Pathogens and Disease ISSN 2049-632X

RESEARCH ARTICLE escapes from the phagosomes of infected human osteoclasts reprograms osteoclast development via dysregulation of cytokines and chemokines Akiyoshi Hoshino1,2,3, Sanshiro Hanada3, Hiroyuki Yamada4, Shinji Mii2, Masahide Takahashi2, Satoshi Mitarai4, Kenji Yamamoto3 & Yoshinobu Manome1

1 Department of Molecular Cell Biology, Institute of DNA Medicine, Jikei University School of Medicine, Tokyo, Japan 2 Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya, Japan 3 Vice Director’s Lab, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 4 Department of Mycobacterium Reference and Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Tokyo, Japan

In the context of spinal tuberculosis a study of the inflammatory responses of human multinucleated osteoclasts infected with virulent Mtb is of interest. Intracellular Mtb infection resulted in the rapid growth of Mtb and production of proinflammatory cytokines. In contrast, highly-fused multinucleated osteoclasts incapacitated the production of these cytokines, Mtb escaped from the endosome/phagosome, and led to a different pattern of osteoclast activation with the production of a set of chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation of cytokines and chemokines.

Keywords Abstract chemokines; chemokine receptors; host defense; inflammation; Mycobacterium Spinal tuberculosis is a condition characterized by massive resorption of the spinal tuberculosis; osteoclast. vertebrae due to the infection with Mycobacterium tuberculosis (Mtb). However, the pathogenesis of spinal tuberculosis has not been established because it was Correspondence almost completely eradicated by the establishment of antibiotic treatment in the Kenji Yamamoto, Vice Director’s Lab, mid-20th century. In this study, we investigated the inflammatory responses of Research Institute, National Center for Global human multinucleated osteoclasts infected with virulent Mtb strain. We found that Health and Medicine. Toyama 1-21-1, the intracellular Mtb infection of multinuclear osteoclasts resulted in the rapid Shinjuku-ku Tokyo 162-8655, Japan. growth of Mtb and an osteolytic response, rather than inflammation. In response to Tel.: +81-3-3202-7181 ext2856 Mtb infection, the mononuclear osteoclast precursors produced proinflammatory fax: +81-3-3202-7364 cytokines including tumor necrosis factor (TNF)-a, an intrinsic characteristic they e-mail: [email protected] share with macrophages. In contrast, highly fused multinucleated osteoclasts incapacitated the production of these cytokines. Instead, the intracellular Mtb Received 15 February 2013; revised 3 July inside multinuclear osteoclasts escaped from the endosome/phagosome, leading 2013; accepted 30 July 2013. Final version to a different pattern of osteoclast activation, with the production of chemokines published online 10 September 2013. such as CCL5, CCL17, CCL20, CCL22, CCL24, and CCL25. Moreover, intracel- lular infection with an avirulent Mtb strain resulted in diminished production of doi:10.1111/2049-632X.12082 these chemokines. These findings indicate that intracellular Mtb infection in multinuclear osteoclasts reprograms osteoclast development via the dysregulation Editor: Patrick Brennan of cytokines and chemokines.

Introduction inflammatory destruction (Haynes, 2004), fracture, and collapse of the vertebrae, resulting in massive kyphotic Inflammatory bone diseases are characterized by the deformities. Finally, the spinal canal may become narrower infiltration of immune cells, including lymphocytes, mono- because of , granulation tissue or direct dural cytes, polymorphonuclear leukocytes, and even activated invasion, leading to and neurolog- osteoclasts. Spinal tuberculosis (also called Pott’s disease) ical deficits. is defined as a chronic inflammatory destruction of spinal The mortality from tuberculosis has decreased dramati- , mainly induced by the mycobacterial infection of the cally in developed countries following the introduction of spinal cavity, and is believed to be initiated by the abnormal effective chemotherapeutic agents in the mid-20th century, activation of osteoclasts in the bone tissue, which leads to including , , , ,

28 Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved A. Hoshino et al. Osteoclast dysregulation by M. tuberculosis infection and , before the molecular biology-based path- whereas other studies reported that bacterial stimulation ological mechanism of the disease had been completely suppressed the generation of osteoclast precursors revealed. However, Mycobacterium tuberculosis (Mtb) now (Takami et al., 2002; Ji et al., 2009). Thus, it is still not affects one-third of the world population, and causes almost clear whether bacterial stimulation promotes osteoclasto- 2 million deaths per year; 30% of new cases can be found in genesis. India, China, Africa, and South America (WHO, 2012). In Recently, chemokines have been recognized to be major addition, current treatments are becoming obsolete because factors involved in osteolysis and pathological osteoclasto- of the emergence of drug-resistant strains of Mtb. Thus, the genesis (Oba et al., 2005; Kim et al., 2006). CCL3/MIP-1a development of a new, improved vaccine and/or new drugs induces ectopic osteoclastogenesis in osteolytic lesions of that tackle the emergence of antibiotic resistance are sorely rheumatoid arthritis (Haringman et al., 2006; Menu et al., needed. 2006) and multiple myeloma (Choi et al., 2000; Han et al., There is a question remaining as to whether osteoclastic 2001; Haringman et al., 2006), implying that CCL3 and its activation and differentiation are facilitated at the inflamma- receptor CCR1 act as a crucial chemokine for communica- Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 tory sites of spinal tuberculosis. It is well known that tion between osteoclasts and osteoblasts (Hoshino et al., activated osteoclasts play an important role in inflammatory 2010). The participation of other axes of chemokines– bone destruction, such as that occurring due to rheumatoid chemokine receptors such as CCL2-CCR2 axis (Kim et al., arthritis and inflammatory osteolysis. Similarly, spinal tuber- 2006; Li et al., 2007; Binder et al., 2009), CCL5-CCR5 axis culosis is believed to develop as a result of the activation of (Oba et al., 2005; Menu et al., 2006; Hoshino et al., 2009), osteoclasts in bone tissue in response to Mtb infection, and CX3CL1-CX3CR1 axis (Saitoh et al., 2007; Koizumi as an enormous amount of multinuclear osteoclast-like et al., 2009; Hoshino et al., 2013) also play roles in bone cells surrounding granulomatous caseous necrosis were remodeling. Nowadays, some types of chemokines observed around destroyed bones in a histological analysis. are responsible for pathological bone destruction through In fact, the development of spinal tuberculosis seems to be the regulation of osteoclasts and their precursor cells that related to the infiltration of inflammatory immune cells into are derived from common progenitor cells in bone marrow, the bone tissue. However, there are no data available thus suggesting that several chemokine antagonists provide concerning whether Mtb directly infects the multinuclear a strong rationale for further development of the therapeutic osteoclasts intracellularly. Thus, we assumed that the targets of associated osteolytic bone disease such as source of Mtb-activated osteoclasts in spinal tuberculosis multiple myeloma and rheumatoid arthritis (Oba et al., was as follows; (1) abnormal activation of Mtb-infected 2005; Menu et al., 2006; Dairaghi et al., 2012). These macrophages or their precursors in the inflammatory sites findings are referenced in favor of therapeutic treat- following a local infection, and subsequent activation of the ments targeting chemokines to prevent pathogenic bone RANK-RANKL axis, (2) abnormal induction and extravasa- resorption. tion of Mtb-infected mononuclear osteoclast precursors from To clarify the pathological mechanism underlying the circulating cells into the inflammatory sites due to systemic overactivation of osteoclasts observed in tuberculosis- infection, resulting in abnormal activation, and (3) unex- related osteolytic lesions, we stimulated osteoclasts and pected pathogenic activation and reprogramming of their precursors with virulent living Mtb and investigated the multinuclear osteoclasts resident in the tissue in response expression profiles of chemokines specific for tuberculosis to Mtb infection, followed by the dysregulation of cytokines infection. and chemokines, causing the pathological destruction of the bone tissue. Materials and methods Generally, proinflammatory cytokines, such as tumor necrosis facter (TNF)-a, interleukin (IL)-1b, and IL-6, are Ethical guidelines for human studies produced by macrophages in response to bacterial infec- tion; proinflammatory cytokines also promote osteoclasto- All human experiments were performed with the approval of genesis (Boyle et al., 2003). In the case of rheumatoid the local ethics committees of the Research Institute of arthritis, the RANK-RANKL axis plays a principal role in the National Center for Global Health and Medicine (No. promotion of osteoclast differentiation. This is because the H21-785). higher expression of RANKL in synovial fibroblasts, which is strongly induced by the autoreactive T cells that Cells, materials, and bacteria infiltrated into the synovial tissue, directs pathological osteoclast differentiation via the production of proinflam- Mycobacterium tuberculosis H37Rv virulent laboratory matory cytokines (Takayanagi et al., 2000a, b). Bacterial strain (ATCC 27294), Mycobacterium tuberculosis H37Ra pathogens surged RANKL via Toll-like receptor (TLR)-med- avirulent strain (ATCC 25177), and iated signaling (Kikuchi et al., 2001; Jiang et al., 2002; BCG-Tokyo vaccine strain were cultured at exponential Suda et al., 2002); however, several reports suggested that growth phase in (BD, Sparks, MD) proinflammatory cytokines (Boyle et al., 2003) and several with ADC enrichment (BD) and 0.05% Tween 80 at 37 °C bacterial molecules act as promoters of osteoclastogenesis for 7–10 days. Lipopolysaccharide (LPS) from Escherichia via the enhancement of RANKL through several TLRs coli O55B5 and peptidoglycan (PGN) from Staphylococcus (Kikuchi et al., 2001; Jiang et al., 2002; Suda et al., 2002), aureus were purchased from Sigma Aldrich (St. Louis, MO)

Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 29 Osteoclast dysregulation by M. tuberculosis infection A. Hoshino et al. and Fluka Chemical (St. Gallen, Switzerland), respectively. buffer (PB, pH7.4) for 1 h at 4 °C, rinsed three times with Recombinant human M-CSF and RANKL were purchased PB, and post-fixed with 1% osmium tetroxide. Then, the from R&D Systems Inc. (Minneapolis, MN). Normal human samples were dehydrated with a graded ethanol series, natural mononuclear osteoclast precursors cells (PoieticsTM embedded with Spurr’s resin and polymerized at 70 °C for Osteoclast Precursor Cell System) were purchased from 16 h. Ultrathin sections were cut, stained with uranyl acetate Lonza Walkersville, Inc. (Walkersville, MD) and maintained and lead citrate, and examined with a JEOL JEM-1230 with osteoclast precursor cell basal medium in the presence microscope (Yamada et al., 2001). of 10 ng mLÀ1 M-CSF (without RANKL) and used as mononuclear osteoclast precursors (precursor mononuclear Real-time PCR analysis cells; pMCs). The multinuclear osteoclasts were induced according to the manufacturer’s instructions. Human periph- A real-time quantitative PCR analysis was performed using eral blood monocytes were collected from a healthy volun- an ABI 7700 sequence detector system with the purchased â teer, separated using CD11b MicroBeads (Miltenyi Biotec probe sets (Taqman gene expression analysis system, Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 Inc., Bergisch Gladbach, Germany), and stored at –80 °C Applied Biosystems, Foster City, CA). The sequences were prior to use. amplified for 40 cycles under the following two-step param- eters: denaturation at 95 °C for 15 s, annealing, and extension at 60 °C for 60 s. The relative gene expression Osteoclast culture and intracellular Mtb infection levels were normalized by the expression of GAPDH by the Mononuclear pMCs were cultured with osteoclast precursor 2-D(Ct) method. cell basal medium (Lonza), and the culture medium was replaced every 3 days. The highly fused multinuclear oste- Measurement of cytokines and chemokines, and TRAP oclasts (multinuclear osteoclasts; mOCs) were induced from À À activity mononuclear cells with 10 ng mL 1 M-CSF and 20 ng mL 1 RANKL for 5 days. Intracellular Mtb infection was performed The production of chemokines was determined using â by the co-culture of mononuclear pMCs and multinuclear DuoSet ELISA kits (R&D systems) for CCL17, CCL20, mOCs (5 9 104 cells per well, 1 mL) with 0.5 lLofMtb CCL22, CCL24, and CCL25. The cytokines [TNF-a, IL-1b, H37Rv broth (equivalent to 1.0 9 105 CFU per wells) for IL-6, IL-8, and interferon (IFN)-c] and chemokines (CCL2, 24 h to minimize the influence of bacterial broth, and the CCL3, and CCL5) were measured using the human excess bacteria were rinsed off with phosphate-buffered 27-plex multiple cytokine detection system (Bio-Rad Corp., saline (PBS). The culture supernatants of stimulated oste- Hercules, CA) according to the manufacturer’s instructions. oclasts were harvested, irradiated with UV-C (254-nm Tartrate-resistant acid phosphatase (TRAP) activity in the â wavelength) for 30 s, and filtered with Ultrafree-FC culture supernatant was measured by the Osteolinks TRAP 0.1-lm membrane filters (Merck Millipore, Darmstadt, Ger- assay kit (DS pharma biochemical, Osaka, Japan). many) to remove living Mtb. For immunohistochemical staining, osteoclasts were fixed with 4% paraformaldehyde, Immunohistochemistry permeabilized and stained with the indicated specific Abs or Alexa488-labeled phalloidin (Molecular Probes). The images Paraffin-embedded tissue sections of tuberculosis patients were captured using an IX-81 fluorescent microscopy were deparaffinized in xylene, rehydrated in a graded series (Olympus Corp, Tokyo, Japan) equipped with a confocal of ethanol, and immersed in Target Retrieval Solution pH microscopy DSU unit (Olympus) and analyzed by MetaMor- 9.0 (Dako, Glostrup, Denmark) for 20 min boiling for antigen ph (Universal Imaging corporation, Molecular Devices, retrieval. Then sections were incubated with 10% normal Downingtown, PA). goat serum to avoid nonspecific reaction, and stained with primary antibodies for 2 h. After inactivating the endoge- nous peroxidase activity of tissue sections by 3% hydrogen Ziehl–Neelsen staining peroxide, the signals were visualized using HRP-conjugated For acid-fast bacterial staining, the culture slides (LabTechâ anti-rabbit IgG secondary antibody system (EnVisionTM+ chamber slide system, Nunc-NalgeneTM, Thermo Fisher system, Dako) for 15 min incubation, followed by DAB Scientific, Waltham, MA) were fixed, stained with steps of substrate chromogen system (Dako) with nuclear counter- 0.3% carbol-fuchsin solution, decolorization with a 3% staining using hematoxylin. hydrochloric acid/95% ethanol solution, and counterstaining with Loffrel’s€ methylene blue. The slides were rinsed, Statistical analysis allowed to dry, and examined under a binocular microscope with oil immersion. The data are presented as the means Æ SE and statistical significance was determined with a one-factor factorial ANOVA and Tukey–Kramer’s HSD test in the case of multiple Electron microscopic examination of infected â comparisons, using the KALEIDAGRAPH 4.0 program for osteoclasts WINDOWS (Synergy Software, Reading, PA). A significant Osteoclasts cultured and infected with the Mtb H37Rv strain difference (P < 0.05) is indicated with an asterisk and NS were fixed with 2.5% glutaraldehyde in 100 mM phosphate indicates nonsignificant differences.

30 Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved A. Hoshino et al. Osteoclast dysregulation by M. tuberculosis infection

that induces osteoclast recruitment and inhibits the prolifer- Results and discussion ation of osteoblast precursor cells (Meghji et al., 1997). However, it remains to be elucidated whether Mtb has the Mtb Living were incorporated and expanded inside ability to intracellularly infect multinucleated osteoclasts, multinuclear osteoclasts although osteoclasts may play an important role in the It is well known that macrophages are designed to kill immunopathogenesis of inflammatory bone diseases. pathogens inside the phagosome. After the uptake of To investigate whether living Mtb were incorporated and pathogens into the macrophages by receptor-mediated expanded inside multinuclear osteoclasts, we cultured living phagocytosis, the resulting phagosome undergoes a series Mtb with mature multinuclear osteoclasts (mOCs) and their of fusion and fission events via the endocytosis pathways. It precursor mononuclear pre-osteoclasts (pMCs) (Fig. 1a). is believed that osteoclast precursors could be infected with After co-culture with Mtb, large numbers of bacteria were Mtb intracellularly, which would be followed by activation in incorporated into both mononuclear pMCs and multinuclear the inflammatory lesions of spinal tissues, as well as mOCs at day 3; in addition, Mtb-infected pMCs have ability Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 activation of a conditional immune response in macrophag- to multinucleate at day 5 (Fig. 1a). The results indicated es. Generally, the pathogenesis of spinal tuberculosis has that multinuclear osteoclasts also have ability to uptake been reported to rely on an Mtb-derived chaperonin protein living Mtb inside, as well as mononuclear cells. Next we

(a) Precursor mononuclear cells (pMC) Multinuclear osteoclasts (mOCs) (c)

N day 3 N

40 m N

N

5 μm day 5

(d) N 40 m

(b) 10 Mtb-infected pMCs 8 Mtb-infected mOCs * CFU/well) 6 6 (×10 4 * * 2

M.tuberculosis 0 day 1 day 3 day 5 0.5 μm

Fig. 1 Intracellular growth of Mtb in human mononuclear and multinuclear osteoclasts. (a) Human pMCs and mOCs with living Mtb (6.0 9 106 CFU per well) for 3 or 5 days were stained red with Ziehl–Neelsen stain. Nuclei were counterstained with methylene blue. Magnification 91000 with oil immersion. Scale bar: 40 lm. (b) The CFU numbers of intracellularly infected Mtb by pMCs (open column) and mOCs (filled column) were quantified by bacterial culture using a mycobacteria growth indicator glass tube with Ogawa media. (c,d) Electron microscopic images of multinuclear osteoclasts infected with living Mtb. The low magnification electron micrograph shows Mtb phagocytosed by an mOC (c), and an enlarged image (d) of the area indicated by the rectangle, where five tubercle bacilli reside in the osteoclast cytoplasm without phagosome membrane (black arrrows). Free lysosomes are also distributed in the cytoplasm near tubercle bacilli (white arrows). N, nucleus of the osteoclast.

Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 31 Osteoclast dysregulation by M. tuberculosis infection A. Hoshino et al. investigated the numbers of intracellular Mtb, since Mtb was Living Mtb have the ability to facilitate osteolytic reported previously to grow rapidly inside macrophages (van response by multinuclear osteoclasts rather than der Wel et al., 2007). The numbers of intracellular Mtb proinflammatory responses increased after a five day culture of the osteoclasts. Notably, Mtb grew better in multinuclear osteoclasts than in We investigated whether proinflammatory responses mononuclear cells; there were four times as many intracel- were induced by intracellular Mtb-infected multinuclear lular Mtb in the mOCs than in pMCs on day 5 of culture osteoclasts. To assess the productivity of proinflammatory (Fig. 1b). These results suggest that living Mtb has a greater cytokines during osteoclastogenesis in response to living ability to expand intracellularly following the infection of Mtb, the production of proinflammatory cytokines (TNF-a multinuclear osteoclasts than mononuclear osteoclast and IL-1b) and proinflammatory chemokines (MCP-1/CCL2 precursors. and MIP-1a/CCL3) in response to intracellular Mtb infection We next evaluated the intracellular localization of the living by mOCs were measured (Fig. 2a and b). We found that

Mtb in multinuclear osteoclasts using electron microscopic Mtb-infected mOCs lost the ability to produce proinflam- Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 analysis, because the intracellular localization of Mtb inside matory cytokines (TNF-a and IL-1b) and proinflammatory the macrophage has been a matter of debate in recent years chemokines (MCP-1/CCL2 and MIP-1a/CCL3), whereas (Welin & Lerm, 2012). Mtb has the ability to grow in Mtb-infected pMCs retained the ability to produce these macrophages by arresting the normal process of phago- proinflammatory factors. Surprisingly, the production level some maturation. As part of their strategy for intracellular of another proinflammatory cytokine IL-6 decreased by half, survival, mycobacteria prevent the maturation of the phago- suggesting that the basal level of IL-6 production of pMCs somes, in which they reside inside macrophages. Notably, was dampened by the early phase of Mtb infection. we observed no significant phagosome membrane around Notably, we detected no IFN-c production during osteocl- the intracellular Mtb in our study (Fig. 1c and d). These data astogenesis (data not shown), although IFN-c is involved in demonstrated that Mtb escapes from osteoclastic phago- Th1-mediated immune response and is followed by the somes, whereas the bacteria were arrested in phagosomes formation of granulomatous caseous necrosis observed of macrophages. Escape from phagosomes could drive the around destroyed bone tissue (Schluger & Rom, 1998). more rapid expansion of Mtb in osteoclasts. During our The data demonstrate that the production patterns of electron microscopic analysis, we did not observed any proinflammatory cytokines in inflammatory sites with bone significant phagosome membrane formation around intra- destruction in tuberculosis lesions might be different from cellular Mtb (Fig. 1d). It is well known that Mtb survives those of other inflammatory conditions, such as rheumatoid inside macrophage phagosomes, whose normal maturation arthritis. process is arrested by the bacteria (Fratti et al., 2001; Pethe In addition, the enzymatic activity of tartrate-resistant acid et al., 2004). Thus, Mtb interrupts the acidification of phosphatase (TRAP), which is the principal osteolytic endosomes, followed by the fusion of acidic endosomes enzyme secreted in the culture supernatant, was measured and lysosomes. As a result, the immature endosomes (Fig. 2c). In pMCs, elevated TRAP activity was observed in maintain a high pH (> 6.4) (Pethe et al., 2004), which leads unstimulated pMCs, although Mtb-infected pMCs showed to a failure of the cells to supply hydrolytic enzymes and decreased TRAP production. This was in contrast to several antimicrobial peptides (Rohde et al., 2007a, b). Our previ- previous reports suggesting that TNF-a is an enhancer of ous report also provided support for the hypothesis that Mtb inflammatory osteolysis in bone destructive lesions (Abu- resides inside endosomes to form multilocal necrotic lesions Amer et al., 1997; Lam et al., 2002). As reported previously, in the lung, liver, and spleen tissues (Yamada et al., 2002), bacterial products, LPS (a ligand for TLR4) and Pam3CSK4 and that phagolysosomal fusion incorporating many tubercle (a TLR2 ligand), has the ability to promote TRAP secretion bacilli is prominent (Yamada et al., 2001). In contrast, our by pMCs (Fig. 2d), indicating that osteoclastogenesis is current results demonstrated that there are Mtb-containing accelerated not only by TNF-a but also by TLR-ligands. phagosomes/endosomes inside the multinuclear osteo- However, intracellular Mtb infection in mOCs sustained, clasts (see Fig. 1d). The fact that Mtb escapes from the whereas TLR-ligands obstructed, the TRAP secretion (Fig. 2c endosome/phagosome might be due to the endosomal and d). These findings are in agreement with previous acidification; osteoclasts expressed higher amounts of reports that the ligands for TLR4 inhibit osteoclast differen- acid-producing vacuolar type H+-ATPase (V-ATPase) for tiation (Itoh et al., 2003; Chang et al., 2007). osteolysis. Similar bacterial evacuation from the endosomes In contrast to TRAP activity, the transcriptional level of is observed for other mycobacteria species. An acid-fast another osteolytic enzyme, cathepsin K, was enhanced by bacillus, , which causes a systemic pMCs in response to Mtb (Fig. 2e). Thus, intracellular tuberculosis-like disease in its natural hosts, such as fish infection of Mtb resulted in a different osteolytic response and frogs, was reported to escape from phagosomes than typical microbial responses via TLR-mediated signals. (Stamm et al., 2003). In addition, intracellular pathogens, We confirmed that the expression levels of TLR2 and including Listeria monocytogenes, Shigella flexneri, and TLR4 were not significantly changed during osteoclasto- Rickettsia rickettsii (Goldberg, 2001) also share the ability to genesis (data not shown). Inflammatory activation signals enter the host cell cytoplasm, induce actin polymerization, in response to intracellular Mtb, which seems to be and use actin-based motility to spread between host cells independent of the TLR-mediated pathways, could inter- during intracellular infection. rupt the osteoclast development.

32 Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved A. Hoshino et al. Osteoclast dysregulation by M. tuberculosis infection

(a) (b) (c) (d) * * * 250 none 3.0 * 800 1.2 * ) 3000 160 * MCP-1 LPS * –1 ) 700 α ) * MIP-1 2.5 ) 1.0 2500 200 Pam3CSK4 –1 –1 600 –1 * 120 2.0 0.8 2000 500 * 150 * 1.5 400 0.6 1500 80 100 300 0.4 1.0 IL-6 (ng mL 1000 IL-1 β (pg mL TNF- α (ng mL NS * 200 40 50 TRAP5b (mUnit/dL) TRAP5b (mUnit/dL) 0.5 0.2 Chemokines (pg mL 500 100 0 0 0 0 0 0 Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb Mtb none none none none none none none none none none

(e) (f)Unstimulated (g)

] 600 250

pMCs mOCs Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 * 2 80 * * 500 [ μ m 200

* –6 60 400 * 150 300 100 m 40 * 100 200 * Intracellular M.tuberculosis -infected * 20 pMCs mOCs 100 50

Osteoclast area x10 0 0 0 LPS Pam3 Osteoclast numbers/24 well Plates LPS Pam3 none Mtb none Mtb Relative CTSK expression/ GAPDH Mtb Mtb none none (h) 0.25 0.06 NS LPS (10 ng/ml) Pam3CSK4 (100 ng/ml) * 0.05 0.20 0.04 0.15 NS 0.03 * 0.10 0.02

0.05 CSF-1R expression /GAPDH 0.01

Relative RANK expression /GAPDH 0 0 Relative Mtb Mtb Mtb Mtb none none none none

Fig. 2 The production of pro-inflammatory cytokines, chemokines and an osteoclast-specific enzyme by pre-osteoclasts and multinuclear osteoclasts after Mtb infection. (a,b) The production of (a) the proinflammatory cytokines TNF-a, IL-1b and IL-6, and (b) the proinflammatory chemokines MCP-1/ CCL2 and MIP-1a/CCL3 for 24 h after Mtb infection, were measured by an ELISA. The data show the means Æ SD (n = 6). (c,d) The levels of Trap5b 5 À1 À1 À1 in pMCs and mOCs stimulated with Mtb (10 CFU mL ), LPS (10 ng mL ) and Pam3CSK4 (100 ng mL ) were measured by an ELISA. The data show the means Æ SD (n = 6). (e) The relative expression levels of an osteolytic enzyme, cthepsin K (CTSK), produced by pMCs and mOCs, stimulated with Mtb, were measured by real-time Q-PCR. The data show the means Æ SD (n = 4). (f,g) In (f) the area and the numbers of osteoclasts 5 À1 À1 À1 after Mtb (10 CFU mL ), LPS (10 ng mL ), and Pam3CSK4 (100 ng mL ) stimulation were visualized by immunohistochemical staining using an anti-cathepsin K antibody conjugated with Alexa594 (red). F-actin and nuclei were counterstained by phalloidin–AlexaFluor 488 (green) and hoechst33258 (blue), respectively. Magnification 9400. (g) Histograms of the area distribution of multinuclear osteoclasts delimited with phalloidin and of the number of multinuclear osteoclasts in (f). The data are presented as the means Æ SD (n = 3). (h) The relative expression levels of the RANKL receptor (RANK) and M-CSF receptor (CSF1R) by pMCs and mOCs stimulated with Mtb were measured by real-time Q-PCR. The data show the means Æ SD (n = 4).

We next investigated whether Mtb infection exerts a osteoclast numbers were decreased by Mtb stimulation facilitatory effect on osteoclast formation, because bacterial (Fig. 2g), indicating that intracellular Mtb infection facilitates products are reported to utilize two different pathways via osteoclast formation but abrogates typical osteoclastic TLR4 in both survival and cytokine production of osteoclasts activation. Therefore, intracellular Mtb infection induces (Itoh et al., 2003). Compared with mOCs, Mtb infection irregular osteoclastogenesis by shifting the properties of by pMCs did not facilitate the formation of multinuclear osteoclasts from osteolytic to inflammation. osteoclasts (Fig. 2f and g). Stimulation of pMCs by We then investigated whether the changes in the prop- Pam3CSK4 also resulted in formation of large numbers of erties of Mtb-infected osteoclasts were due to the dysfunc- large multinuclear osteoclasts. In contrast, in response to tion of the RANK-RANKL axis, which is the principal LPS, pMCs increased the number of osteoclasts with osteoclastic regulator. Mtb-infected pMCs exhibited diminished osteoclast area. The result indicates that LPS decreased expression levels of RANK and c-fms, which facilitates the osteoclast formation but not osteoclastic encodes CSF-1R, the receptor for M-CSF (Fig. 2h). These activation directly via TLR4. Notably, we found that the data indicated that Mtb-infected pMCs lost their ability to osteoclast area increased in response to Mtb, although the differentiate into osteoclasts in response to osteoclastogenic

Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 33 Osteoclast dysregulation by M. tuberculosis infection A. Hoshino et al. growth factors M-CSF and RANKL as a result of downre- Menu et al., 2006; Hoshino et al., 2009, 2010), which is gulation of their receptors on osteoclasts. Consequently, induced by the combination of M-CSF and RANKL. The fact Mtb infection by pMCs leads to dysfunction in the physio- that intracellular Mtb infection disturbs the physiological logical osteoclastogenesis process and facilitates the osteoclastogenesis prompted us to investigate whether inflammatory osteoclastic activation. However, it is still intracellular Mtb infection could disrupt the chemokine unknown why the development of osteoclasts from the profiles. Therefore, we compared the chemokine expression precursor cells was inhibited by intracellular Mtb infection profiles in response to intracellular Mtb infection. We noted but not by the TLR-ligands. Various reports have suggested that various chemokine ligands are selectively produced by that there are differences in the biological responses osteoclasts at mRNA transcription level (Supporting Infor- induced by TLRs and RANK, although both TLRs and mation, Fig. S1). Among these ligands, CCL5 (also called RANK share a common downstream signaling molecule, RANTES) was highly upregulated during osteoclastogene- TRAF-6 (Mansell et al., 2004; Takeda & Akira, 2005). One sis (Fig. 3a), consistent with the chemokine expression possible reason for this is that the early secreted antigen, profile in murine osteoclastogenesis (Hoshino et al., 2010). Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 ESAT-6, facilitates the recognition of Mtb in cooperation with In addition, pMC expressed small amounts of chemokines, TLR2 in macrophages (Pathak et al., 2007). namely, CCL17, CCL20, CCL22, CCL24, and CCL25 (Fig. 3a). However, mature mOCs failed to produce these proinflammatory chemokines in response to Mtb infection. Specific expression profiles of chemokine ligands and To confirm that the immune response to virulent Mtb their counterpart receptors are induced by Mtb-infected strains is different from the response to non-virulent strains, osteoclasts we measured the transcription of the chemokines by pMCs Several reports suggested the participation of chemokines and mOCs in response to an avirulent Mtb strain (H37Ra) during physiological osteoclastogenesis (Oba et al., 2005; and a BCG-Tokyo strain. Both the avirulent H37Ra strain

(a) CCL5 CCL17 CCL20 (c) CTSK 4.5 8 0.10 120 4.0 * 7 * 0.08 * 100 3.5 6 3.0 80 5 0.06 2.5 4 60 2.0 0.04 3 1.5 40 2 1.0 0.02 20 0.5 1 0 0 0 0 None H37Rv H37Ra BCG None H37Rv H37Ra BCG None H37Rv H37Ra BCG CCL22 CCL24 CCL25 MMP9 60 6 0.00010 30

50 * 5 * 25 0.00008 * 40 4 20 0.00006 30 3 Relative enzyme expression/GAPDH 15 Relative chemokine expression/GAPDH 0.00004 20 2 10 0.00002 * 10 1 5

0 0 0 0 None H37Rv H37Ra BCG None H37Rv H37Ra BCG None H37Rv H37Ra BCG

(b) CCR1 CCR4 CCR5 CCR7 CCR9 0.0020 0.05 0.0006 0.30 * 0.05 0.0005 0.25 * 0.04 0.04 * * 0.0015 0.20 0.0004 0.03 0.03 0.15 0.0010 0.0003 0.02 0.02 0.10 0.0002 0.0005 0.01 0.01 expression/GAPDH 0.05 0.0001 Relative chemokineR

0 0 0 0 0 None H37Rv H37Ra BCG None H37Rv H37Ra BCG None H37Rv H37Ra BCG None H37Rv H37Ra BCG None H37Rv H37Ra BCG

Fig. 3 The expression levels of chemokine ligands and their receptors by osteoclasts after Mtb infection. (a–c) The relative expression levels of CC-chemokine ligands by pMCs (a), and CC-chemokine receptors by mOCs (b), and osteolytic enzymes cathepsin K (CTSK) bone-specific matrix metalloproteinase (MMP9) (c), stimulated with a virulent strain Mtb H37Rv (c. 6.0 9 105 CFU mLÀ1), an avirulent strain Mtb H37Ra À À (c. 8.7 9 105 CFU mL 1) or a BCG strain (c. 3.4 9 105 CFU mL 1) were measured by real-time Q-PCR. *Statistically significant differences (P < 0.05); N.D., not detected.

34 Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved A. Hoshino et al. Osteoclast dysregulation by M. tuberculosis infection and the BCG strain had a decreased ability to induce the Infection of osteoclasts with Mtb produces specific production of the chemokine ligands by osteoclasts chemokines (Fig. 3a). We next investigated the expression profile of C-C To further investigate whether these chemokine responses chemokine receptors during human osteoclastogenesis. In are specific for Mtb infection, we measured and compared previous murine experiments we reported that the selective the production of these chemokines after Mtb infection, and upregulation of two chemokine receptors, CCR1 and CCR5, LPS stimulation by pMCs and mOCs (Fig. 5). As previously which are required for osteolytic enzyme production (Hoshino mentioned, inflammatory chemokines CCL2/MCP-1 and et al., 2009), occurred with the downregulation of other CCL3/MIP-1a are produced in limited numbers by Mtb- chemokine receptors during murine osteoclastogenesis infected pMCs (see Fig. 1b). Instead, abundant production (Hoshino et al., 2009, 2010). Among the chemokine recep- of several chemokines by mOCs was observed: (1) a tors evaluated, CCR1 and CCR5 were both upregulated common proinflammatory response to both LPS and during human osteoclastogenesis (Fig. 3b). Besides the intracellular Mtb infection, (2) a Mycobacteria-specific Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 virulent Mtb strain, H37Rv infection also upregulated the chemokine response, and (3) an intracellular virulent Mtb expression of CCR1, CCR7, and CCR9, whereas there was infection -specific chemokine response. The first group of diminished expression of CCR2, CCR3, CCR6, and CX3CR1 chemokines, CCL5, CCL24, and CCL17, are commonly (Fig. 3b, Fig. S2). The avirulent Mtb strain (H37Ra) had produced in response to both LPS and intracellular Mtb sustained expression of CCR1 during osteoclastogenesis, stimulation (Fig. 5a–c), suggesting that the activation of the whereas the expression of other chemokine receptors CCR5-CCL5, CCR4-CCL17, and CCR3-CCL24 axes is a decreased. The osteolytic enzyme cathepsin K (CTSK)is common infectious response of multinuclear osteoclasts. also expressed only following infection with the virulent Mtb Notably, the CCL17 production was limited to mOCs strain (Fig. 3c). An enzyme matrix metalloproteinase-9 (Fig. 5c), implying that the activation of the CCR4-CCL17 (MMP9), which was secreted mainly by epithelial cells axis reflects abnormal osteoclast activation. The second surrounding the growing granuloma and promoted the group of chemokines, including CCL22, was enhanced by recruitment of new macrophages to the granuloma (Volkman virulent Mtb H37Rv infection, as well as avirulent H37Ra et al., 2010), was also expressed with the virulent Mtb infection (Fig. 5d). CCL22 was physiologically produced by stimulation. As CCR1 is the most abundant chemokine unstimulated mOCs, and its production was pathologically receptor during osteoclastogenesis, we next investigated the enhanced by pMCs in response to both LPS and Mtb expression of CCR1 and CCR5 in multinuclear cells in a stimulation, indicating that the aberrant production of CCL22 granuloma from a tissue section of the Mtb-infected patient by pMCs might be a facilitator for the abnormal osteoclastic (Fig. 4), and the results suggested that the expression of activation. Interestingly, the production of the third group of chemokine receptors plays a role during multinuclear cell chemokines, CCL20 and CCL25, which was enhanced by formation in the pathological condition. Therefore, intracellu- virulent Mtb infection, was observed only by mOCs (Figs. 5e lar Mtb infection induces the evacuation of Mtb into the and f). The data suggested that CCL20 and CCL25 might cytosol, which leads to irregular osteoclastogenesis as a play pivotal roles in the osteolytic response of virulent result of the dysregulation of chemokines, chemokine Mtb-infected bone tissue. These findings suggest that the receptors, and osteoclastic molecules, and finally results in pivotal role of chemokines in pathological osteoclastogen- a shift in the properties of osteoclasts from osteolytic to esis was via the combined production of osteoclast-specific inflammatory. chemokines, such as CCL17, and Mtb-specific chemokines,

H&E CCR1 CCR5

100 µm 100 µm

Fig. 4 Expression of chemokine receptors CCR1 and CCR5 in Mtb-infected multinuclear granuloma in a tuberculosis patient. (Center panel) Immunohistochemical staining of chemokine receptor CCR1 visualized by an anti-human CCR1 antibody and followed by DAB chromogen. Nuclei were counterstained using hematoxylin. Magnification 9400. (Right panel) Immunohistochemical staining of chemokine receptor CCR5 visualized by â an anti-human CCR5 antibody conjugated with phycoerythrin (red). Nuclei were counterstained by Cyto13 green fluorescent dye in green. Magnification 9400. (Left panel) H&E stain of counterpart section. Scale bar: 100 lm.

Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 35 Osteoclast dysregulation by M. tuberculosis infection A. Hoshino et al.

(a) (d) 2500 * 800 * * * ) )

2000 * –1 –1 600 * * * 1500 400 1000 hCCL5 (pg mL hCCL22 (pg mL 200 500

0 0 none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra

pMCs mOCs pMCs mOCs Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021

(b) (e) 2500 * * 1000 * )

) *

2000 –1 800 –1 * * 1500 600 * 1000 400 hCCL20 (pg mL hCCL24 (pg mL 500 200

0 0 none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra pMCs mOCs pMCs mOCs

(c) (f) 300 40 * ) ) 250 –1 –1 * 30

200 mL

150 20

100 hCCL17 (pg mL

hCCL25 (pg 10 50

0 0 none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG none LPS Mtb Mtb BCG H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra H37Rv H37Ra pMCs mOCs pMCs mOCs

Fig. 5 The production of several chemokines by multinuclear osteoclasts stimulated with Mtb. The production of chemokines CCL5 (a), CCL24 (b), CCL17 (c), CCL22 (d), CCL20 (e), and CCL25 (f) in pMCs and mOCs stimulated with the bacterial product LPS (100 ng mLÀ1), virulent Mtb H37Rv À À À strain (c. 6.0 9 105 CFU mL 1), avirulent Mtb H37Ra strain (c. 8.7 9 105 CFU mL 1), and BCG strain (c. 3.4 9 105 CFU mL 1) were measured by an ELISA. The data are presented as the means Æ SE of duplicate samples (n = 3). ND, not detected. including CCL20, CCL22, and CCL25, in response to ligands, namely CCL5, CCL17, CCL19, CCL20, CCL22, evacuated Mtb in the cytosol. CCL24, and CCL25 (Fig. 3a, Fig. S1), which was followed The findings of the present study demonstrate that the by the induction of their corresponding chemokine recep- intracellular infection of multinuclear osteoclasts by Mtb tors, such as CCR1, CCR4, CCR5, CCR7, and CCR9 (see failed to induce the secretion of typical proinflammatory Fig. 3b). Thus, these chemokine axes could be an important cytokines and proinflammatory chemokines, such as TNF-a, chemokine-mediated response induced by intracellular Mtb IL-1b, CCL2/MCP-1 or CCL3/MIP-1a (Fig. 2), but resulted in infection. Among them, the chemokine axes of CCR1/ the selective expression of osteoclast-specific chemokines CCR5-CCL5, CCR4-CCL17/CCL22, and CCR9-CCL25 and their receptors (Fig. 3). However, the combination of could play roles in the chemokine-mediated response to chemokine ligands and their receptors do not have a intracellular Mtb infection. In support of this, both CCR1 and one-to-one correspondence; the induction ratio of chemoki- CCR5 were highly expressed in multinuclear granuloma ne receptors after virulent Mtb infection revealed that Mtb cells by a Mtb-infected patient (Fig. 4). In addition, several infection activates a very limited number of chemokine clinical papers indicated the involvement of the chemokine

36 Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved A. Hoshino et al. Osteoclast dysregulation by M. tuberculosis infection system in intracellular Mtb infection; CCR7 is related to the eases. The dysregulation of cytokines and chemokines progression of several inflammatory bone diseases, such as appears to play a pivotal role in the abnormal differentiation rheumatoid arthritis (Bugatti et al., 2005; Pickens et al., of macrophage-lineage cells via reprogramming of the 2011) and a murine model of tuberculosis (Kahnert et al., inflammatory responses involved in pathological bone 2007; Khader et al., 2009). The involvement of the CCR9– metabolism. Further studies are needed to clarify the CCL25 axis in rheumatoid arthritis was also reported mechanism of destruction following intracellular infections (Endres et al., 2010; Schmutz et al., 2010). CCL22 and its with pathogens. receptor, CCR4, are highly expressed in the lungs, espe- cially in tuberculous lungs (Volpe et al., 2006; Okamoto Acknowledgements et al., 2007; Wu et al., 2010), and it has also been reported to be produced by human osteoclasts in response to foreign The author is grateful to Prof. Yoshichika Arakawa and substances, such as titanium particles (Cadosch et al., Dr. Keiko Yamada (Dept. Bacteriology/Drug Resistance and

2010). These data provide new aspects of how CCR4 Pathogenesis, Nagoya University Graduate School of Med- Downloaded from https://academic.oup.com/femspd/article/70/1/28/450245 by guest on 27 September 2021 switches its ligands, CCL17 and CCL22. The mechanism icine) for kindly providing a vaccine strain of Mycobacterium leading to the switch in the specific chemokine axes could bovis strain BCG-Tokyo/Nagoya. This work was mainly play a pivotal role during pathological osteoclastogenesis supported by a KAKENHI Grant-in-aid for young scientists B via virulent Mtb infection. Further investigations will be (2010–2011, #22790359), and by a research fellowship from needed to clarify the osteolytic mechanism of skeletal the Japan Society for the Promotion of Science for Young tuberculosis. Scientists (2007–2009) to A.H. In conclusion, we have characterized the pathological activation of osteoclasts in response to intracellular Mtb Authors’ contributions infection. Our data indicate that the inflammatory osteocl- astogenesis by Mtb infection is facilitated not only by A.H. and S.H. performed the experiments and analyzed the proinflammatory chemokines such as TNF-a, and by the data for H.Y., Sa.M., and Y.M.; H.Y. and Sa.M. provided the activation of the RANK-RANKL pathways, but also by the equipment, the Bio Safety Level-3 facility for Mtb experi- production of specific chemokines in response to intracel- ments, and virulent Mtb strain H37Rv; Sh.M. and M.T. lular Mtb infection. Notably, we did not detect any TNF-a provided the paraffin-embedded tissue section of tubercu- production by Mtb-infected multinuclear osteoclasts (see losis patients; A.H., Y.M., and K.Y. designed the research; Fig. 2a). TNF-a is responsible for granuloma formation and A.H. wrote the paper. Contact authors: Yoshinobu Manome maintenance, and the deficiency of TNF-a resulted in ([email protected]), Akiyoshi Hoshino (hoshinoa@nih. hypersusceptibility to tuberculosis (Ramakrishnan, 2012). go.jp; [email protected]). Thus, the unresponsiveness of TNF-a might cause the defective immune response during the early stages of Mtb References infection. Although early infection of macrophages might be predicted to promote initial host immunity, Mtb impairs Abu-Amer Y, Ross FP, Edwards J & Teitelbaum SL (1997) antigen presentation and an effective adaptive immune Lipopolysaccharide-stimulated osteoclastogenesis is mediated response. Furthermore, we found that the intracellular Mtb by tumor necrosis factor via its P55 receptor. J Clin Invest 100: – inside multinuclear osteoclasts escaped from the endo- 1557 1565. some/phagosome, and led to dysregulation of osteoclast Binder NB, Niederreiter B, Hoffmann O, Stange R, Pap T, Stulnig TM, Mack M, Erben RG, Smolen JS & Redlich K (2009) activation. Aberrant production of chemokines is due to the Estrogen-dependent and C-C chemokine receptor-2-dependent evacuation of Mtb from the endosome/phagosome, which pathways determine osteoclast behavior in osteoporosis. Nat accomplishes to avoid acidification. Evacuated Mtb in Med 15: 417–424. cytosol might lead to dysregulation of cytokines and Boyle WJ, Simonet WS & Lacey DL (2003) Osteoclast differenti- chemokines, which promotes atypical osteoclast activation, ation and activation. Nature 423: 337–342. and finally causes pathological bone destruction in the bone Bugatti S, Caporali R, Manzo A, Vitolo B, Pitzalis C & Montecucco C tissue. Consequently, our data suggest that the source of (2005) Involvement of subchondral bone marrow in rheumatoid Mtb-activated osteoclasts in spinal tuberculosis could be arthritis: lymphoid neogenesis and in situ relationship to sub- derived from tissue-resident multinuclear osteoclasts that chondral bone marrow osteoclast recruitment. Arthritis Rheum – were unexpectedly activated in response to Mtb infection. 52: 3448 3459. Cadosch D, Gautschi OP, Chan E, Simmen HP & Filgueira L (2010) Our findings provide novel information about an atypical Titanium induced production of chemokines CCL17/TARC and type of inflammation that is independent of proinflammatory CCL22/MDC in human osteoclasts and osteoblasts. J Biomed cytokine production due, in the case of osteoclast activation Mater Res A 92: 475–483. to spinal tuberculosis. Chang EJ, Kim HJ, Ha J et al. (2007) Hyaluronan inhibits The present observations provide further evidence that osteoclast differentiation via Toll-like receptor 4. J Cell Sci 120: the chemokine/chemokine receptor axes in bone metabo- 166–176. lism play pathological roles, including the functional differ- Choi SJ, Cruz JC, Craig F, Chung H, Devlin RD, Roodman GD & entiation of osteoclasts following intracellular infection. Our Alsina M (2000) Macrophage inflammatory protein 1-alpha is a current findings emphasize the relevance of the chemokine potential osteoclast stimulatory factor in multiple myeloma. Blood – axes as exacerbating factors for bone destruction dis- 96: 671 675.

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Pathogens and Disease (2014), 70, 28–39, © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 39