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• W2612773113 abstract "•Mycobacterium leprae infection of zebrafish damages nerves, causing demyelination•Nerve damage requires M. leprae phenolic glycolipid (PGL-1) and host macrophages•PGL-1 induces macrophages to produce excess nitric oxide•Excess nitric oxide damages nerves by damaging their mitochondria Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy. Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy. Leprosy, like tuberculosis, presents as a granulomatous disease. These granulomas are usually cutaneous, reflecting the ∼30°C growth optimum of M. leprae, similar to that of the human skin (∼34°C) (Bierman, 1936Bierman W. The temperature of the skin surface.J. Am. Med. Assoc. 1936; 106: 1158-1162Crossref Scopus (55) Google Scholar, Renault and Ernst, 2015Renault C.A. Ernst J.D. Mycobacterium leprae (Leprosy).in: Bennett J.E. Dolin R. Blaser M.J. Mandell, Douglas, and Bennett’s Infectious Disease Essentials. Elsevier, 2015: 2819-2831Google Scholar, Truman and Krahenbuhl, 2001Truman R.W. Krahenbuhl J.L. Viable M. leprae as a research reagent.Int. J. Lepr. Other Mycobact. Dis. 2001; 69: 1-12PubMed Google Scholar). M. leprae is the only mycobacterial infection that causes widespread demyelinating neuropathy, which results in the main morbidities of leprosy, including autoamputation of digits and blindness (Renault and Ernst, 2015Renault C.A. Ernst J.D. Mycobacterium leprae (Leprosy).in: Bennett J.E. Dolin R. Blaser M.J. Mandell, Douglas, and Bennett’s Infectious Disease Essentials. Elsevier, 2015: 2819-2831Google Scholar). Understanding the pathogenesis of leprosy neuropathy has been stymied by the inability to culture M. leprae, which has undergone severe reductive evolution of its genome to become an obligate intracellular pathogen (Cole et al., 2001Cole S.T. Eiglmeier K. Parkhill J. James K.D. Thomson N.R. Wheeler P.R. Honoré N. Garnier T. Churcher C. Harris D. et al.Massive gene decay in the leprosy bacillus.Nature. 2001; 409: 1007-1011Crossref PubMed Scopus (1370) Google Scholar, Scollard et al., 2006Scollard D.M. Adams L.B. Gillis T.P. Krahenbuhl J.L. Truman R.W. Williams D.L. The continuing challenges of leprosy.Clin. Microbiol. Rev. 2006; 19: 338-381Crossref PubMed Scopus (590) Google Scholar). The lack of genetic tools for studying M. leprae is compounded by the lack of genetically tractable animal models that mimic the human disease. The athymic mouse footpad is used to grow M. leprae for research purposes, but mice do not manifest neurological disease (Scollard et al., 2006Scollard D.M. Adams L.B. Gillis T.P. Krahenbuhl J.L. Truman R.W. Williams D.L. The continuing challenges of leprosy.Clin. Microbiol. Rev. 2006; 19: 338-381Crossref PubMed Scopus (590) Google Scholar). While the nine-banded armadillo develops neuropathy following infection with M. leprae, it suffers from a paucity of molecular and genetic tools (Truman et al., 2014Truman R.W. Ebenezer G.J. Pena M.T. Sharma R. Balamayooran G. Gillingwater T.H. Scollard D.M. McArthur J.C. Rambukkana A. The armadillo as a model for peripheral neuropathy in leprosy.ILAR J. 2014; 54: 304-314Crossref PubMed Scopus (31) Google Scholar). Consequently, our understanding of the pathogenesis of leprosy neuropathy in vivo largely comes from studies of patients; however, the 4- to 10-year delay in the onset of symptoms largely precludes studies of the early events that lead to neuropathy (Noordeen, 1994Noordeen S. The epidemiology of leprosy.in: Hastings R.C. Leprosy. Churchhill Livingstone, 1994: 29-48Google Scholar). Leprosy can present as a clinical spectrum; at the poles of this spectrum are paucibacillary (or tuberculoid) and multibacillary (or lepromatous) disease. The former is characterized by a vigorous immune response, while the latter, an ineffective one (Scollard et al., 2006Scollard D.M. Adams L.B. Gillis T.P. Krahenbuhl J.L. Truman R.W. Williams D.L. The continuing challenges of leprosy.Clin. Microbiol. Rev. 2006; 19: 338-381Crossref PubMed Scopus (590) Google Scholar). Neuropathy features prominently in both forms of the disease. Hence, bacterial determinants and host immune responses likely play roles in leprosy neuropathy, although the relative importance and mechanisms by which each contributes to nerve injury are poorly understood. In vitro studies suggest a model wherein M. leprae directly causes demyelination by infecting and dedifferentiating the Schwann cells that myelinate peripheral nerves (Rambukkana et al., 2002Rambukkana A. Zanazzi G. Tapinos N. Salzer J.L. Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.Science. 2002; 296: 927-931Crossref PubMed Scopus (164) Google Scholar, Truman et al., 2014Truman R.W. Ebenezer G.J. Pena M.T. Sharma R. Balamayooran G. Gillingwater T.H. Scollard D.M. McArthur J.C. Rambukkana A. The armadillo as a model for peripheral neuropathy in leprosy.ILAR J. 2014; 54: 304-314Crossref PubMed Scopus (31) Google Scholar). These studies identified an M. leprae outer membrane lipid, phenolic glycolipid 1 (PGL-1), that is critical for binding to laminin α2, an interaction thought to promote infection of the Schwann cells (Ng et al., 2000Ng V. Zanazzi G. Timpl R. Talts J.F. Salzer J.L. Brennan P.J. Rambukkana A. Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.Cell. 2000; 103: 511-524Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). However, this model fails to explain the neuropathy in paucibacillary leprosy, in which bacteria are seldom observed within nerve lesions (Shetty and Antia, 1996Shetty V.P. Antia N.H. A semi quantitative analysis of bacterial load in different cell types in leprous nerves using transmission electron microscope.Indian J. Lepr. 1996; 68: 105-108PubMed Google Scholar). Rather, a pathogenic CD4 T cell response, possibly acting through secreted cytokines, is implicated in paucibacillary disease (Renault and Ernst, 2015Renault C.A. Ernst J.D. Mycobacterium leprae (Leprosy).in: Bennett J.E. Dolin R. Blaser M.J. Mandell, Douglas, and Bennett’s Infectious Disease Essentials. Elsevier, 2015: 2819-2831Google Scholar). Further, the specific contributions of macrophages in leprosy neuropathy are unknown, although they are commonly infected and almost universally present in affected nerves (Job, 1973Job C.K. Mechanism of nerve destruction in tuberculoid-borderline leprosy. An electron-microscopic study.J. Neurol. Sci. 1973; 20: 25-38Abstract Full Text PDF PubMed Scopus (17) Google Scholar, Shetty and Antia, 1996Shetty V.P. Antia N.H. A semi quantitative analysis of bacterial load in different cell types in leprous nerves using transmission electron microscope.Indian J. Lepr. 1996; 68: 105-108PubMed Google Scholar). The developing zebrafish is an effective model for studying mycobacterial pathogenesis using M. marinum, a close genetic relative of the M. tuberculosis complex and the agent of fish tuberculosis (Ramakrishnan, 2004Ramakrishnan L. Using Mycobacterium marinum and its hosts to study tuberculosis.Curr. Sci. 2004; 86: 82-92Google Scholar). The genetic tractability of the zebrafish, coupled with the optical transparency of its larva, allows host-bacterium interactions to be monitored in real-time, providing critical insights into disease pathogenesis (Ramakrishnan, 2004Ramakrishnan L. Using Mycobacterium marinum and its hosts to study tuberculosis.Curr. Sci. 2004; 86: 82-92Google Scholar). Furthermore, adaptive immunity is not yet present at the larval developmental stage, permitting study of host-pathogen interactions in the sole context of innate immunity (Davis et al., 2002Davis J.M. Clay H. Lewis J.L. Ghori N. Herbomel P. Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.Immunity. 2002; 17: 693-702Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Here, we exploit the optical transparency of larval zebrafish to directly visualize the earliest interactions of M. leprae with macrophages (Davis et al., 2002Davis J.M. Clay H. Lewis J.L. Ghori N. Herbomel P. Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.Immunity. 2002; 17: 693-702Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), and the initial events in nerve injury (Czopka, 2016Czopka T. Insights into mechanisms of central nervous system myelination using zebrafish.Glia. 2016; 64: 333-349Crossref PubMed Scopus (32) Google Scholar). We use M. marinum as a comparator for these studies because, like M. leprae, it grows at ∼30°C and produces cutaneous granulomatous infections in humans (Ramakrishnan, 2004Ramakrishnan L. Using Mycobacterium marinum and its hosts to study tuberculosis.Curr. Sci. 2004; 86: 82-92Google Scholar). However, it does not cause neuropathy. Our studies reveal that M. leprae interacts with macrophages and incites granulomas similar to M. marinum (Madigan et al., 2017Madigan C.A. Cameron J. Ramakrishnan L. A zebrafish model for Mycobacterium leprae granulomatous infection.J Infect Dis. jix329. 2017; https://doi.org/10.1101/127639Crossref Google Scholar), but is unique in its ability to produce demyelination and axonal damage. We show that the innate macrophage response to PGL-1 triggers demyelination in vivo, even before bacilli have detectably infected the glia. Finally, we determine the mechanism of nerve damage using M. leprae and M. marinum engineered to synthesize PGL-1. To determine if zebrafish larvae might be a useful model for studying early M. leprae infection, we first examined the earliest interactions of M. leprae with phagocytes, by injecting bacteria into the caudal vein or the hindbrain ventricle (Figure 1A), where phagocytes are rarely observed in the absence of infection (Davis et al., 2002Davis J.M. Clay H. Lewis J.L. Ghori N. Herbomel P. Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.Immunity. 2002; 17: 693-702Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Aggregates of infected macrophages formed within 4 days (Figure 1B), similar to the case with M. marinum infection (Davis et al., 2002Davis J.M. Clay H. Lewis J.L. Ghori N. Herbomel P. Ramakrishnan L. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos.Immunity. 2002; 17: 693-702Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Prior studies indicate that phagocyte recruitment to M. marinum infection is unique in two respects: (1) neutrophils are not recruited to the initial site of infection (Yang et al., 2012Yang C.-T. Cambier C.J. Davis J.M. Hall C.J. Crosier P.S. Ramakrishnan L. Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages.Cell Host Microbe. 2012; 12: 301-312Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), and (2) macrophage recruitment is independent of TLR-signaling, but dependent on the monocyte chemokine CCL2 and its receptor CCR2 (Cambier et al., 2014Cambier C.J. Takaki K.K. Larson R.P. Hernandez R.E. Tobin D.M. Urdahl K.B. Cosma C.L. Ramakrishnan L. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.Nature. 2014; 505: 218-222Crossref PubMed Scopus (318) Google Scholar). M. leprae shared both of these features with M. marinum: neutrophils were not recruited, whereas macrophages were (Figures 1C and 1D). Further, this recruitment was TLR/MyD88 independent and CCL2/CCR2 dependent (Figure 1D) (Cambier et al., 2014Cambier C.J. Takaki K.K. Larson R.P. Hernandez R.E. Tobin D.M. Urdahl K.B. Cosma C.L. Ramakrishnan L. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.Nature. 2014; 505: 218-222Crossref PubMed Scopus (318) Google Scholar). The M. marinum phenolic glycolipid (PGL-mar) induces CCL2 expression and mediates CCL2/CCR2-dependent macrophage recruitment (Cambier et al., 2014Cambier C.J. Takaki K.K. Larson R.P. Hernandez R.E. Tobin D.M. Urdahl K.B. Cosma C.L. Ramakrishnan L. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.Nature. 2014; 505: 218-222Crossref PubMed Scopus (318) Google Scholar, Cambier et al., 2017Cambier C.J. O’Leary S.M. O’Sullivan M.P. Keane J. Ramakrishnan L. Phenolic glycolipid facilitates mycobacterial escape from microbicidal tissue-resident macrophages.Immunity. 2017; 47https://doi.org/10.1101/147421Crossref Google Scholar), suggesting that PGL-1 may play a similar role in M. leprae infection. Macrophages play a dichotomous role in controlling M. marinum infection: they restrict bacterial numbers, while promoting dissemination of bacteria from the infection site into deeper tissues (Clay et al., 2007Clay H. Davis J.M. Beery D. Huttenlocher A. Lyons S.E. Ramakrishnan L. Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.Cell Host Microbe. 2007; 2: 29-39Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Similar to the case observed for M. marinum, M. leprae-infected animals depleted of macrophages using the pu.1 morpholino (Clay et al., 2007Clay H. Davis J.M. Beery D. Huttenlocher A. Lyons S.E. Ramakrishnan L. Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.Cell Host Microbe. 2007; 2: 29-39Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) displayed higher bacterial burdens (Figures 1E and 1F). The increased bacterial burden in the pu.1 morphants is likely due to the lack of bacterial killing, rather than bacterial replication. The doubling time of M. leprae is approximately 12 days (Levy and Ji, 2006Levy L. Ji B. The mouse foot-pad technique for cultivation of Mycobacterium leprae.Lepr. Rev. 2006; 77: 5-24PubMed Google Scholar); therefore, most bacteria would not have replicated in the larvae during the 2-day infection. In addition, we assessed the role of macrophages in M. leprae dissemination, by infecting animals with fluorescent vascular endothelial cells (kdrl:dsRed). By 2 days post-infection (dpi) (4 dpf), M. leprae escaped the vasculature and entered peripheral tissues in the majority of wild-type, but not macrophage-depleted, larvae (Figures 1G and 1H). Furthermore, in wild-type animals, M. leprae resided in macrophages (apparent by Nomarski imaging in Figure S1), suggesting these cells carried M. leprae from the circulation into tissues. This is reminiscent of zebrafish infected with M. marinum, in which infected macrophages disseminate bacteria from the initial infection site into the body (Clay et al., 2007Clay H. Davis J.M. Beery D. Huttenlocher A. Lyons S.E. Ramakrishnan L. Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish.Cell Host Microbe. 2007; 2: 29-39Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). In sum, M. leprae displays interactions with macrophages, from initial recruitment through granuloma formation, that resemble those seen for M. marinum. The presence of M. leprae-infected macrophages in the circulation of larvae mirrors findings in human leprosy (Drutz et al., 1972Drutz D.J. Chen T.S.N. Lu W.-H. The continuous bacteremia of lepromatous leprosy.N. Engl. J. Med. 1972; 287: 159-164Crossref PubMed Scopus (69) Google Scholar). We next investigated the interactions of M. leprae with cells of the zebrafish nervous system, to determine if infection produced demyelination. Transgenic mbp (myelin basic protein) larvae express membrane-localized GFP that labels the myelinating membrane of glia in both the peripheral nervous system (Schwann cells) and central nervous system (oligodendrocytes) (Jung et al., 2010Jung S.-H. Kim S. Chung A.-Y. Kim H.-T. So J.-H. Ryu J. Park H.-C. Kim C.-H. Visualization of myelination in GFP-transgenic zebrafish.Dev. Dyn. 2010; 239: 592-597Crossref PubMed Scopus (19) Google Scholar). Oligodendrocytes express all Schwann cell determinants that have been reported to interact with M. leprae (Table S1), and myelin structure is similar in the central and peripheral nervous systems (Morell and Quarles, 1999Morell P. Quarles R.H. Characteristic composition of myelin.in: Siegel G.J. Agranoff B.W. Albers R.W. Fisher S.K. Uhler M.D. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. Lippincott-Raven, 1999: 56-67Google Scholar). Therefore, we studied M. leprae interactions with nerves in the spinal cord rather than peripheral nerves because of their easier accessibility. We injected fluorescent M. leprae into the dorsal spinal cord of larvae at 2–4 days post-fertilization (dpf), and imaged nerves at 4–8 dpf, a developmental stage at which these tracts have become myelinated (Czopka, 2016Czopka T. Insights into mechanisms of central nervous system myelination using zebrafish.Glia. 2016; 64: 333-349Crossref PubMed Scopus (32) Google Scholar) (Figures 2A and 2B ). At 2 dpi (4 dpf), we observed cellular protrusions from an otherwise intact myelin sheath, clustered around M. leprae in the nerve (Figure 2C). M. marinum injected into the dorsal spinal cord did not alter the myelinating membrane structure, even though the M. marinum burdens at the injection sites were higher than those in M. leprae infections (Figures 2C–2E). The M. leprae-induced myelin protrusions increased in size and number with time but always remained next to the bacteria (Figure 2F). Three-dimensional rendering showed that protrusions were doughnut shaped, not spherical, suggesting that these structures were not cell bodies but rather protrusions of myelinating membrane (Figure 2G; Movie S1). In vitro studies suggest that M. leprae interacts with glial determinants through a surface-localized long chain lipid, known as PGL-1 (m/z 2,043.75), which carries a unique trisaccharide (Ng et al., 2000Ng V. Zanazzi G. Timpl R. Talts J.F. Salzer J.L. Brennan P.J. Rambukkana A. Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.Cell. 2000; 103: 511-524Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Renault and Ernst, 2015Renault C.A. Ernst J.D. Mycobacterium leprae (Leprosy).in: Bennett J.E. Dolin R. Blaser M.J. Mandell, Douglas, and Bennett’s Infectious Disease Essentials. Elsevier, 2015: 2819-2831Google Scholar) (Figure S2A). The phenolic glycolipid of M. marinum contains a monosaccharide and shorter lipid chains that renders it detectable at a lower mass value (m/z 1,567.44) (Figures 3A and S2B). We wondered if the trisaccharide that is normally found on M. leprae PGL-1 would be sufficient to render M. marinum capable of altering myelin. We transformed M. marinum with the six M. leprae genes responsible for assembly of PGL-1’s terminal disaccharide (Tabouret et al., 2010Tabouret G. Astarie-Dequeker C. Demangel C. Malaga W. Constant P. Ray A. Honoré N. Bello N.F. Perez E. Daffé M. Guilhot C. Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.PLoS Pathog. 2010; 6: e1001159Crossref PubMed Scopus (52) Google Scholar). Ion chromatograms (Figures 3A and 3B ) and collision-induced dissociation mass spectrometry (Figure S2) of total lipid from the transformant, M. marinum:PGL-1, proved that it produced triglycosylated PGL-1. PGL-1 expression conferred on M. marinum the ability to cause myelin protrusions, indistinguishable from those of M. leprae in both morphology and their invariable co-localization with the bacteria (Figures 3C–3E).Figure 3Phenolic Glycolipid 1 Triggers Myelin DissociationShow full caption(A) Normal phase high-performance liquid chromatography mass spectrometry measured at the known mass-to-charge ratios (m/z) for triglycosylated and monoglycosylated forms of PGL, leading to the separate detection of PGL-mar (m/z 1,567.44, upper structure) and PGL-1 (m/z 1,903.58, lower structure) in total lipid extracts of the indicated strains (B).(B) Chromatograms of the ions depicted in (A), showing the increased retention time of PGL-1 from M. marinum:PGL-1 (Mm:PGL1) compared to that of PGL-mar from WT M. marinum.(C) Representative confocal images, like in Figure 2C, of 2 dpi (4 dpf) larvae infected with ∼200 CFU M. marinum or M. marinum:PGL-1; myelin protrusions are quantified in (D). Scale bar, 10 μm.(D) Mean number of myelin protrusions per animal in uninjected larvae (unt) or after injection with PBS vehicle (veh), M. marinum, or M. marinum:PGL-1 (∼200 CFU each; ∗p < 0.05, one-way ANOVA with Bonferroni’s post-test).(E) Mean bacterial burden at the injection site of larvae from (D).(F) Representative confocal image of a 6 dpf larva with fluorescently labeled nuclei, 4 dpi with M. marinum:PGL-1 (∼100 CFU). Asterisk indicates an aggregate of infected cells. Scale bar, 10 μm.(G) Stills from time-lapse imaging of an mbp larva injected with M. marinum:PGL-1, showing myelin protrusions forming from apparently intact myelin. Arrow, intact myelin sheath; arrowheads, myelin protrusions. Relative time code. Scale bar, 10 μm.See also Figures S2 and S3 and Movie S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Normal phase high-performance liquid chromatography mass spectrometry measured at the known mass-to-charge ratios (m/z) for triglycosylated and monoglycosylated forms of PGL, leading to the separate detection of PGL-mar (m/z 1,567.44, upper structure) and PGL-1 (m/z 1,903.58, lower structure) in total lipid extracts of the indicated strains (B). (B) Chromatograms of the ions depicted in (A), showing the increased retention time of PGL-1 from M. marinum:PGL-1 (Mm:PGL1) compared to that of PGL-mar from WT M. marinum. (C) Representative confocal images, like in Figure 2C, of 2 dpi (4 dpf) larvae infected with ∼200 CFU M. marinum or M. marinum:PGL-1; myelin protrusions are quantified in (D). Scale bar, 10 μm. (D) Mean number of myelin protrusions per animal in uninjected larvae (unt) or after injection with PBS vehicle (veh), M. marinum, or M. marinum:PGL-1 (∼200 CFU each; ∗p < 0.05, one-way ANOVA with Bonferroni’s post-test). (E) Mean bacterial burden at the injection site of larvae from (D). (F) Representative confocal image of a 6 dpf larva with fluorescently labeled nuclei, 4 dpi with M. marinum:PGL-1 (∼100 CFU). Asterisk indicates an aggregate of infected cells. Scale bar, 10 μm. (G) Stills from time-lapse imaging of an mbp larva injected with M. marinum:PGL-1, showing myelin protrusions forming from apparently intact myelin. Arrow, intact myelin sheath; arrowheads, myelin protrusions. Relative time code. Scale bar, 10 μm. See also Figures S2 and S3 and Movie S2. The protrusions, like those produced by M. leprae, did not colocalize with a histone marker that labels cell nuclei. This suggested they did not simply represent an accumulation of oligodendrocyte cell bodies, but rather were composed of myelinating membrane (Figure 3F). Using time-lapse imaging to observe the formation of protrusions in real time, we observed that an intact myelin sheath near the M. marinum:PGL-1 injection site began to condense and then bulge (Figure 3G). Protrusions formed by 10 hr post-infection and expanded over time (Figure 3G). To further test if myelin protrusions represent recruitment or proliferation of oligodendrocyte cell bodies, we generated larvae with a single GFP-labeled oligodendrocyte. Time-lapse movies of these larvae showed that individual oligodendrocytes form myelin protrusions by retracting portions of myelinating membrane from previously intact sheaths (Movie S2A). This occurred after injection with M. marinum:PGL-1, but not with PBS (compare Movies S2A and S2B). These findings strongly suggested that the protrusions arise from previously intact myelin sheaths, consistent with early demyelination. Similar to human leprosy (de Freitas and Said, 2013de Freitas M.R.G. Said G. Leprous neuropathy.in: Gérard S. Christian K. Handbook of Clinical Neurology. Elsevier, 2013: 499-514Crossref Scopus (21) Google Scholar), myelin dissociation occurred in discrete areas, with the surrounding myelin sheaths remaining intact (Figures S3A–S3D). Demyelination can be imaged in detail by transmission electron microscopy (TEM). We compared TEM images of transverse sections through areas of myelin protrusions at 2 days after infection to identical sections through the injection site of PBS-injected fish (Figures 4A–4C). TEMs from animals injected with M. leprae or M. marinum:PGL-1 revealed a selective decrease in myelinated axons, while the total number of axons was preserved (Figures 4D, 4E, S3E, and S3F). Higher-magnification images revealed apparently intact axons surrounded by disorganized myelin, with large spaces in between the individual lamellae (Figure S3G); this myelin decompaction is characteristic of early demyelination in human leprosy (Figure 4F) (Job, 1973Job C.K. Mechanism of nerve destruction in tuberculoid-borderline leprosy. An electron-microscopic study.J. Neurol. Sci. 1973; 20: 25-38Abstract Full Text PDF PubMed Scopus (17) Google Scholar, Shetty et al., 1988Shetty V.P. Antia N.H. Jacobs J.M. The pathology of early leprous neuropathy.J. Neurol. Sci. 1988; 88: 115-131Abstract Full Text PDF PubMed Scopus (66) Google Scholar). The condensed, fragmented myelin, which was no longer associated with axons, was observed scattered throughout the extracellular space (Figures 4A–4C and S3G). In vitro studies have focused on M. leprae-induced demyelination as a mechanism of nerve injury (Rambukkana et al., 2002Rambukkana A. Zanazzi G. Tapinos N. Salzer J.L. Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.Science. 2002; 296: 927-931Crossref PubMed Scopus (164) Google Scholar, Scollard, 2008Scollard D.M. The biology of nerve injury in leprosy.Lepr. Rev. 2008; 79: 242-253Crossref PubMed Google Scholar). However, the peripheral neuropathy of human leprosy involves both myelinated and nonmyelinated axons (Medeiros et al., 2016Medeiros R.C.A. Girardi K.D. Cardoso F.K.L. Mietto B.S. Pinto T.G.T. Gomez L.S. Rodrigues L.S. Gandini M. Amaral J.J. Antunes S.L.G. et al.Subversion of Schwann cell glucose metabolism by Mycobacterium leprae.J. Biol. Chem. 2016; 291: 24803Crossref PubMed Scopus (0) Google Scholar, Shetty and Antia, 1996Shetty V.P. Antia N.H. A semi quantitative analysis of bacterial load in different cell types in leprous nerves using transmission electron microscope.Indian J. Lepr. 1996; 68: 105-108PubMed Google Scholar, Shetty et al., 1988Shetty V.P. Antia N.H. Jacobs J.M. The pathology of early leprous neuropathy.J. Neurol. Sci. 1988; 88: 115-131Abstract Full Text PDF PubMed Scopus (66) Google Scholar). To test if nonmyelinated axons were also affected in zebrafish, we selected an area of the spinal cord containing only one myelinated axon surrounded by many nonmyelinated axons. We observed swelling of nonmyelinated axons, as evidenced by their increased area compared to PBS-injected control (Figures 4G and 4H). Thus, M. leprae and M. marinum:PGL-1 rapidly induce damage to both myelinated and nonmyelinated axons in the zebrafish, similar to the pathological changes found in human leprosy. Contrary to the previous model (Rambukkana, 2000Rambukkana A. How does Mycobacterium leprae target the peripheral nervous system?.Trends Microbiol. 2000; 8: 23-28Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), our findings in vivo did not support cont" @default.
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• W2612773113 title "A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy" @default.
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