Immunometabolic circuits in host defense Mihai G. Netea IY31CH17-OGarra ARI 15 February 2013 4:46 Draining Lung lymph node M. tuberculosis 2 IL-12(p40)2/IL-12p70 1c DC CCL19/CCL21 DC Alveolar 8–12 days 1a macrophage Uptake IL-12p70 PGE2 E Antimicrobial peptides, ferocytosis IL-1α/β, TNF-α, Naive T cell Apoptosis IL-12p40, IL-6, Lipoxins chemokines (LXA4) 3 Necrosis Uninfected 4 1b macrophage Th1 cell Antimicrobial peptides CXCL10/CCL19/CCL21 (e.g., cathelicidins), Necrosis 14–17 days chemokines, IL-1β Neutrophil γ Th1 cells IL-12p70 IFN- iNOS 5 IL-12p40 TNF-α Macrophage DC M. tuberculosis dissemination? O’Garra et al, Ann Rev Imm 2013 Figure 4 The cellular immune response to M. tuberculosis. Following aerosol infection with M. tuberculosis, resident lung alveolar macrophages by Radboud Universiteit Nijmegen on 09/06/13. For personal use only. (1a), neutrophils (1b)andlungDCs(1c) can become infected, leading to the production and secretion of antimicrobial peptides, cytokines, and chemokines. The balance of lipid mediators, such as prostaglandin E2 (proapoptotic) or lipoxin (LX) A4 (pronecrotic), Annu. Rev. Immunol. 2013.31:475-527. Downloaded from www.annualreviews.org within infected macrophages plays a major role in determining downstream pathways leading to the induction of either apoptosis or necrosis. Infected apoptotic cells can be taken up by resident lung DCs or efferocytosed by uninfected lung macrophages (1c). M. tuberculosis–infected DCs migrate to the local lung-draining lymph nodes by 8–12 days post infection. DCs migrate to the lymph nodes under the influence of IL-12(p40)2 and IL-12p70 and that of the chemokines CCL19 and CCL21 (2), to drive naive T cell differentiation toward a Th1 phenotype (3). Protective antigen-specific Th1 cells migrate back to the lungs in a chemokine-dependent manner 14–17 days after the point of initial infection/exposure (4) and produce IFN-γ, leading to macrophage activation, cytokine production, the induction of microbicidal factors including iNOS (5), and bacterial control. nodes and occurs earlier in resistant C57BL/6 dissemination of M. tuberculosis may aid in mice than in susceptible C3H mice, result- the initiation of an appropriate and timely ing in an earlier immune response in the adaptive immune response, although this re- C57BL/6 mice (134). This finding suggests sponse may be under strict host genetic control that instead of only spreading infection, early (134). 488 O’Garra et al. Immune cell TLR stimulation glucose glucose autophagy glucose HIF-1α mTOR Akt glycolysis PDH Oxidative 2ATP pyruvate Lipid synthesis phosphorylation O2 ACC present AcetylCoA O2 not Krebs cycle present NAD+ HIF-1α Lactic acid Electron transport Histone acetyl NADH transferase 36 ATP Active cells Naïve cells RESEARCH ARTICLE These phenomena were not observed in our experiments either at early CFU in lung and spleen when compared with mice that received ETH time points (4 hours) or after prolonged MET exposure (24 and 48 hours), alone (Fig. 2, D and E, and fig. S8, C and D). The efficacy of MET was indicating that the drug’smechanismofactionisnotcausingcelldeath further evaluated in the chronic model of Mtb infection. In this experi- of host cells (fig. S7, A to D). ment, mice treated with MET or INH + MET starting from day 42 after infection showed decreased bacillary load in the lungs compared with MET enhances the efficacy of conventional anti-TB drugs untreated or INH-treated mice, respectively (Fig. 2F). Together, these We next evaluated the in vivo efficacy of MET treatment in a mouse experiments suggest that MET represents a potential adjunct drug that model of acute and chronic TB (24). Mtb-infected mice were admin- can enhance Mtb clearance by conventional treatment. istered MET alone or MET in combination with either isoniazid (INH) or ethionamide (ETH), starting on day 7 or 42 after infection. In five dif- MET reduces TB-induced tissue pathology and enhances ferent acute model experiments, mice treated with MET (500 mg/kg) immune response alone had reduced bacillary load in both lung and spleen (Fig. 2, A To significantly improve current TB therapy, anti-Mtb drugs should to E, and fig. S8, A and C). This dose is equivalent to a MET dose of also promote resolution of tissue pathology in addition to accelerating 2430 mg/day for a 60-kg human (http://www.naturalhealthresearch. bacillary clearance (4). Lungs and spleens from Mtb-infected mice treated org/extrapolation-of-animal-dose-to-human/) (25) and lower than with MET were smaller than those of untreated mice at 35 days after the maximum daily dose for MET therapy in diabetic patients infection (Fig. 3, A and B). As expected, mice treated with the conven- (3000 mg/day; http://www.medicines.org.uk/emc/medicine/20952/spc; tional anti-TB drug INH or ETH exhibited a clear reduction in organ http://www.australianprescriber.com/magazine/37/1/2/5). Furthermore, size and tissue lesions, and combination therapy of MET with INH or MET administration enhanced the efficacy of the conventional first- ETH further reduced tissue pathology. Histopathological evaluation line anti-TB drug INH, as demonstrated by decreased bacillary load in of the infected lungs of untreated control mice revealed diffuse coales- the lungs of mice cotreated with INH + MET when compared with mice cent lung lesions with large numbers of infiltrating macrophages and that received INH alone (Fig. 2C and fig. S8, A and B). Indeed, in some lymphocytes and scatteredintracellularacid-fastbacilli(AFB)(Fig.3C). experiments, we were unable to detect any CFU in the lungs of mice In contrast, MET treatment was associated with reduced numbers of that received combined INH + MET therapy (table S3). We next eval- AFB and increased lymphocyte infiltration of the infected tissues uated the efficacy of MET adjunctive therapy when used in combi- (Fig. 3C), which has previously been linked with improved Mtb control nation with the second-line anti-TB drug ETH. In an acute model, in mice (26–28). Mice treated with INH alone had few residual lesions on November 24, 2014 Mtb-infectedMetformin mice cotreated and withhost-directed ETH + MET exhibited therapy decreased in tuberculosisin the lungs with areas of only increased cellularity. No granulomas were observed in the lungs of mice treated with INH + MET, with some areas of the lungs appearing completely normal. Mor- phometric analysis of the histological sec- tions indicated that the percentage of total area of lung tissue involved in TB pathology at 35 days after infection was stm.sciencemag.org reduced in MET-treated mice compared with untreated control animals (3.8% ver- sus 9.9%, respectively; Fig. 3D) and that combination therapy with MET and INH further reduced areas of lung tissue dam- age compared to INH-alone treatment (0.13% versus 0.70%, respectively; Fig. Downloaded from 3D). We next evaluated MET effects on Thelpercell1(TH1) immune responses in lung tissues because TH1 immunity participates in controlling Mtb infec- tion (29). MET-treated Mtb-infected mice showed a trend of larger CD4+ and RESEARCH ARTICLE CD8+ T cell numbers in the lung com- Fig. 2. Efficacy of MET monotherapy and combinationTUBERCULOSIS therapy in a mouse model of TB. (A) Mtb-infected pared to untreated mice (fig. S9, A to C). mice were treated with MET [250 mg/kg (M250) orMetformin 500 mg/kg as (M500)] adjunct starting antituberculosis 7 days after infection. therapy Bacillary In Mtb-infected mice, MET treatment was loads (CFU) were enumerated in the lungs on daysAmit Singhal, 1, 7, 121,* Liu and Jie,1† Pavanish 35 after Kumar, infection.1† Gan Suay UNT, Hong,2 untreatedMelvin Khee-Shing infected Leow,3,4 associated with an increased percent- Bhairav Paleja,1 Liana Tsenova,5,6 Natalia Kurepina,5 Jinmiao Chen,1 Francesca Zolezzi,1 mice. (B)SpleenbacillaryloadinMtb-infected mice treated5 with MET as1,7 described in2 (A). (C)EfficacyofINH5,8 Barry Kreiswirth, Michael Poidinger, Cynthia Chee, Gilla Kaplan, age and number of mycobacteria-specific Yee Tang Wang,2 Gennaro De Libero1,9* monotherapy (5 mg/kg, I5) and INH + M500 therapy in the lungs of Mtb-infected mice. (D)EfficacyofETH interferon-g (IFN-g)–secreting CD8+ Tcells monotherapy (15 mg/kg, E15) and ETH + M500 therapyThe global burden in the of tuberculosis lungs (TB) of morbidityMtb-infected and mortality mice. remains immense. (E)Spleenbacillary A potential new approach to TB therapy is to augment protective host immune responses. We report that the antidiabetic drug metformin (MET)compared with untreated control animals load in Mtb-infected mice treated as describedreduces in (D). the intracellular (F) Mtb growth-infected of Mycobacterium mice tuberculosis were treated(Mtb) in an AMPK daily (adenosine with monophosphate MET – activated protein kinase)–dependent manner. MET controls the growth of drug-resistant Mtb strains, increases pro-(Fig. 3, E and F). The number, but not the (250 mg/kg, M250), INH (10 mg/kg, I10), and I10duction + M250 of mitochondrial in drinking reactive oxygen water species, and starting facilitates phagosome-lysosome 42 days after fusion. infection. In Mtb-infected mice, + use of MET ameliorated lung pathology, reduced chronic inflammation, and enhanced thespecificimmuneresponsepercentage, of IFN-g–secreting CD4 Tcells Bacillary loads (CFU) were enumerated in theand lungs the efficacy on of conventionaldays 1, TB 21, drugs. 42, Moreover, and in two100 separate after human infection. cohorts, MET treatment In these was associated experiments, each group per time point consistedwith improved of four control of toMtb nineinfection mice. and decreased Data disease are severity. expressed Collectively, these as data means indicate that ± MET isalso a showed an increasing trend in the lungs promising candidate host-adjunctive therapy for improving the effective treatment of TB. SEM. P values are provided in table S12, two-tailed Student’s t test.