Bone Marrow Mesenchymal Stem Cells Provide an Antibiotic-Protective Niche for Persistent Viable Mycobacterium tuberculosis that Survive Antibiotic Treatment

During tuberculosis (TB), some Mycobacterium tuberculosis bacilli persist in the presence of an active immunity and antibiotics that are used to treat the disease. Herein, by using the Cornell model of TB persistence, we further explored our recent finding that suggested that M. tuberculosis can escape therapy by residing in the bone marrow (BM) mesenchymal stem cells. We initially showed that M. tuberculosis rapidly disseminates to the mouse BM after aerosol exposure and maintained a stable burden for at least 220 days. In contrast, in the lungs, the M. tuberculosis burden peaked at 28 days and subsequently declined approximately 10-fold. More important, treatment of the mice with the antibiotics rifampicin and isoniazid, as expected, resulted in effective clearance of M. tuberculosis from the lungs and spleen. In contrast, M. tuberculosis persisted, albeit at low numbers, in the BM of antibiotic-treated mice. Moreover, most viable M. tuberculosis was recovered from the bone marrow CD271+CD45−-enriched cell fraction, and only few viable bacteria could be isolated from the CD271−CD45+ cell fraction. These results clearly show that BM mesenchymal stem cells provide an antibiotic-protective niche for M. tuberculosis and suggest that unraveling the mechanisms underlying this phenomenon will enhance our understanding of M. tuberculosis persistence in treated TB patients. During tuberculosis (TB), some Mycobacterium tuberculosis bacilli persist in the presence of an active immunity and antibiotics that are used to treat the disease. Herein, by using the Cornell model of TB persistence, we further explored our recent finding that suggested that M. tuberculosis can escape therapy by residing in the bone marrow (BM) mesenchymal stem cells. We initially showed that M. tuberculosis rapidly disseminates to the mouse BM after aerosol exposure and maintained a stable burden for at least 220 days. In contrast, in the lungs, the M. tuberculosis burden peaked at 28 days and subsequently declined approximately 10-fold. More important, treatment of the mice with the antibiotics rifampicin and isoniazid, as expected, resulted in effective clearance of M. tuberculosis from the lungs and spleen. In contrast, M. tuberculosis persisted, albeit at low numbers, in the BM of antibiotic-treated mice. Moreover, most viable M. tuberculosis was recovered from the bone marrow CD271+CD45−-enriched cell fraction, and only few viable bacteria could be isolated from the CD271−CD45+ cell fraction. These results clearly show that BM mesenchymal stem cells provide an antibiotic-protective niche for M. tuberculosis and suggest that unraveling the mechanisms underlying this phenomenon will enhance our understanding of M. tuberculosis persistence in treated TB patients. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an intracellular pathogen known to infect primarily macrophages and dendritic cells.1Cooper A.M. Cell-mediated immune responses in tuberculosis.Annu Rev Immunol. 2009; 27: 393-422Crossref PubMed Scopus (861) Google Scholar, 2Flynn J.L. Chan J. Lin P.L. Macrophages and control of granulomatous inflammation in tuberculosis.Mucosal Immunol. 2011; 4: 271-278Crossref PubMed Scopus (265) Google Scholar, 3Bodnar K.A. Serbina N.V. Flynn J.L. Fate of Mycobacterium tuberculosis within murine dendritic cells.Infect Immun. 2001; 69: 800-809Crossref PubMed Scopus (168) Google Scholar However, the viability of M. tuberculosis in these intracellular niches is poor,4Keane J. Balcewicz-Sablinska M.K. Remold H.G. Chupp G.L. Meek B.B. Fenton M.J. Kornfeld H. Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis.Infect Immun. 1997; 65: 298-304Crossref PubMed Google Scholar and no evidence exists indicating that these cells can maintain live nonreplicating M. tuberculosis for long periods of time. Therefore, it is unlikely that these cell populations can harbor viable M. tuberculosis during the chronic phase of the disease, which lasts for months or years, as well as during the latent TB infection, which can last for decades. We have recently shown that M. tuberculosis can reside in bone marrow (BM) within the CD271+CD45− mesenchymal stem cells (BM-MSCs) of individuals treated for pulmonary TB and in mice experimentally infected with M. tuberculosis.5Das B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar MSCs can provide an ideal protective niche for M. tuberculosis because these cells have several properties that may promote the pathogen's long-term persistence and survival: i) MSCs are present in TB granulomas of infected mouse and human lung tissue6Raghuvanshi S. Sharma P. Singh S. Van Kaer L. Das G. Mycobacterium tuberculosis evades host immunity by recruiting mesenchymal stem cells.Proc Natl Acad Sci U S A. 2010; 107: 21653-21658Crossref PubMed Scopus (86) Google Scholar; ii) stem cells possess the capacity for self-renewal7Sacchetti B. Funari A. Michienzi S. Di Cesare S. Piersanti S. Saggio I. Tagliafico E. Ferrari S. Robey P.G. Riminucci M. Bianco P. Self-renewing osteoprogenitors in BM sinusoids can organize a hematopoietic microenvironment.Cell. 2007; 131: 324-336Abstract Full Text Full Text PDF PubMed Scopus (1756) Google Scholar; iii) stem cells express drug efflux pumps, such as ABCG2, that could contribute to drug evasion by M. tuberculosis5Das B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar; iv) stem cells have low production of reactive oxygen species,8Das B. Antoon R. Tsuchida R. Lotfi S. Morozova O. Farhat W. Malkin D. Koren G. Yeger H. Baruchel S. Squalene selectively protects mouse BM progenitors against cisplatin and carboplatin-induced cytotoxicity in vivo without protecting tumor growth.Neoplasia. 2008; 10: 1105-1119Abstract Full Text PDF PubMed Scopus (78) Google Scholar which may favor the viability of nonreplicating M. tuberculosis; v) although MSCs have the capacity of self-renewal, they are relatively quiescent,9Aria F. Suda T. Quiscent stem cells in the niche. StemBook. Harvard Stem Cell Institute, Cambridge, MA2008: 1-11Google Scholar and reside in the immune-privileged niche of the BM10Fujisaki J. Wu J. Carlson A.L. Silberstein L. Putheti P. Larocca R. Gao W. Saito T.I. Lo Celso C. Tsuyuzaki H. Sato T. Cote D. Sykes M. Strom T.B. Scadden D.T. Lin C.P. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche.Nature. 2011; 474: 216-219Crossref PubMed Scopus (423) Google Scholar, 11Tormin A. Li O. Brune J.C. Walsh S. Schutz B. Ehinger M. Ditzel N. Kassem M. Scheding S. CD146 expression on primary nonhematopoietic BM stem cells is correlated with in situ localization.Blood. 2011; 117: 5067-5077Crossref PubMed Scopus (327) Google Scholar; and vi) MSCs do not normally express major histocompatibility complex (MHC) class II on their cell surface and their MHC class I molecules are not functionally active (ie, these molecules do not trigger effector functions of cytotoxic T lymphocytes).12Jacobs S.A. Roobrouck V.D. Verfaillie C.M. Van Gool S.W. Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells.Immunol Cell Biol. 2013; 91: 32-39Crossref PubMed Scopus (180) Google Scholar Therefore, it is logical that BM-MSCs constitute a host cell capable of supporting long-term persistence of viable nonreplicating M. tuberculosis. However, many fundamental questions regarding the survival of virulent M. tuberculosis in BM-MSCs remain unanswered. Herein, we confirmed in vivo in the mouse model of TB that virulent M. tuberculosis disseminates rapidly to the BM within 2 weeks after infection with aerosolized organisms and preferentially resides within BM-MSCs. In addition, and more important, we show that antibiotics appeared to efficiently clear the infectious process within the lungs and spleens but fail to do so in the BM. Six- to eight-week-old C57BL/6 female mice (Charles River Laboratories, Kingston, NY) were exposed (nasally only) to virulent M. tuberculosis Erdman strain using a CH Technologies (Westwood, NJ) aerosol-generating machine contained within a class III biosafety cabinet in the New England Regional Biosafety Laboratory (Grafton, MA). The low-dose exposure deposited approximately 80 bacilli in the lungs. After 28 days of infection, half of the mice received 100 mg/L rifampicin (RIF) with 250 mg/L isoniazid (INH) in the drinking water, and the other half received sterile acidified water. At the time points indicated, mice were euthanized by CO2, and lungs, spleens, and BM were harvested, homogenized in sterile phosphate-buffered saline (PBS), and plated onto oleate-albumin-dextrose-catalase–supplemented 7H11 agar. BM was harvested from both femurs and tibia from each mouse by flushing with sterile PBS, homogenized by pressing through 70-μm nylon mesh screens to yield single-cell suspensions, and resuspended in 2 mL of PBS. In initial experiments, the entire 2 mL of BM from each mouse was plated onto agar for colony-forming unit (CFU) recovery. In subsequent experiments, 500 μL of the 2-mL BM from each mouse was plated onto agar. The remaining 1.5 mL from four to six mice was pooled, and red blood cells were lysed, counted (yielding a calculated average of approximately 3.5 × 107cells per mouse), and enriched for BM-MSCs. The enriched/depleted/unfractioned cells were plated onto agar in duplicate or triplicate at the maximum number possible for BM-MSCs or 1 to 1.25 × 107 cells for the depleted and unfractioned cells. In some experiments, single-cell suspensions were obtained from the BM, and enriched for CD271+CD45− BM-MSCs using Stemcell Technologies (Vancouver, BC, Canada) EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (catalog number 19771) for mouse MSCs, and fractions plated onto agar. Lung and spleen homogenates were serially diluted in sterile PBS before plating. M. tuberculosis CFUs were counted after at least 3 weeks at 37°C. To confirm the quality control of the kit before using it inside the biosafety level 3 facility, BM fractions were obtained from noninfected mice (outside the biosafety level 3 facility) by flushing the marrow out of femur and tibia, followed by softening of bone chips by incubation in solution containing 0.25% collagenase type I in PBS + 20% fetal bovine serum for 45 minutes at 37°C. The mononuclear cells were then filtered through a 70-μm cell strainer and washed twice in PBS containing 2% fetal bovine serum. The CD45− fractions were obtained by performing magnetic sorting, and the isolated cell population was stained for anti-CD45 (17-0451-83, activated protein C labeled; eBiosciences, San Diego, CA), anti-CD271 (ab62122, fluorescein isothiocyanate labeled; Abcam, Cambridge, MA), or isotype-matched controls. Flow cytometry analysis was performed on an AccuriC6 cytometer (BD Biosciences, San Jose, CA). We were able to collect a calculated average of 2.8 × 106 of CD45− cells from 3 × 107 BM cells (n = 5); 33.5% of CD45− cells showed expression of CD271. This analysis confirmed CD271+ cell enrichment using the Stemcell Technologies EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment Kit (catalog number 19771) for mouse MSCs. All experiments were approved by Tufts University (Grafton, MA) Institutional Biosafety Committee (GRIA04) and Institutional Animal Care and Use Committee (G2012-151). Our initial observation that showed that M. tuberculosis could infect and reside in vivo in the mouse BM-MSCs was done using the i.v. route of infection and an avirulent strain of M. tuberculosis.5Das B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar To confirm those findings after a natural route of M. tuberculosis transmission, mice were initially infected with a low dose (approximately 80 CFUs) of aerosolized virulent M. tuberculosis (Erdman strain). Mice were then euthanized at different time points after the infection, and the BM target cells of the infectious process were evaluated. After infection, typical bacillary growth for C57BL/6 mice was observed, peaking at 3 to weeks and then by approximately to a stable burden J. M. P. A.M. Immunological for of tuberculosis in Immun. 2001; 69: PubMed Scopus Google Scholar, G.L. B. J. blood lung responses and Mycobacterium tuberculosis in Immunol. 2008; PubMed Scopus Google Scholar The of the growth and in burden days 28 and in the lungs to the for the and of tuberculosis type 1 A.M. Cell-mediated immune responses in tuberculosis.Annu Rev Immunol. 2009; 27: 393-422Crossref PubMed Scopus (861) Google Scholar, G.L. B. J. blood lung responses and Mycobacterium tuberculosis in Immunol. 2008; PubMed Scopus Google Scholar days and after aerosol exposure to M. tuberculosis, the bacteria from the lungs to the BM This is to that in spleen and A.M. Cell-mediated immune responses in tuberculosis.Annu Rev Immunol. 2009; 27: 393-422Crossref PubMed Scopus (861) Google Scholar, J.L. Chan J. of tuberculosis.Annu Rev Immunol. 2001; PubMed Scopus Google Scholar it that M. tuberculosis the BM before the of Moreover, and in to the lungs, and shown in this the of the M. tuberculosis burden in the BM do not the of a and in these but in the BM, the burden and stable for at least days These results are with our observation to BM as a target of the infectious process that the lung of virulent aerosolized M. tuberculosis. To the BM cell target of M. tuberculosis, mice were with aerosolized bacteria and their BM cells were subsequently harvested at different time These experiments to confirm that viable M. tuberculosis preferentially target BM-MSCs. BM cells were by Technologies EasySep Mouse Mesenchymal Stem/Progenitor Cell Enrichment of that CD271+CD45− (BM-MSCs) and a population of cells that are To which cell population was preferentially infected with M. tuberculosis, the fractions of BM cells were in agar and at for at least 3 followed by of the the infection the burden of M. tuberculosis infection (CFU) was in the CD271+CD45−-enriched BM cell (BM-MSCs) in the cell population This BM by M. tuberculosis as as days after lung and is maintained the chronic phase of the expected, the CD271+CD45− depleted cell population a relatively level of infection because this population of cells other cell a population of cells that are normally infected by M. tuberculosis. Moreover, the not infected cells because the CFUs recovered from the population of cells not from the cell population the other it is possible that the depleted CD271+ cells CD271+CD45− cells because enrichment not the cells. these results clearly confirm that virulent M. tuberculosis the host through the and subsequently MSCs in a the BM. Our initial observation in that TB treated and viable M. tuberculosis in their B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar is possible that this of to therapy could be a of efflux present in these B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar, C. The of in the 2011; PubMed Scopus Google Scholar, T. S. H. L. is not for of primary and by the human protein J. PubMed Scopus Google Scholar To to a for BM-MSCs as a protective niche M. tuberculosis to therapy by we of a model of dormant In this known for as the Cornell H. Chan J. Flynn J.L. of latent on the Cornell murine Immun. Google Scholar, R. Fate of Mycobacterium tuberculosis in mouse as by the the persistence of bacilli in the Med. PubMed Scopus Google Scholar, W. R. The of Mycobacterium tuberculosis in mouse as by the the of infection to the latent by the of and a Med. PubMed Scopus Google Scholar mice are infected with virulent M. tuberculosis and the infection is treated for weeks with the which bacilli from the lungs, and However, the can or be by and M. tuberculosis can be from lungs and spleens of approximately of the This that in some bacilli were present and from the BM is a major niche of MSCs, we that viable M. tuberculosis could be present in BM after of the lungs and spleen. In M. which received treatment with and 28 days after infection, viable M. tuberculosis were present in BM for at least 100 days after no more viable M. tuberculosis could be recovered from the lungs and spleens after the of the was a and of the viable M. tuberculosis that could be recovered from lungs and spleens, which to of viable M. tuberculosis In contrast, no in the BM, although was a in viable M. tuberculosis that the maximum at after infection weeks of then M. tuberculosis bacilli at low the of the at 220 after infection months of The level of in our was for the lungs, spleens, and BM. To the cell population in the BM that and protects the M. tuberculosis from mice were infected for 28 followed by treatment with the antibiotics for BM cells were obtained and as CD271+CD45− cells and CD271−CD45+ cells. M. tuberculosis bacilli were 100 more in the CD271+CD45− cell population in the CD271−CD45+ cell These results clearly that BM-MSCs constitute a in the Cornell model of M. tuberculosis persistence, protects the bacteria from properties of antibiotics that are used to treat Our that BM in the CD271+CD45− harbor viable M. tuberculosis B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar However, these not the of from the lungs and persistence of virulent M. tuberculosis in the BM or growth of the M. tuberculosis in the BM, with the primary of infection and or other these fundamental in vivo is because the results have for M. tuberculosis latent TB infection, which of the P.L. Flynn J.L. latent a Immunol. 2010; PubMed Scopus Google Scholar these experiments be performed in we used the most mouse strain in M. tuberculosis C57BL/6 mice. C57BL/6 mice are to be relatively to low-dose aerosol infection of virulent M. tuberculosis we used with survival E. of some mouse to Mycobacterium tuberculosis and to major histocompatibility complex and PubMed Scopus Google Scholar to immunity to and T J. M. P. A.M. Immunological for of tuberculosis in Immun. 2001; 69: PubMed Scopus Google Scholar, Wu Mycobacterium T cells to target cells and provide in Immunol. 2008; PubMed Scopus Google Scholar we used C57BL/6 mice as a model for M. tuberculosis in the BM of capable of type 1 tuberculosis Herein, we after lung infection of mice with virulent M. tuberculosis, the infectious process disseminates not only to spleen and but to BM as the of growth and control of the M. tuberculosis in the lungs, and A.M. Cell-mediated immune responses in tuberculosis.Annu Rev Immunol. 2009; 27: 393-422Crossref PubMed Scopus (861) Google Scholar, J. M. P. A.M. Immunological for of tuberculosis in Immun. 2001; 69: PubMed Scopus Google Scholar, G.L. B. J. blood lung responses and Mycobacterium tuberculosis in Immunol. 2008; PubMed Scopus Google Scholar, J.L. Chan J. of tuberculosis.Annu Rev Immunol. 2001; PubMed Scopus Google Scholar is not the to that with M. tuberculosis in the BM. In to the and growth is and burden because of the growth of the bacteria in the BM not during the chronic phase of we have not the and mechanisms that contribute to this it is possible that M. tuberculosis within the BM is from the host immune because this is an immune-privileged J. Wu J. Carlson A.L. Silberstein L. Putheti P. Larocca R. Gao W. Saito T.I. Lo Celso C. Tsuyuzaki H. Sato T. Cote D. Sykes M. Strom T.B. Scadden D.T. Lin C.P. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche.Nature. 2011; 474: 216-219Crossref PubMed Scopus (423) Google Scholar, 11Tormin A. Li O. Brune J.C. Walsh S. Schutz B. Ehinger M. Ditzel N. Kassem M. Scheding S. CD146 expression on primary nonhematopoietic BM stem cells is correlated with in situ localization.Blood. 2011; 117: 5067-5077Crossref PubMed Scopus (327) Google Scholar and this is by our observation that no granulomas within the BM More important, the present clearly that M. tuberculosis, in to the resides in the BM within the population The results that this that the CD271+CD45− cells were more infected the population of cells. However, it is to in that during the in vivo infectious the cell population a major target of the M. tuberculosis infection because these cells are more the CD271+CD45− cell because of the properties of the MSCs, the results the that infection of these cells an M. tuberculosis escape of survival within the At this is known the of of M. tuberculosis the BM-MSCs. is possible that the of the bacteria within these cells may be a process (ie, the M. tuberculosis may be initially host followed by subsequent stem this of recently and to be the for Mycobacterium T. J. J. R. A. adult cells to stem cells by bacilli promotes of 2013; Full Text Full Text PDF PubMed Scopus Google Scholar This that the the adult the host cells for M. to their to a of cells by cell and of this is an to our that M. tuberculosis can be and persist within BM-MSCs. The persistence of M. tuberculosis may an escape from both the host immune and may from the of The evasion of the immune is to within the BM-MSCs because these cells do not normally express MHC class II on their cell surface and their MHC class I molecules are not functionally active and do not trigger effector functions of cytotoxic T S.A. Roobrouck V.D. Verfaillie C.M. Van Gool S.W. Immunological characteristics of human mesenchymal stem cells and multipotent adult progenitor cells.Immunol Cell Biol. 2013; 91: 32-39Crossref PubMed Scopus (180) Google Scholar from is to be because BM-MSCs have efflux of B. Kashino S.S. Pulu I. Kalita D. Swami V. Yeger H. Felsher D.W. Campos-Neto A. CD271(+) BM mesenchymal stem cells may provide a niche for dormant Mycobacterium tuberculosis.Sci Transl Med. 2013; 5: 170ra13PubMed Google Scholar an that can a of the out of the C. The of in the 2011; PubMed Scopus Google Scholar, T. S. H. L. is not for of primary and by the human protein J. PubMed Scopus Google Scholar our to this using the Cornell mouse model to M. tuberculosis persistence after drug for the our results clearly showed that with and treatment for months with and the could be recovered from the BM. In and as from the Cornell no M. tuberculosis could be isolated from the lungs or spleen. These results a of the and viable M. tuberculosis present in mice in the Cornell which for More important, our results clearly that within the BM cells of antibiotic-treated the pathogen was isolated primarily from the CD271+CD45− In contrast, only few M. tuberculosis could be isolated from the CD271−CD45+ cells and other However, because the CD271+CD45− enriched cell population was obtained by other cells BM-MSCs may be present in this BM cell fraction. Therefore, other possible of infection by M. tuberculosis that could the pathogen escape treatment be However, the that the depleted contained few organisms the that the BM-MSCs can M. tuberculosis from long-term of These are because they may it can be to of TB with In by using a in vivo model of these results the that BM-MSCs are target cells of the infection process that a antibiotic-protective niche for M. tuberculosis to therapy within In addition, these suggest that unraveling the mechanisms underlying the M. tuberculosis and will enhance our understanding of M. tuberculosis persistence immune and in TB patients. We and England Regional Biosafety Tufts University of MA) for providing their

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