Mycobacterium tuberculosis Impairs Dendritic Cell Functions through the Serine Hydrolase Hip1 This information is current as of December 5, 2016. Ranjna Madan-Lala, Jonathan Kevin Sia, Rebecca King, Toidi Adekambi, Leticia Monin, Shabaana A. Khader, Bali Pulendran and Jyothi Rengarajan Supplementary Material References Subscriptions Permissions Email Alerts http://www.jimmunol.org/content/suppl/2014/03/21/jimmunol.130318 5.DCSupplemental.html This article cites 58 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/192/9/4263.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscriptions Submit copyright permission requests at: http://www.aai.org/ji/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/cgi/alerts/etoc The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 J Immunol 2014; 192:4263-4272; Prepublished online 21 March 2014; doi: 10.4049/jimmunol.1303185 http://www.jimmunol.org/content/192/9/4263 The Journal of Immunology Mycobacterium tuberculosis Impairs Dendritic Cell Functions through the Serine Hydrolase Hip1 Ranjna Madan-Lala,* Jonathan Kevin Sia,* Rebecca King,* Toidi Adekambi,* Leticia Monin,† Shabaana A. Khader,† Bali Pulendran,*,‡ and Jyothi Rengarajan*,x T he immense success of Mycobacterium tuberculosis as a pathogen can be largely attributed to its ability to subvert host innate and adaptive immune responses (1–6). Upon infection with M. tuberculosis, the majority of infected individuals mount robust CD4 T cell responses involving Th1 cytokines such as IFN-g and TNF-a, which are critical for activating macrophages and inducing microbicidal responses. Several studies have shown increased susceptibility to mycobacterial diseases in IFN-g–deficient mice and in humans with IL-12 or IFN-g receptor abnormalities (7–9). Although Th1 responses are required to control M. tuberculosis infection, they are not sufficient for eradicating the pathogen from the host. This is because M. tuberculosis has evolved multiple strategies to resist host defenses. These include interfering with the ability of IFN-g to effectively activate antimicrobial responses in M. tuberculosis– *Emory Vaccine Center, Emory University, Atlanta, GA 30329; †Division of Infectious Diseases, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15224; ‡Department of Pathology, Emory University School of Medicine, Atlanta, GA 30329; and xDivision of Infectious Diseases, Department of Medicine, Emory University School of Medicine, Atlanta, GA 30329 Received for publication November 26, 2013. Accepted for publication February 22, 2014. This work was supported by National Institutes of Health Grants R00TW008043, 5R01AI083366-02 (to J.R.), R37AI48638, and R37DK057665 (to B.P.) and Yerkes National Primate Center Base Grant RR000165. Address correspondence and reprint requests to Dr. Jyothi Rengarajan, Emory University School of Medicine, 954 Gatewood Road, Room 1022, Atlanta, GA 30329. E-mail address: jrengar@emory.edu The online version of this article contains supplemental material. Abbreviations used in this article: BMDC, bone marrow–derived dendritic cell; DC, dendritic cell; ESAT-6, early secreted antigenic target 6; Hip1, hydrolase important for pathogenesis 1; MDC, monocyte-derived DC; MHC II, MHC class II; MOI, multiplicity of infection; PRR, pattern recognition receptor; TB, tuberculosis; Tg, transgenic. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303185 infected macrophages, inhibition of phagosome acidification and maturation, resistance to reactive oxygen and nitrogen intermediates, impairing Ag presentation (1, 10), and preventing optimal activation of pattern recognition receptor (PRR)–dependent pathways in macrophages (11–18). M. tuberculosis has been shown to inhibit macrophage activation and cytokine induction through secreted and cell envelope–associated factors (12–14, 17–19). We have shown that the cell envelope–associated serine hydrolase, hydrolase important for pathogenesis 1 (Hip1), a protein critical for M. tuberculosis virulence, hinders optimal TLR2- and inflammasome-dependent activation in macrophages and promotes dampening of proinflammatory responses (11, 20–23). Thus, Hip1 prevents robust macrophage responses to M. tuberculosis infection. In addition to macrophages, it is increasingly appreciated that dendritic cells (DCs) also serve as an important intracellular niche for M. tuberculosis (24–28). DCs are the primary APCs of the immune system and strategically located at sites of pathogen entry. Immature DCs recognize pathogen-associated molecular patterns via PRRs, and concomitant with phagocytosis and internalization of microbes, these events lead to a process of maturation. Mature DCs are characterized by high surface expression of MHC class II (MHC II), costimulatory molecules such as CD40, CD80, and CD86, and secretion of key cytokines, such as the Th1-polarizing cytokine IL-12 (29). Mature DCs can migrate into secondary lymphoid organs, where they present pathogenderived Ags to naive T cells, initiate activation and differentiation of these T cells, and play a critical role in determining the types of Th subsets that are generated in response to infection (27, 30–32). Thus, DCs play a central role in immunity to microbial pathogens by effectively linking innate and adaptive immune responses (31, 33). Recent studies have demonstrated that M. tuberculosis infects human and mouse DCs at high frequencies in vitro and in vivo, and there is growing evidence that DCs play a critical role in immunity to tuberculosis (TB) (25, 34–37). In Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 Mycobacterium tuberculosis is a highly successful human pathogen that primarily resides in host phagocytes, such as macrophages and dendritic cells (DCs), and interferes with their functions. Although multiple strategies used by M. tuberculosis to modulate macrophage responses have been discovered, interactions between M. tuberculosis and DCs are less well understood. DCs are the primary APCs of the immune system and play a central role in linking innate and adaptive immune responses to microbial pathogens. In this study, we show that M. tuberculosis impairs DC cytokine secretion, maturation, and Ag presentation through the cell envelope–associated serine hydrolase, Hip1. Compared to wild-type, a hip1 mutant strain of M. tuberculosis induced enhanced levels of the key Th1-inducing cytokine IL-12, as well as other proinflammatory cytokines (IL-23, IL-6, TNF-a, IL-1b, and IL-18) in DCs via MyD88- and TLR2/9-dependent pathways, indicating that Hip1 restricts optimal DC inflammatory responses. Infection with the hip1 mutant also induced higher levels of MHC class II and costimulatory molecules CD40 and CD86, indicating that M. tuberculosis impairs DC maturation through Hip1. Further, we show that M. tuberculosis promotes suboptimal Ag presentation, as DCs infected with the hip1 mutant showed increased capacity to present Ag to OT-II– and early secreted antigenic target 6–specific transgenic CD4 T cells and enhanced Th1 and Th17 polarization. Overall, these data show that M. tuberculosis impairs DC functions and modulates the nature of Ag-specific T cell responses, with important implications for vaccination strategies. The Journal of Immunology, 2014, 192: 4263–4272. 4264 Materials and Methods Ethics statement All animal experiments were approved by the Institutional Animal Care and Use Committee at the Emory University School of Medicine. Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. Bacterial strains and media M. tuberculosis H37Rv (wild-type), hip1::tn (hip1 mutant), and hip1 mutant–complemented strains were grown at 37˚C in Middlebrook 7H9 broth or 7H10 agar supplemented with 10% oleic acid/albumin/dextrose/ catalase, 0.5% glycerol, and 0.05% Tween 80 (for broth), with the addition of 25 mg/ml kanamycin (Sigma-Aldrich, St. Louis, MO) for the hip1 mutant and 10 mg/ml streptomycin (Sigma-Aldrich) for the hip1 mutant–complemented strain. For inactivation of M. tuberculosis strains, bacteria were grown in Middlebrook 7H9 until midlog phase, washed twice with PBS, and heat killed by incubating at 80˚C for 2 h. Mice All mice were housed under specific pathogen-free conditions in filtertop cages within the vivarium at the Yerkes National Primate Center, Emory University, and provided with sterile water and food ad libitum. C57BL/6J mice were purchased from The Jackson Laboratory. MyD882/2 and TLR22/2 mice originally generated in the laboratory of Dr. S. Akira (Osaka University, Osaka, Japan) and OTII-transgenic (Tg) mice specific for OVA323–339 peptide originally generated in the laboratory of Dr. F. Carbone (University of Melbourne, Melbourne, VIC, Australia) were bred at the Yerkes animal facility; bone marrow cells from TLR2/92/2 mice were a kind gift from Dr. Padmini Salgame (University of Medicine and Dentistry of New Jersey, Newark, NJ); and TCR-Tg mice specific for early secreted antigenic target 6 (ESAT-6)1–20/I-Ab epitope were obtained from Dr. Andrea Cooper (Trudeau Institute). Murine DC infection and cytokine assays For generating murine bone marrow–derived DCs (BMDCs), bone marrow cells from C57BL/6J mice were grown in RPMI 1640 medium (Lonza, Walkersville, MD) with 10% heat-inactivated FBS (HyClone, Logan, UT), 2 mM glutamine, 13 2-ME, 10 mM HEPES, 1 mM sodium pyruvate, 13 nonessential amino acids, and 20 ng/ml murine recombinant GM-CSF (R&D Systems, Minneapolis, MN). Incubations were carried out at 37˚C with 5% CO2. Fresh medium with GM-CSF was added on days 3 and 6, and cells were used on day 7 for all experiments. We routinely obtained ∼75% CD11c+CD11b+ cell purity by flow cytometry. BMDCs were further purified by using magnetic beads coupled to CD11c+ mAb and passed through an AutoMACS column as per the manufacturer’s instructions, where indicated (Miltenyi Biotec, Auburn, CA). For all experiments, cells were maintained throughout in medium containing GM-CSF. For infection, BMDCs were plated onto 24-well plates (3 3 105 per well). Bacteria were filtered through 5-mM filters, resuspended in complete medium containing 20 ng/ml GM-CSF, and sonicated twice for 5 s each before addition to the adherent monolayers. Each bacterial strain was used for infection (in duplicate or triplicate) at a multiplicity of infection (MOI) of 5 or as indicated. Infection of BMDCs was carried out for 4 h, after which monolayers were washed four times with PBS before replacing with RPMI 1640 medium containing 20 ng/ml GM-CSF. To determine intracellular CFU, one set of DCs was lysed in PBS containing 0.5% Triton X-100 and plated on 7H10 agar plates containing the appropriate antibiotics. Alternatively, BMDCs were infected with heat-killed M. tuberculosis at an MOI of 5 or as indicated in RPMI medium containing 20 ng/ml GMCSF. Cell-free supernatants from DC monolayers were isolated at indicated points and assayed for cytokines by ELISA using Duo Set kits for IL-12p40, IL-12p70, IL-6, TNF-a, and IL-1b (BD Biosciences, San Jose, CA); IL-23 from BioLegend (San Diego, CA); and IL-18 (MBL International, Woburn, MA). Assays were carried out according to the manufacturers’ instructions. Uninfected BMDCs were used as controls for each experiment. Flow cytometry and Abs Murine anti-CD11c allophycocyanin (clone N418) and anti-CD11b FITC (clone M1/70) were obtained from BioLegend; anti-CD40 PE (clone 3//23), anti-CD86 PE (clone GL1), and anti–MHC II PE (clone M5/ 114.15.2) were purchased from BD Biosciences. Staining for cell-surface markers was done by resuspending ∼1 3 106 cells in 200 ml PBS with 2% FBS containing the Ab mixture. Cells were incubated at 4˚C for 30 min and then washed with PBS containing 2% FBS. Data were immediately acquired using an FACSCalibur (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, San Carlos, CA). Ag-specific CD4 T cell Ag-presentation assays CD4 T cells were purified from single-cell suspensions of spleen and lymph nodes from 6–8-wk-old ESAT-6–Tg and OTII-Tg mice using the CD4 T cell isolation kit and AutoMACS column as per the manufacturer’s instructions (Miltenyi Biotec). BMDCs were incubated in 24-well plates (3 3 105/well) with 10 mg/ml ESAT-61–20 peptide or OVA323–339 peptides for 6 h, washed with PBS, and infected with heat-killed wild-type, hip1 mutant, or medium alone for 24 h. Infected DCs were washed twice with PBS and cocultured with Ag-specific CD4 T cells at a 1:4 ratio for 72 h. Supernatants collected from these cells were analyzed for IFN-g (Mabtech, Cincinnati, OH) and IL-2 (BD Biosciences) by ELISA according to the manufacturers’ instructions. CD4 T cell polarization assays CD4 T cells were purified from single-cell suspensions of spleen and lymph nodes from 6–8-wk-old C57BL/6J mice as described above. BMDCs infected with wild-type, hip1 mutant, or medium alone for 24 h as described above were cocultured with CD4 T cells at a 1:4 ratio for 72 h. Cell-free supernatants collected from these cells were analyzed for IFN-g (Mabtech) and IL-17 (eBioscience, San Diego, CA) by ELISA according to the manufacturers’ instructions. Aerogenic infection of mice with M. tuberculosis strains M. tuberculosis strains, H37Rv, and hip1 mutant were grown to early log phase (OD600 of ∼0.6–0.8), washed two times in 13 PBS, and 1-ml aliquots were frozen at 280˚C and used for infection after thawing. Singlecell suspensions of these aliquots were used to deliver ∼100 CFU H37Rv or the hip1 mutant into 8–10-wk-old C57BL/6J mice using an aerosol apparatus manufactured by In-Tox Products (Moriarty, NM). Bacterial burden was estimated by plating serial dilutions of the lung homogenates on 7H10 agar plates on day 1. Tissue harvest and cell preparation Lungs from infected mice were harvested at 3 wk postinfection and digested with 1 mg/ml collagenase D (Worthington) at 37˚C for 30 min. The Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 the aerogenic mouse model of TB, M. tuberculosis–infected DCs have been shown to be important for transporting bacteria from the lungs to the draining mediastinal lymph nodes, where they initiate T cell–mediated immune responses (36–38). Depletion of CD11c+ cells in mice, which includes DCs, caused a delay in CD4 T cell responses and impaired control of M. tuberculosis (39). However, M. tuberculosis has also been shown to interfere with DC migration and Ag presentation in vivo (36), which likely impact the priming of Th1 responses. Thus, interactions between M. tuberculosis and DCs during early stages of infection will directly influence the onset and development of adaptive immunity. Although M. tuberculosis employs a number of cell wall–associated and extracellularly secreted bacterial factors to modulate innate immune cells, factors that interfere with DC functions are poorly understood. In this study, we show that M. tuberculosis infection impairs key aspects of DC functions through Hip1 (Rv2224c) and thereby impacts adaptive immune responses. Infection of DCs by a hip1deficient mutant induced significantly higher levels of IL-12 and other proinflammatory cytokines compared with wild-type M. tuberculosis and enhanced surface expression of MHC II, CD40, and CD86. This enhanced DC maturation induced by the hip1 mutant was dependent largely on MyD88 and partially on TLR2/9 pathways. Further, we provide evidence that DCs matured by the hip1 mutant were more efficient in presenting Ags to CD4 T cells and priming Th1 and Th17 responses. Overall, our data demonstrate that M. tuberculosis Hip1 impairs DC functions and modulates the nature of Ag-specific T cell responses. Enhancing adaptive immune responses by boosting DC activation and Ag presentation has important implications for developing better vaccines for TB. M. TUBERCULOSIS INHIBITS DENDRITIC CELL FUNCTIONS The Journal of Immunology upper right lobe of the lung was used for determining CFU. Homogenized single-cell lung suspensions were filtered through a 70-mm cell strainer (BD Biosciences), treated with RBC lysis buffer for 3–5 min, and washed twice with cell culture media. Cells were counted and stimulated with 10 mg/ml ESAT-61–20 peptide for 48 h. Cell-free supernatants were isolated and assayed for IFN-g and IL-17 by ELISA. Human DC infection and Th cell differentiation assays Statistical analysis The statistical significance of data were analyzed using the Student unpaired t test (GraphPad Prism 5.0; GraphPad). Data are shown as mean 6 SD of one representative experiment from two to five independent experiments. Results M. tuberculosis limits DC production of IL-12 and other proinflammatory cytokines through the serine hydrolase Hip1 Although M. tuberculosis has been shown to infect DCs and impair their functions in vivo (36), the M. tuberculosis factors that modulate DC responses during infection are not well understood. Based on the recently identified role for Hip1 in modulating macrophage functions, we investigated whether Hip1 impacts DC functions. We first assessed the ability of wild-type (H37Rv) and the hip1 mutant strains of M. tuberculosis to induce IL-12, a cytokine that is critical for inducing the differentiation of naive T cells into the IFN-g–secreting Th1 phenotype. We infected BMDCs from C57BL/6J mice with the wild-type or hip1 mutant strains of M. tuberculosis at an MOI of 5 and measured the levels of IL-12p40 and p70 subunits at 24 h postinfection in the cell-free supernatants. The intracellular bacterial counts in wild-type and hip1 mutant–infected BMDCs at 4 h and 8 d postinfection were comparable (data not shown). The hip1 mutant induced significantly higher levels of IL-12p40 and IL-12p70 in infected DCs compared with wild-type (Fig. 1A), indicating that M. tuberculosis limits the production of the key Th1-polarizing cytokine upon infection of DCs. To address whether the viability of M. tuberculosis is necessary for the enhanced secretion of IL-12 seen in the absence of Hip1, we infected BMDCs with heat-killed strains of wild-type and the hip1 mutant. The heat-killed hip1 mutant induced significantly higher levels of IL-12p40 and IL-12p70 compared with heat-killed wild-type M. tuberculosis (Fig. 1B), indicating that bacterial viability is not necessary for eliciting enhanced levels of IL-12 in DCs. In addition, IL-12 induced by the hip1 mutant was restored to wild-type levels upon infection with the complemented strain (Fig. 1A, 1B), confirming that M. tuberculosis limits IL-12 production through Hip1. Although IL-12 is a major Th1polarizing cytokine secreted by myeloid DCs upon microbial stimulation, DCs also secrete other proinflammatory cytokines, which serve as early triggers of inflammation. In response to infection with the hip1 mutant, BMDCs secreted high levels of IL-23, IL-6, TNF-a, IL-1b, and IL-18 compared with wild-type, and these levels were restored to wild-type levels by the complemented strain (Fig. 1C, Supplemental Fig. 1). We did not detect significant amounts of IL-10 or IFN-b secretion from infected DCs under these conditions (data not shown). Taken together, these results indicate that M. tuberculosis limits the magnitude of IL-12 production, as well as that of additional proinflammatory cytokines in infected DCs, in a Hip1-dependent manner. M. tuberculosis impairs DC maturation through Hip1 Following phagocytosis and Ag capture at the site of infection by immature DCs, interactions between pathogen-associated molecular patterns and PRRs induce maturation of DCs and migration into the local draining lymph nodes, where they prime T cells through cellsurface expression of costimulatory molecules such as MHC II and secretion of cytokines such as IL-12. To determine whether Hip1 influences DC maturation, we infected BMDCs with the wild-type or hip1 mutant at an MOI of 5 for 24 h and monitored the surface expression of CD40, CD86, and MHC II by flow cytometry. Although wild-type M. tuberculosis induced all three markers on CD11c+ BMDCs, the expression levels were much lower than that induced by LPS from Salmonella (Fig. 2). In contrast, hip1 mutant induced higher surface expression of CD40, CD86, and MHC II (Fig. 2). This robust maturation of DCs infected with the hip1 mutant was restored to wild-type levels upon complementation with Hip1 (data not shown). These results indicate that M. tuberculosis impairs optimal DC maturation through Hip1. To investigate whether the impaired maturation of DCs upon M. tuberculosis infection is due to direct inhibition of host pathways by Hip1, we asked if M. tuberculosis could block DC maturation induced by an exogenous stimulus, such as LPS. We exposed BMDCs to 1 mg/ml LPS or wild-type M. tuberculosis at an MOI of 5, either independently or together, and measured the surface expression of CD40 by flow cytometry after 24 h (Fig. 3A). The median fluorescence intensity (MFI) of LPS-induced CD40 on the cell surface was not diminished by the addition of M. tuberculosis, demonstrating that wild-type M. tuberculosis does not actively inhibit LPS-induced expression of CD40 on BMDCs (Fig. 3A). We next exposed BMDCs to mixed cultures of wild-type and hip1 mutant strains (1:1) and compared CD40 expression to single infections of either strain. Surface expression of CD40 in the mixed infection setting was comparable to that induced by the hip1 mutant alone (Fig. 3B), suggesting that the hip1 mutant phenotype is dominant and that wild-type M. tuberculosis does not hinder hip1 mutant–induced DC maturation. Thus, these data suggest that the presence of Hip1 in wild-type M. tuberculosis prevents optimal DC maturation, whereas in the absence of Hip1, interactions between the hip1 mutant and DCs promote robust DC maturation. Inhibition of DC functions by M. tuberculosis is dependent on MyD88 and TLR2/9 pathways We have previously demonstrated that Hip1-dependent modification of the M. tuberculosis cell envelope dampens macrophage proinflammatory responses by limiting interactions between TLR2 agonists on M. tuberculosis and TLR2 on macrophages, leading to suboptimal TLR2 activation. Because studies have shown that M. tuberculosis engages different TLRs on macrophages and DCs (40), we sought to determine which pathways are engaged by the hip1 mutant and lead to enhanced cytokine secretion and maturation Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 PBMCs were isolated from the blood of healthy donors by centrifugation in CPT tubes (BD Biosciences). CD14+ monocytes were purified from PBMCs by positive selection using CD14+ microbeads (Miltenyi Biotec). Cell purity was .80% as assessed by flow cytometry using an FACSCalibur (BD Biosciences). To generate immature monocyte-derived DCs (MDCs), CD14 + cells were cultured at 1 3 106 cells/ml in RPMI 1640 (Lonza) supplemented with 10% heat-inactivated FBS (HyClone), 13 nonessential amino acids, 20 ng/ml human rGM-CSF (PeproTech, Rocky Hill, NJ), and 40 ng/ml IL-4 (Pepro Tech). Incubations were carried out at 37˚C with 5% CO2. Fresh medium with rGM-CSF and IL-4 was added every alternate day. MDCs were harvested on day 6 or 7 for experiments. For infection, human MDCs were plated onto 24-well plates (3 3 105/ well) and infected with heat-killed H37Rv or hip1 mutant at an MOI of 10. Cell-free supernatants from DC monolayers were isolated at 24 h postinfection and assayed for cytokines by ELISA using Duo Set kits for IL12p40 and IL-6 (R&D Systems and BD Biosciences, respectively). Assays were carried out according to the manufacturers’ instructions. For T cell polarization assays, infected DCs were coincubated with autologous lymphocytes at a ratio of 1:5 at 37˚C with 5% CO2. Cell-free supernatants were isolated at day 3 and assayed for IFN-g and IL-17 by ELISA (BD Biosciences and R&D Systems, respectively). 4265 4266 M. TUBERCULOSIS INHIBITS DENDRITIC CELL FUNCTIONS of DCs. We infected BMDCs derived from C57BL/6J and MyD882/2 mice with the wild-type or hip1 mutant and assayed the supernatants for IL-12p40, IL-12p70, and IL-6 by ELISA. As seen in Fig. 4A, production of all three cytokines was largely abolished in the MyD882/2 BMDCs. Next, we infected BMDCs derived from TLR22/2 mice with wild-type or hip1 mutant and assayed the supernatants for cytokines. As seen in Fig. 4B, IL-6 and IL-12p70 levels are largely abolished in TLR22/2 BMDCs, and IL-12p40 levels are significantly reduced compared with BMDCs from C57BL/6J mice. Because engagement of TLR9 on DCs has been implicated in IL-12 production, we tested the involvement of TLR9 in the enhanced IL-12 production induced by FIGURE 2. Enhanced surface expression of CD40, CD86, and MHC II on hip1 mutant–infected DCs. C57BL/6J BMDCs were exposed to medium alone (ui), heat-killed wildtype (wt), or hip1 mutant (mut) at MOI of 5 or 1 mg/ml LPS for 24 h. DCs were labeled with anti–CD11callophycocyanin and anti–CD40-PE, anti–CD86-PE, or anti–MHC II–PE. Representative histograms and PE MFI for CD11c+ cells are shown. Isotype control is shown as gray-shaded area. Data are representative of three independent experiments. Values are presented as mean 6 SD. **p , 0.01, ***p , 0.001. Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 FIGURE 1. Enhanced inflammatory response in hip1 mutant–infected DCs. (A) Purified BMDCs derived from C57BL/6J mice were exposed to medium alone (ui) or infected with the wild-type (wt), hip1 mutant (mut), or hip1 mutant complemented with Hip1 (comp) M. tuberculosis at MOI of 5. At 24 h postinfection, cell-free supernatants were assayed for IL12p40 and IL-12p70 by ELISA. Purified C57BL/6J BMDCs were infected with heat-killed wild-type (wt), hip1 mutant, or comp strain at MOI of 5. At 24 h postinfection, cell-free supernatants were assayed for IL-12p40 and IL-12p70 (B) and IL-23, IL-6, TNF-a, IL-1b, and IL-18 (C) by ELISA. Data are representative of three independent experiments. Values are presented as mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001. ud, undetectable. The Journal of Immunology 4267 mutant (Fig. 5A). The higher IFN-g and IL-2 production induced by hip1 mutant–infected DCs was also observed using an exogenous Ag. Coculture of hip1 mutant–infected DCs with naive TCR-Tg CD4 T cells isolated from OT-II mice and OVA323–339 showed enhanced induction of IL-2 and the Th1 cytokine IFN-g compared with their wild-type counterparts (Fig. 5B). Thus, the absence of Hip1 enhanced the capacity of DCs to present Ag to CD4 T cells and induce Th1 cytokine responses. These data show that suboptimal DC maturation and Ag presentation by M. tuberculosis is dependent on Hip1. M. tuberculosis Hip1 modulates CD4 T cell differentiation in vitro and in vivo the hip1 mutant. We infected BMDCs from mice doubly deficient in TLR2 and TLR9 with wild-type or hip1 mutant and assayed the supernatants for IL-12p40, IL-12p70, and IL-6. As seen in Fig. 4C, IL-12 levels are almost completely abrogated in TLR22/2 /TLR92/2 BMDCs. These results indicate that the enhanced cytokine secretion in the absence of Hip1 is dependent on activation of the TLR2 and TLR9 pathways. We next examined whether M. tuberculosis regulates the cellsurface expression of costimulatory markers in a MyD88-TLR– dependent manner. For this, we infected BMDCs with wild-type or hip1 mutant at an MOI of 5 for 24 h and monitored the surface expression of CD40 by flow cytometry. As seen by the MFI of CD11c+ cells, CD40 expression was largely abolished in MyD882/2 DCs while being mostly independent of TLR2 and TLR9 (Fig. 4D). Overall, these data show that Hip1-mediated enhanced DC maturation is dependent on MyD88–TLR2/9 pathways. M. tuberculosis interferes with DC Ag presentation in a Hip1-dependent manner DCs are the most effective APCs for activating naive CD4 T cells. Expression of high levels of costimulatory molecules and MHC II on the cell surface is essential for efficient Ag presentation and T cell activation. We therefore hypothesized that enhanced expression of costimulatory molecules and MHC II in DCs infected with the hip1 mutant would impact Ag presentation to naive CD4 T cells. To test this hypothesis, we infected BMDCs with wild-type or hip1 mutant at an MOI of 10 for 24 h followed by a coculture with naive TCR-Tg CD4 T cells that were specific for the M. tuberculosis ESAT-61–20 peptide, in the presence of ESAT-61–20 peptide. Supernatants were collected 72 h after coculture and assayed for IFN-g and IL-2 by ELISA. We found that wild-type– infected DCs elicited significantly lower IFN-g and IL-2 from ESAT-61–20–specific CD4 T cells as compared with the hip1 M. tuberculosis interacts with human DCs to impair T cell differentiation To address whether Hip1 also plays a role in impairing human DC– T cell interactions, we isolated PBMCs from healthy donors and differentiated them in vitro in the presence of GM-CSF and IL-4. These MDCs were infected with the wild-type or hip1 mutant at an MOI of 10 for 24 h. We assayed for representative Th cell–polarizing cytokines IL-12 and IL-6 in supernatants and found that hip1 mutant–infected MDCs from each donor induced significantly higher levels of IL-12p40 and IL-6 compared with wild-type M. tuberculosis (Fig. 7A). To investigate whether MDCs infected by the hip1 mutant also promote IFN-g and IL-17 production by T cells, infected DCs were cocultured with autologous lymphocytes from the respective donors for 3 d, and supernatants were assayed for IFN-g and IL-17 by ELISA. As seen in Fig. 7B, the hip1 mutant–infected DCs induced increased production of IFN-g and IL-17 from human lymphocytes in each donor. Overall, these data extend our observations in mice to human cells and demonstrate that the interaction of M. tuberculosis with DCs impairs their capacity to initiate optimal adaptive immunity. Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 FIGURE 3. Wild-type M. tuberculosis does not block LPS- or hip1 mutant–induced DC maturation. C57BL/6J BMDCs were exposed to 1 mg/ ml LPS or heat-killed wild-type M. tuberculosis (wt) at MOI of 5 either independently or together (A) or infected with heat-killed wt, hip1 mutant (mut), or wt + hip1 mutant (1:1) at MOI of 5 (B) for 24 h. DCs were labeled with anti–CD11c-allophycocyanin and anti–CD40-PE. Representative histograms and PE MFI for CD11c+ cells are shown. Isotype control is shown as gray-shaded area. Data are representative of two independent experiments. Values are presented as mean 6 SD. *p , 0.05, **p , 0.01. The increased induction of IFN-g by DCs matured with the hip1 mutant is likely due to the enhanced IL-12p70 levels, which synergize with costimulatory molecules like CD40 to induce Th1 differentiation. Because the hip1 mutant also induced enhanced production of the cytokines IL-6, IL-1b, and IL-23, which are known to promote differentiation to the Th17 phenotype, we sought to determine whether the interactions between BMDCs and the hip1 mutant–infected DCs induced IL-17–secreting CD4 T cells. We infected BMDCs with wild-type or hip1 mutant at an MOI of 10 for 24 h followed by a coculture with purified CD4 T cells from uninfected C57BL/6J mice. After 72 h, supernatants were assayed for IFN-g and IL-17 by ELISA. As seen in Fig. 6A, BMDCs infected with the hip1 mutant elicited enhanced IFN-g and IL-17 levels from CD4 T cells as compared with wild-type M. tuberculosis, indicating that Hip1 controls Th cell differentiation by DCs. To test whether Hip1 influences Th cell differentiation in vivo, we infected C57BL/6J mice with ∼100 CFU of wild-type or the hip1 mutant by the aerosol route. We harvested lungs at 3 wk postinfection, because at this time point, Ag-specific IFN-g–producing CD4 T cells have been shown by multiple groups to be present in the lungs of M. tuberculosis–infected mice. Single-cell lung suspensions were stimulated with 10 mg/ml ESAT-61–20 peptide for 48 h, and cell-free supernatants were assayed for IFN-g and IL-17 by ELISA. As shown in Fig. 6B, lung cells from the hip1 mutant–infected mice show higher levels of IFN-g and IL-17 in response to ESAT-61–20 peptide stimulation compared with wildtype–infected mice. These data suggest that wild-type M. tuberculosis limit IFN-g and IL-17 production in lungs early in infection and that Hip1 mediates this effect. 4268 M. TUBERCULOSIS INHIBITS DENDRITIC CELL FUNCTIONS Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 FIGURE 4. M. tuberculosis impairment of DC activation and maturation requires MyD88- and TLR2/9-dependent pathways. Purified BMDCs from C57BL/6J and MyD882/2 (A), TLR22/2 (B), or TLR2/92/2 (C) mice were exposed to medium alone (ui) or infected with heat-killed wild-type (wt) or the hip1 mutant (mut) at MOI of 5 for 24 h, and cell-free supernatants were assayed for IL12p40, IL-12p70, and IL-6 by ELISA. (D) Infected BMDCs from MyD882/2 and TLR2/92/2 were labeled with anti–CD11c-allophycocyanin and anti– CD40-PE. Representative histograms and PE MFI for CD11c+ cells are shown. Data are representative of three (A, B) or two (C, D) independent experiments. Values are presented as mean 6 SD. *p , 0.05, ***p , 0.001. ud, undetectable. Discussion The findings reported in this study reveal new insights into the interactions between DCs and M. tuberculosis and their impact on the initiation of CD4 T cell responses. Although the ability of M. tuberculosis to inhibit macrophage activation and antimicrobial functions has been well studied, the mechanisms by which M. tuberculosis modulates DC functions are poorly defined. By infecting murine and human DCs with a hip1 mutant strain of M. tuberculosis that induced enhanced DC responses, we found that wild-type virulent M. tuberculosis prevents optimal IL-12 production and DC maturation and impairs DC Ag presentation to CD4 T cells. Thus, we show that the M. tuberculosis serine The Journal of Immunology hydrolase Hip1 plays a significant role in limiting DC functions that may have important consequences on T cell responses and disease development. We found that wild-type M. tuberculosis prevents robust maturation of infected DCs and limits the secretion of key proinflammatory cytokines such as IL-12. These results support and extend previous reports suggesting that M. tuberculosis does not permit optimal DC maturation and thus limits their functions (36, 41, 42). A study using human MDCs showed that M. tuberculosis induces minimal upregulation of surface maturation markers as compared with a potent cytokine-maturation mixture, and M. tuberculosis–infected DCs were compromised in their ability to induce allogeneic lymphoproliferation (41). M. tuberculosis has also been shown to interfere with DC migration and Ag presentation in vivo (36). Our finding that M. tuberculosis prevents optimal expression of IL-12 and CD40 has important implications for the initiation and amplification of M. tuberculosis–specific adaptive immune responses. Our studies support the idea that downmodulation of CD40 expression on DCs and restricting IL-12 production through Hip1 is an important strategy employed by M. tuberculosis to restrict the delivery of DC-derived signals required for inducing optimal Th1 responses. Our studies implicating a role for the serine hydrolase Hip1 in preventing optimal DC maturation and IL-12 production extend our previous results showing that M. tuberculosis prevents robust proinflammatory cytokine and chemokine responses in macrophages through Hip1. Our previous data suggested that Hip1-mediated FIGURE 6. M. tuberculosis–DC interactions modulate CD4 T cell differentiation in vitro and in vivo. (A) Purified C57BL/6J BMDCs in medium alone (ui) or infected with heat-killed wild-type (wt) or the hip1 mutant (mut) at MOI of 10 for 24 h were cocultured with CD4 T cells from C57BL/6J mice for 3 d. Cell-free supernatants were collected and assayed for IFN-g and IL-17 by ELISA. (B) Single-cell suspensions were prepared from lungs of mice aerogenically infected with live wt or the hip1 mut at 3 wk postinfection, and cells were stimulated with 10 mg/ml ESAT-61–20 peptide for 48 h. Supernatants were collected and assayed for IFN-g and IL-17 by ELISA. Data are representative of three (A) or two (B) independent experiments. Values are presented as mean 6 SD. **p , 0.01, ***p , 0.001. ud, undetectable. remodeling of the M. tuberculosis cell wall hinders optimal macrophage activation by limiting interactions between TLR2 agonists on M. tuberculosis and TLR2 on macrophages and promotes a hypoimmune response that delays detection of M. tuberculosis by the host (11). In contrast, hip1-deficient M. tuberculosis induced robust MyD88- and TLR2-dependent activation of macrophages and enhanced proinflammatory responses. In this study, we show that the enhanced IL-12 produced by DCs infected with the hip1 mutant is dependent on TLR2 as well as TLR9 pathways (Fig. 4). The additional requirement for TLR9 is consistent with the increased engagement of TLR9 reported in DCs relative to macrophages (40, 43). Although we do not conclude from these data that Hip1 is directly suppressing MyD88–TLR2/9 pathways, we speculate that Hip1-mediated modification of the M. tuberculosis cell envelope prevents optimal MyD88–TLR2/9 activation on DCs during wild-type infection and that the absence of Hip1 enhances MyD88-TLR2/9 activation, resulting in robust DC activation. Although the enhanced surface expression of costimulatory markers by the hip1 mutant is dependent on MyD88 pathways, it appears to be largely independent of TLR2/4/9 pathways (Fig. 4 and data not shown), suggesting that yet-unknown MyD88-dependent pathways may be involved. These studies support use of specific TLR agonists as adjuvants to augment DC maturation, cytokine production, and Ag presentation as a strategy Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 FIGURE 5. hip1 mutant augments Ag presentation by BMDCs. Purified C57BL/6J BMDCs in medium alone (ui) or infected with heat-killed wildtype (wt) or hip1 mutant (mut) at MOI of 10 for 24 h were cocultured with ESAT-61–20 peptide and ESAT-6–specific Tg CD4 T cells (A) or OVA323–339 peptide and OT-II–specific Tg CD4 T cells for 3 d (B). Cell-free supernatants were collected and assayed for IFN-g and IL-2 by ELISA. Data are representative of three independent experiments. Values are presented as mean 6 SD. *p , 0.05, **p , 0.01, ***p , 0.001. 4269 4270 for improving vaccination against TB. Recent data showing that nanoparticles containing TLR4 and TLR7 ligands boost the magnitude and persistence of vaccine-elicited Ab responses, improving vaccine-mediated protection against influenza virus, demonstrate that these approaches are feasible and efficacious in the setting of infectious diseases (44). To test how the enhanced DC maturation, MHC II expression, and IL-12 production induced by the hip1 mutant affect M. tuberculosis– specific CD4 T cell responses, we studied the Ag presentation capacity of DCs and compared the ability of DCs infected with wildtype versus hip1 mutant M. tuberculosis to present the ESAT-61–20 peptide to ESAT-6 TCR-Tg CD4 T cells in vitro. We found that the hip1 mutant promoted increased IFN-g and IL-2 production upon coculture of DCs and CD4 T cells in the presence of ESAT-61–20 peptide, demonstrating that early interactions between DCs and CD4 T cells are likely to influence the kinetics and magnitude of Th1 cell responses to M. tuberculosis (Fig. 5A). Several studies have shown that TLR2 signaling induces DCs to stimulate a Th2 or regulatory T pathway (45–49). The present results indicating a role for Th1 induction by enhanced TLR2 signaling in the hip1 mutant– infected DCs suggest that the context in which TLR2 signaling occurs may play a role in the outcome. Higher levels of IFN-g were also observed in vivo within the lungs of hip1 mutant–infected mice at 3 wk postinfection (Fig. 6B). One of the hallmarks of M. tuberculosis infection is a delayed Th1 response in the lungs, which is in part due to delayed DC migration and Ag presentation to T cells (38, 50–53). This delay, in combination with inadequate tempering of the ensuing inflammatory response, contributes to the damage sustained by the host in TB. Our studies suggest that the presence of Hip1 contributes to this delay by hindering Ag presentation and is an immune evasion mechanism employed by M. tuberculosis to manipulate the onset and magnitude of adaptive immune responses. In addition to increased IFN-g production, we also observed that hip1 mutant infection led to increased levels of IL-17 in murine and human DC–T cell coculture experiments in vitro as well as in vivo in M. tuberculosis–infected mice (Figs. 6, 7). This is consistent with the enhanced production of the cytokines IL-6, IL-1b, and IL-23, which are known to be crucial for driving differentiation of Th17 cells. Although the significance of earlier IL-17 production in the case of the hip1 mutant infection is not entirely clear, it has been suggested that early induction of IL-17 promotes recruitment of IFN-g–producing T cells into the lungs via chemokine signals and improves bacterial killing. IL-17 has also been implicated in protective immunity to TB; intratracheal M. tuberculosis infection of mice deficient in IL-17A showed poor control of M. tuberculosis infection, and mycobacteria-exposed healthy adults harbored IL-17–producing CD4 T cells in their peripheral blood (54, 55). However, IL-17 production during chronic infection or unchecked Th17 responses may be detrimental by mediating immune pathology (56). Thus, a finely tuned balance between Th1 and Th17 subsets is likely to be required for protective immunity to M. tuberculosis infection. Because mice infected with the hip1 mutant exhibit severely reduced lung pathology relative to wild-type despite high bacterial burdens (21–23), we speculate that robust proinflammatory responses and more efficient Ag presentation during early, acute stages of infection will promote adaptive responses that are less pathologic and may confer protection to the host. Our studies demonstrating a role for Hip1 in dampening DC responses adds significance to a small but growing body of data showing that M. tuberculosis–derived factors modulate DC functions. A few purified M. tuberculosis Ags have been implicated in inhibiting DC maturation and functions. The M. tuberculosis Ag ESAT-6 inhibited LPS/CD40L-induced maturation of human PBMC-derived DCs and reduced IFN-g production from T cells (57), and the M. tuberculosis cell wall component mannose-capped lipoarabinomannan inhibited LPS-induced DC maturation by targeting DC-specific intercellular adhesion molecule-3–grabbing nonintegrin (58). However, the role of these factors in the context of whole M. tuberculosis remains unclear. In another study, an Ag85A-deficient mutant strain of M. tuberculosis, DfbpA, induced higher expression of MHC II on murine BMDCs as well as higher levels of IL-12p70; these DCs primed T cells to produce more IFN-g as compared with wild-type M. tuberculosis (42). Further, DfbpA-vaccinated mice showed better protection against M. tuberculosis challenge compared with those vaccinated with bacillus Calmette-Guérin. Similar studies are ongoing with the hip1 mutant to assess whether the hip1 mutant in M. tuberculosis or bacillus Calmette-Guérin has potential as a vaccine candidate. In summary, we have shown that M. tuberculosis serine hydrolase Hip1 impairs DC maturation and functions, highlighting its important role in modulating DC–pathogen interactions. Wild-type M. tuberculosis induces suboptimal DC maturation and restricts the secretion of IL-12 and other key proinflammatory cytokines. This inhibition of DC maturation and cytokine secretion compromises Ag presentation to CD4 T cells and results in lower IFN-g and IL-17 compared with the hip1 mutant. Overall, these findings show that optimal activation of DCs should result in a more efficient T cell response against M. tuberculosis and have important implications for vaccine design. Acknowledgments We thank Dr. Padmini Salgame for TLR2/9 double knockout bone marrow, Paul Hakimpour for breeding and maintaining several knockout mice strains, Dr. Chris Ibegbu for help with human MDC experiments, and Dr. David Weiss for helpful discussions. Downloaded from http://www.jimmunol.org/ by guest on December 5, 2016 FIGURE 7. M. tuberculosis interacts with human DCs to impair T cell differentiation. (A) Human MDCs were infected with heat-killed wild-type (wt) or hip1 mutant (mut) M. tuberculosis at MOI of 10 for 24 h. Cell-free supernatants were assayed for IL-12p40 and IL-6 by ELISA. 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