Recombinant Mycobacterium smegmatis expressing antigen 85B for immunotherapy of Mycobacterium tuberculosis-infected mice

In the current study, we determined the effects of chronic alcohol consumption on the mortality of young and old mice and immune responses during Mycobacterium tuberculosis (Mtb) infection. Eighty percent of Mtb H37Rv infected alcohol-fed young mice died in five months compared to twenty-five percent in Mtb infected alcohol-fed old mice. There is no significant difference in lung bacterial burden of control and alcohol diet fed young and old mice. IFN-α levels were significantly higher in the lungs of Mtb infected alcohol-fed young mice and treatment with anti-IFNAR-1 antibody enhanced their survival. There are significantly higher numbers of CD11b+Ly6G+ neutrophils in the lungs of Mtb infected alcoholic young mice compared to Mtb infected alcohol-fed old mice and Mtb infected control diet-fed young mice. CD11b+Ly6G+ neutrophils are the major source of IFN-α in Mtb-infected alcohol-fed young mice and IFN-α enhanced the expression of RIP-1 and RIP-3 molecules, which are known to be involved in necroptosis. Alcohol-fed old Mtb infected mice and Mtb infected control diet-fed old and young mice expressed low level of IFN-α and RIP-1 and RIP-3 in their lungs. In response to Mtb stimulation, peripheral blood mononuclear cells (PBMC) from young healthy alcoholic individuals with latent tuberculosis infection (LTBI) produced significantly higher amounts of IFN-α compared to non-alcoholic young, alcoholic and non-alcoholic old healthy LTBI+ individuals. Our findings demonstrate that in Mtb infected young mice, alcohol enhances CD11b+Ly6G+ neutrophil infiltration in lungs and excess IFN-α production by neutrophils causes lung macrophage necroptosis and enhanced mortality.

Novel vaccines against M. tuberculosis

Mycobacterium tuberculosis (Mtb) infection remains one of society's greatest human health challenges. Recognition of pathogens such as Mtb by phagocytes via pattern recognition receptors (PRRs) is critical for immune defense. Phagocytes and other innate immune cells must successfully discriminate between pathogen infection or noisy signals derived from transient non-pathogenic exposures to pattern recognition receptor ligands. Previous studies have underscored the importance of toll-like receptor 2 (TLR2) in the recognition of Mtb, and other studies have demonstrated that toll-like receptor 4 (TLR4) exhibits sigmoidal dynamics in inflammatory protein production in response to soluble ligands. How these dynamics extend to TLR2 in response to Mtb or soluble TLR2 ligands is not known. Here, we address a challenging question essential to the pathogenesis of tuberculosis (TB) disease: What is the quality of the early innate response to Mtb infection at low multiplicity of infection and how does the response to soluble TLR2 ligand stimulation reflect the intact pathogen response? Using soluble TLR2 ligands, we demonstrate that inflammatory protein production in response to TLR2 stimulation similarly follows sigmoidal activation. We further show that inflammatory protein production following Mtb infection operates in this subthreshold regime and cannot be attributed to irreversible disruption of inflammatory signaling pathways in Mtb-infected cells. Using highly sensitive assays to interrogate signaling in Mtb-infected primary human macrophages, we further show that Mtb infection results in limited phosphorylation of p38, a key regulator of inflammatory pathways. We further demonstrate that this poor activation is not driven by irreversible inhibition of p38 signaling by Mtb. Lastly, we demonstrate that the quality of canonical inflammatory signal input is decoupled from the ability to control Mtb growth. Together, these data demonstrate that pathways downstream of TLR signaling are differentially activated following Mtb infection and that barriers to activation of these pathways may contribute to functional partitioning of the inflammatory state of Mtb infected cells. Importantly, these studies provide a roadmap to dissect heterogeneous innate responses to Mtb infection as well as provide a tractable experimental system to simulate the early innate immune response to paucibacillary infection in humans.

Cellular immunity to Mycobacterium tuberculosis (Mtb) requires coordinated cell migration and localization to form granulomas. In addition to chemokines and adhesion proteins, we found that Mtb infection of mouse bone marrow-derived DC or MØ upregulated expression of members of a family of neuronal guidance molecules: netrins, semaphorins, slits, and ephrins, which have been implicated in inflammation and immunity. Since netrin-1 and its receptor Unc5b cause immobilization of MØ in atherosclerotic lesions, we hypothesized that up-regulation of these molecules during Mtb infection contributes to MΦ and DC sequestration in granulomas. Using RT-qPCR, we found that netrin-1 and its receptors: Unc5b, Adora 2a, and Adora2b are up-regulated in Mtb-infected cells. Immunoblotting revealed that netrin-1 is secreted by Mtb-infected MΦ, and immunofluorescence showed netrin-1 is expressed in the lungs of Mtb infected mice. Since netrin-1 is secreted, we studied the migration of peritoneal macrophages in response to CCL19 in the presence of supernatants from Mtb-infected or uninfected MΦ and DCs. Supernatants from Mtb-infected cells inhibited MΦ migration in a netrin-1-dependent fashion. Based on these results, we hypothesize that neuronal guidance molecules contribute to granuloma formation and/or maintenance in tuberculosis.

In this study, to determine whether diabetic (DM) mice are susceptible to Mycobacterium tuberculosis (Mtb) infection, we developed experimentally induced diabetes in wild type C57BL/6 mice with streptozotocin & nicotinamide. We then infected non-DM and DM mice with Mtb H37Rv by aerosol. Around 50% of Mtb infected DM (p=0.05) and 25% of uninfected DM (p=0.07) mice died in 5 to 6 months compared to no deaths in infected control mice. Six months after Mtb infection, there was a statistically significant but marginal increase in bacterial burden in the lungs of Mtb infected DM mice compared to non-DM infected mice (3.1 ± 0.4 x106 vs 0.9 ± 0.1 x 106, p=0.001) suggesting increased mortality was not due to increased bacterial burden. Real time PCR analysis of Mtb infected DM lungs indicated significantly increased pro- and anti-inflammatory cytokine gene expression compared to uninfected DM and infected control mice, including increased levels of IL-6 mRNA. Anti-IL-6 antibody treatment of Mtb infected DM mice enhanced the survival (100% vs 60% survival in isotype control antibody treated, p=0.02) and reduced pro- and anti-inflammatory cytokine production. Dendritic cells (DC), but not other immune cells were the major source for IL-6 in Mtb infected DM mice. NK cells of Mtb infected DM mice further enhanced IL-6 production by autologous DCs. Our results suggest NK-DC interaction enhance IL-6 production which drives the immune pathology and mortality of Mtb infected diabetic mice

A CD4+ TNF+ monofunctional memory T-cell response to BCG vaccination is associated with Mycobacterium tuberculosis infection in infants exposed to HIV.

ABSTRACTImmune networks that control antimicrobial and inflammatory mechanisms have overlapping regulation and functions to ensure effective host responses. Genetic interaction studies of immune pathways that compare host responses in single and combined knockout backgrounds are a useful tool to identify new mechanisms of immune control during infection. For disease caused by pulmonary Mycobacterium tuberculosis (Mtb) infections, which currently lacks an effective vaccine, understanding the genetic interactions between protective immune pathways may identify new therapeutic targets or disease-associated genes. Previous studies have suggested a direct link between the activation of NLRP3-Caspase1 inflammasome and the NADPH-dependent phagocyte oxidase complex during Mtb infection. Loss of the phagocyte oxidase complex alone resulted in increased activation of Caspase1 and IL-1β production during Mtb infection, resulting in failed disease tolerance during the chronic stages of disease. To better understand this interaction, we generated mice lacking both Cybb, a key subunit of the phagocyte oxidase, and Caspase1/11. We found that ex vivo Mtb infection of Cybb−/−Caspase1/11−/− macrophages resulted in the expected loss of IL-1β secretion but an unexpected change in other inflammatory cytokines and bacterial control. Mtb infected Cybb−/−Caspase1/11−/− mice rapidly progressed to severe TB, succumbing within 4 weeks to disease characterized by high bacterial burden, increased inflammatory cytokines, and the recruitment of granulocytes that associated with Mtb in the lungs. These results uncover a key genetic interaction between the phagocyte oxidase complex and Caspase1/11 that controls protection against TB and highlight the need for a better understanding of the regulation of fundamental immune networks during Mtb infection.

Previously, we developed a mouse model of type 2 diabetes mellitus (T2DM) using streptozotocin and nicotinamide and found that T2DM mice are susceptible to Mycobacterium tuberculosis (Mtb) infection. We also found that alveolar macrophages from T2DM mice were more permissive to Mtb growth ex vivo compared to non-diabetic controls. In the current study, we determined the defective mechanisms that make T2DM mice alveolar macrophages more susceptible to Mtb infection. Mtb infected alveolar macrophages from T2DM mice produced more TNF-α (973.8 ± 13.3 pg/ml vs. 614.6 ± 27.2 pg/ml, p<0.003) and less apoptotic (4.7 ± 1.8% vs. 28.6 ± 2.3%, p<0.001) compared to Mtb infected non-diabetic control mice. Mtb infected alveolar macrophages from T2DM mice expressed higher levels of TNFR1 (12.8 ± 0.5 vs. 2.09 ± 0.01, p<0.001) and markers of necroptosis RIPK1 (11.5 ± 0.57 vs. 2.94 ± 0.20, p<0.002), RIPK3 (16.1 ± 0.58 vs. 2.89 ± 0.2, p<0.001) and MLKL (9.2 ± 0.56 vs. 1.58 ± 0.72, p<0.02) compared to Mtb infected alveolar macrophages from non-diabetic control mice as determined by real-time PCR. This finding was again proved by Western blot and confocal microscopy. Anti-TNFR1 antibody treatment of alveolar macrophages from T2DM mice before or after Mtb infection reduced RIPK1, RIPK3 and MLKL expression as determined by RT-PCR. Our findings demonstrate that T2DM induces necroptosis of alveolar macrophages upon Mtb infection. Enhanced TNFR1 signaling in T2DM mice alveolar macrophages is responsible for enhanced necroptosis. We are also determining the subpopulations of T2DM mice alveolar macrophages that express higher levels of TNFR1 upon Mtb infection. Studies are underway to determine the in vivo relevance of our current findings to Mtb growth in T2DM mice.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract How Mycobacterium tuberculosis (Mtb) rewires macrophage energy metabolism to facilitate survival is poorly characterized. Here, we used extracellular flux analysis to simultaneously measure the rates of glycolysis and respiration in real time. Mtb infection induced a quiescent energy phenotype in human monocyte-derived macrophages and decelerated flux through glycolysis and the TCA cycle. In contrast, infection with the vaccine strain, M. bovis BCG, or dead Mtb induced glycolytic phenotypes with greater flux. Furthermore, Mtb reduced the mitochondrial dependency on glucose and increased the mitochondrial dependency on fatty acids, shifting this dependency from endogenous fatty acids in uninfected cells to exogenous fatty acids in infected macrophages. We demonstrate how quantifiable bioenergetic parameters of the host can be used to accurately measure and track disease, which will enable rapid quantifiable assessment of drug and vaccine efficacy. Our findings uncover new paradigms for understanding the bioenergetic basis of host metabolic reprogramming by Mtb. https://doi.org/10.7554/eLife.39169.001 Introduction Mechanisms underlying the pathogenesis induced by Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), are poorly understood, and increasing evidence suggests that Mtb subverts the host’s immune response to establish a persistent infection (Cambier et al., 2014; Hmama et al., 2015; Józefowski et al., 2008). Crucial to the success of the immune system to control microbial infection is the metabolic plasticity of immune cells to activate antimicrobial mechanisms in macrophages and activate T cells in response to microbial invasion. Precise coordination between diverse metabolic pathways underlies this plasticity (Ganeshan and Chawla, 2014; Loftus and Finlay, 2016; Mathis and Shoelson, 2011), which is disrupted by pathogenic bacteria. Hence, host-directed therapies are increasingly considered for adjunctive treatment of tuberculosis (Guler and Brombacher, 2015; Mahon and Hafner, 2015; Wallis and Hafner, 2015). Studies suggest that Mtb pathogenicity is reinforced with participation of metabolic pathways from the host, including evidence suggesting that Mtb adaptation to the host environment requires catabolism of host-derived lipids (Daniel et al., 2011; Muñoz-Elías and McKinney, 2005; Pandey and Sassetti, 2008; Rohde et al., 2012; Lee et al., 2013). This is assumed to be induced through Mtb regulating metabolic thresholds of the host macrophage (Mehrotra et al., 2014). Recent studies suggested that there is a shift from oxidative phosphorylation towards glycolysis in macrophages infected with an avirulent strain (H37Ra) or dead γ-irradiated Mtb (Gleeson et al., 2016), and in Mtb (H37Rv)-infected mouse lungs using transcriptomic profiling and confocal imaging (Shi et al., 2015). Lachmandas et al. (Lachmandas et al., 2016) demonstrated that the switch to aerobic glycolysis observed in human peripheral blood mononuclear cells stimulated with dead Mtb lysate is TLR2-dependent, and is mediated in part through the AKT-mTOR (mammalian target of rapamycin) pathway. While this evidence supports the conclusion that dead Mtb reprograms host energy metabolism, the actual underlying mechanisms with live virulent Mtb infection enabling it to persist in humans remain elusive. Furthermore, the metabolic health of the Mtb-infected cell is poorly defined as there is a lack of knowledge on exactly what metabolic health comprises, and what should be measured. Thus, development of a technological advance to address these gaps in our knowledge is expected to uncover the fundamental role of host energy metabolism in allowing Mtb to persist for decades without causing disease. Aberrant cellular bioenergetics have been associated with, and are often the cause of, diseases such as diabetes, cancer, neurodegeneration, and cardiac disease. The dysfunctional energy metabolism in these diseases has been successfully investigated using extracellular flux (XF) analysis (Devarajan et al., 2011; Hill et al., 2009; Salabei et al., 2016; Wu et al., 2007; Lee et al., 2017; Cronin-Furman et al., 2013). XF analysis monitors the rate of oxygen consumed by cells (oxygen consumption rate, OCR) and the release of protons from the cells into the extracellular medium (extracellular acidification rate, ECAR) non-invasively in real time (Figure 1A). Measurements of cellular respiration and acidification form the foundation of our understanding of bioenergetics because cells use two main pathways to produce ATP, namely oxidative phosphorylation (OXPHOS) and glycolysis. This technology is largely unexplored in the field of bacterial pathogenesis, with a few studies focused on Helicobacter pylori infections (Hammond et al., 2015; Saha et al., 2010), but studies on live virulent Mtb pathogenesis are lacking. Figure 1 Download asset Open asset Schematic illustration of cellular metabolism pathways and XF assays used to analyze metabolic pathways. (A) The XF measures oxygen consumption rate (OCR) of the cell, which is mostly consumed at complex IV of the electron transport chain (ETC) in the mitochondria, and extracellular acidification rate (ECAR), which is generated from lactic acid produced from pyruvate, the end-product of glycolysis, and carbonic acid produced from CO2 released during the TCA cycle. Assays performed on the XF include: (B) mitochondrial respiration test, (C) extracellular acidification test, (D) glycolytic rate assay, (E) mitochondrial fuel test, (F) fatty acid oxidation assay and (G) real-time ATP rate assay. Oligo, oligomycin; FCCP, cyanide-4-[trifluoromethoxy]phenylhydrazone; AntiA and Rot, antimycin A and rotenone; 2-DG, 2-Deoxyglucose; G-6-P, glucose-6-phosphate; G-3-P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; α-KG, α-ketoglutarate; OAA, oxaloacetate. https://doi.org/10.7554/eLife.39169.002 In this study, we used extracellular flux analysis to explore the modulation of the energy metabolism of differentiated THP-1 macrophages and human monocyte derived macrophages (hMDM) infected with live virulent Mtb, the slow-growing non-pathogenic vaccine strain, M. bovis BCG (BCG) and dead-Mtb. We examined how mycobacterial burden affects OXPHOS and the glycolysis of macrophages, we investigated ATP production by glycolysis and OXPHOS during mycobacterial infection, and assessed the capacity, dependency and flexibility of mitochondria on glucose, glutamine or fatty acids during infection. Lastly, we confirmed our findings with [U-13C]glucose stable isotope tracing experiments. By adapting a real-time, non-invasive bioenergetic platform to study the bioenergetics of the Mtb-infected host cell, we have generated new knowledge that may contribute towards a better understanding of Mtb persistence and development of novel approaches for host-directed therapeutic interventions. Results Mtb infection depresses the rate of mitochondrial respiration in macrophages Mitochondria are regarded as the energy factory of the cell that generates ATP through OXPHOS. It is reasonable to expect that on infection with Mtb, host energy metabolism is rewired, which has implications for understanding how Mtb causes disease. To examine the effect of mycobacterial infection on host OXPHOS, we made use of an extracellular flux analyzer (XF, Agilent Seahorse, Santa Clara, CA) and the mitochondrial respiration test (Nicholls et al., 2010) to determine key respiratory parameters in mammalian cells. These include basal respiration (Basal Resp), which is the initial OCR measured before addition of any inhibitors minus the non-mitochondrial respiration; ATP-linked respiration (ATP-linked OCR), which is determined after addition of oligomycin that inhibits ATP synthase (Complex V) and thus approximates the respiration required to drive ATP synthesis; proton leak, which is the difference between the ATP-linked OCR and the non-mitochondrial respiration; maximal respiration (Max Resp), which is induced after addition of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) that uncouples ATP synthesis from electron transport; spare respiratory capacity (SRC), which is the difference between maximal respiration and basal respiration; and non-mitochondrial respiration (Non-Mito Resp), which is the OCR after addition of rotenone, a complex I inhibitor, and antimycin A, a complex III inhibitor (Figure 1B). SRC is an important parameter that reflects the ability of the cell to increase respiration to increase the supply of ATP in scenarios when the energy demand exceeds supply under conditions of stress or increased work load. Several lines of evidence preclude any contribution of the infecting Mtb to the measured OCR of infected macrophages. Firstly, we have previously demonstrated that 106 Mtb consumed 10–20 pmoles O2/min (Lamprecht et al., 2016) in contrast to 100–200 pmoles O2/min consumed by 80 000 to 100 000 macrophages under the basal conditions measured in this study. Thus, at a multiplicity of infection (MOI) of 1, 105 Mtb would result in a negligible contribution (<1 pmoles O2/min) to the OCR of the uninfected macrophages. Secondly, this basal OCR of Mtb was measured in media favourable for Mtb respiration and growth, whereas the intracellular macrophage environment is not conducive to ‘healthy’ Mtb respiration. Thirdly, Mtb infection at MOI of 1 and 2.5 progressively decreases the basal respiration (OCR) of both THP-1 macrophages and hMDMs (Figure 2—figure supplement 1) relative to that of uninfected macrophages. Should Mtb contribute to basal respiration (OCR), we should see an increase in OCR with increasing number of Mtb infecting the macrophages. Fourthly, the growth media of the infected macrophages after the time of infection and treatment was removed and the cells were washed in the XF assay medium before the XF assay, to remove most extracellular mycobacteria. When the washes of the macrophages were plated out on 7H11 agar plates, less than 200 CFU were obtained per well from the washes of the infected hMDMs (MOI 5), and less than 100 CFU per well from the infected THP-1 cells (MOI 5). To demonstrate that these extracellular mycobacteria do not contribute to the OCR readings of the infected macrophages, the final wash was transferred to a separate XF cell culture microplate and a separate mitochondrial respiration assay was performed on any extracellular bacteria present in the washes. The OCR and ECAR readings obtained were below 0 pmol/min and at 0 mpH/min, respectively, and the extracellular bacteria did not respond to the sequential injections of oligomycin, FCCP and rotenone and antimycin A (Figure 2—figure supplement 2A–D). Thus, the infecting mycobacteria do not contribute to the measured OCR of the infected macrophages under our conditions. It is not possible to ensure that every cell will be infected in in vitro infections, thus the percentage of uninfected cells will contribute to the resulting XF profiles. We infected the macrophages with Mtb-green fluorescent protein reporter strain (Mtb-GFP) and used bright-field and fluorescence microscopy to determine the percentage of cells that were infected. We found that there was an increase in the percentage of infected cells with an increase in MOI of both the THP-1 cells and the hMDMs (Figure 2—figure supplement 2E–G). Although the percentage of uninfected cells will contribute to the readout of the XF profiles, previous studies have demonstrated that lipids shed by intracellular mycobacteria, such as TDM and PIM2, spread via the endocytic network throughout the macrophage, and via exocytic vesicles to neighboring uninfected cells (Beatty et al., 2000; Xu et al., 1994) and can elicit the production of proinflammatory cytokines (Rhoades et al., 2003). Consequently, the bioenergetic metabolism of the ‘by-stander’ uninfected cells will also be modulated. Thus, the XF profiles are providing collective data of a mixed population of macrophages. Overall, our data demonstrated that strain pathogenicity and burden have distinct effects on virtually all respiratory parameters. Figure 2A and B shows that infection of THP-1 macrophages with Mtb or BCG significantly decreased the respiratory parameters: Basal Resp, ATP-linked OCR, proton leak and Max Resp (and SRC in the case of Mtb), and increased Non-Mito Resp. Similar patterns were observed at lower MOIs of 1 and 2.5, but to lesser degrees (Figure 2—figure supplement 1A–D). Infection with the dead Mtb only significantly reduced the respiratory parameters at MOIs of 2.5 and 5 (Figure 2—figure supplement 1C–D and Figure 2A–B). Figure 2 with 3 supplements see all Download asset Open asset Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI. Respiratory profiles (OCR) and respiratory parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and ∆Dead Mtb (heat-killed Mtb) at MOIs of 5 for 24 h. Refer to Figure 2—figure supplement 1 for profiles of lower MOIs. After obtaining basal respiration, cells were subjected to oligomycin (Oligo, 1.5 µM), which inhibits ATP synthase and demonstrates the mitochondrial ATP-linked OCR, followed by FCCP (cyanide-4-[trifluoromethoxy]phenylhydrazone), which uncouples mitochondrial respiration and maximizes OCR (1 µM for THP-1 and hMDMs), and finally antimycin A and rotenone (AntiA and Rot), which inhibit complex III and I in the ETC, respectively, and shut down respiration (0.5 µM of each for THP-1; 2.5 µM of each for hMDMs). Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. https://doi.org/10.7554/eLife.39169.003 Mtb infection of hMDMs dramatically reduced the respiratory parameters of the macrophage, while significantly increasing Non-Mito Resp (Figure 2C–D). Smaller reductions in the respiratory parameters were observed at lower Mtb MOIs of 1 and 2.5 with an increase in Non-Mito Resp (Figure 2—figure supplement 1E–H). Notably, contrary to Mtb, BCG infection increased the Max Resp and SRC of the hMDMs at all MOIs investigated. At a MOI of 5, BCG decreased the Basal Resp, ATP-linked OCR and proton leak, while increasing the Non-Mito Resp (Figure 2C–D). At lower MOIs, BCG had little effect on the other respiratory parameters (Figure 2—figure supplement 1E–H). Dead Mtb did not affect the respiratory parameters of the hMDMs at MOIs of 1 and 5, but a MOI of 2.5 increased the Max Resp and SRC of the macrophages as in the BCG infection (Figure 2C–D and Figure 2—figure supplement 1E–H). In Mtb-infected macrophages, the oxidative burst (via NADPH oxidase, which consumes O2) induced by a combination of infection, uncoupling with FCCP and inhibition of the ETC after treatment with antimycin A and rotenone increases the OCR above the initial OCR before treatment with oligomycin (Figure 2A and C). This results in the calculated basal respiration having a negative value (Figure 2B and D). As we have used standard equations to calculate the basal respiration (Nicholls et al., 2010), we propose that when the non-mitochondrial respiration is greater than the initial OCR readings before the addition of any inhibitors, an additional mitochondrial respiration assay should be performed without the addition of the FCCP to determine the non-mitochondrial respiration, to obtain the values of basal respiration and proton leak. Using this format, the true (positive) values for basal respiration and proton leak are obtained (Figure 2—figure supplement 3). The basal respiration of both Mtb and BCG infections at a MOI of 2.5 and 5 were less than that of uninfected THP-1 cells, and the proton leak of BCG was less than that of the uninfected THP-1 cells (Figure 2—figure supplement 3A–D). The non-mitochondrial respiration was increased in both of these infections at a MOI of 5. Similar patterns were observed with the Mtb- and BCG-infected hMDMs at a MOI of 5 (Figure 2—figure supplement 3E,F). In sum, there are profound contrasting respiratory differences among Mtb, BCG and dead Mtb infection of the macrophages. In particular, Max Resp, SRC and Non-Mito Resp are strongly influenced by the mycobacterial strain, burden and macrophage type. Mtb infection of hMDMs decreases Max Resp and SRC in contrast to BCG increasing Max Resp and SRC, and both strains increase Non-Mito Resp. SRC has consequences on how the macrophage responds to environmental stresses such as nutrient availability, redox state and changes in pH. Thus, an increase in the SRC of hMDMs following infection with potential vaccine candidates may aid identification of promising candidates. Strikingly, dead Mtb infection still alters the bioenergetic metabolism of the macrophage, in particular that of the THP-1 cells. This has implications for pharmacological killing of Mtb, as killing intracellular Mtb will not fully restore the macrophage’s bioenergetic metabolism to that of the uninfected macrophage. However, pharmacological killing will improve the bioenergetic profile of the live Mtb-infected macrophages, in particular, the ATP-linked Resp and the Non-Mito Resp. Therefore, improvements in these parameters of the infected macrophages can be used as indicators of effective pharmacological killing of Mtb during screening of potential anti-TB drug leads in Mtb-infected macrophages. Mtb infection reduces the extracellular acidification rate of the macrophage Glycolysis is the second pathway used to supply ATP for the energy requirements of the cell, in addition to anabolic intermediates. Here, we measured the glycolytic parameters of mycobacterial infected cells, including the glucose metabolism extracellular acidification rate after addition of glucose; the maximal glycolytic capacity (Gly capacity) following inhibition of OXPHOS ATP synthesis with oligomycin; and the non-glycolytic extracellular acidification measured after treatment of the mycobacterial infected cells with 2-deoxyglucose (2-DG), an inhibitor of hexokinase II, which catalyzes the first step of glycolysis (Figure 1C). The difference between the extracellular acidification rate of glucose metabolism rate and the maximal glycolytic capacity of the cells defines the spare glycolytic reserve. Mtb strikingly decreased the glycolytic parameters of both types of macrophages after 24 h (Figure 3). In THP-1 cells, all the mycobacterial strains reduced the glycolytic parameters at MOIs of 5 (Figure 3A–B) and 2.5 (Figure 3—figure supplement 1C,D), with dead Mtb having the least effects. At a MOI of 1, Mtb decreased the glycolytic parameters, BCG increased the glycolytic parameters and dead Mtb had no effect (Figure 3—figure supplement 1A–B). Figure 3 with 1 supplement see all Download asset Open asset Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI. ECAR profiles and glycolytic parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and dead Mtb at MOI of 5 for 24 h. Refer to Figure 3—figure supplement 1 for profiles at lower MOIs. After obtaining non-glycolytic acidification, glucose (Glc, 10 mM) was added to the cells, followed by oligomycin (1.5 µM), which inhibits ATP synthase inducing maximal glycolysis to compensate for loss of mitochondrial generated ATP, and finally 2-deoxyglucose (2-DG, 100 mM) to inhibit glycolysis and demonstrate that the prior acidification was generated by glycolysis. Profiles and glycolytic parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. https://doi.org/10.7554/eLife.39169.007 In hMDMs, Mtb at a MOI of 2.5 had little effect and at a MOI of 1 increased glucose metabolism extracellular acidification. (Figure 3—figure supplement 1E–H). Contrary to THP-1 cells, BCG and dead Mtb infection of hMDMs increased glucose metabolism acidification and the glycolytic capacity at all MOIs investigated. Increases in the non-glycolytic acidification were observed in the BCG and dead Mtb infections, probably as a result of the carbonic acid produced from CO2 generated by the tricarboxylic acid cycle (TCA). These results underscore the different modulations of dead and live Mtb on macrophage bioenergetics. In sum, marked glycolytic differences were observed between the virulent and non-virulent infections, with Mtb infection significantly reducing glucose metabolism extracellular acidification in the macrophages. BCG and dead-Mtb infections induced contrasting effects dependent on macrophage cell type, with a decrease in THP-1 glucose metabolism extracellular acidification versus an increase in hMDM extracellular acidification. Mtb infection shifts the bioenergetic phenotype of the macrophage towards quiescence To determine how mycobacterial infection shifts the energy metabolism of the macrophage, basal OCR was plotted as a function of ECAR to form a bioenergetic phenogram that the energy phenotypes of the macrophages. The energy phenotype of cells can be as glycolytic or quiescent (Figure Figure Download asset Open asset demonstrate that increasing MOI of Mtb shifts macrophages towards quiescent energy Basal OCR and ECAR from the respiratory assay (Figure before addition of oligomycin were plotted to of THP-1 cells and hMDMs infected with Mtb, BCG and ∆Dead Mtb at MOIs of 1, 2.5 and 5. Data are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. In THP-1 cells and hMDMs, infection with Mtb the most shift from an phenotype towards that of a phenotype with increasing MOI (Figure and D). In contrast, BCG and dead Mtb infections of THP-1 cells 2.5 and induced shifts towards quiescence (Figure and whereas in hMDMs, only BCG infection at a MOI of 5 a shift towards Dead Mtb did not affect the OCR of the hMDMs but decreased the ECAR at lower MOIs (Figure the differences between live and dead Mtb. In sum, our data demonstrate that Mtb infection shifts the energy phenotypes of human macrophages towards a metabolic quiescent Mtb decreases the glycolytic proton rate of macrophages studies using Mtb (Lachmandas et al., 2016), Mtb and (Gleeson et al., 2016), or profiling (Shi et al., to the that Mtb aerobic glycolysis for ATP as the In XF acidification of the extracellular as measured by is not for glycolysis as the mitochondrial TCA CO2 that is in the extracellular medium and to the acidification of the extracellular medium et al., In the glycolytic rate assay and inhibition of mitochondrial respiration after addition of rotenone and antimycin A of the contribution of the mitochondrial respiration to the rate of proton (Figure of the mitochondrial proton rate from the proton rate the glycolytic proton rate (Figure supplement 1A–D). To the is added to inhibit glycolytic acidification (Figure glycolysis to the ability of the cell to increase glycolysis after OXPHOS has been with rotenone and antimycin Figure 5 with 1 supplement see all Download asset Open asset Mtb infection reduces the glycolytic proton rate of macrophages. (A) Extracellular acidification can be by both and protons produced from pyruvate, the final of glycolysis, in addition to carbonic acid generated from CO2 from oxidation in the proton rate the glycolytic to be from the mitochondrial (Figure supplement 1A–D). Basal and glycolytic of THP-1 cells and hMDMs infected with Mtb, BCG and ∆Dead Mtb at MOI of 5 for h. Refer to Figure supplement for profiles at lower MOIs. basal of ECAR and OCR, to determine basal glycolytic rotenone and antimycin A were added to determine This was followed by addition of to ensure that the observed was by glycolysis. Profiles and are representative of two independent experiments. Data shown are the mean ± SD (n = Student’s t test relative to the uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. rate of ATP production was calculated as the of glycolytic ATP rate to and ATP rate that was from the ATP-linked OCR, a of of ATP in (F) THP-1 cells and (G) hMDM cells infected with Mtb, BCG or ∆Dead Mtb at MOI for h. Refer to Figure supplement for contribution of glycolysis and OXPHOS to the rate of ATP are SD (n = 6 biological replicates). Student’s t #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. The proton rates in Figure are the calculated values of the glycolytic without the acidification contribution from mitochondrial respiration. Basal glycolysis and glycolysis of the macrophages, which is induced when mitochondrial ATP synthase is the cell to use glycolysis to the ATP are as is that Mtb significantly reduces the basal and glycolytic rates of THP-1 cells (MOI 2.5 and 5, Figure Figure supplement 1E–H). In contrast, BCG and dead Mtb infections increased the basal glycolytic rates of THP-1 cells at all MOIs Furthermore, glycolysis was increased in BCG-infected cells at all MOIs, and in dead Mtb infections at a MOI of differences were observed between the live and dead Mtb In hMDMs, Mtb (MOI decreased both the glycolytic rate and the glycolytic rate significantly (Figure but lower MOIs (1 and did not affect the glycolytic rates of hMDMs (Figure supplement BCG infection increased both the glycolytic rate and the glycolytic rates at all MOIs, whereas dead Mtb had no effect (Figure Figure supplement In sum, profound differences were observed in the of infections with virulent versus non-virulent mycobacterial Mtb decreases the glycolytic in human macrophages at mycobacterial whereas BCG increases the glycolytic However, dead Mtb infection increases the in THP-1 cells and has no effect in When we the which is considered a of the glycolytic rate and with ECAR data of the infected macrophages, we found that the observed were dependent on the infecting mycobacterial To the reduced basal and of Mtb-infected THP-1 cells (Figure Figure supplement the observed decreased glycolytic ECAR and glycolytic capacity (Figure Figure 3—figure supplement 1A–D). In contrast, distinct differences were observed in the BCG infection of the THP-1 cells, increases in both basal and (Figure Figure supplement were to reductions in the glucose metabolism ECAR and glycolytic capacity (Figure Figure 3—figure supplement These were not observed in the BCG infection of hMDMs, increased basal and (Figure Figure supplement were by increased glucose metabolism ECAR and glycolytic capacity (Figure Figure 3—figure supplement 1E–H). However, between and ECAR were observed with dead Mtb infection of THP-1 cells with increased basal (Figure Figure supplement to or no reductions in glycolytic ECAR (Figure Figure 3—figure supplement 1A–D). in hMDMs, the changes in basal and in Mtb infection 1 and the

Objective To identify the cross-reactive antigens shared by Mycobacteria smegmatis(MS) and Mycobacteria tuberculosis(MTB) and to analyze their antigenicity. Methods Bacterial antigens were extracted from strains of MS and MTB by ultrasonication. Western blot assay was performed to analyze common antigens that reacted with both of the antiserum samples against MS and MTB. The extracted bacterial antigens were mixed with incomplete Freund′s adjuvant and then were injected into muscles of mice. Cytokines secreted by murine spleen lymphocytes following stimulation with various antigens of MS and MTB were determined by ELISPOT and flow cytometry on the 7th day. IgG levels in serum samples were detected by ELISA 7 days after injection. Results There were cross-reactive antigens shared by MS and MTB. Potent humoral immune responses and cellular immunity against both MS and MTB could be induced by those cross-reactive antigens after sensitization the mice by either MS or MTB antigens. Cytokines of IL-2 and IFN-γ in CD4+ and CD8+ T cells of mice stimulated with MS or MTB antigens were significantly increased as compared with those of non-sensitization group and those of Brucella antigens stimulation group. Conclusion Cross-reactive antigens shared by MS and MTS can effectively promote specific immune reactions to the infection of MTB, which provides a scientific basis for the development of tuberculosis vaccines. Key words: Mycobacteria tuberculosis; Mycobacteria smegmatis; Cross-reactive antigen; Immune response

Tuberculosis (TB) is a highly infectious bacterial disease that primarily attacks the lungs. TB is manifested either as latent TB infection (LTBI) or active TB disease, the latter posing a greater threat to life. The risk of developing active TB disease from LTBI is three times higher in individuals with type 2 diabetes mellitus (T2DM). The association between TB and T2DM is becoming more prominent as T2DM is rapidly increasing in settings where TB is endemic. T2DM is a chronic metabolic disorder characterized by elevated blood glucose, insulin resistance, and relative insulin deficiency. Insulin resistance and stress-induced hyperglycemia have been shown to be increased by TB and to return to normal upon treatment. Previously, we demonstrated that adipocytes (or fat tissue) regulate pulmonary pathology, inflammation, and Mycobacterium tuberculosis (Mtb) load in a murine model of TB. Metabolic disturbances of adipose tissue and/or adipocyte dysfunction contribute to the pathogenesis of T2DM. Thus, pathological adipocytes not only regulate pulmonary pathology, but also increase the risk for T2DM during TB infection. However, the cellular and molecular mechanisms driving the interaction between hyperglycemia, T2DM and TB remain poorly understood. Here, we report the impact of Mtb infection on the development of insulin resistance in mice fed on a regular diet (RD) versus high-fat diet (HFD) and, conversely, the effect of hyperglycemia on pulmonary pathogenesis in juvenile and adult mouse models. Overall, our study demonstrated that Mtb persists in adipose tissue and that Mtb infection induces irregular adipocyte lipolysis and loss of fat cells via different pathways in RD- and HFD-fed mice. In RD-fed mice, the levels of TNFα and HSL (hormone sensitive lipase) play an important role whereas in HFD-fed mice, ATGL (adipose triglyceride lipase) plays a major role in regulating adipocyte lipolysis and apoptosis during Mtb infection in adult mice. We also showed that Mtb infected adult mice that were fed an RD developed insulin resistance similar to infected adult mice that were overweight due to a HFD diet. Importantly, we found that a consequence of Mtb infection was increased lipid accumulation in the lungs, which altered cellular energy metabolism by inhibiting major energy signaling pathways such as insulin, AMPK and mToR. Thus, an altered balance between lipid metabolism and glucose metabolism in adipose tissue and other organs including the lungs may be an important component of the link between Mtb infection and subsequent metabolic syndrome.

Cellular immunity in Mycobacteria tuberculosis (Mtb) infection is important for the pathogenesis and final clearance of intracellular Mtb infection. In addition, it is valuable for the diagnosis of tuberculosis. In this pioneering work, we tested in vitro and in vivo antigen presentation and diagnostic application of a recombinant overlapping peptide-protein derived from two Mtb RD1 antigens ESAT-6 and CFP-10 (ROP-TB). The overlapping peptide sequence of ROP-TB is cleaved by the cathepsin S enzyme and covers the entire length of the two proteins. ROP-TB can be expressed and purified from E. coli. Once taken in by antigen-presenting cells, ROP-TB can be cleaved into a peptide pool by cathepsin S within the cells. We found that in dendritic cells, ROP-TB can be processed in 6 hours of co-culture, while the ESAT-6/CFP-10 fusion protein remained in the endosomal compartment. In Mtb-infected mice, ROP-TB stimulated stronger specific T cell responses than pooled synthetic peptides derived from ESAT-6 and CFP-10. With regard to the presentation of in vivo antigens, in a guinea pig model infected with Mtb, ROP-TB induced delayed type hypersensitivity (DTH) responses comparable to those of the tuberculin purified protein derivative (PPD) and ESAT-6/CFP-10 fusion protein. In Mycobacterium bovis (Bovine TB)-infected cattle, ROP-TB elicited DTH responses. Finally, in Mtb infected patients, ROP-TB stimulated cellular immune responses in majority of patients (16/18) of different HLA phenotypes while a single peptide derived from the same proteins did not elicit the immune responses in all patients. In summary, in vitro and in vivo data suggest that ROP-TB stimulates a strong cellular immune response irrespective of HLA phenotypes and is therefore suitable for use in vitro and in vivo diagnostics.

Exosomal co-delivery of CircRNA-TRAPPC6B and rifampicin for enhanced immunotherapy against tuberculosis.

Tuberculosis (TB) is a chronic inflammatory disease that is often associated with alterations in systemic and cellular metabolism that resolves following successful antimicrobial drug treatment. We hypothesized that altered systemic glucose metabolism as a consequence of Mycobacterium tuberculosis (Mtb) infection, contributes to TB pathogenesis, and when normalized with anti-glycemic drugs would improve clinical outcomes. To test this hypothesis, guinea pigs were treated daily with the anti-diabetic drug metformin starting 4 weeks prior or concurrent with aerosol exposure to the H37Rv strain of Mtb. In the chronic stages of infection, Mtb infected metformin-treated animals had restored systemic insulin sensitivity but remained glucose intolerant as determined by oral glucose tolerance testing. Despite persistent glucose intolerance, metformin-treated guinea pigs had a 2.8-fold reduction in lung lesion burden and a 0.7 log decrease in CFUs. An alternative hypothesis that metformin treatment improved clinical disease by having a direct effect on immune cell energy metabolism was tested using extracellular flux analysis and flow cytometry. The proinflammatory immune response to Mtb infection in untreated guinea pigs was associated with a marked increase in energy metabolism (glycolysis and mitochondrial respiration) of peripheral blood mononuclear cells (PBMCs), which was normalized in metformin-treated guinea pigs. Moreover, both CD4+ and CD8+ T lymphocytes from Mtb infected, metformin treated animals maintained a more normal mitochondrial membrane potential while those isolated from untreated animals had persistent mitochondrial hyperpolarization. These data suggest that metformin promotes natural host resistance to Mtb infection by maintaining immune cell metabolic homeostasis and function during the chronic stages of active TB disease.

Mesenchymal stromal cells (MSC) have strong immunomodulatory properties and therefore can be used to control inflammation and tissue damage. It was suggested recently that MSC injections can be used to treat multi-drug resistant tuberculosis (TB). However, MSC trafficking and immunomodulatory effects of MSC injections during Mycobacterium tuberculosis (Mtb) infection have not been studied. To address this issue we have analyzed MSC distribution in tissues and local immunological effects of MSC injections in Mtb infected and uninfected mice. After intravenous injection, MSC accumulated preferentially in the lungs where they were located as cell aggregates in the alveolar walls. Immunological analysis of MSC effects included detection of activated, IFN-γ and IL-4 producing CD4+ lymphocytes, the frequency analysis of dendritic cells (CD11c+F4/80) and macrophages (CD11c-F4/80+) located in the lungs, the expression of IA/IE and CD11b molecules by these cells, and evaluation of 23 cytokines/chemokines in lung lysates. In the lungs of uninfected mice, MSC transfer markedly increased the percentage of IFN-γ+ CD4+ lymphocytes and dendritic cells, elevated levels of IA/IE expression by dendritic cells and macrophages, augmented local production of type 2 cytokines (IL-4, IL-5, IL-10) and chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1), and downregulated type 1 and hematopoietic cytokines (IL-12p70, IFN-γ, IL-3, IL-6, GM-CSF). Compared to uninfected mice, Mtb infected mice had statistically higher “background” frequency of activated CD69+ and IFN-γ+ CD4+ lymphocytes and dendritic cells, and higher levels of cytokines in the lungs. The injections of MSC to Mtb infected mice did not show statistically significant effects on CD4+ lymphocytes, dendritic cells and macrophages, only slightly shifted cytokine profile, and did not change pathogen load or slow down TB progression. Lung section analysis showed that in Mtb infected mice, MSC could not be found in the proximity of the inflammatory foci. Thus, in healthy recipients, MSC administration dramatically changed T-cell function and cytokine/chemokine milieu in the lungs, most likely, due to capillary blockade. But, during Mtb infection, i.e., in the highly-inflammatory conditions, MSC did not affect T-cell function and the level of inflammation. The findings emphasize the importance of the evaluation of MSC effects locally at the site of their predominant post-injection localization and question MSC usefulness as anti-TB treatment.