase of infection). The transfer of BMM was performed repeatedly at 1 × 106 cells/mouse from days 8 to 10 p.i. and then at 2 × 106 cells/mouse from days 12 to 14 p.i.

Detection of serum cytokines.Blood was harvested from each mouse by cardiac puncture, and serum was obtained by centrifugation for 15 min at 5,000 rpm. The concentrations of individual sample cytokines were determined with enzyme-linked immunosorbent assay (ELISA) kits using respective standard curves prepared with known concentrations of mouse recombinant IFN-γ, TNF-α, interleukin-2 (IL-2), IL-4, IL-10, and IL-12 (Pierce Biotechnology, Rockford, IL, USA), according to the manufacturer's instructions.

Measurement of nitric oxide (NO).The levels of nitrate and nitrite production in mouse sera were measured using a nitrate/nitrite assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.

Determination of the antibody response.Bacterial recombinant B. microti P32 was used as an antigen in an ELISA to detect parasite-specific antibodies (20). Briefly, 96-well microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 50 μl/well of coating buffer (50 mM carbonate-bicarbonate buffer [pH 9.6]) with 0.2 μg/well of recombinant B. microti P32. The plates were washed once with 0.05% Tween 20–phosphate-buffered saline (PBST) and then incubated with 100 μl/well of 3% skim milk-PBS (SM-PBS) for 1 h at 37°C. The plates were washed with PBST and then incubated with 50 μl of mouse serum diluted at 1:100 in SM-PBS for 1 h at 37°C. After washing six times with PBST, the plates were incubated with 50 μl/well of horseradish peroxidase-conjugated goat anti-mouse IgG1 or IgG2a (Bethyl Laboratories, Montgomery, TX, USA) diluted at 1:4,000 in SM-PBS for 1 h at 37°C. Thereafter, the plates were washed six times with PBS-T, and 100 μl/well of the substrate solution [0.1 M citric acid, 0.2 M sodium phosphate, 0.3 mg/ml of 2,2-azide-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma), and 0.01% of 30% H2O2] was then added to each well and incubated for 1 h at room temperature. Finally, the optical density (OD) at a wavelength of 415 nm was measured using an MTP-500 microplate reader (Corona Electric, Tokyo, Japan).

Real-time PCR assays for mRNA quantifications of IFN-γ and TNF-α.Mouse spleens were aseptically removed, and total RNA was extracted using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA). The concentration of total RNA was adjusted to 50 ng/μl, and the RNA was then used as a template for real-time quantitative PCR assays (21) with IFN-γ- and TNF-α-specific-labeled probes with assay identification numbers Mm00439616_m1 and Mm00443258_m1, respectively (Applied Biosystems, Carlsbad, CA, USA). Data were analyzed using SDS 2.1 software (Applied Biosystems), and the relative expressions of the IFN-γ and TNF-α genes were normalized with the β-actin gene as an internal control.

Hematology and serum biochemistry.Hematological examination for blood cell count was made using an automatic cell counter (Celltac α; Nihon Kohden, Tokyo, Japan). Serum samples for biochemical analyses were examined by a clinical chemistry automated analyzer (Toshiba Medical Systems Co., Tochigi, Japan) for measuring the concentrations of total protein, glucose, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase, T-cholesterol (T-cho), creatinine (CRE), and blood urea nitrogen (BUN) with specific detection reagents (Denka Seiken, Tokyo, Japan).

Histopathology of spleen sections.Spleens were harvested from mice, and the tissues were fixed in 10% (vol/vol) buffered formalin, embedded in paraffin, and then stained with hematoxylin and eosin (22).

Statistical analysis.The significant differences (GraphPad Prism 5; GraphPad Software, San Diego, CA, USA) between the means of all variables were examined by one-way analysis of variance, followed by Tukey's multiple-comparison test for pairwise comparison of data from the multiple groups. Survival analyses for statistically significant differences were done using a Kaplan-Meier nonparametric model. Results were considered to be statistically significant when the P value was <0.05.

RESULTS

Depletion of macrophages at different times alters the outcome of B. microti infection.To investigate the involvement of macrophages in the defense mechanism against B. microti infection, we initially examined the effects of CLL treatment at different times during the course of infection in BALB/c mice. For depletion of the macrophages in the early phase of infection, mice were repeatedly injected on days −2 and +2 p.i., and the parasitemia and survival were monitored for a period of 30 days. While the kinetics of parasitemia were almost identical throughout the period of ascending parasitemia, macrophage-depleted mice exhibited significantly prolonged and greater magnitudes of peak parasitemia than did controls treated with PL or not treated (Fig. 1A). Subsequently, one mouse (6.6%) succumbed to the infection with high parasitemia (>80%), and the remaining mice resolved the parasitemia in the third week of infection (Fig. 1A and B). Next, the macrophages were depleted at the acute phase of infection by injecting the BALB/c mice with CLL on days 7 and 11 p.i. Interestingly, these treated mice also demonstrated higher and prolonged parasitemia peaks (Fig. 1C), and only 3 mice (20%) survived and resolved their parasitemias (Fig. 1D). These results were encouraging for studying the importance of macrophages in the resolving and latent phases of infection. Therefore, mice were injected with CLL on days 14 and 18 p.i. for depletion of macrophages at the resolving phase and on days 45 and 49 p.i. at the latent phase of infection. Surprisingly, the depletion of macrophages at the resolving phase of the infection resulted in an immediate and pronounced exacerbation of parasitemia and 60% mortality in mice (Fig. 1E and F). Likewise, treatment with CLL at the latent phase of infection resulted in a temporal increase of parasitemias coupled with 13% mortality (Fig. 1G and H). Collectively, our data show the crucial role of macrophages, not only in resolving the parasitemia but also in preventing the parasites' recrudescence. To further ascertain the need of macrophages for the control of and resistance to B. microti infection, adoptive transfer of BMM from naive mice was performed to CLL-treated mice at the acute phase of infection. Strikingly, CLL-treated mice that received BMM showed improved survival coupled with better patterns of recovery from parasitemia than CLL-treated mice that received no cells (Fig. 2A and B). Nevertheless, the differences in the survival of these mice were not statistically significant. Moreover, PL-treated mice that received BMM showed a slight reduction in parasitemia during the descending phase of infection compared to CLL-treated mice that received no cells (Fig. 2A and B). These results show that reconstitution of macrophages slightly improves survival in macrophage-depleted mice. Furthermore, hematological analyses of blood samples revealed a gradual decline in the hematocrit (HCT) values and the numbers of peripheral platelets, coinciding with the peaks of parasitemia. However, these values gradually became normal in the recovery phase of the infection coincident with the reduction of parasitemia (Fig. 2C and D). Of note, mice displayed significantly greater numbers of platelets after CLL injection than after injection with PL (Fig. 2C and D). The HCT values of CLL-treated mice were significantly lower than those of PL-treated mice in the resolving phase of the infection (Fig. 2C). These results demonstrate that the depletion of macrophages minimizes the progression of thrombocytopenia and prolongs the anemia caused by the infection. Taken together, our data clearly indicate the crucial role of macrophages in the resistance to and control of the B. microti infection.

FIG 1
FIG 1

Efficacy of the depletion of macrophages at different phases of infection with B. microti in BALB/c mice. Parasitemia (A, C, E, and G) and survival rates (B, D, F, and H) of infected mice treated with clodronate liposomes (CLL) or PBS liposomes (PL) at different times during the course of infection with B. microti or not treated (NT) are shown. Mice received CLL or PL on days −2 and 2 p.i. (A and B), on days 7 and 11 p.i. (C and D), on days 14 and 18 p.i. (E and F), and on days 45 and 49 p.i. (G and H). Data are results (average for n = 15 mice ± standard deviation [SD]) pooled from three independent experiments. P values for survival comparison were determined by the log rank (Mantel-Cox) test.

FIG 2
FIG 2

Efficacy of the adoptive transfer of BMM from naive to macrophage-depleted BALB/c mice at the acute phase of infection with B. microti. The infected mice were treated with clodronate liposomes (CLL) or PBS liposomes (PL) alone or with BMM. (average for n = 7 mice ± SD). (A) Parasitemia; (B) survival rates; (C) hematocrit values; (D) number of total blood platelets. P values for survival comparison were determined by the log rank (Mantel-Cox) test.

Depletion of macrophages impairs the architectural structure of the spleen.Due to the fact that the spleen plays a critical role in host defense, including removing pRBCs from the blood and regulating host cellular immune functions (1), we studied the pathological changes in the spleens of B. microti-infected mice after treatment with CLL at different phases of infection. We first compared splenic weight and architectural structure changes for all mice. Of note, spleen weight was significantly increased after infection in all mice, whereas the weights of the spleens collected from macrophage-depleted mice were significantly lower than those of spleens from control mice (see Fig. S1 in the supplemental material). The increased spleen weight after infection was most likely due to the expansion of red pulp and white pulp compartments in infected mice. Indeed, infected mice exhibited a marked extramedullary hematopoiesis that could be seen as a diffused hyperplasia in the red pulp, and enlargement in the follicles appeared to be fused to the white pulp (data not shown). Histopathology examination of the spleens of macrophage-depleted mice revealed a marked architectural disorganization evidenced by a loss of cells in the marginal zone and red pulp regions (see Fig. S2 in the supplemental material). Collectively, the loss of macrophages in the spleen caused by CCL treatment might explain the impairment of the host defense mechanism needed for eliminating parasites and pRBCs from circulation. Thereafter, the pathological changes in the spleens of B. microti-infected mice that received either CLL or PL at the acute phase of infection were examined after adoptive transfer of BMM. All CLL-treated mice showed a loss of cells in the marginal zone and red pulp regions; however, mice that received BMM showed a slight increase in the cells of the marginal zone and red pulp associated with significant elevation of spleen F4/80+ cells (see Fig. S3 in the supplemental material). Consistently, spleen weights of mice that received BMM were not significantly increased compared to those of mice that received no cells (see Fig. S3 in the supplemental material). These results suggest the partial recovery of the macrophage population in the spleen after adoptive transfer of BMM from naive mice to CLL-treated mice at the acute phase of infection.

Depletion of macrophages modulates the immune response to B. microti infection.To assess the immune responses of infected mice lacking their macrophages, BALB/c mice treated with CLL at different times during the course of infection were sampled on days 9, 15, 22, and 53 p.i., and the serum levels of cytokines and NO, the gene expression levels of splenic cytokines, and the parasite-specific antibody titers were determined. Of note, the early-phase depletion of macrophages significantly reduced the serum levels of IFN-γ, IL-10, and TNF-α on day 9 p.i. compared to those in the control mice (Fig. 3A). On the other hand, mice that had received CLL at the acute, resolving, and latent phases of infection exhibited marked reductions of serum IFN-γ and TNF-α, which were correlated with elevation of IL-10, compared to level in the control mice (Fig. 3 B to D). Moreover, a significant reduction of serum NO was observed only after the depletion of macrophages at the acute phase of infection (Fig. 3E to H). The level of serum IL-12 was not significantly affected by the depletion of macrophages over the time course of treatment and infection (data not shown). Likewise, serum IL-4 cytokines were only detected at the acute phase of infection, with no significant difference among the groups of mice (data not shown). Furthermore, we examined the mRNA expression of IFN-γ and TNF-α in the spleen. Significant decreases of IFN-γ and TNF-α expression were observed in the macrophage-depleted mice only at the acute phase and not at the early or latent phases of infection (Fig. 3J to L). These results demonstrate that macrophage-depleted mice have impaired production of Th1-related cytokines, including IFN-γ and TNF-α, during the course of infection with B. microti. To further assess the changes of humoral immune responses to infection, the specific antibody to B. microti P32 was examined. Notably, the depletion of macrophages at the early phase of infection resulted in a marked reduction of the antibody response on day 9 p.i. compared to that in the control mice (Fig. 4A). In contrast, a significant increase in the levels of IgG1 titers was observed in mice that had received CLL at the acute, resolving, and latent phases of infection with B. microti (Fig. 4B to D). These results suggest that the host macrophages might contribute to the regulation of the immune response to B. microti infection.

FIG 3
FIG 3

Detection of cytokines and nitric oxide in sera (A to H) and relative gene expression of IFN-γ and TNF-α in spleens (I to L) of B. microti-infected mice treated with clodronate liposomes (CLL) or PBS liposomes (PL) or not treated (NT). (A, E, and I) Mice were sampled at 9 days p.i. after depletion of macrophages at the early phase of infection. (B, F, and J) Mice were sampled at 15 days p.i. after depletion of macrophages at the acute phase of infection. (C, G, and K) Mice were sampled at 22 days p.i. after depletion of macrophages at the resolving phase of infection. (D, H, and L) Mice were sampled at 53 days p.i. after depletion of macrophages at the latent phase of infection. Data are results (average for n = 6 mice ± SD) pooled from two independent experiments. Asterisks indicate a significant difference between the groups.

FIG 4
FIG 4

Antibody responses of B. microti-infected mice treated with clodronate liposomes (CLL) or PBS liposomes (PL) or not treated (NT). (A) Mice were sampled at 9 days p.i. after depletion of macrophages at the early phase of infection. (B) Mice were sampled at 15 days p.i. after depletion of macrophages at the acute phase of infection. (C) Mice were sampled at 22 days p.i. after depletion of macrophages at the resolving phase of infection. (D) Mice were sampled at 53 days p.i. after depletion of macrophages at the latent phase of infection. Data are results (average for n = 6 mice ± SD) pooled from two independent experiments. Asterisks indicate a significant difference between the groups.

Biochemical analyses of serum samples were performed to assess the correlation between mouse mortality and organ dysfunction. However, there were no significant increases in the serum levels of AST, ALT, ALP, and T-cho as indictors of liver function and of CRE and BUN as indictors of kidney function between the CLL- and PL-treated mice over the course of treatment and infection (see Table S4 in the supplemental material). These results reveal that the dying mice have no severe dysfunction in the liver and kidneys, suggesting that the mortality is independent of dysfunction of these organs.

Depletion of macrophages in IFN-γ−/− and SCID mice exaggerates the severity of B. microti infection.Since IFN-γ acts as a key inducer of the immune effector mechanisms for initial control of the rodent Babesia infection (8), we further examined the kinetics of parasitemia and mortality in IFN-γ−/− mice after the depletion of macrophages. Mice were treated with CLL on days −2 and +2 p.i. for the early phase, on days 7 and 11 p.i. for the acute phase, and on days −14 and 18 p.i. for the late phase of infection (Fig. 5A to F). Importantly, all of the macrophage-depleted IFN-γ−/− mice succumbed to the infection, with average parasitemia approaching 85% (Fig. 5A to F). In contrast, no mortality was observed in mice that received PL over the monitoring time of 30 days. Next, we examined the kinetics of parasitemia and mortality in SCID mice after treatment with CLL at different phases of infection to clarify that the mortality in macrophage-depleted mice was independent of lymphocyte responses. Mice were treated with CLL following the same time course as used for the IFN-γ−/− mice. Consistently, macrophage-depleted mice at the acute phase of infection exhibited high parasitemia coupled with 100% mortality (see Fig. S5 in the supplemental material). Moreover, treating the SCID mice with CLL on days −2 and +2 p.i. for the early phase and on days 18 and 22 p.i. for the late phase of infection resulted in increased parasitemia coupled with 40 to 60% mortality (see Fig. S5 in the supplemental material). Together, these results emphasize the importance of host macrophages in mouse control of and resistance to B. microti infection.

FIG 5
FIG 5

Efficacy of macrophage depletion at different phases of infection with B. microti in IFN-γ−/− mice. Parasitemia (A, C, and E) and survival rates (B, D, and F) of mice treated with clodronate liposomes (CLL) or PBS liposomes (PL) at different times during the course of infection are shown. Mice received CLL on days −2 and 2 p.i. (A and B), on days 7 and 11 p.i. (C and D), and on days 14 and 18 p.i. (E and F). Data are results (average for n = 8 mice ± SD) pooled from two independent experiments. P values for survival comparison were determined by the log rank (Mantel-Cox) test.

DISCUSSION

Increased health concerns about human babesiosis caused by B. microti demand an urgent preventive strategy for controlling the infection. However, understanding the immunological pathways that will lead to controlling the infection is important for future vaccine development (1, 6). Until recently, research on the immunological events and vaccine development for Babesia infection has focused on adaptive immunity (6, 9). Accumulating evidence suggests that innate immune defenses are critical for determining the outcome of the infection and should be considered in future vaccine design (7, 10). Innate immune mechanisms represent the first line of defense against invading pathogens, whereas macrophages are the major effector cells needed for killing the invading pathogens and regulating the consequent adaptive immune responses (7, 14, 15). However, the function of those cells in the control of and resistance to B. microti infection is poorly defined. Therefore, we investigated the protective function of macrophages against B. microti infection at different times during the course of infection in the mouse model.

In the current study, the depletion of macrophages impaired the resistance of BALB/c mice to infection, as reflected by enhanced parasite outgrowth and high mortality. The exacerbating parasitemia immediately after depletion in acutely and chronically infected mice suggests a crucial role of macrophages in resolving the parasitemia of B. microti. The substantial recovery of platelets after treatment with CLL suggests that macrophages may be involved in the removal of platelets during the infection with B. microti. Moreover, the improved survival rate observed in macrophage-depleted mice that received BMM emphasizes the importance of these cells in determining the outcome of infection. However, the partial protection observed in macrophage-depleted mice that received BMM can be explained by incomplete recovery of the macrophage population in spleen and may also suggest the involvement of other types of cells in the protection. In apparent agreement with our observation, depletion of macrophages by CLL results in elevated susceptibility and impaired resistance of C57BL/6 mice to infection with Babesia duncani (10). Moreover, depletion of macrophages impairs the protection induced by B. microti immunization against Babesia rodhaini challenge infection (16). In regard to closely related parasites, administration of silica for macrophage depletion 6 days p.i. with Plasmodium chabaudi AS results in fulminant parasitemia and high mortality in C57BL/6 mice (23). However, another study has shown that macrophage depletion results in a slight increase in parasitemia, with no significant change in the survival of mice infected with lethal or nonlethal Plasmodium yoelii during the acute phase of infection (18). Likewise, a recent study noted that the depletion of macrophages did not significantly alter the parasitemia of Plasmodium falciparum in humanized mice (24). The discrepancy with our observation might be explained based on the differences in the experimental designs of those studies, as we depleted the cells at different phases of infection but not at one consistent time point during infection.

Macrophages have been identified as acting as the predominant inducers of resistance and protection not only by phagocytizing and killing the pathogens, including in protozoan infections, but also by initiating the inflammatory response (6, 14, 25). In our study, the reductions of IFN-γ and TNF-α cytokines coupled with the elevation of IL-10 cytokines and the level of IgG1 in CLL-treated mice suggest a crucial role of macrophages in regulating Th1 response during B. microti infection. Changes in cytokines and antibody responses might be caused by the increase of peripheral pRBCs, allowing more interaction with the lymphocytes that elevate the production of IgG1 and IL-10 and suppress IFN-γ production. Another possible reason for the decline of IFN-γ production might be the toxic effects of CLL on dendritic cells in the liver and spleen, which are known to produce type I IFN cytokines, which upregulate expression of type II IFN cytokines (26, 27). The differences in the levels of IFN-γ and TNF-α in sera and their transcripts in spleens at the resolving and latent stages suggest that, in addition to the spleen as a major contributor to cytokinemia, other lymph nodes and circulating immune cells might participate in the production of circulating cytokines. In general, macrophages respond to IFN-γ secretion of natural killer and T cells by producing proinflammatory cytokines, such as TNF-α and IL-6, leading to a predominant Th1 response and pathogen elimination. It is well established that IFN-γ acts as the key inducer of immune effector mechanisms by priming the macrophage responses, leading to the destruction of intracellular protozoan parasites (7, 13, 28) and regulating T cell expansion, differentiation, and contraction (29). Nevertheless, there is evidence that the IFN-γ level needs to be carefully balanced in both babesiosis and malaria infections to avoid immune pathology. Indeed, rapid innate production of IFN-γ may predispose to overproduction of inflammatory cytokines, leading to severe infection (30, 31). In contrast, the production of IL-10 by dendritic cells and lymphocytes inhibits the secretion of proinflammatory cytokines and limits the differentiation and activation of T cells, resulting in either fulminant and rapidly fatal or chronic nonhealing infection. The excessively mistimed production of IL-10 leads to poor outcomes in various protozoal infections, including those with B. bovis, Plasmodium, Leishmania, and Toxoplasma (32–34). Moreover, the high level of serum nitric oxide observed in macrophage-depleted mice despite of the suppression of IFN-γ and TNF-α might derive from vascular endothelial cells (35) as a response to the increase of peripheral pRBCs.

The mechanisms by which the host controls the blood-stage parasites are proposed to be through the capability of activated macrophages of the spleen to phagocytize the parasites and the pRBCs (1). In fact, patients and mice with splenectomy exhibit high susceptibility to the infection and suffer from severe infection with high mortality (1). Our data suggest that a lack of marginal zone and red pulp macrophages might be associated with failure to control parasitemia in BALB/c mice. These cells are specialized macrophages that are strategically positioned to phagocytize pathogens and cell debris entering the spleen from the circulation and to regulate the activation of T cell responses (36). Importantly, the loss of these cells increases susceptibility to a broad range of pathogen infections, including malaria (36, 37). Phagocytic macrophages produce oxygen and nitrogen intermediates that are thought to mediate the lysis of pRBCs and the parasites (1, 38). Soluble factors derived from activated macrophages inhibit the replication of intracellular Babesia and induce degeneration of the parasites inside the RBCs (39, 40).

In conclusion, we have demonstrated that macrophages are the key effector cells that mediate the clearance of pRBCs and regulate the consequent immune response. These findings may help in the development of an effective Babesia vaccine or novel therapeutics that can potentially modulate macrophage responses for better outcomes of infection. Identifying the effector molecules of macrophages used to kill Babesia parasites may offer important clues for a future strategy for control of the infection.

ACKNOWLEDGMENTS

This study was supported by a grant from the Global COE Program from the Japanese Ministry of Education, Culture, Sports, Science and Technology and by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (JSPS). M.A.T. was supported by a research grant fellowship from JSPS for young scientists.

FOOTNOTES

    • Received 3 June 2014.
    • Returned for modification 22 June 2014.
    • Accepted 11 September 2014.
    • Accepted manuscript posted online 13 October 2014.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02128-14.

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