infectious disease plasmodium products...

Lee et al., Sci. Immunol. 2, eaam8093 (2017) 2 June 2017

S C I E N C E I M M U N O L O G Y | R E S E A R C H A R T I C L E

1 of 11

I N F E C T I O U S D I S E A S E

Plasmodium products persist in the bone marrow and promote chronic bone lossMichelle S. J. Lee,1 Kenta Maruyama,2 Yukiko Fujita,1 Aki Konishi,1 Patrick M. Lelliott,1 Sawako Itagaki,3 Toshihiro Horii,3 Jing-wen Lin,4,5 Shahid M. Khan,4 Etsushi Kuroda,6,7 Shizuo Akira,2 Ken J. Ishii,6,7 Cevayir Coban1*

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retarda-tion as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results high-light the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment.

INTRODUCTIONMalaria caused by Plasmodium parasites is a life-threatening infec-tious disease that kills at least half a million people annually while causing more than 200 million new infections. In some cases, com-plications can quickly develop, such as cerebral malaria, respiratory distress, and severe anemia, often leading to death. Despite these se-vere complications, most patients recover from the disease. However, there is evidence that malaria survivors experience long-term “hidden” pathologies that are as yet poorly defined. For instance, physical growth retardation in young children in Africa is associated with in-fectious diseases, with a high prevalence among malaria-infected children regardless of nutritional status (1, 2). An increased incidence of porous bone lesions has also been reported in malaria-endemic regions, suggesting that infection may compromise bone integrity (3). Despite the importance of bone tissue in health and development, and the known interaction between Plasmodium parasites and bone marrow cells whereby parasites circulate, reside (4), and infect (5), the pathology of malaria in bone is poorly understood.

Bone tissue constantly undergoes formation by osteoblasts (OBs) and resorption by osteoclasts (OCs) in a tightly regulated process. OC differentiation is initiated when receptor activator of nuclear factor ĸB (NFB) ligand (RANKL; encoded by Tnfsf11), constitutively expressed on OBs, binds to the RANK receptor on the surface of OC precur-sors (OCPs) from monocyte/macrophage lineage (6). The RANKL-RANK interaction leads to recruitment of the adaptor molecule tumor

necrosis factor (TNF) receptor–associated factor 6 (TRAF6) and down-stream activation of NFB, c-fos, and AP-1, leading to transcription of the master regulator of osteoclastogenesis nuclear factor of acti-vated T cells 1 (NFATc1). This results in the formation of mature OCs, which express tartrate-resistant acid phosphatase (TRAP) (7). Aside from its role in osteoclastogenesis, RANKL is an important immu-noregulatory molecule (8). Up-regulation of RANKL expression on OBs and immune cells leads to bone destruction during bacterial in-fections, such as osteomyelitis and gingivitis, and osteoporosis in HIV, implicating a tight relationship between immune activation and bone remodeling (9–11). Plasmodium infection induces robust immune activation and invasion of parasites into the bone marrow, further suggesting the harmful potential of malaria on bone.

Here, we addressed the direct effect of malaria infection on bone. We reported that the Plasmodium parasite and its associated prod-ucts produced during and after malaria infection cause chronic in-flammation leading to bone loss. We identified alfacalcidol, a vitamin D3 (VitD3) analog, as a successful drug for the treatment of bone problems caused by malaria infection.

RESULTSPlasmodium infection causes bone loss and bone growth retardationTo address the effect of Plasmodium infection on bone, we used two mouse malaria models, Plasmodium yoelii nonlethal (PyNL) and Plasmodium chabaudi chabaudi (Pcc). PyNL infection, which resem-bles Plasmodium vivax infection in humans and preferentially invades reticulocytes (12), was used to determine the effect of a single self-cleared Plasmodium infection on bone homeostasis, whereas Pcc in-fection, which more closely resembles Plasmodium falciparum infection in humans (12), was used to evaluate the effect of low-level chronic infection on bone homeostasis. Six-week-old adult mice were infected with these parasite species, and changes in bone were assessed during the acute (peak parasitemia, day 8 or 14), convalescent (clearance, day 30), and chronic (day 90) phases of infection (Fig. 1A and fig. S1A).

1Laboratory of Malaria Immunology, Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. 2Laboratory of Host Defense, IFReC, Osaka University, Suita, Osaka 565-0871, Japan. 3Department of Mo-lecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. 4Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical Centre, 2333 ZA Leiden, Netherlands. 5Division of Pediatric Infectious Diseases, State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University and Collaboration Innovation Centre, Chengdu 610041, China. 6Laboratory of Vaccine Science, IFReC, Osaka University, Suita, Osaka 565-0871, Japan. 7Laboratory of Adjuvant Innovation, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.*Corresponding author. Email: [email protected]

2017 © The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science.

by guest on June 4, 2017http://im

munology.sciencem

ag.org/D

ownloaded from



2 of 11

Microcomputed tomography (CT) analysis showed that PyNL- and Pcc-infected mice had significantly reduced trabecular bone volume and number and increased trabecular bone spacing, which continued for over 60 days after parasite clearance, as confirmed by PyNL 18S ribosomal RNA (rRNA) quantitative polymerase chain reaction (qPCR) (Fig. 1, B to D, and fig. S1, B and C). Recovery from anemia by day 30 excluded anemia’s contribution to further bone loss (Fig. 1E).

Increasing evidence suggests that Plasmodium infection may re-sult in growth stunting of young children in malaria-endemic regions

(summarized in table S1) (1, 2, 13–19). To assess the effect of infection on bone growth at young age, we infected 3-week-old mice with PyNL (fig. S1D). At this age, mice are undergoing active bone development before epiphyseal closure. Young mice sacrificed 3 weeks after infec-tion, at 6 weeks old, had significantly shorter femurs and lower trabec-ular bone volume compared with naïve littermates (Fig. 1, F to H). In mice sacrificed 6 weeks after infection, 3 weeks after parasite clear-ance, femur length had recovered to normal levels (fig. S1E); however, trabecular bone volume remained significantly reduced (Fig. 1I).

Fig. 1. Plasmodium infection results in bone loss and bone growth retardation. (A) Parasitemia in peripheral blood of PyNL-infected mice (n = 6 to 16 mice per group). (B) Gene expression of PyNL 18S rRNA in whole bone (n = 5 per group). (C) Representative CT images of distal femurs. (D) Bone morphometric analysis of distal femurs by CT. Bv/Tv, bone volume/total volume; Tb.N, trabecular bone number; Tb.Th, trabecular bone thickness; Tb.Sp, trabecular bone spacing. w.o., weeks old. (E) Whole-blood count. RBC, red blood cell.; n.s., not significant. (F) Femurs of 6-week-old naïve and PyNL-infected mice. (G) Length measurement of young mice femurs. (H and I) Rep-resentative CT images and bone morphometric analysis of distal femurs of naïve and PyNL-infected young mice at 6 (H) and 9 (I) weeks old after recovery from infection. Each data point represents individual mouse and are represented as means ± SD. Experiments were repeated at least twice. *P < 0.05, **P < 0.01, Mann-Whitney test (A, D, G, H, and I) and Kruskal-Wallis with Dunn’s posttest (E).


munology.sciencem

ag.org/D

ownloaded from



3 of 11

Together, these findings suggest that both chronic low-level infection and a single self-cleared infection result in a substantial chronic effect on bone tissue, leading to bone loss and bone development defects.

Bone homeostasis is disrupted after Plasmodium infectionTo further investigate bone dynamics at a cellular level, we used the PyNL infection model for the following studies. Bone histomorpho-metric analysis revealed total suppression of both OC and OB num-bers and significantly reduced bone formation rate during acute PyNL infection (Fig. 2, A and B). The OC marker TRAP and the OB marker alkaline phosphatase (ALP) were also significantly reduced during acute infection, supporting the notion that bone remodeling is sup-pressed, and presumably, bone growth is halted during this phase of infection (Fig. 2C). In contrast, after the clearance of parasites on day 30, osteoblastic and osteoclastic parameters returned to normal levels, whereas by day 90 after infection, bone resorption rate was elevated compared with naïve conditions (Fig. 2, A to C). To further examine the effect of Plasmodium infection on OC differentiation, we stimulated bone marrow cells from naïve and PyNL-infected mice with RANKL in vitro. Cells from day 14 PyNL-infected mice rarely differentiated into TRAP+ multinucleated OCs based on cell number and expression of Acp5 (the gene encoding TRAP) (Fig. 2D), whereas cells taken from day 30 recovered mice differentiated into OCs more readily than cells taken from naïve mice (Fig. 2E). Together, these results suggest that both OBs and OCs were suppressed during acute infection leading to bone loss and attenuation of bone growth, whereas after parasite clearance, bone loss was most likely attributable to en-hanced OC activity rather than OB impairment.

TRAF6, Jdp2, and NFATc1 signaling are disrupted during acute Plasmodium infectionThe RANK-RANKL signaling pathway is essential for the differen-tiation of OCs; we therefore investigated whether modulation of this pathway could explain changes in osteoclastogenesis observed during infection (Fig. 2F). The expression of Tnfrsf11a (encoding RANK) was comparable between OCPs derived from naïve and PyNL-infected mice at all time points tested (Fig. 2G). However, Traf6 expression was down-regulated in cells derived from acutely infected mice, with concomitant suppression of downstream signaling molecules Jdp2, Nfatc1, and Acp5 (Fig. 2, D and G).

We next addressed possible Plasmodium-related factors involved in the suppression of OCs in vivo. Several cytokines, as well as hemin, have been reported to be involved in the inhibition of OC differen-tiation (20). We detected increased interferon- (IFN), interleukin-4 (IL-4), IL-10, and IL-13 levels in the serum during acute infection compared with naïve controls (Fig. 2H). In addition, acute hemoly-sis during Plasmodium infection results in substantial heme release from ruptured erythrocytes (20, 21). We confirmed in vitro that OC differentiation was directly suppressed by hemin as indicated by re-duced Acp5 expression, highlighting a potential role for heme in the suppression of osteoclastogenesis during acute infection (Fig. 2I).

To further evaluate the role of OCs in bone remodeling during malaria infection, we infected Jdp2−/− mice with PyNL. As previous-ly reported, Jdp2−/− mice were osteopetrotic due to osteoclastogen-esis defect (22). Despite similar course of infection, bone loss was not observed in these mice during or after infection (Fig. 2, J and K). More-over, mice exhibited increased bone volume during convalescence (Fig. 2K). This highlights a key role for OCs in malaria-induced bone loss. In accordance with this, OC markers and inflammatory cyto-

kines were up-regulated in the bone during convalescence, indicating OCs activation (Fig. 2L and fig. S2). Together, these findings sug-gest that Plasmodium infection causes suppression of bone remod-eling during acute infection due to increased cytokines and acute heme release, whereas elevated Jdp2, Nfatc1, and Acp5 expression in the bone during convalescence leads to enhanced OC activity and chronic bone loss.

Accumulation of Plasmodium products into the bone marrow alters bone marrow nicheWe next investigated possible factors contributing to increased os-teoclastogenesis during convalescence. Black discoloration of femurs was prominent during the convalescent and chronic phases of in-fection, but not during the acute phase, suggesting that Plasmodium products gradually accumulate in the bone and remain there long term (Fig. 3, A and B). To further understand the interaction of these Plasmodium products with bone marrow cells, we cultured OCs and calvarial OBs in vitro with P. falciparum crude extract (PfCE), which consists of parasite proteins, nucleic acid, and hemozoin. PfCE did not inhibit OC maturation on the basis of TRAP expression (Fig. 3C); however, both OCPs and primary calvarial OB precursors (OBPs) responded strongly to PfCE and PyCE, with dose-dependent induc-tion of the inflammatory cytokines IL-1, IL-1, IL-6, and TNF (Fig. 3, D and E, and fig. S3). These inflammatory cytokines robustly pro-mote OC formation via OB RANKL induction (23, 24). PfCE sig-nificantly induced Tnfsf11 expression (encoding RANKL) in mature OBs (Fig. 3F and fig. S3). Together, these data suggest that sustained infiltration of Plasmodium products in the bone after parasite clear-ance directly induces proinflammatory and osteoclastogenic cytokines from both OCPs and OBPs, which in turn may promote osteoclas-togenesis via amplification of RANKL.

MyD88 controls Plasmodium-induced chronic bone inflammationGiven the role of Plasmodium products in inducing inflammatory cytokines and RANKL expression, consequently leading to the acti-vation of OBs, OCs, and their precursors, we investigated the possi-ble involvement of Plasmodium-immune recognition machinery in bone loss. Plasmodium products have been reported to induce im-mune responses involving MyD88, TLR9 (Toll-like receptor 9), NLRP3, and IL-1R (25, 26). Infection of mice deficient in these genes showed that Myd88−/− mice, but not Tlr9−/−, Il1r−/−, or Nlrp3−/− mice, were protected from PyNL-induced bone loss (Fig. 4, A and B). Although bone volume negatively correlated with parasitemia, such correlation was not found in Myd88−/− mice (fig. S4A). The lack of bone loss in Myd88−/− mice was unlikely because of osteoclastogenesis or osteo-blastogenesis impairment because Myd88−/− OCs and OBs formed normally in vitro (fig. S4B). Rather, we found that Plasmodium product–induced proinflammatory cytokine expression in OCPs and OBPs was Myd88-dependent (Fig. 4, C and D), leading to a lack of RANKL expression in OBs under stimulatory conditions (Fig. 4E). This in-dicates that MyD88 signaling is essential for Plasmodium-induced bone loss through its role in bone cell inflammation and concomi-tant RANKL expression.

Plasmodium products are involved in malaria-induced bone pathologyBecause it is difficult to determine the full moiety of accumulated Plasmodium products in the bone, we further investigated one of the


munology.sciencem

ag.org/D

ownloaded from



4 of 11

components, hemozoin, a heme detoxification by-product of Plasmodium, which we identified abundantly in the bone (Fig. 3B). In contrast, parasites and parasite nucleic acids could not be detected in the bone during convalescence (Fig. 1B). To investigate the involvement of hemo-zoin in chronic bone loss, we used a mutant Plasmodium berghei ANKA (PbA) parasite strain, pm4bp2, which is deficient in the pm4 and bp2 genes encoding the plasmepsin-4 and bergheipain-2 hemoglobinases, respectively (27). This mutant has impaired hemoglobin digestion and produces little or no hemozoin. Unlike the PbA strain, this mutant strain loses virulence and mice are able to clear infection (27); therefore, its course of infection resembles that of PyNL (Fig. 5A). To compare bone loss in PbA versus pm4bp2 infection, it was necessary to treat PbA-infected mice with chloroquine to control parasitemia to prevent death. Bone marrow smears taken during convalescence confirmed

a marked reduction in hemozoin accumulation in PbApm4bp2- infected mice (Fig. 5A). Compared with PbA-infected mice that had significant bone loss despite chloroquine treatment (fig. S5), pm4bp2 infection did not cause bone loss, despite a higher parasitemia during acute infection (Fig. 5, B and C). To assess the exact role of Plasmodium products but not other factors such as cytokines or anemia on the bone pathology, we used Rag2−/− mice that were unable to resolve the in-fection because of lack of T and B cells. Similar to the observation in wild-type (WT) mice, pm4bp2 infection in Rag2−/− mice did not cause bone loss despite having similar parasitemia, chronic anemia, and cytokines with PbA infection (Fig. 5, D to F, and figs. S6 and S7). Furthermore, in vitro studies showed that pm4bp2 crude extract in-duced lower inflammatory responses and RANKL expression in OB cul-ture compared with PbA crude extract (PbACE) stimulation (Fig. 5G).

Fig. 2. Bone remodeling is suppressed during acute Plasmodium infection but is highly activated after parasite clearance. (A and B) Bone histomorphometric analysis of tibia sections and OB and OC numbers per bone surface (A) and bone formation and bone resorption rates (B). (C) Serum levels of TRAP and ALP. OD405nm, optical density at 405 nm. (D and E) Number of OCs and mRNA expression of Acp5 in OC culture of naïve and PyNL-infected mice on day 14 (D) and on day 30 (E). (F) Schematic diagram of OC signaling. (G) Gene expression of cultured OCs. (H) Serum cytokine levels. (I) Acp5 mRNA expression of a 3-day differentiated OC culture incubated with hemin for 6 hours. (J) Course of PyNL infection in Jdp2+/− and Jdp2−/− mice (n = 7 to 8 mice per group). (K) Representative CT images and bone morphometric analysis of distal femurs of naïve and PyNL-infected Jdp2+/− and Jdp2−/− mice. (L) Gene expressions of Jdp2, Nfatc1, and Acp5 in indicated mouse tibia. Naïve control groups were indicated as day 0 PyNL infection. Data are means ± SD; each point represents individual mouse and is repeated at least twice with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis with Dunn’s posttest (A to C, G, H, K, and L) and Mann-Whitney test (D and E).


munology.sciencem

ag.org/D

ownloaded from



5 of 11

To specifically investigate the direct role of hemozoin in bone, we used synthetic hemozoin (sHZ) crystals (28) as a proxy for Plasmodium- produced hemozoin. We found that both OCPs and mature OCs were capable of phagocytizing sHZ in vitro (Fig. 5H). sHZ stimulated in-flammatory cytokine production in OCPs and OBPs, which further induced RANKL expression in OBs in a Myd88-dependent manner (Fig. 5, I to K). Overall, these data suggest the involvement of hemo-zoin in the MyD88-dependent bone inflammation and resultant chronic bone loss after malaria infection.

Malaria-induced chronic bone loss is rescued by alfacalcidol supplementationVitD is an important molecule for bone metabolism and calcium homeostasis and is also involved in immunomodulation (29). VitD insufficiency occurs in human malaria (30), and possible beneficial role of VitD supplementation for cerebral malaria has been reported

(31). To address whether VitD supplementation would alleviate bone loss caused by Plasmodium infection, we treated mice with alfacalcidol, a VitD3 derivative used to treat osteoporosis because of its high ef-ficacy and safety (32, 33). Treatment of alfacalcidol orally at 100 to 400 ng/kg was sufficient to prevent malaria-induced bone loss, whereas higher doses (400 to 1000 ng/kg) resulted in enhanced bone growth despite infection (Fig. 6, A to C). VitD treatment also suppressed PfCE-induced inflammation in OCPs and OBPs in vitro, suggesting that it may act by preventing overactivation of osteoclastogenesis during infection (Fig. 6, D and E). Alfacalcidol treatment not only improved bone status, it also suppressed parasite growth at high-er doses (Fig. 6F). In vitro P. falciparum inhibition assay showed that alfacalcidol reduced parasite growth dose-dependently (Fig. 6G). Together, these results suggest that alfacalcidol administration might be beneficial to reduce parasite burden while preventing bone loss.

Fig. 3. Persistence of Plasmodium products in the bone elicits inflammation and amplifies RANKL. (A) Images of femurs from naïve and PyNL-infected mice. (B) Repre-sentative images of bone histology sections and bone marrow smears of naïve and PyNL-infected mouse tibias. Scale bars, 20 m (B). Arrows show the accumulated bire-fringent brownish hemozoin. (C) Acp5 expression of a 3-day differentiated OC culture with PfCE containing 0, 4, 10, and 20 × 106 infected RBCs (iRBCs) for 24 hours. (D) Cytokine expression in OCP and a 3-day differentiated OC culture after a 6-hour stimulation with PfCE containing 0, 4, 10, and 20 × 106 iRBCs. (E) Cytokine expression in OBP and differentiated OB culture after a 6-hour stimulation with PfCE containing 0, 10, and 20 × 106 iRBCs. (F) Tnfsf11 expression in OB culture after a 24-hour stimulation with PfCE containing 20 × 106 iRBCs. All experiments were repeated at least twice. Data are means ± SD. *P < 0.05, Mann-Whitney test.


munology.sciencem

ag.org/D

ownloaded from



6 of 11

DISCUSSIONThe long-term pathological consequences of chronic ma-laria infection are poorly un-derstood. Recent study has suggested that chronic expo-sure to Plasmodium parasites promotes prolonged immune activation and enhances the risk of lymphoma formation (34). Another unforeseen out-come of malaria is the evidence that chronically infected birds experience enhanced telomere degradation (35). These are likely only few among many potential deleterious outcomes triggered by chronic malaria infection. In particular, the possible association between growth retardation and malaria infection in humans (table S1) suggests that infection may have a negative impact on bone remodeling and growth. However, there is a limitation in studying the direct effect of malaria infection on bone in humans due to various coexisting factors such as malnutrition and other infections. Using well-established mouse models mimicking various aspects of human Plasmodium infection, we showed here that infection causes significant and long-term bone loss in adult mice and growth retardation in young mice. Bone remodeling is completely suppressed during acute infection but is highly activated immediately after parasite clearance, with increased osteoclastic activity skewing the balance toward bone resorption. OCs are activated by the key oste-clastogenic cytokine RANKL, which was up-regulated in OBs through MyD88-dependent inflammation, triggered by the persistence of para-site products in the bone marrow (Fig. 7).

The alteration of bone remodeling has been observed in other in-fectious and inflammatory conditions (36), suggesting that the mech-anism of bone loss in malaria might be multifactorial. Induction of

IL-4, IL-10, IL-13, and IFN by activated T cells in response to in-fection likely disrupts OC signaling. The suppression of RANK-RANKL signaling from the upstream TRAF6 during acute Plasmodium infection could be due to increased IFN (37). An additional factor unique to Plasmodium infection might be the release of heme due to malaria- induced hemolysis (21), which may directly contribute to the impairment of OC and OB differentiation (38–40). Hence, multiple infection- related factors likely cooperate to cause suppression of bone remodel-ing during acute malaria.

A key finding of our study is that, although Plasmodium infection is resolved systemically, the bone marrow evolves into a state of chronic inflammation that could be associated with the persistent accumula-tion of Plasmodium products in the bone marrow. Although low-level chronic infection is a common feature across many pathological condi-tions, persistence of pathogen by-products in tissues is a unique feature of malaria (41). Plasmodium products (i.e., proteins, hemozoin, and nucleic acids) are biologically active compounds capable of modulating immune responses (25, 26, 42). Here, we showed that bone marrow OCs and OCPs engulf Plasmodium products including hemozoin,

Fig. 4. MyD88 controls malaria- induced chronic bone inflamma-tion and bone loss. (A and B) Bone morphometric analysis of femurs of naïve and PyNL-infected Myd88+/− and Myd88−/− (A) and Tlr9−/−, Il1r−/−, and Nlrp3−/− (B) mice. (C) Relative mRNA expression of cytokines in Myd88+/− and MyD88−/− OCP culture stimulated with PfCE at 0, 200, 500, and 1000 g/ml for 6 hours. (D) Cy-tokine expressions in Myd88+/− and Myd88−/− OBP culture stimulated with PfCE at 0, 200, 500, and 1000 g/ml for 24 hours. (E) Tnfsf11 expression in Myd88+/− and Myd88−/− OB culture stimulated with PfCE at 0, 200, 500, and 1000 g/ml for 24 hours. All ex-periments were repeated at least twice. Each data point represents individual mouse. Data are means ± SD. *P < 0.05, **P < 0.01, Kruskal-Wallis test and Dunn’s post-test (A) and Mann-Whitney test (B).


munology.sciencem

ag.org/D

ownloaded from



7 of 11

thereby eliciting strong inflam-matory cytokine production. Although secretion of IL-1, IL-1, IL-6, and TNF is vital in the regulation of immune cell infiltration and activation for the elimination of patho-gens, persistent pro duction of these cytokines can lead to in-flammatory osteoporosis by in creasing OC activity (43). These cytokines act synergis-tically with minimal amounts of RANKL to promote robust osteoclastogenesis (23, 24, 44) and may additionally induce osteoclastogenesis indepen-dently of RANK-RANKL sig-naling (44). In support of the hypothesis that enhanced os-teoclastogenesis is a key factor in malaria-induced bone loss,

Fig. 5. Involvement of Plasmodium products in malaria-induced bone disorder. (A) Bone marrow smears of PbA- and pm4bp2-infected mice during convalescence. Scale bars, 20 m. Arrows show the accu-mulated birefringent hemozoin. (B) Course of infection with WT and mutant parasites. Red arrows indi-cate the time of chloroquine (CQ) treatment. (C) Representative CT images and bone morphometric analysis of distal femurs of WT mice. (D to F) Bone volume of distal femurs (D), blood parasitemia (E), and blood count (F) of Rag2−/− mice upon sac-rifice. (G) Cytokine and Tnfsf11 expres-sions in OB culture after a 16-hour stimulation with crude extracts con-taining 1, 2, and 5 × 107 infected erythrocytes. (H) Transmission elec-tron microscopy (TEM) images of phagocytosis of sHZ by OCP and ma-ture OC in in vitro cultures. Red ar-rows show the phagocytosed sHZ. (I and J) Cytokines expression in Myd88+/− and Myd88−/− OCP and OC cultures (I) and OBP and OB cultures (J) after a 6-hour stimulation with sHZ at 20, 50, and 100 g/ml. (K) Tnfsf11 expression in OB culture after a 24-hour stimulation with sHZ. Data are means ± SD, repeated at least twice with similar results. *P < 0.05, **P < 0.01, ***P < 0.001, Kruskal-Wallis test and Dunn’s post-test (C, D, and F) and Mann-Whitney test (E). by guest on June 4, 2017

http://imm

unology.sciencemag.org/

Dow

nloaded from



8 of 11

Jdp2−/− mice, which lack mature OCs (22, 45), did not experience bone loss during infection. Overall, phagocytosis of Plasmodium products, even in the absence of active infection, can lead to chronic proinflam-matory cytokine release from OCPs and OBs in bone. These cytokines may synergistically amplify RANKL expression, leading to overproduction of OCs and an imbalance in bone resorption and bone formation, ulti-mately resulting in bone loss.

Our data further suggest that Myd88 is involved in this bone pa-thology. Plasmodium products fail to elicit an inflammatory response in Myd88−/− OBs and OCPs, and Myd88−/− mice had intact bones. TLR9, NLRP3, or IL-1 signaling alone had a minimal role in bone loss, suggesting that an as yet unknown Plasmodium recognition ma-chinery upstream of MyD88 may be involved or possibly several simultaneous signaling pathways may act through MyD88 to induce bone loss. One possible Plasmodium component that contributes to

this pathological bone loss is hemozoin. The absence of bone loss in mice infected with a mutant Plasmodium parasite with strongly re-duced hemozoin production and reduced virulence (27) gives rise to two possible mechanisms: (i) a direct effect of hemozoin on bone loss or (ii) the involvement of Plasmodium antigens as virulence fac-tors in bone loss. Plasmepsin-4 and bergheipain-2 proteases are involved in the degradation of hemoglobin. Deficiency of these two proteases causes a significant reduction of hemozoin production due to inef-ficient hemoglobin digestion, yet the parasites can still survive by possibly acquiring proteins from other sources (27). Besides the activa-tion of bone cells by hemozoin, the concurrent recognition of parasite proteases by another receptor may also involve Myd88 signaling (46), which may further synergize to contribute to the pathologic bone dis-order. Because hemozoin persists in the bone marrow, it can possi-bly sustain the immune response initiated by the parasite antigens,

Fig. 6. Malaria-induced bone loss can be prevented by alfacalcidol treatment. (A) Experimental design of alfacalcidol treatment after PyNL infection. (B and C) Rep-resentative CT images (B) and bone morphometric analysis (C) of distal femur of naïve and PyNL-infected mice with and without alfacalcidol treatment. (D and E) Cyto-kine expressions of OCP (D) and OBP (E) culture stimulated with PfCE at 1 mg/ml) for 6 hours in the presence of 10, 50, and 100 M alfacalcidol. (F) Parasitemia of PyNL-infected mice with and without alfacalcidol treatment. (G) In vitro antimalarial effect of alfacalcidol on P. falciparum (3D7) culture at indicated doses. Each data point represents individual mouse. Data are means ± SD, repeated at least twice. *P < 0.05, **P < 0.01, Kruskal-Wallis test with Dunn’s posttest.


munology.sciencem

ag.org/D

ownloaded from



9 of 11

given that our group had previously shown the adjuvanticity of hemo-zoin (28, 42). Further work is needed to determine which mechanism is involved.

Although chloroquine is a common antimalarial and has a pro-tective effect on bone (47), it failed to prevent malaria-induced bone loss. Alternatively, we showed that alfacalcidol, a VitD3 derivative, protects Plasmodium-infected mice from losing bone. Alfacalcidol has been safely and effectively used to treat osteoporosis by promot-ing osteoblastic activity and suppressing OC formation (32), possi-bly by decreasing the RANKL expression on OB (48), and restricting the pool of OCPs in the bone marrow (49). VitD has also been shown to have immunomodulatory effects, including suppression of inflam-mation (50). Consistent with this, our data showed the suppression of Plasmodium product–induced inflammatory cytokines in vitro. VitD derivatives, including alfacalcidol, can restrict parasite growth directly (51). Our in vivo and in vitro data support this notion, but further work is needed to understand the mechanism(s) involved in the antiplasmodial effect of alfacalcidol.

On the basis of our current findings, we postulate that malaria infection and resultant Plasmodium products accumulation in the bone marrow modulate both the immune response and bone homeo-stasis, which eventually leads to bone loss. Sustained bone loss does not seem to ameliorate even after recovery from malaria, suggesting that malaria-infected patients are likely at risk of bone deterioration. These observations suggest that antimalarials coupled with bone ther-

apy may be beneficial in improving bone health in malaria-infected individuals.

MATERIALS AND METHODSStudy designThis study aimed at the characterization of the effects of malaria infection on bone by using various Plasmodium spp. in mice. Ani-mal experiments were carried out according to the guidelines of the Immunology Frontier Research Center and Research Institute for Microbial Diseases of Osaka University and the National Institutes of Biomedical Innovation, Health and Nutrition. Age- and gender- matched littermates of the same strain were used in each experiment. Experimental replicates were indicated in the figure legends. Blinded bone histomorphological analysis was conducted by the Niigata Bone Science Institute. No other randomization was performed in animal experiments.

MiceFemale BALB/c and male C57BL/6J WT mice were purchased from CLEA Japan. Female BALB/c Myd88−/− and Tlr9−/− mice as well as male C57BL/6J Rag2−/−, Il1r−/−, and Nlrp3−/− and female Jdp2−/− mice were used as described (22, 52).

ReagentssHZ was produced as described (28). Hemin (Fluka) was dissolved in 0.01 M NaOH. PfCE was prepared from schizont stage–infected eryth-rocytes (42). PbACE and PbApm4bp2 crude extract (pm4bp2CE) were prepared from infected mice blood at high parasitemia. Leukocytes and platelets were removed using Plasmodipur filters (EuroProxima). Crude extracts were subjected to five cycles of freeze-thaw to release the Plasmodium components. Alfacalcidol (Wako) was dissolved in absolute ethanol to 20 mg/ml and further diluted in medium-chain triglyceride (Nisshin OilliO).

Plasmodium infection and treatmentsMice were inoculated intraperitoneally with 1 × 105 PyNL-, Pcc-, PbA- (12, 53), or PbApm4bp2-infected erythrocytes (27). PbA-infected mice were treated with chloroquine (50 mg/kg) (Sigma-Aldrich) in-traperitoneally, as indicated. PyNL-infected mice were sacrificed on days 14, 30, and 90 after infection; Pcc-infected mice on days 8, 30, and 90 after infection; and PbA- and PbApm4bp2-infected WT mice on day 30 after infection. PbA- and PbApm4bp2-infected Rag2−/− mice were sacrificed on days 27 and 33 after infection, re-spectively, at similar parasitemia. EDTA-treated blood was analyzed by complete blood counter (Horiba LC-662). Three-week-old PyNL- infected mice were treated with chloroquine (25 mg/kg), as indicated. Femur length was measured using a digital micrometer caliper (As One Japan). Mice were orally administered with alfacalcidol (400 and 1000 ng/kg) daily from day 2 to day 14 after infection, and the doses were then reduced to 100 and 400 ng/kg from day 11 up to day 30.

Bone morphometric analysisEthanol-fixed femurs were scanned by a three-dimensional CT using Scan-Xmate RB080SS110 scanner (Comscan) and analyzed using TRI/3D-Bon software (Ratoc System Engineering). For bone histo-morphometric analysis, mice were injected intraperitoneally with cal-cein (16 mg/kg) (Dojindo) in 2% sodium hydrogen carbonate at 96 and 24 hours before sacrifice. Methylmethacrylate-embedded tibia

Fig. 7. Proposed schematic mechanism of malaria-induced bone loss. During acute Plasmodium infection, several malaria infection–related factors may have a deleterious effect on bone tissue, resulting in the suppression of both OC and OB differentiation. One example molecule is heme, which is released abundantly during hemolysis of infected and noninfected erythrocytes and strongly inhibits both OC and OB. Also, cytokines such as IL-4, IL-10, IL-13, and IFN released from activated T cells in response to Plasmodium infection disrupt the signaling in os-teoclastogenesis. During convalescent and chronic phase of Plasmodium infection, although parasites are cleared systemically, parasite products remain and gradually accumulate in the bone marrow, leading to MyD88-dependent chronic inflamma-tion in OCPs, OBPs, and mature OBs. Plasmodium product–induced inflammatory responses synergistically up-regulate RANKL expression on OBs, which further pro-motes osteoclastogenesis. The robust activation of OCs skews bone remodeling toward bone resorption and causes chronic bone loss.


munology.sciencem

ag.org/D

ownloaded from



10 of 11

samples were sectioned and analyzed by the Niigata Bone Science Institute, Japan.

In vitro culture of bone marrow OCBone marrow–derived macrophages (OCPs) were maintained with macrophage colony-stimulating factor (M-CSF) (10 ng/ml) (PeproTech) in -minimum essential medium (-MEM) supplemented with 10% fetal calf serum (54). OCs were formed after a 4-day stimulation with M-CSF (10 ng/ml) and RANKL (50 ng/ml) (R&D Systems). Cells were fixed and stained with an acid phosphatase staining kit (Sigma-Aldrich). TRAP+ multinucleated OCs (more than three nu-clei) were counted under light microscope, and images were captured at ×10 magnification. Cells were stimulated for 6 hours, as indicated. In some experiments, alfacalcidol was added into the culture 1 hour before stimulation.

In vitro calvarial OB cultureCalvarial OB was isolated from newborn mice by sequential diges-tion in -MEM containing 0.1% collagenase and 0.2% dispase at 37°C with continuous shaking (54). Calvarial cells were cultured in RPMI 1640 supplemented with an OB inducer reagent (Takara). Culture medium was replenished every 3 days. After 20 days, cells were stim-ulated for 6 or 24 hours as indicated.

Gene expression analysis by quantitative reverse transcription PCRRNAs were extracted from cultured cells and whole bone. Gene ex-pressions were analyzed by qPCR and normalized to the expression level of 18S rRNA, as described previously (12). The expression level of P. yoelii 18S rRNA (Py18S) was measured using a customized probe and primers (53, 55).

Statistical analysesData are expressed as means ± SD. Data points represent individual mouse. Statistical significance between two groups was analyzed using Mann-Whitney test. Kruskal-Wallis test and Dunn’s posttest were performed when more than two groups were compared. Significances are represented as *P < 0.05, **P < 0.01, and ***P < 0.001. All statis-tical analyses were performed using GraphPad Prism 5.0.

SUPPLEMENTARY MATERIALSimmunology.sciencemag.org/cgi/content/full/2/12/eaam8093/DC1Materials and MethodsFig. S1. Bone loss in P. chabaudi infection and suppression of bone growth in PyNL-infected young mice.Fig. S2. Gene expression of inflammatory cytokines in the bone.Fig. S3. PyNL crude extract elicits inflammation in OCPs, OBPs, and OBs.Fig. S4. Comparison between Myd88+/− and Myd88−/− mice.Fig. S5. Chloroquine does not rescue Plasmodium-infected mice from bone loss.Fig. S6. Serum cytokines of Rag2−/− mice infected with WT PbA and mutant PbApm4bp2.Fig. S7. Bone marrow infection of PbA-infected and mutant pm4bp2-infected mice.Table S1. Possible association between malaria infection and child growth.Table S2. Excel file containing source data in tabular format for all figures.

REFERENCES AND NOTES 1. C. P. Lewis, C. B. D. Lavy, W. J. Harrison, Delay in skeletal maturity in Malawian children.

J. Bone Joint Surg. Br. 84, 732–734 (2002). 2. A. D. LaBeaud, M. Nayakwadi Singer, M. McKibben, P. Mungai, E. M. Muchiri, E. McKibben,

G. Gildengorin, L. J. Sutherland, C. H. King, C. L. King, I. Malhotra, Parasitism in children aged three years and under: Relationship between infection and growth in rural coastal Kenya. PLOS Negl. Trop. Dis. 9, e0003721 (2015).

3. N. E. Smith-Guzmán, The skeletal manifestation of malaria: An epidemiological approach using documented skeletal collections. Am. J. Phys. Anthropol. 158, 624–635 (2015).

4. R. Joice, S. K. Nilsson, J. Montgomery, S. Dankwa, E. Egan, B. Morahan, K. B. Seydel, L. Bertuccini, P. Alano, K. C. Williamson, M. T. Duraisingh, T. E. Taylor, D. A. Milner, M. Marti, Plasmodium falciparum transmission stages accumulate in the human bone marrow. Sci. Transl. Med. 6, 244re5 (2014).

5. T. Imai, H. Ishida, K. Suzue, T. Taniguchi, H. Okada, C. Shimokawa, H. Hisaeda, Cytotoxic activities of CD8+ T cells collaborate with macrophages to protect against blood-stage murine malaria. eLife 4, e04232 (2015).

6. H. Yasuda, N. Shima, N. Nakagawa, K. Yamaguchi, M. Kinosaki, S.-i. Mochizuki, A. Tomoyasu, K. Yano, M. Goto, A. Murakami, E. Tsuda, T. Morinaga, K. Higashio, N. Udagawa, N. Takahashi, T. Suda, Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl. Acad. Sci. U.S.A. 95, 3597–3602 (1998).

7. M. Asagiri, K. Sato, T. Usami, S. Ochi, H. Nishina, H. Yoshida, I. Morita, E. F. Wagner, T. W. Mak, E. Serfling, H. Takayanagi, Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J. Exp. Med. 202, 1261–1269 (2005).

8. M. M. Guerrini, H. Takayanagi, The immune system, bone and RANKL. Arch. Biochem. Biophys. 561, 118–123 (2014).

9. T. Kawai, T. Matsuyama, Y. Hosokawa, S. Makihira, M. Seki, N. Y. Karimbux, R. B. Goncalves, P. Valverde, S. Dibart, Y.-P. Li, L. A. Miranda, C. W. O. Ernst, Y. Izumi, M. A. Taubman, B and T lymphocytes are the primary sources of RANKL in the bone resorptive lesion of periodontal disease. Am. J. Pathol. 169, 987–998 (2006).

10. K. Titanji, A. Vunnava, A. N. Sheth, C. Delille, J. L. Lennox, S. E. Sanford, A. Foster, A. Knezevic, K. A. Easley, M. N. Weitzmann, I. Ofotokun, Dysregulated B cell expression of RANKL and OPG correlates with loss of bone mineral density in HIV infection. PLOS Pathog. 10, e1004497 (2014).

11. A. Widaa, T. Claro, T. J. Foster, F. J. O’Brien, S. W. Kerrigan, Staphylococcus aureus protein a plays a critical role in mediating bone destruction and bone loss in osteomyelitis. PLOS ONE 7, e40586 (2012).

12. H. Zhao, A. Konishi, Y. Fujita, M. Yagi, K. Ohata, T. Aoshi, S. Itagaki, S. Sato, H. Narita, N. H. Abdelgelil, M. Inoue, R. Culleton, O. Kaneko, A. Nakagawa, T. Horii, S. Akira, K. J. Ishii, C. Coban, Lipocalin 2 bolsters innate and adaptive immune responses to blood-stage malaria infection by reinforcing host iron metabolism. Cell Host Microbe 12, 705–716 (2012).

13. B. Shikur, W. Deressa, B. Lindtjørn, Association between malaria and malnutrition among children aged under-five years in Adami Tulu District, south-central Ethiopia: A case-control study. BMC Public Health 16, 174 (2016).

14. I. U. N. Sumbele, O. S. M. Bopda, H. K. Kimbi, T. R. Ning, T. Nkuo-Akenji, Nutritional status of children in a malaria meso endemic area: Cross sectional study on prevalence, intensity, predictors, influence on malaria parasitaemia and anaemia severity. BMC Public Health 15, 1099 (2015).

15. M. A. A. Alexandre, S. G. Benzecry, A. M. Siqueira, S. Vitor-Silva, G. C. Melo, W. M. Monteiro, H. P. Leite, M. V. G. Lacerda, M. d. G. C. Alecrim, The association between nutritional status and malaria in children from a rural community in the Amazonian region: A longitudinal study. PLOS Negl. Trop. Dis. 9, e0003743 (2015).

16. D. P. Mathanga, K. E. Halliday, M. Jawati, A. Verney, A. Bauleni, J. Sande, D. Ali, R. Jones, S. Witek-McManus, N. Roschnik, S. J. Brooker, The high burden of malaria in primary school children in southern Malawi. Am. J. Trop. Med. Hyg. 93, 779–789 (2015).

17. H. Kang, B. Kreuels, O. Adjei, R. Krumkamp, J. May, D. S. Small, The causal effect of malaria on stunting: A Mendelian randomization and matching approach. Int. J. Epidemiol. 42, 1390–1398 (2013).

18. F. O. Ter Kuile, D. J. Terlouw, S. K. Kariuki, P. A. Phillips-Howard, L. B. Mirel, W. A. Hawley, J. F. Friedman, Y. P. Shi, M. S. Kolczak, A. A. Lal, J. M. Vulule, B. L. Nahlen, Impact of permethrin-treated bed nets on malaria, anemia, and growth in infants in an area of intense perennial malaria transmission in western Kenya. Am. J. Trop. Med. Hyg. 68, 68–77 (2003).

19. E. Villamor, R. Mbise, D. Spiegelman, E. Hertzmark, M. Fataki, K. E. Peterson, G. Ndossi, W. W. Fawzi, Vitamin A supplements ameliorate the adverse effect of HIV-1, malaria, and diarrheal infections on child growth. Pediatrics 109, e6 (2002).

20. A. Ferreira, J. Balla, V. Jeney, G. Balla, M. P. Soares, A central role for free heme in the pathogenesis of severe malaria: The missing link? J. Mol. Med. 86, 1097–1111 (2008).

21. A. J. Cunnington, J. B. de Souza, M. Walther, E. M. Riley, Malaria impairs resistance to Salmonella through heme- and heme oxygenase–dependent dysfunctional granulocyte mobilization. Nat. Med. 18, 120–127 (2011).

22. K. Maruyama, M. Fukasaka, A. Vandenbon, T. Saitoh, T. Kawasaki, T. Kondo, K. K. Yokoyama, H. Kidoya, N. Takakura, D. Standley, O. Takeuchi, S. Akira, The transcription factor Jdp2 controls bone homeostasis and antibacterial immunity by regulating osteoclast and neutrophil differentiation. Immunity 37, 1024–1036 (2012).

23. J. Lam, S. Takeshita, J. E. Barker, O. Kanagawa, F. P. Ross, S. L. Teitelbaum, TNF- induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J. Clin. Invest. 106, 1481–1488 (2000).


munology.sciencem

ag.org/D

ownloaded from



11 of 11

24. S. Wei, H. Kitaura, P. Zhou, F. P. Ross, S. L. Teitelbaum, IL-1 mediates TNF-induced osteoclastogenesis. J. Clin. Invest. 115, 282–290 (2005).

25. C. Coban, K. J. Ishii, T. Horii, S. Akira, Manipulation of host innate immune responses by the malaria parasite. Trends Microbiol. 15, 271–278 (2007).

26. M. Olivier, K. Van Den Ham, M. T. Shio, F. A. Kassa, S. Fougeray, Malarial pigment hemozoin and the innate inflammatory response. Front. Immunol. 5, 25 (2014).

27. J.-w. Lin, R. Spaccapelo, E. Schwarzer, M. Sajid, T. Annoura, K. Deroost, R. B. G. Ravelli, E. Aime, B. Capuccini, A. M. Mommaas-Kienhuis, T. O’Toole, F. Prins, B. M. D. Franke-Fayard, J. Ramesar, S. Chevalley-Maurel, H. Kroeze, A. J. Koster, H. J. Tanke, A. Crisanti, J. Langhorne, P. Arese, P. E. Van den Steen, C. J. Janse, S. M. Khan, Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance. J. Exp. Med. 212, 893–903 (2015).

28. M. S. J. Lee, Y. Igari, T. Tsukui, K. J. Ishii, C. Coban, Current status of synthetic hemozoin adjuvant: A preliminary safety evaluation. Vaccine 34, 2055–2061 (2016).

29. S. Christakos, M. Hewison, D. G. Gardner, C. L. Wagner, I. N. Sergeev, E. Rutten, A. G. Pittas, R. Boland, L. Ferrucci, D. D. Bikle, Vitamin D: Beyond bone. Ann. N. Y. Acad. Sci. 1287, 45–58 (2013).

30. S. E. Cusick, R. O. Opoka, T. C. Lund, C. C. John, L. E. Polgreen, Vitamin D insufficiency is common in Ugandan children and is associated with severe malaria. PLOS ONE 9, e113185 (2014).

31. X. He, J. Yan, X. Zhu, Q. Wang, W. Pang, Z. Qi, M. Wang, E. Luo, D. M. Parker, M. T. Cantorna, L. Cui, Y. Cao, Vitamin D inhibits the occurrence of experimental cerebral malaria in mice by suppressing the host inflammatory response. J. Immunol. 193, 1314–1323 (2014).

32. A. Shiraishi, S. Takeda, T. Masaki, Y. Higuchi, Y. Uchiyama, N. Kubodera, K. Sato, K. Ikeda, T. Nakamura, T. Matsumoto, E. Ogata, Alfacalcidol inhibits bone resorption and stimulates formation in an ovariectomized rat model of osteoporosis: Distinct actions from estrogen. J. Bone Miner. Res. 15, 770–779 (2000).

33. M. Rix, P. Eskildsen, K. Olgaard, Effect of 18 months of treatment with alfacalcidol on bone in patients with mild to moderate chronic renal failure. Nephrol. Dial. Transplant. 19, 870–876 (2004).

34. D. F. Robbiani, S. Deroubaix, N. Feldhahn, T. Y. Oliveira, E. Callen, Q. Wang, M. Jankovic, I. T. Silva, P. C. Rommel, D. Bosque, T. Eisenreich, A. Nussenzweig, M. C. Nussenzweig, Plasmodium infection promotes genomic instability and AID-dependent B cell lymphoma. Cell 162, 727–737 (2015).

35. M. Asghar, D. Hasselquist, B. Hansson, P. Zehtindjiev, H. Westerdahl, S. Bensch, Hidden costs of infection: Chronic malaria accelerates telomere degradation and senescence in wild birds. Science 347, 436–438 (2015).

36. V. Walker Harris, T. T. Brown, Bone loss in the HIV-infected patient: Evidence, clinical implications, and treatment strategies. J. Infect. Dis. 205, S391–S398 (2012).

37. H. Takayanagi, K. Ogasawara, S. Hida, T. Chiba, S. Murata, K. Sato, A. Takaoka, T. Yokochi, H. Oda, K. Tanaka, K. Nakamura, T. Taniguchi, T-cell-mediated regulation of osteoclastogenesis by signalling cross-talk between RANKL and IFN-. Nature 408, 600–605 (2000).

38. R. Moreau, D. Tshikudi Malu, M. Dumais, E. Dalko, V. Gaudreault, H. Roméro, C. Martineau, O. Kevorkova, J. S. Dardon, E. L. Dodd, D. S. Bohle, T. Scorza, Alterations in bone and erythropoiesis in hemolytic anemia: Comparative study in bled, phenylhydrazine-treated and Plasmodium-infected mice. PLOS ONE 7, e46101 (2012).

39. T.-H. Lin, C.-H. Tang, S.-Y. Hung, S.-H. Lui, Y.-M. Lin, W.-M. Fu, R.-S. Yang, Upregulation of heme oxygenase-1 inhibits the maturation and mineralization of osteoblasts. J. Cell. Physiol. 222, 757–768 (2010).

40. J. Zwerina, S. Tzima, S. Hayer, K. Redlich, O. Hoffmann, B. Hanslik-Schnabel, J. S. Smolen, G. Kollias, G. Schett, Heme oxygenase 1 (HO-1) regulates osteoclastogenesis and bone resorption. FASEB J. 19, 2011–2013 (2005).

41. R. Frita, D. Carapau, M. M. Mota, T. Hänscheid, In vivo hemozoin kinetics after clearance of Plasmodium berghei infection in mice. Malar. Res. Treat. 2012, 373086 (2012).

42. C. Coban, Y. Igari, M. Yagi, T. Reimer, S. Koyama, T. Aoshi, K. Ohata, T. Tsukui, F. Takeshita, K. Sakurai, T. Ikegami, A. Nakagawa, T. Horii, G. Nuñez, K. J. Ishii, S. Akira, Immunogenicity of whole-parasite vaccines against Plasmodium falciparum involves malarial hemozoin and host TLR9. Cell Host Microbe 7, 50–61 (2010).

43. K. Redlich, J. S. Smolen, Inflammatory bone loss: Pathogenesis and therapeutic intervention. Nat. Rev. Drug Discov. 11, 234–250 (2012).

44. K. Kobayashi, N. Takahashi, E. Jimi, N. Udagawa, M. Takami, S. Kotake, N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shima, H. Yasuda, T. Morinaga, K. Higashio, T. J. Martin,

T. Suda, Tumor necrosis factor stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL–RANK interaction. J. Exp. Med. 191, 275–286 (2000).

45. R. Kawaida, T. Ohtsuka, J. Okutsu, T. Takahashi, Y. Kadono, H. Oda, A. Hikita, K. Nakamura, S. Tanaka, H. Furukawa, Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factor, mediates osteoclast differentiation induced by RANKL. J. Exp. Med. 197, 1029–1035 (2003).

46. P. Rallabhandi, Q. M. Nhu, V. Y. Toshchakov, W. Piao, A. E. Medvedev, M. D. Hollenberg, A. Fasano, S. N. Vogel, Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: A novel paradigm for receptor cooperativity. J. Biol. Chem. 283, 24314–24325 (2008).

47. A. A. Al-bari, M. Shinohara, Y. Nagai, H. Takayanagi, Inhibitory effect of chloroquine on bone resorption reveals the key role of lysosomes in osteoclast differentiation and function. Inflamm. Regen. 32, 222–231 (2012).

48. N. Takahashi, N. Udagawa, T. Suda, Vitamin D endocrine system and osteoclasts. Bonekey Rep. 3, 495 (2014).

49. T. Shibata, A. Shira-Ishi, T. Sato, T. Masaki, A. Masuda, A. Hishiya, N. Ishikura, S. Higashi, Y. Uchida, M.-O. Saito, M. Ito, E. Ogata, K. Watanabe, K. Ikeda, Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow. J. Bone Miner. Res. 17, 622–629 (2002).

50. Y. Zhang, D. Y. M. Leung, B. N. Richers, Y. Liu, L. K. Remigio, D. W. Riches, E. Goleva, Vitamin D inhibits monocyte/macrophage proinflammatory cytokine production by targeting MAPK phosphatase-1. J. Immunol. 188, 2127–2135 (2012).

51. H. J. Vial, M. J. Thuet, J. R. Philippot, Inhibition of the in vitro growth of Plasmodium falciparum by D vitamins and vitamin D-3 derivatives. Mol. Biochem. Parasitol. 5, 189–198 (1982).

52. M. Onishi, K. Ozasa, K. Kobiyama, K. Ohata, M. Kitano, K. Taniguchi, T. Homma, M. Kobayashi, A. Sato, Y. Katakai, Y. Yasutomi, E. Wijaya, Y. Igarashi, N. Nakatsu, W. Ise, T. Inoue, H. Yamada, A. Vandenbon, D. M. Standley, T. Kurosaki, C. Coban, T. Aoshi, E. Kuroda, K. J. Ishii, Hydroxypropyl--cyclodextrin spikes local inflammation that induces Th2 cell and T follicular helper cell responses to the coadministered antigen. J. Immunol. 194, 2673–2682 (2015).

53. H. Zhao, T. Aoshi, S. Kawai, Y. Mori, A. Konishi, M. Ozkan, Y. Fujita, Y. Haseda, M. Shimizu, M. Kohyama, K. Kobiyama, K. Eto, J. Nabekura, T. Horii, T. Ishino, M. Yuda, H. Hemmi, T. Kaisho, S. Akira, M. Kinoshita, K. Tohyama, Y. Yoshioka, K. J. Ishii, C. Coban, Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria. Cell Host Microbe 15, 551–563 (2014).

54. K. Maruyama, T. Kawagoe, T. Kondo, S. Akira, O. Takeuchi, TRAF family member-associated NF-B activator (TANK) is a negative regulator of osteoclastogenesis and bone formation. J. Biol. Chem. 287, 29114–29124 (2012).

55. S. Schussek, P. L. Groves, S. H. Apte, D. L. Doolan, Highly sensitive quantitative real-time PCR for the detection of Plasmodium liver-stage parasite burden following low-dose sporozoite challenge. PLOS ONE 8, e77811 (2013).

Acknowledgments: We thank K. Honjo for insights into public data evaluation, and K. Matsuda and H. Omori for help with TEM. Funding: This work was supported by the Grants-in-Aid for Scientific Research (B) (grant no. 16H05181 to C.C.), the Japan Agency for Medical Research and Development (to C.C. and K.J.I.), the Uehara Memorial and Yamada Science Foundations (to C.C.), the Grant-in-Aid for Young Scientists (A) (grant no. 15H05686 to K.M.). M.S.J.L. is the recipient of the Japanese Government Scholarship (Ministry of Education, Culture, Sports, Science and Technology). Author contributions: M.S.J.L. conducted all experiments and statistical analyses. K.M. helped with bone studies; Y.F. with qPCR; and A.K., P.M.L., and S.I. with infections. K.M., T.H., E.K., S.A., K.J.I., J.-w.L., and S.M.K. contributed critical reagents. M.S.J.L. and C.C. wrote the manuscript, and C.C. provided overall supervision. All authors contributed to critical revision of the manuscript. Competing interests: The authors declare that they have no competing financial interests.

Submitted 19 January 2017Accepted 28 April 2017Published 2 June 201710.1126/sciimmunol.aam8093

Citation: M. S. J. Lee, K. Maruyama, Y. Fujita, A. Konishi, P. M. Lelliott, S. Itagaki, T. Horii, J.-w. Lin, S. M. Khan, E. Kuroda, S. Akira, K. J. Ishii, C. Coban, Plasmodium products persist in the bone marrow and promote chronic bone loss. Sci. Immunol. 2, eaam8093 (2017).


munology.sciencem

ag.org/D

ownloaded from

doi: 10.1126/sciimmunol.aam80932, (2017) Sci. Immunol.

Cevayir CobanShahid M. Khan, Etsushi Kuroda, Shizuo Akira, Ken J. Ishii andPatrick M. Lelliott, Sawako Itagaki, Toshihiro Horii, Jing-wen Lin, Michelle S. J. Lee, Kenta Maruyama, Yukiko Fujita, Aki Konishi,bone lossbone marrow and promote chronic

products persist in thePlasmodium

bone therapies with antimalarial drugs may prevent bone loss in infected individuals. with alfacalcidol, a vitamin D3 analog, could prevent this bone loss, suggesting that combiningin osteoclast and osteoblast precursors, resulting in bone resorption. Treating infected animals bone loss. Mechanistically, these products induced MyD88-dependent inflammatory responsesloss. Infection with a mutant Plasmodium that lacked the by-product hemozoin did not induce et al. now report that Plasmodium by-products retained in the bone marrow lead directly to boneinfection may develop long-term consequences, such as bone loss and growth retardation. Lee Editor's Summary Plasmodium leftovers cause bone lossIndividuals who recover from malarial

You might find this additional info useful...

55 articles, 19 of which you can access for free at: ’This article citeshttp://immunology.sciencemag.org/content/2/12/eaam8093.full#BIBL

including high resolution figures, can be found at:Updated information and serviceshttp://immunology.sciencemag.org/content/2/12/eaam8093.full

can be found at:Science Immunology about Additional material and informationhttp://www.sciencemag.org/journals/immunology/mission-and-scope

This information is current as of June 4, 2017.

trademark of AAASthe Advancement of Science; all rights reserved. Science Immunology is a registered York Avenue NW, Washington, DC 20005. Copyright 2016 by The American Association forpublished by the American Association for the Advancement of Science (AAAS), 1200 New

(ISSN 2375-2548) publishes new articles weekly. The journal isScience Immunology


munology.sciencem

ag.org/D

ownloaded from

infectious disease plasmodium products...

Documents