Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish

RESEARCH ARTICLE Identification of protective postexposure mycobacterial vaccine antigens using an immunosuppression-based reactivation model in the zebrafish Henna Myllyma ki 1, *, Mirja Niskanen 1, *, Hanna Luukinen 1 , Mataleena Parikka 1,2 and Mika Ra met 1,3,4,5,‡ ABSTRACT Roughly one third of the human population carries a latent Mycobacterium tuberculosis infection, with a 5-10% lifetime risk of reactivation to active tuberculosis and further spreading the disease. The mechanisms leading to the reactivation of a latent Mycobacterium tuberculosis infection are insufficiently understood. Here, we used a natural fish pathogen, Mycobacterium marinum,to model the reactivation of a mycobacterial infection in the adult zebrafish (Danio rerio). A low-dose intraperitoneal injection (∼40 colony-forming units) led to a latent infection, with mycobacteria found in well-organized granulomas surrounded by a thick layer of fibrous tissue. A latent infection could be reactivated by oral dexamethasone treatment, which led to disruption of the granuloma structures and dissemination of bacteria. This was associated with the depletion of lymphocytes, especially CD4 + T cells. Using this model, we verified that ethambutol is effective against an active disease but not a latent infection. In addition, we screened 15 mycobacterial antigens as postexposure DNA vaccines, of which RpfB and MMAR_4207 reduced bacterial burdens upon reactivation, as did the Ag85-ESAT-6 combination. In conclusion, the adult zebrafish-M. marinum infection model provides a feasible tool for examining the mechanisms of reactivation in mycobacterial infections, and for screening vaccine and drug candidates. This article has an associated First Person interview with the first author of the paper. KEY WORDS: Tuberculosis, Reactivation, Zebrafish, Vaccine antigens INTRODUCTION Tuberculosis (TB) remains one of the major global health problems. Mycobacterium tuberculosis (Mtb), the causative agent of TB, led to 1.4 million deaths and 10.4 million new infections in 2015 (WHO, 2017). The World Health Organization (WHO) estimates that one third of the human population carries a latent TB infection, and therefore has up to a 10% lifetime risk of it reactivating into an active disease. Both vaccines and antibiotics have their limitations in combating TB. Curative antibiotic treatments are lengthy and further complicated by the emergence of multidrug-resistant Mtb strains (WHO, 2017). The only available TB vaccine, Bacillus Calmette Guérin (BCG), is still widely used. Although the BCG vaccine can protect infants from disseminated TB, its ability to induce long-term cell-mediated immune responses is limited, and therefore it does not properly prevent the reactivation of a latent TB infection (Tang et al., 2016). To reach the ambitious goal of eliminating TB by the year 2050, innovative approaches are needed. As nonhuman primates are the only animal models that fully replicate all phases seen in human TB, it has been challenging to study this area, especially the latency and reactivation of mycobacterial infections (Capuano et al., 2003; Myllymäki et al., 2015). It is known that reactivation of a latent TB infection is often associated with immunosuppression, such as human immunodeficiency virus (HIV), chemotherapy or immunosuppressive drugs (Ai et al., 2016), but the more detailed mechanisms of reactivation remain largely elusive on both the bacterial and host side (Dutta and Karakousis, 2014; Peddireddy et al., 2017). Considering the number of latent TB carriers, preventing the reactivation of latent TB would be a key step in the battle against TB (Matteelli et al., 2017). During the past couple of decades, the zebrafish (Danio rerio)has proven an applicable alternative for modeling TB (Myllymäki et al., 2016). For this purpose, a natural fish pathogen and a close relative of Mtb, Mycobacterium marinum, is used (Stinear et al., 2008). Depending on the infectious dose, a M. marinum infectioninadult zebrafish can lead either to an active or to a naturally latent form of the disease (Parikka et al., 2012; Swaim et al., 2006). In the latent form of human TB, the bacteria are contained in structures termed granulomas, which are surrounded by immune cells and a fibrotic capsule. Although granulomas were long thought to be a protection method elicited solely by the host, it has been found that they also promote persistence of the bacteria (O’Garra et al., 2013; Davis and Ramakrishnan, 2009). Upon a M. marinum infection, both adult zebrafish and larvae form granulomas that are highly similar in structure to those in humans (Parikka et al., 2012; Davis et al., 2002), and the immune responses they elicit against mycobacteria share similarities to those in humans (Parikka et al., 2012; Swaim et al., 2006; Hammarén et al., 2014; Clay et al., 2008; Torraca et al., 2015; Pagán et al., 2015; Yang et al., 2012). This has facilitated the translation of some of the results from fish studies to humans (Tobin et al., 2010; Adams et al., 2014; Berg et al., 2016; Thuong et al., 2017). In the zebrafish, a latent mycobacterial infection can spontaneously reactivate into an active disease (Hammarén et al., Received 22 November 2017; Accepted 14 February 2018 1 BioMediTech Institute and Faculty of Medical and Life Sciences, University of Tampere, Tampere FI-33014, Finland. 2 Oral and Maxillofacial Unit, Tampere University Hospital, Tampere FI-33521, Finland. 3 Department of Pediatrics, Tampere University Hospital, Tampere FI-33521, Finland. 4 Department of Children and Adolescents, Oulu University Hospital, Oulu FI-90220, Finland. 5 PEDEGO Research Unit, and, Medical Research Center, University of Oulu, Oulu FI-90014, Finland. *These authors contributed equally to this work ‡ Author for correspondence (mika.r[email protected]) H.M., 0000-0002-6936-4879 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 © 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms 2014). In the current study, we have developed an experimental method to reactivate a latent mycobacterial infection in the adult zebrafish by feeding them the glucocorticoid dexamethasone. We use this model for the characterization of the cellular and molecular mechanisms associated with the reactivation of a mycobacterial infection. In addition, we show that the model can be used in screening for antibiotics and novel vaccine candidates against the reactivation of a latent mycobacterial infection. RESULTS Dexamethasone treatment leads to an elevated bacterial burden in zebrafish with a latent M. marinum infection In humans, the reactivation of a latent TB infection often follows immunosuppression. Similarly, immunosuppression by gamma irradiation leads to the reactivation of a mycobacterial infection in zebrafish (Parikka et al., 2012). To further study the mechanisms of the reactivation of a mycobacterial infection in zebrafish, we tested the effects of various immunosuppressive medicines on a latent M. marinum infection (Fig. 1A). The fish were first infected with a low dose of M. marinum, which leads to a latent infection in most fish, and the immunosuppressive treatments were started 5 weeks later. For this, we selected chemicals that are commonly used in humans to alleviate excessive immune reactions associated with various medical situations, such as organ transplantation, arthritis and inflammatory bowel disease, and have been reported to increase risk of the reactivation of TB in individuals with a latent infection (Jick et al., 2006; Maltzman and Koretzky, 2003). The drugs included azathioprine, dexamethasone, methylprednisolone and prednisolone. To minimize additional stress, the drugs were administered orally, as a gelatin mix that was used to coat the fish food pellets. The experimental fish were treated with the drugs, 10 µg/fish/day, for 4 weeks, whereas the control fish received gelatin-coated food with no chemicals. After the treatment, the fish were euthanized and the DNA extracted from the intraperitoneal cavity of each fish was used to determine the number of mycobacteria. We found that feeding with dexamethasone led to an increase in bacterial burdens (P=0.04, one-tailed Mann–Whitney test); azathioprine produced a similar trend, though the increase was not statistically significant (Fig. 1A). Within the anticipated dose of 10 µg/day, prednisolone and methylprednisolone did not lead to elevated bacterial burdens. One fish from the control group and five fish from the group fed with dexamethasone were euthanized according to the humane endpoint criteria during the treatment – these fish were also included in the analysis. Consistently, a high number of mycobacteria [average of 2.9×10 6 ±4.8×10 5 colonyforming units (cfu)] was detected in the euthanized fish (Fig. 1A), suggesting that the signs of discomfort were due to the progression of the M. marinum infection rather than the immunosuppression itself. In line with this, the feeding of dexamethasone did not cause signs of discomfort in uninfected fish. Therefore, the increase in the bacterial counts following a dexamethasone treatment is likely to be caused by the progression of a latent mycobacterial infection into an active phase. The kinetics of this progression were analyzed by measuring the bacterial numbers at several time points following a dexamethasone treatment in two independent experiments (Fig. 1B; Fig. S1A). An increase in the bacterial burden was observed 3 days after the onset of the feeding of dexamethasone (P=0.007, one-tailed Mann– Whitney test), eventually leading to an approximately 1000-fold increment at 4 weeks. A higher dose of dexamethasone (20 µg/day/ fish) had an essentially identical effect on bacterial numbers (Fig. S1B). Therefore, the lower dose of dexamethasone (10 µg/day) was selected for subsequent experiments. A pretreatment with dexamethasone prior to infection did not affect the outcome of a lowdose infection (Fig. S1C), suggesting that dexamethasone is more likely to affect the host’s ability to control a latent infection than to suppress the responses against mycobacteria during an early infection. To obtain a more mechanistic insight into this, we next investigated the cellular and molecular events associated with the reactivation of a latent mycobacterial infection in the adult zebrafish. Reactivation of a latent mycobacterial infection alters the quality and quantity of granulomas The formation of granulomas is defined as the hallmark of tuberculosis, and granulomas also form in the adult zebrafish during the course of an M. marinum infection. To demonstrate the presence of granulomas during reactivation, we used Ziehl–Neelsen staining to visualize mycobacteria in histological sections, and trichrome staining to visualize the fibrous capsule typically found around mature granulomas (Parikka et al., 2012). In fish with a latent infection, the granulomas were generally well organized and surrounded by a thick layer of fibrous tissue confining the mycobacteria inside the granulomas (Fig. 2A). After 2 weeks of dexamethasone treatment, the granulomas appeared larger and looser in structure, showing thinning and disruption of the Fig. 1. Dexamethasone treatment increases bacterial loads in adult zebrafish with a latent M. marinum infection. (A) Zebrafish with a latent mycobacterial infection were treated with immunosuppressants. Each dot shows the bacterial count in one fish after 4 weeks of exposure to immunosuppressive medication (10 µg/fish/day), (n=17-19 fish/group). (B) Dots represent bacterial loads after 3, 7, 4 and 28 days of dexamethasone treatment of fish with a latent mycobacterial infection (n=9-10 fish/group). Horizontal lines represent the median bacterial count of each group. Statistical significance was analyzed with the one-tailed Mann–Whitney test. See also Fig. S1. 2 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms surrounding fibrous layer. After 3 weeks of dexamethasone treatment, further loss of granuloma integrity was observed, together with bacteria escaping from the granulomas and spreading into tissues, indicating progression of the infection into an active state. To quantify the dexamethasone-induced changes in the number and types of granulomas, we counted the total number of granulomas per fish at different time points during the treatment (4, 14 and 21 days, n=3-6) (Fig. 2B). In the control group, the number of granulomas remained relatively constant (average of 917 granulomas/fish), while in the dexamethasone-treated group, the number of granulomas increased from an average of 17 granulomas at day 4 to an average of 57 granulomas per fish at 21 days. To further characterize the process, we calculated the number of Fig. 2. Dexamethasone treatment leads to disruption of the granuloma structure, increase in the number of granulomas and loss of hypoxia in zebrafish with a latent mycobacterial infection. (A) Ziehl–Neelsen staining (ZN) of mycobacteria (purple). Black arrowheads indicate the outline of a granuloma. Trichrome staining (TC) of fibrous tissue around granulomas (blue) (black arrows). (B) Quantification of the total number of granulomas and the proportion of necrotic and non-necrotic granulomas (left), and the number of nascent granulomas and sites with non-capsulated mycobacteria (right) per fish after 4, 14 and 21 days of dexamethasone treatment or normal feeding (n=3-6 fish/group). Data are presented as mean±s.d. Statistical significance was assessed by unpaired Student’st-test. (C) Hypoxic staining shows hypoxic lesions inside granulomas in dark brown (pink stars). The proportions of hypoxic and non-hypoxic granulomas during a latent infection, and after 2 weeks of dexamethasone treatment, are quantified in the pie chart. See also Fig. S2. 3 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms necrotic (Fig. 2B) and multicentric granulomas separately (Fig. S2A-C). Both are subtypes of mature granulomas that contain a high number of mycobacteria, indicating an advanced stage of a mycobacterial disease (Parikka et al., 2012). We found that after 3 weeks of dexamethasone treatment, the number of necrotic granulomas had increased from an average of 4.5 at day 4 to 27 at day 21 (P=0.02, one-tailed Mann–Whitney test) (Fig. 2B), while there was also an increase in the number of multicentric granulomas (Fig. S2B) and the proportion of necrotic versus non-necrotic granulomas (Fig. S2C). Consistent with the qualitative analysis, there was also a clear increase in the number of nascent granulomas and sites with noncapsulated mycobacteria, from a mean of 11 granulomas or sites at day 4 to 51 at day 21 (Fig. 2B). This suggests that the treatment leads to the escape of mycobacteria from existing granulomas and the seeding of new ones. Hypoxia has been shown to have an important role in mycobacterial pathogenesis. A low-oxygen environment induces metabolic adaptation in bacteria, which allows the bacteria to persist inside the host during a latent infection. To visualize hypoxic areas in granulomas, pimonidazole treatment and Hypoxyprobe staining were used (Fig. 2C) (Matty et al., 2015). Small hypoxic areas were found adjacent to 25% of the granulomas in the fish with a latent infection. Following a 21-day dexamethasone treatment, hypoxic lesions were observed in only 8% of all granulomas (P<0.0001, Fisher’s exact test) (Fig. 2C). Altogether, these data suggest that during the reactivation of a latent disease, mycobacteria first replicate within existing granulomas. This leads to bacteria escaping and forming new granulomas, as well as the appearance of free bacteria, which is associated with the disruption of the granuloma structure and the loss of hypoxia inside the granulomas. Dexamethasone treatment leads to a decrease in the amount of lymphocytes Next, we investigated the alterations caused by dexamethasone in immune cell populations in the kidney, which is the site of hematopoiesis in the adult zebrafish. For this, we first treated wildtype fish with dexamethasone for 1, 2 and 6 weeks and dissected their kidneys for a flow cytometric [fluorescence-activated cell sorting (FACS)] analysis. For the analysis, the cells in the live gate were separated into populations based on size [defined by the forward scatter (FSC)] and granularity [side scatter (SSC)]. These included lymphocytes, granulocytes and monocytes, and blood cell precursors. Upon dexamethasone treatment, the proportion of lymphocytes decreased from 19.3±3.5% to 12.4±1.6% (P<0.01, two-way ANOVA), while the relative amount of granulocytes and monocytes and blood cell precursors remained unchanged (Fig. S3). To study the effect of dexamethasone treatment on lymphocytes in more detail, we used the Tg:lck(lck-EGFP) fish line, which expresses GFP under the tyrosine kinase promoter specific for mature T cells, NK-like cells and myeloid-like cells in the kidney of adult zebrafish (Langenau et al., 2004; Carmona et al., 2017). lckGFP fish were treated with dexamethasone for 1, 2 and 4 weeks, followed by FACS analysis of the kidney cell populations as above (Fig. 3; Fig. S4B). As expected, the GFP + cell population mainly fell into the lymphocyte gate (Fig. 3A; Fig. S4B,C) (Langenau et al., 2004). Again, upon dexamethasone treatment, the proportion of lymphocytes decreased from 30.1±4.0% to 18.2±3.1% (P<0.001, two-way ANOVA), while there was a slightly increasing trend in the relative amount of granulocytes and monocytes and blood cell precursors (Fig. 3B,C; Fig. S4B,C). More specifically, the proportion of GFP + lymphocytes decreased during the first week of dexamethasone treatment, from the original 12.5±3.4% of live cells in untreated fish to 6.3±2.4% (P<0.001, two-way ANOVA), after which it remained the same throughout the treatment (Fig. 3D). In addition, the GFP – lymphocyte population, which contains B cells and immature T cells (Langenau et al., 2004), decreased from 18.5±2.8% to 10.3±1.7% of live cells (P<0.001, two-way ANOVA) after 4 weeks of treatment (Fig. 3D). The proportion of GFP + lymphocytes compared to GFP – ones decreased slightly at the 1and 2-week time points, but by 4 weeks, the GFP + :GFP – ratio had returned to the same as that in the control group (40.2% and 43.0% of GFP + lymphocytes) (Fig. S4D). Dexamethasone treatment alters the expression of T cell markers upon infection To further characterize the effects of dexamethasone treatment on the immune response against mycobacteria, we compared the kidney lymphocyte population of uninfected and infected wild-type zebrafish after 1 week of treatment with dexamethasone (Fig. 4; Fig. S5). A latent mycobacterial infection led to an increase in the lymphocyte population (from 21.7±4.5% to 28.3±7.3%; P<0.01, two-way ANOVA with Bonferroni posttest). Treating fish with a latent infection with dexamethasone led to a decrease in the proportion of lymphocytes, to the level seen in uninfected fish (24.6±3.2%, P<0.05, two-way ANOVA with Bonferroni posttest) (Fig. 4A). To investigate the effect of dexamethasone on lymphocyte function, we studied the expression levels of marker genes of different types of T and B cells from the isolated kidney cells using quantitative reverse transcription PCR (qRT-PCR) (Fig. 4B-G; Fig. S5B-G). Of the studied inflammation markers, both tnfa and interferon gamma 1-2 (ifng1-2;ifng-1) were induced in the fish carrying a latent infection, with the former being slightly upregulated (Fig. S5B), and the latter slightly downregulated (Fig. 4B), by dexamethasone. The expression of tnfa correlated with the bacterial burden (Spearman r 0.8180, P<0.0001), which likely explains its relative elevation in the dexamethasone-treated individuals, whereas no such correlation was observed for ifng1-2 expression (r –0.1740). Zebrafish have CD4 + and CD8 + lymphocyte populations with functions similar to those in humans (Langenau et al., 2004; Yoon et al., 2015; Renshaw and Trede, 2012). Consistently, the expression of both cd4-1 and cd4-2.1 was induced upon infection (P=0.004 and P=0.0002, respectively), whereas their expression was reduced following dexamethasone treatment: cd4-1 was suppressed following reactivation (P=0.0002) (Fig. 4C), and cd4-2.1 was reduced in both uninfected (P=0.02) and infected (P=0.088) fish (Fig. 4D). Expression of cd8a, a marker gene for cytotoxic T cells, was induced in infected fish (P<0.0001), but its expression was not altered by the dexamethasone treatment (Fig. 4E). The balance between the CD4 + Th1 and Th2 cells has been shown to play a role in the control of a mycobacterial infection (Hammarén et al., 2014). To study the CD4 + subpopulation more specifically, we used the expression of the tbx21 transcription factor as a marker for the Th1 population, and the transcription factor gata3 and il4,il10 and il13 as markers for Th2 cells. tbx21 expression was induced upon infection (P<0.0001) (Fig. 4F), as was the expression of il10 (P=0.004) (Fig. S5C), while the expression of the other Th2 markers tested remained relatively constant (Fig. S5D,E). Dexamethasone treatment suppressed the expression of tbx21 in uninfected fish (P=0.052) and in fish with a latent infection (P=0.02) (Fig. 4F). Of the Th2 markers, a decreasing expression for gata3 was seen after dexamethasone treatment in 4 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms uninfected fish (P=0.03) and a corresponding trend was observed in infected fish (Fig. 4G). The expression of IgM (ighm), which was used as a marker for B cells, remained relatively constant in both infected and dexamethasone-treated fish (Fig. S5F), whereas tgfb1b, a marker for regulatory T cells (Tregs), was suppressed upon infection in the immunocompetent fish (P=0.0007), and upregulated in the dexamethasone-treated group (P=0.03) (Fig. S5G). However, the expression of foxp3a, a transcription factor characteristic for Tregs was not affected by dexamethasone treatment (Fig. S4H). Overall, these data indicate that a dexamethasone treatment leads to the general depletion of lymphocytes. The most prominent effect is seen in the expression of cd4-1,cd4-2.1 and the Th1 marker tbx21. Although dexamethasone-treated fish are able to induce an IFN-γresponse against mycobacteria, the suppression of specific T cell populations, together with the induction of the inhibitory cytokine tgfb1b, are associated with compromised ability to control infection. The zebrafish model can be utilized to assess the efficacy of drugs at different phases of a mycobacterial infection Owing to their small size and fast reproduction rate, zebrafish are well suited for large-scale pharmaceutical screening studies (Lohi et al., 2013). On this premise, we investigated whether the dexamethasone-based reactivation model can be used for testing the effectiveness of antimicrobial medicines against reactivated mycobacteria. We chose four different antimicrobial drugs that have been used for treating mycobacterial diseases, including ethambutol, isoniazid, amikacin and metronidazole. Each of the antibiotics Fig. 3. Dexamethasone treatment leads to a depletion of lymphocytes in the adult zebrafish. (A) Sorting of the kidney cell populations of lck-GFP fish based on size (side scatter, SSC-A) and granularity (forward scatter, FSC-A) (left). The lymphocyte population is further separated into GFP + and GFP – subpopulations (middle), and the majority of the GFP – cells fall into the lymphocyte gate by the SSC-A and FSC-A. (B) After a 1-week dexamethasone treatment, the kidney cells fall into the same gates, but the number of both GFP + and GFP – lymphocytes is depleted (right), as quantified in C and D. n=12. Data are presented as mean±s.d. Statistical significance is analyzed by twoway ANOVA with Bonferroni posttest, *P<0.05, **P<0.01, ***P<0.001. Also see Figs S3 and S4. 5 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms was first tested for their effect against M. marinum in vitro using a bioluminescent strain, which allowed the quantification of bacterial growth at different time points with a luminometer. Two different doses were tested for each antimicrobial drug. During the 7-day follow-up period, all antibiotics, excluding isoniazid, inhibited the growth of M. marinum in a dosedependent manner, compared to bacteria cultured in medium without antibiotics (Fig. 5A). Higher doses of ethambutol (2.4 µg/ml), and amikacin (45.5 µg/ml), completely prevented bacterial growth (P<0.05, respectively, one-way ANOVA with Friedman’s test). Next, zebrafish with a latent M. marinum infection were treated for 3 weeks with dexamethasone, followed by 4 weeks of treatment with antibiotics (50 µg/fish/day each). Metronidazole (160 µg/ml), Fig. 4. Dexamethasone treatment decreases the expression of CD4 + T lymphocyte markers in uninfected and infected zebrafish. (A) Lymphocytes are expanded in the zebrafish kidney upon a latent M. marinum infection and depleted by dexamethasone treatment. The kidney cell populations of AB fish were analyzed with FACS. Bars show the mean percentage of lymphocytes±s.d. P-values were calculated using two-way ANOVA with Bonferroni posttest, *P<0.05, **P<0.01, ***P<0.001. (B-G) Relative expression levels of inflammatory genes and T cell markers. Each dot represents the expression level of the marker gene relative to the housekeeping gene (EF1a). Horizontal lines show the median value in each group. n=10-11 fish/group. P-values were calculated using the two-tailed Mann– Whitney test. (B) ifng1-2, (C) cd4-1, (D) cd4-2.1, (E) cd8a, (F) tbx21, (G) gata3. See also Fig. S5. 6 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms which is designed to target anaerobic microbes, inhibited the growth of M. marinum in vitro by 48% compared to controls, but did not limit the bacterial burden in vivo in a latent (Fig. S6A) or reactivated infection (Fig. 5B). However, ethambutol decreased the median bacterial counts in dexamethasone-treated zebrafish by 96% compared to untreated controls (P=0.01, Mann–Whitney test), which is close to the level seen in a latent infection (Fig. 5B). Of note, a 2-week treatment with ethambutol did not affect bacterial counts in zebrafish with a latent M. marinum infection (Fig. S6B), consistent with the knowledge that ethambutol acts by preventing the replication of mycobacteria (Forbes et al., 1962). This further demonstrates that dexamethasone treatment causes the reactivation of a latent mycobacterial infection in zebrafish, leading to the active replication of the bacteria and susceptibility to ethambutol. Identification of novel protective postexposure vaccine antigens against the reactivation of TB in the zebrafish model We have previously shown that the zebrafish-M. marinum infection model is suitable for the preclinical screening of novel vaccine candidates against a mycobacterial infection (Oksanen et al., 2013, 2016). The zebrafish can be partially Fig. 5. Ethambutol inhibits the growth of M. marinum both in vitro and upon dexamethasone treatment in vivo.(A) The effects of selected antibiotics on the growth of a bioluminescent strain of M. marinum in vitro. The graphs represent the detected relative bioluminescence (RLU) of bacteria at the indicated time points after exposing them to an antibiotic. n=5-6. One-way ANOVA with Friedman’s test and Dunn’s multiple comparison test was used for statistical analysis, *P<0.05, **P<0.01. (B) Adult zebrafish with a latent mycobacterial infection were treated for 3 weeks with dexamethasone (10 µg/fish/day) followed by 4 weeks of treatment with selected antibiotics. Each dot represents the bacterial burden per fish. Horizontal lines show the median value of each group. The statistical analysis was performed with the two-tailed Mann–Whitney test. 7 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms protected against a primary M. marinum infection by BCG, or by prophylactic DNA-based vaccines consisting of a combination of previously studied mycobacterial antigens (Ag85 and ESAT-6), as well as with some novel antigens (Myllymäki et al., 2017). Based on this, we studied the applicability of the dexamethasonebased reactivation model for the preclinical screening of new DNAbased vaccines by examining the therapeutic effect of the selected mycobacterial antigens against the reactivation of a latent M. marinum infection. The zebrafish were first infected with a low dose of M. marinum, and vaccinated 5 weeks postinfection. Four weeks after the vaccinations, the fish were subjected to dexamethasone treatment (10 µg/fish/day) for 4 weeks, after which their bacterial burdens were quantified by qPCR (the protocol is outlined in Fig. 6). The antigens showing a significant reduction in bacterial counts in the initial screen were subjected to a replicate experiment to confirm the effect. We first tested the effect of the Ag85-ESAT-6 antigen combination against reactivation, and found that this immunization decreased the bacterial counts by 56% or 0.35 log 10 (P=0.02, two-tailed Mann–Whitney test) compared to the control group immunized with an empty GFP plasmid (Fig. 7). The other antigens tested involved genes from different functional categories, including the four M. marinum Resuscitation promoting factors (Rpf proteins), three members of the PE/PPE protein family, four conserved membrane proteins, one secreted factor and three metabolic enzymes. Compared to the GFPimmunized control group, most of these did not affect the bacterial burden of the fish upon reactivation. Noteworthy, immunization with the antigens consisting of RpfB and MMAR_4207, led to a significant reduction in bacterial counts (by 63% or 0.53 log 10 and 85% or 0.81 log 10 , respectively) (Fig. 7). Altogether, these data show that the dexamethasone-based reactivation model provides an amenable tool for the preclinical screening of both novel antimicrobials and therapeutic vaccines against the reactivation of TB. For the latter, we identified two promising candidates, RpfB and the conserved membrane protein MMAR_4207. DISCUSSION TB remains a major health problem, and new antimicrobial medicines and vaccines are needed to prevent new infections and to prevent the reactivation of a latent TB infection (WHO, 2017). During the past decade, the zebrafish, with its natural pathogen M. marinum, has become a powerful model for studying the mechanisms associated with mycobacterial defense as well as new therapeutic strategies to fight mycobacterial infections. Some of the results have been already translated into combatting human TB infections (Adams et al., 2014; Tobin et al., 2013, 2012; Oehlers et al., 2017). In the current study, we utilized the adult zebrafish-M. marinum model to establish a tool for studying the control and reactivation of latent mycobacterial infections. This was achieved by the administration of the glucocorticoid dexamethasone in the fish food. In humans, glucocorticoids are used as immunosuppressants to treat various diseases, such as arthritis, and long-term use at a high dose has been associated with the reactivation of a latent TB infection, but the exact mechanisms underlying the reactivation are poorly understood (Jick et al., 2006). TB remains one of the major killers of HIV-coinfected individuals: people with HIV are 20-30 times more likely to develop active TB; and there were 0.4 million of such deaths in 2015 (WHO, 2017). In humans and macaques, an HIV or a Simian immunodeficiency virus (SIV) infection is associated with the loss of CD4 + T cells, which has long been thought to be the main factor promoting the reactivation of a latent TB infection (Barnes et al., 1991; Diedrich et al., 2010). This is also supported by data from the mouse model (Kupz et al., 2016a,b). Using the adult zebrafish, we observed a depletion of the lymphocyte population by dexamethasone treatment in uninfected fish, and a failure to induce lymphocyte expansion in infected fish. This is consistent with the depletion of T cells by dexamethasone seen in zebrafish larvae (Langenau et al., 2004). Moreover, our results suggest that dexamethasone, in particular, decreases the amount of conventional CD4 + lymphocytes, including both Th1 and Th2 cells, whereas the expression of tgf-b1b, induced by the immunosuppressive Treg cell population, is upregulated. Dexamethasone has been shown to alleviate allergic symptoms in mice by upregulation of Treg cells (Zhang et al., 2016), but the exact role of these cells and the associated cytokines in the control of mycobacterial infections remains to be studied. Considering the functional similarities between human and zebrafish CD4 + and CD8 + T cells (reviewed in Renshaw and Trede, 2012), CD4 + cells could be a key player in controlling a latent mycobacterial infection also in the zebrafish. However, the susceptibility to the reactivation of a latent TB varies even among HIV-coinfected individuals, and in a macaque model, CD8 + and B cells have been shown to elicit a protective effect in some of the coinfected individuals (Foreman et al., 2016). In the zebrafish, the expression levels of cd8a and the B cell marker IgM remained constant during the dexamethasone treatment, suggesting that these cells are left unaffected by the treatment. Furthermore, the individual zebrafish within a single line have been shown to differ in their ability to control a mycobacterial infection, probably due to genetic heterogeneity, leading to differences in the immune response (Hammarén et al., 2014). We also observed that some fish managed to maintain a relatively low mycobacterial count despite the dexamethasone treatment, which could be explained by the protective effect of CD8 and/or B cells, mimicking the situation in humans and macaques (Foreman et al., 2016; van Meijgaarden et al., 2015). The number of granulocytes and monocytes was not decreased by dexamethasone. Macrophages and neutrophils are needed for the formation of granulomas and Fig. 6. Schematic representation of the experimental protocol for testing vaccine candidates against the reactivation of a latent mycobacterial infection. Fish with a latent M. marinum infection are immunized with a DNA plasmid carrying a GFP-tagged mycobacterial antigen, which allows detection of successful immunizations by fluorescent microscopy. After 5 weeks, the fish are subjected to dexamethasone treatment (10 μg/fish/day) for 4 weeks, followed by determination of bacterial burdens by qPCR. 8 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms for the early control of a mycobacterial infection (Davis et al., 2002; Yang et al., 2012; Davis and Ramakrishnan, 2009). Although treatment with dexamethasone prior to an infection does not increase susceptibility to mycobacteriosis, but rather compromises the control of a chronic infection, confirming that dexamethasone does not affect the properties of macrophages or Fig. 7. Ag85-ESAT-6, RpfB and MMAR_4207 antigens decrease bacterial burdens upon reactivation of a latent M. marinum infection in the adult zebrafish. Fish with a latent M. marinum infection were immunized with the GFP control or experimental vaccines and 5 weeks later treated for 4 weeks with dexamethasone. Each dot represents the bacterial load in one fish. Horizontal lines show the median values of pooled experiments (n=11-29 fish/group). Results from different experiments are marked with different symbols. A two-tailed Mann–Whitney test was used to calculate P-values. 9 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033175. doi:10.1242/dmm.033175 Disease Models & Mechanisms