Caloric Restriction Promotes Immunometabolic Reprogramming Leading to Protection from Tuberculosis

•Controlled caloric restriction (CR) protects from pulmonary MTB infection•CR enhances immune cell intracellular killing, thus reducing lung bacterial load•CR induces an immunometabolic reprogramming leading to reduction of collateral damage•CR tightness intercellular junctions and extracellular matrix in the lungs There is a strong relationship between metabolic state susceptibility Mycobacterium tuberculosis (MTB) infection, with energy metabolism setting basis for exaggerated immuno-inflammatory response, which concurs pathogenesis. Herein, we show that controlled (CR), not malnutrition, susceptible DBA/2 mice against infection by load, immunopathology, generation foam cells, reservoir granulomas. Mechanistically, CR induced shift toward glycolysis, decreased both fatty acid oxidation mTOR activity associated induction autophagy cells. An integrated multi-omics approach revealed specific CR-induced metabolomic, transcriptomic, proteomic signature reduced damage protective remodeling interstitial able limit spreading. Our data propose as feasible manipulation control this offers unexpected strategy boost immunity MTB. accounts almost 2 million deaths annually worldwide major threat public health, particularly low- middle-income countries (Glaziou et al., 2018Glaziou P. Floyd K. Raviglione M.C. Global epidemiology tuberculosis.Semin. Respir. Crit. Care Med. 2018; 39: 271-285Crossref PubMed Scopus (63) Google Scholar; World Health Organization, 2017World OrganizationGlobal report 2017.https://www.who.int/tb/publications/global_report/gtbr2017_main_text.pdf?ua=1Date: 2017Google Zwerling 2016Zwerling A. Hanrahan C. Dowdy D.W. Ancient disease, modern epidemiology: century progress understanding fighting tuberculosis.Am. J. Epidemiol. 2016; 183: 407-414Crossref (4) Scholar). It well established multiple determinants, including genetic environmental, can elevate risk (TB). Among environmental undernutrition known increase hence developing TB, causing low protein-energy intake, fat mass, micronutrient deficiencies, all factors determine secondary immunodeficiency (Cegielski McMurray, 2004Cegielski J.P. McMurray D.N. The malnutrition tuberculosis: evidence studies humans experimental animals.Int. Tuberc. Lung Dis. 2004; 8: 286-298PubMed Koethe von Reyn, 2016Koethe J.R. Reyn C.F. Protein-calorie macronutrient supplements, tuberculosis.Int. 20: 857-863Crossref (25) Sinha 2019Sinha Davis Saag L. Wanke Salgame Mesick Horsburgh Jr., C.R. Hochberg N.S. Undernutrition health implications.J. Infect. 2019; 219: 1356-1363Crossref (21) Food Agriculture Organization (FAO) estimates about 800 individuals are undernourished, sub-Saharan Africa Southeast Asia, where high rates TB incidence indeed correlate intake (Bhargava, 2016Bhargava Undernutrition, nutritionally acquired immunodeficiency, control.BMJ. 355: i5407Crossref (12) United Nations 2017Food (FAO), International Fund Agricultural Development, Children’s Fund, Programme, OrganizationThe food security nutrition world 2017. Building resilience peace security. FAO, This geographical overlap provides crucial intimate among immunity, nutrition/metabolism, TB. Surprisingly, however, more affluent developed countries, appropriate sustains host immunocompetence supports building early anti-mycobacterial defense, response nonetheless often inadequate inefficacious eliminating pathogen, spite inflammatory reaction (Dorhoi 2011Dorhoi Reece S.T. Kaufmann S.H. For better or worse: balances pathology protection.Immunol. Rev. 2011; 240: 235-251Crossref (124) In addition, immune-inflammatory process causes extensive damage, also utilized pathogen itself further inside (Maertzdorf 2018Maertzdorf Tönnies M. Lozza Schommer-Leitner S. Mollenkopf H. Bauer T.T. S.H.E. invasion human lung: first contact.Front. Immunol. 9: 1346Crossref (17) Meunier 2017Meunier I. E. Downey Divangahi Unravelling networks dictating resistance versus tolerance during infections.Cell Tissue Res. 2017; 367: 525-536Crossref (11) Palma 2007Palma Iona Giannoni F. Pardini Brunori Orefici G. Fattorini Cassone Ag85B protein may turn Ag85B-DNA vaccine into potent but non-protective Th1 mice.Cell. Microbiol. 2007; 1455-1465Crossref (28) Scholar, 2008Palma Del Giudice LTK63 adjuvant improves protection conferred DNA-protein prime-boosting vaccination dampening IFN-gamma response.Vaccine. 2008; 26: 4237-4243Crossref (0) Studies on immunometabolism have demonstrated cells influenced systemic processes, changes nutritional status alter effector functions outcome, since responses pathogens costly terms demand, changes, nutrients (Geltink 2018Geltink R.I.K. Kyle R.L. Pearce E.L. Unraveling complex interplay T function.Annu. 36: 461-488Crossref (175) Matarese 2002Matarese La Cava Sanna V. Lord G.M. Lechler R.I. Fontana Zappacosta Balancing autoimmunity: role leptin?.Trends 2002; 23: 182-187Abstract Full Text PDF (167) 2005Matarese Moschos Mantzoros C.S. Leptin immunology.J. 2005; 174: 3137-3142Crossref Van den Bossche 2017Van O’Neill L.A. Menon D. Macrophage immunometabolism: (going)?.Trends 38: 395-406Abstract (300) Compelling has suggested pathogenicity linked its own capacity regulate metabolism. Taken together, these findings argue importance mounting effective at same time harmful (Koeken 2018Koeken V.A.C.M. van Crevel R. Netea M.G. evolved tolerate tuberculosis.Cell Metab. 28: 332-333Abstract Tzelepis 2018Tzelepis Blagih Khan N. Gillard Mendonca Roy D.G. Ma E.H. Joubert Jones R.G. Mitochondrial cyclophilin D regulates disease tuberculosis.Sci. 3: eaar4135Crossref (35) Since been immunosuppression increased MTB, nobody ever tested whether moderate (Fontana Partridge, 2015Fontana Partridge Promoting longevity through diet: model organisms humans.Cell. 2015; 161: 106-118Abstract (571) Piccio 2008Piccio Stark J.L. Cross A.H. Chronic calorie attenuates autoimmune encephalomyelitis.J. Leukoc. Biol. 84: 940-948Crossref (142) Scholar) represent possible convey improved anti-MTB immunity. We herein investigated cellular molecular level CR, does incur undernutrition, positively impact outcome highly immunocompetent (North Jung, 2004North R.J. Jung Y.J. Immunity tuberculosis.Annu. 22: 599-623Crossref (533) results intriguing springboard boosting without uncontrolled damage. To investigate immunocompetent, were either ad libitum (AL)-fed (dietary ranging 25% 35% relative AL-fed mice; Figure 1A STAR Methods details) 28 days. Subsequently, they challenged intravenously (i.v.) virulent H37rv maintained feeding regimen 40 days post-infection (Figure 1A). body weight (23% day compared AL mice) serum leptin (88% 28; S1A), expected (La Matarese, 2004La immunity.Nat. 4: 371-379Crossref Analyses performed comparing four study groups: respectively infected infected. Forty post-infection, sacrificed clinical outcomes spleens. Measurement colony-forming units (CFU) showed, AL, significant load (>1 log10) 1B), marked improvement MTB-lung immunopathology. Indeed, area was consolidated granulomas (Figures 1C 1I), characterized pronounced degeneration neutrophil infiltration 1C). some enlarged granulomas, central showed necrosis, hallmark progressive 1I, upper panel). On contrary, agreement lower burden, minimal signs majority preserved healthy structure middle mice, few, small, structured, predominantly around small vessels, macrophages surrounded mantle lymphocytes, rarer presence neutrophils Furthermore, number CD3+ fork-head-boxP3 (FOXP3+) regulatory (Treg) significantly consistent lymphocytic 1D). Finally, while levels pro-inflammatory cytokines such interferon (IFN)-?, chemokine (C-C motif) ligand- 4 (CCL-4), interleukin (IL)-1? upregulated cytokines/chemokines only IL-1? enhanced uninfected basal 1E). Although IFN-?, CCL-4, required their elevated active higher load/disease severity animal models (Lyadova 2010Lyadova I.V. Tsiganov E.N. Kapina M.A. Shepelkova G.S. Sosunov V.V. Radaeva T.V. Majorov K.B. Shmitova Ham H.J. Ganusov al.In progression intensive accumulation Gr-1 lungs.PLoS ONE. 2010; 5: e10469Crossref Mishra 2013Mishra B.B. Rathinam V.A. Martens G.W. Martinot A.J. Kornfeld Fitzgerald K.A. Sassetti C.M. Nitric oxide controls immunopathology inhibiting NLRP3 inflammasome-dependent processing IL-1?.Nat. 2013; 14: 52-60Crossref (336) Petruccioli 2016Petruccioli Scriba T.J. Petrone Hatherill Cirillo D.M. Joosten S.A. Ottenhoff T.H. Denkinger Goletti Correlates risk: predictive biomarkers tuberculosis.Eur. 48: 1751-1763Crossref (105) experiments confirmed intranasal route administration S1B–S1G) C57BL/6 resistant than mice. MTB-associated tissue although it did affect S1H–S1P). capable containing spleen colonization exemplified CFU 1F). spleens replication granuloma lesion size 1G 1J), extreme paucity multinucleated giant (Langhans cells) 1J, panels, white arrows), FOXP3+ Treg 1H). At harvesting (corresponding 68 after infection; 1A), smaller general cellularity, previous observations (data shown) (Howard 1999Howard J.K. Vendetti Ghatei Ritter Bloom S.R. starvation-induced lymphoid atrophy increases thymic cellularity ob/ob mice.J. Clin. Invest. 1999; 104: 1051-1059Crossref By fluorescence-activated sorting (FACS) phenotyping CD3+, CD4+, CD8+, CD19+, F4/80+, CD11b+, CD11c+, Ly6G+ shown splenic proportion, affecting CD4+ CD8+ subsets, B lymphocytes monocytes/macrophages S2A). Both activation/proliferation (CD69, CD25, Ki-67) exhaustion (PD-1) markers found surface S2B S2C). Instead, (CD8+FOXP3+ CD4+FOXP3+) frequency S2D) differences cell-associated marker (CD25, CTLA-4, PD-1, Ki67) expression S2E) suppressive S2F). measurement isolated ex vivo culture S2G, left panel) replicated rate per right vitro naive dose evaluation total population capability inhibit 7 (Tanner 2019Tanner Smith S.G. Meijgaarden K.E. Wilkie Gabriele Dockrell H.M. T.H.M. McShane Optimisation, harmonisation standardisation direct mycobacterial growth inhibition assay using cryopreserved peripheral blood mononuclear cells.J. Methods. 469: 1-10Crossref (10) (harvested two different points: 21 days) had greater points S2H S2I ), particular evident shorter (21 days). observed S1O S1P). corroborate notion applied regimens before challenge (preventive group) (curative 1K), animals 47 infection. groups, reflected S2J). Interestingly, preventive inhibited infectivity growth, testified 1L, 1M, 1O, 1P, 1R, 1S). even controlling throughout experiment length. parallel (CD3+, FOXP3+) recruitment group conventional 1N S2K). group, similar trend 1Q S2L). Concerning curative (CR MTB), still reduce extent 1K–1S S2J–S2L). dissect how occurring contribute examined enzymes involved glucose, acids, nutrient-energy sensing biochemical level. MTB-infected key glycolytic pathway, enolase-1 (ENO1) pyruvate kinase1/2 (PKM1/2), upregulated; moreover, phosphorylation Ser79 acetyl-CoA carboxylase (p-ACC) activation enzyme pivotal de novo synthesis 2A 2B) (Fullerton 2013Fullerton M.D. Galic Marcinko Sikkema Pulinilkunnil T. Chen Z.P. Ford Palanivel O’Brien al.Single sites Acc1 Acc2 lipid homeostasis insulin-sensitizing effects metformin.Nat. 19: 1649-1654Crossref (418) Ha 1994Ha Daniel Broyles S.S. Kim K.H. Critical activity.J. Chem. 1994; 269: 22162-22168Abstract nutrient- energy-sensing mechanistic target rapamycin (mTOR) kinase directly (Lachmandas 2016Lachmandas Beigier-Bompadre Cheng S.C. Kumar Laarhoven Wang X. Ammerdorffer Boutens Jong Kanneganti T.D. al.Rewiring via AKT/mTOR pathway contributes defence murine cells.Eur. 46: 2574-2586Crossref Scholar), evaluated activated phospho-mTOR (p-mTOR) downstream phospho-S6 phospho-AKT pro-resolving anti-inflammatory phenotype (Covarrubias 2016Covarrubias Aksoylar H.I. Yu Snyder N.W. Worth Iyer Ben-Sahra Byles Polynne-Stapornkul al.Akt-mTORC1 signaling Acly integrate input macrophage activation.eLife. e11612Crossref (185) Manning Toker, 2017Manning B.D. Toker AKT/PKB signaling: navigating network.Cell. 169: 381-405Abstract (1169) Schlegel 2018Schlegel Körner Kaussen Knausberg U. Gerber Hansmann Jónasdóttir H.S. Giera Mirakaj Inhibition neogenin fosters resolution inflammation regeneration.J. 128: 4711-4726Crossref (6) concomitant PKM1/2 p-ACC p-mTOR suggesting glycolysis 2C 2D). mTOR-dependent Ser757 ULK1, inducing interaction AMPK (Kim 2011Kim Kundu Viollet B. Guan K.L. Ulk1.Nat. Cell 13: 132-141Crossref (3536) prompting autophagy, mechanism used gain degrading components starvation (Klionsky Emr, 2000Klionsky D.J. Emr S.D. Autophagy regulated degradation.Science. 2000; 290: 1717-1721Crossref (2716) finding, Beclin-1 (a well-known autophagy-associated protein), together lipidation microtubule-associated 1 light chain 3 beta (LC3-I) LC3-II form, another 2E 2F). These 2G–2J) paralleled ability 2K). Treatment inhibitor rapamycin, activate autophagic decrease 2K–2M), phenomenon association synergistic effect. evaluate functional immunometabolism, i.v. BCG, attenuated strain, allowed Seahorse analyzer 3A details). BCG 3B), infection-mediated upregulation CCL-4 lowered IFN-? modified 3C–3E). enhance acidification (ECAR) basal, maximal, oxygen consumption (OCR), 3F–3H). selective stimulation (i.e., polyclonal [anti-CD3] MTB-antigen-specific [purified derivative, PPD] stimulation), case, accompanied OCR induction, PPD-specific 3I–3N). examine alters metabolomic landscape, carried out liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based profiling groups (Tables S1A S1B; https://figshare.com/s/a43c3449fc95489ba731). animals, impacted profile splenocytes, affected upon 4A). particular, when striking concerning energetic cofactor production, TCA cycle, 4B 4C). charge ATP NAD+/NADH ratio, pointing sustained production splenocytes 4D). No difference anabolic processes (NADP+/NAPH ratio) se flux, distinctly anaerobic flux intermediates (glyceraldehyde 3-phosphate phosphoenolpyruvate) lactate, rapidly consumed produce 4E). Concomitantly, several cycle (particularly succinate oxaloacetate) ratio palmitic stearic-carnitine-bound acids (C16+C18) free carnitine (C0) dampened following supported lipidomic analysis S3A–S3E; Tables https://figshare.com/s/a43c3449fc95489ba731) (Weiner 2012Weiner 3rd, Parida S.K. Maertzdorf Black G.F. Repsilber Telaar Mohney R.P. Arndt-Sullivan Ganoza C.A. Faé K.C. al.Biomarkers inflammation, stress patients.PLoS One. 2012; 7: e40221Crossref (144) Changes appeared be lysophosphatidylcholines various phosphatidylcholines, sphingomyelins, ceramides, phosphatidylethanolamines S3C–S3E). RNA sequencing (RNA-seq) analyses conditions (Table S2; GEO: GSE127263). unsupervised clustering principal component RNA-seq profiles distinct, being force gene 5A S4A). A genes differentially expressed S4B S4C). Specifically, 264 enriched proteolytical peptidase activity, digestion 5B). increment registered conditions, played synergy S5). proliferation adhesion locomotion, process, stimulus 5C). single-gene level, transcripts belonging apoptosis cytokine-mediated signaling, metabolism, leukocyte chemotaxis 5D). Moreover, implicated transporter cytoskeleton organization, system, Supervised hierarchical plethora prominent spleens, CR-dependent immune-related transcriptional S4D S4E). Examples important defense colony-stimulating factor (Csf1; Sariko 2018Sariko Maro Gratz Houpt Kisonga Mpagama Heysell Mmbaga B.T. Thomas T.A. Evaluation supernatants diagnosis tuberculosis.J. Inflamm. 12: 15-22Crossref (1) Scholar); receptors (Il1rl1, Il2rb, Il12rb1, Il18r1, Il23r) (Keegan 2018Keegan Krutzik Schenk Scumpia P.O. Lu Pang Y.L.J. Russell B.S. Lim K.S. Shell Prestwich al.Mycobacterium transfer I