Mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via mTORC2/HK1-mediated metabolic rewiring

Bacillus Calmette–Guérin (BCG) vaccination induces a type of immune memory known as “trained immunity”, characterized by the immunometabolic and epigenetic changes in innate immune cells. However, the molecular mechanism underlying the strategies for inducing and/or boosting trained immunity in alveolar macrophages remains unknown. Here, we found that mucosal vaccination with the recombinant strain rBCGPPE27 significantly augmented the trained immune response in mice, facilitating a superior protective response against Mycobacterium tuberculosis and non-related bacterial reinfection in mice when compared to BCG. Mucosal immunization with rBCGPPE27 enhanced innate cytokine production by alveolar macrophages associated with promoted glycolytic metabolism, typical of trained immunity. Deficiency of the mammalian target of rapamycin complex 2 and hexokinase 1 abolished the immunometabolic and epigenetic rewiring in mouse alveolar macrophages after mucosal rBCGPPE27 vaccination. Most noteworthy, utilizing rBCGPPE27’s higher-up trained effects: The single mucosal immunization with rBCGPPE27-adjuvanted coronavirus disease (CoV-2) vaccine raised the rapid development of virus-specific immunoglobulin G antibodies, boosted pseudovirus neutralizing antibodies, and augmented T helper type 1-biased cytokine release by vaccine-specific T cells, compared to BCG/CoV-2 vaccine. These findings revealed that mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via reprogramming mTORC2- and HK-1-mediated aerobic glycolysis, providing new vaccine strategies for improving tuberculosis (TB) or coronavirus variant vaccinations, and targeting innate immunity via mucosal surfaces. Bacillus Calmette–Guérin (BCG) vaccination induces a type of immune memory known as “trained immunity”, characterized by the immunometabolic and epigenetic changes in innate immune cells. However, the molecular mechanism underlying the strategies for inducing and/or boosting trained immunity in alveolar macrophages remains unknown. Here, we found that mucosal vaccination with the recombinant strain rBCGPPE27 significantly augmented the trained immune response in mice, facilitating a superior protective response against Mycobacterium tuberculosis and non-related bacterial reinfection in mice when compared to BCG. Mucosal immunization with rBCGPPE27 enhanced innate cytokine production by alveolar macrophages associated with promoted glycolytic metabolism, typical of trained immunity. Deficiency of the mammalian target of rapamycin complex 2 and hexokinase 1 abolished the immunometabolic and epigenetic rewiring in mouse alveolar macrophages after mucosal rBCGPPE27 vaccination. Most noteworthy, utilizing rBCGPPE27’s higher-up trained effects: The single mucosal immunization with rBCGPPE27-adjuvanted coronavirus disease (CoV-2) vaccine raised the rapid development of virus-specific immunoglobulin G antibodies, boosted pseudovirus neutralizing antibodies, and augmented T helper type 1-biased cytokine release by vaccine-specific T cells, compared to BCG/CoV-2 vaccine. These findings revealed that mucosal recombinant BCG vaccine induces lung-resident memory macrophages and enhances trained immunity via reprogramming mTORC2- and HK-1-mediated aerobic glycolysis, providing new vaccine strategies for improving tuberculosis (TB) or coronavirus variant vaccinations, and targeting innate immunity via mucosal surfaces. Trained immunity, also known as innate immune memory, is a functional adaptation of innate immunity whereby an initial insult results in a more effective response upon subsequent encounters with unrelated pathogens (1Kaufmann E. Sanz J. Dunn J.L. Khan N. Mendon?A L.E. Pacis A. et al.BCG educates hematopoietic stem cells to generate protective innate immunity against tuberculosis.Cell. 2018; 172: 176-190Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar). This phenomenon is characterized by elevated levels of pro-inflammatory cytokine release; macrophage reprogramming toward activated, inflammatory phenotypes; epigenetic changes that promote inflammation; and metabolomic changes that enhance glycolysis (2Netea M. Quintin J. Van der Meer J.M. Trained immunity: a memory for innate host defense.Cell Host Microbe. 2011; 9: 355-361Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar). Trained immunity has been observed following viral vaccines, Candida albicans infections, and exposure to certain pathogen-associated molecular pattern molecules (PAMPs) such as zymosan, β-glucan, and LPS. Recent studies have shown that innate immune cells from BCG-vaccinated subjects exhibit enhanced cytokine responses upon restimulation with pathogen-associated molecular pattern molecules and heterologous pathogens ex vivo (3Cirovic B. Bree L. Groh L. Blok B.A. Schlitzer A. BCG vaccination in humans elicits trained immunity via the hematopoietic progenitor compartment.Cell Host Microbe. 2020; 28: 322-334.e5Abstract Full Text Full Text PDF PubMed Google Scholar). Moreover, human peripheral blood mononuclear cells (PBMCs) trained with live BCG produce high levels of IL-6, IL-1β, and TNF upon LPS restimulation in vitro (4Angelidou A. Diray-Arce J. Conti M.G. Netea M.G. Levy O. Human newborn monocytes demonstrate distinct BCG-induced primary and trained innate cytokine production and metabolic activation in vitro.Front. Immunol. 2021; 12674334Crossref PubMed Scopus (11) Google Scholar, 5Brueggeman J.M. Zhao J. Schank M. Yao Z.Q. Moorman J.P. Trained immunity: an overview and the impact on COVID-19.Front. Immunol. 2022; 13837524Crossref PubMed Scopus (27) Google Scholar). Importantly, BCG-induced training extends to hematopoietic stem cells (HSCs), providing a basis for durable immune protection. The underlying mechanisms for BCG-mediated training in the myeloid monocyte/macrophage lineage rely on metabolic and epigenetic reprogramming initiated by pattern recognition receptor (PRR) engagement, particularly via NOD-2, followed by activation of the mammalian target of rapamycin (mTOR)/hypoxia-inducible factor 1-alpha pathway (6Lange C. Aaby P. Behr M.A. Donald P.R. Kaufmann S.H.E. Netea M.G. et al.100 years of Mycobacterium bovis bacille Calmette-Guérin.Lancet Infect Dis. 2022; 22: e2-e12Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 7Arts R. Carvalho A. Rocca C.L. Palma C. Rodrigues F. Silvestre R. et al.Immunometabolic pathways in BCG-induced trained immunity.Cell Rep. 2016; 17: 2562-2571Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). Trained immunity responses stimulated by BCG vaccination have been suggested to provide some degree of protection against tuberculosis (TB) (8Aaby P. Benn C.S. BCG: new life for a centenarian vaccine.Lancet Infect Dis. 2021; 21: 897-898Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar) as well as offer nonspecific innate immune enhancements against various pathogens (9Lobo N. Brooks N.A. Zlotta A.R. Cirillo J.D. Boorjian S. Black P.C. et al.100 years of Bacillus Calmette-Guérin immunotherapy: from cattle to COVID-19.Nat. Rev. Urol. 2021; 18: 611-622Crossref PubMed Scopus (65) Google Scholar). Furthermore, recent studies have indicated that BCG may offer protection against novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections (10Netea M.G. Giamarellos-Bourboulis E.J. Domínguez-Andrés J. Curtis N. Bonten M. Trained immunity: a tool for reducing susceptibility and severity of SARS-CoV-2 infection.Cell. 2020; 181: 969-977Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 11Gong W. Aspatwar A. Wang S. Parkkila S. Wu X. COVID-19 pandemic: SARS-CoV-2 specific vaccines and challenges, protection via BCG trained immunity, and clinical trials.Expert Rev. Vaccines. 2021; 20: 857-880Crossref PubMed Scopus (34) Google Scholar, 12Forni G. Mantovani A. COVID-19 Commission of Accademia Nazionale dei Lincei, RomeCOVID-19 vaccines: where we stand and challenges ahead.Cell Death Differ. 2021; 28: 626-639Crossref PubMed Scopus (523) Google Scholar), while the administration of BCG in elderly populations has shown a statistically significant reduction in all-cause infections when compared to placebo (13Giamarellos-Bourboulis E.J. Tsilika M. Moorlag S. Antonakos N. Kotsaki A. Domínguez-Andrés J. et al.Activate: randomized clinical trial of BCG vaccination against infection in the elderly.Cell. 2020; 183: 315-323.e9Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Additionally, due to its trained immunity responses, BCG has been utilized as a therapeutic for cancer treatment in conditions such as bladder cancer and melanoma (14Hu Z. Lu S.-H. Lowrie D.B. Fan X.-Y. Trained immunity: a Yin-Yang balance.MedComm (2020). 2022; 3: e121PubMed Google Scholar, 15Dagenais A. Villalba-Guerrero C. Olivier M. Trained immunity: a “new” weapon in the fight against infectious diseases.Front. Immunol. 2023; 141147476Crossref PubMed Scopus (3) Google Scholar). Despite the potential for BCG as a highly protective vaccine, there has been little effort to optimize BCG-induced trained immunity levels in innate immune cells, especially in lung-resident macrophages. Our findings suggest that the recombinant strain rBCGPPE27 enhances the induction of trained immune responses as compared to BCG, regardless of mucosal or intradermal delivery routes in lung-resident macrophages. Specifically, rBCGPPE27 appeared to offer increased protection against tuberculosis disease progression, when compared to BCG, in mice infected with an avirulent Mycobacterium tuberculosis strain, H37Ra. The study also demonstrates the potential of rBCGPPE27's enhanced trained immunity effects to offer stronger CoV-2-specific protective immunity via a vaccination strategy. Mechanistically, the study reveals the potential importance of mTORC2/HK1-mediated immunometabolic and epigenetic rewiring in inducing the trained response of alveolar macrophages following mucosal vaccination with rBCGPPE27. Overall, these findings highlight the superior potential for rBCGPPE27 as a recombinant TB vaccine, which in turn provides for improving trained innate against human immune via mucosal surfaces. the of improving trained immunity of BCG study and recombinant BCG that M. tuberculosis and and its the in vitro training for trained immunity to alveolar macrophages and of Trained immunity by cytokine production in response to for after with BCG or recombinant BCG from the study that recombinant BCG boosted the production of IL-1β, IL-6, and trained responses trained effects also found in mouse macrophages and and human monocytes from peripheral blood mononuclear cells (PBMCs) and and Moreover, the revealed that and significantly increased the of IL-6, and in alveolar or human monocytes when compared to BCG. the such as and trained immune responses and and This demonstrates an in trained immunity in alveolar macrophages for some recombinant BCG such as compared to BCG in we mucosal recombinant BCG strain rBCGPPE27 vaccination enhance trained response compared to BCG ex vivo and LPS of and in alveolar macrophages from rBCGPPE27 mice found to the response to or restimulation Additionally, levels of and increased in alveolar macrophages from mice compared to from mice The in trained immune response also observed in The development of trained immunity provides against infections M.A. et al.BCG vaccination induces immunity against Immunol. 2022; Full Text Full Text PDF PubMed Scopus Google Scholar). the study mucosal recombinant vaccination enhance trained immunity in vivo and that mice more to mice and in in Candida infected of rBCGPPE27 trained mice observed and with increased in alveolar macrophages and These findings suggest that mucosal rBCGPPE27 immunization induces levels of trained immunity when compared to BCG vaccination. compared the immune response by mucosal administration the intradermal vaccination The to the of vaccination by a specific after in vitro of with the of The results revealed that for BCG and the response after intradermal mucosal delivery and However, when with recombinant which is from BCG in release from mucosal ex vivo with the levels of T cells in for mucosal BCG vaccination compared to intradermal response observed for rBCGPPE27 and BCG These suggest that stronger trained effects by mucosal rBCGPPE27 vaccination intradermal immunization on immunity. the induction of trained immunity after intradermal mucosal vaccination with BCG or rBCGPPE27. cells of trained immunity and trained immune responses compared ex vivo heterologous of and alveolar macrophages from rBCGPPE27 BCG in the induction of trained immunity response in and alveolar macrophages following mucosal or intradermal vaccination. The results also revealed compared to mucosal intradermal delivery of BCG and rBCGPPE27 in a of trained immunity, as by trained immune cytokine production after LPS restimulation and the routes of production by alveolar macrophages significantly in mucosal vaccination in intradermal delivery and to BCG, the of the rBCGPPE27 vaccine on production more significant after mucosal and intradermal vaccination. a mucosal and intradermal vaccination with rBCGPPE27 alveolar macrophages to a stronger trained immune BCG an in mucosal rBCGPPE27 vaccination the of of trained immunity in and alveolar macrophages from mucosal rBCGPPE27 or BCG-vaccinated mice to The results revealed that the in in and alveolar macrophages from mice that in the BCG and The of that and in these cells increased after rBCGPPE27 vaccination compared to BCG immunization and However, the of and The of and increased after mucosal rBCGPPE27 while production as shown in the of and to increased The study also the of in and alveolar macrophages after mucosal rBCGPPE27 to of which the in and alveolar macrophages from mucosal rBCGPPE27 mice in cells from BCG mice, the in cytokine production the findings suggest that mucosal rBCGPPE27 vaccination enhances the and epigenetic of trained immunity compared to BCG. to metabolic and epigenetic changes on activation in monocytes R. Carvalho A. Rocca C.L. Palma C. Rodrigues F. Silvestre R. et al.Immunometabolic pathways in BCG-induced trained immunity.Cell Rep. 2016; 17: 2562-2571Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). alveolar macrophages trained with rBCGPPE27 a significantly increased and of which is a target of the mammalian target of rapamycin complex 2 the of in trained the study in alveolar macrophages from mice with myeloid of which is a of shown in of to a of and in alveolar macrophages in response to LPS when compared to Moreover, the of and glycolysis, in with a glycolytic as in the in alveolar macrophages However, in cells after rBCGPPE27 training when compared with activation and glycolysis a reduction in upon rBCGPPE27 training Additionally, as from the the in alveolar with the cytokine production the of myeloid in the immunometabolic and epigenetic changes of trained immunity ex alveolar macrophages from and mice trained with rBCGPPE27 with and of trained immunity The study found that production significantly in alveolar macrophages compared with cells Moreover, the enhanced of the of abolished in cells with the compared to in the as a of and epigenetic of trained immunity in alveolar macrophages from mucosal rBCGPPE27 mice, compared to BCG. studies have shown that BCG from the pathway in innate immune cells Z. Z. B. C. Wang et tuberculosis responses via receptor of 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, pathway has been found to with in to glycolysis S. et glycolysis Rep. Full Text Full Text PDF PubMed Scopus Google Scholar), while levels E.J. S. of of Mycobacterium tuberculosis by 2020; PubMed Scopus Google Scholar). on these that rBCGPPE27 training the which is associated with glycolysis is for training immune the we that significantly in and alveolar macrophages trained by in to cells Furthermore, the effects of on the of trained immunity by of in alveolar macrophages This that the immunometabolic pathway is for the boosted trained effects upon rBCGPPE27 training in alveolar macrophages to the of in trained immunity effects in we alveolar macrophages from myeloid of or mice that of significantly and in alveolar macrophages upon LPS compared with also in a in the in cells, glycolysis Additionally, the found to the in alveolar macrophages in with cytokine production Furthermore, after mucosal rBCGPPE27 alveolar macrophages from or mice with and of trained immunity ex vivo that the of the of and cytokine significantly in alveolar macrophages compared with cells This that activation is for a stronger trained in alveolar macrophages mucosal rBCGPPE27 vaccination. The protective response against infection trained immunity N. J. Sanz J. Kaufmann E. B. Pacis A. et tuberculosis hematopoietic stem cells to and trained 2020; 183: Full Text Full Text PDF PubMed Scopus Google Scholar). the of a we an avirulent strain for the of the with 2 of BCG or rBCGPPE27 in of the the mice with of in The results after the rBCGPPE27 vaccination significantly levels of and in BCG The of also revealed a a T helper type 1 response Additionally, we observed an in the mucosal immune as by increased in cells of mice BCG vaccination levels of pro-inflammatory observed in and of mice rBCGPPE27 vaccination BCG a in immune The in the and also in mice with as compared to with BCG. Moreover, of in myeloid cells abolished the enhanced and mucosal immunity and These findings suggest that mucosal rBCGPPE27 vaccination induces stronger and mucosal immunity against infection BCG which on its augmented trained immunity via of the potential of BCG-induced trained effects for vaccine C. A. A. et and development of trained immunity: for vaccine Immunol. PubMed Scopus Google Scholar, G. S. S. 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These results suggest that glycolysis provides an metabolic mechanism for trained immune response in lung-resident macrophages. trained immunity has been in various immune cells, and/or coronavirus immunity for populations in trained immunity, BCG and Immunol. 2020; PubMed Scopus Google Scholar), of innate immune memory in lung-resident macrophages is Here, we found that alveolar macrophages from after mucosal rBCGPPE27 an increased of and upon LPS is that rBCGPPE27 administration via the mucosal memory alveolar macrophages with changes in glycolysis and the of immunometabolic we for the that memory alveolar macrophages metabolic and epigenetic changes that on myeloid activation of is to that the of trained macrophages the of T cells to the respiratory M. S. M. et of memory alveolar macrophages T and is to trained 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). 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R. et immunity: a tool for reducing susceptibility to and the severity of SARS-CoV-2 infection.Cell. 2020; 181: 969-977Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, L.E. A. C. BCG vaccine protection from severe coronavirus disease S. A. 2020; PubMed Scopus Google Scholar). BCG for nonspecific protection against viral infections, particularly provide a therapeutic coronavirus immunity for populations in trained immunity, BCG and Immunol. 2020; PubMed Scopus Google Scholar, S. from Mycobacterium exposure or BCG vaccination and COVID-19 2020; Scopus Google Scholar). we an vaccine when as a single mucosal CoV-2-specific immunity, particularly the of antibodies, neutralizing antibodies, and vaccine-specific T cells that by BCG/CoV-2 vaccination. study to and the mucosal vaccination of trained immunity and protective against infection remains to in Our study that rBCGPPE27 immunization is more effective BCG via intradermal and the mucosal in of more trained Our findings also revealed an of trained immunity where the trained immune response of alveolar macrophages Specifically, stronger trained immunity by rBCGPPE27 in alveolar macrophages on mTORC2/HK1-mediated immunometabolic This enhanced innate immune memory Furthermore, we an vaccine against by the trained immunity These results and of immune protection on trained immunity against a of pathogens and infections and with the of for the and of by and of mice, mice, mice, and mice from of mice and mice with mice to generate and receptor 2 myeloid primary response and from the of mice in specific conditions the of blood mononuclear cells (PBMCs) from human blood as Quintin J. S. et and aerobic glycolysis as metabolic basis for trained Scopus Google Scholar). 1 to 2 on of a and and for The and cells with by of a of and cells from the and from and to the The of cells by and in an with and and of blood from blood for the in vitro the Z. Z. B. C. Wang et tuberculosis responses via receptor of 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). after for 1 in cells stimulated with BCG, or rBCGPPE27 a of for human Z. Z. B. C. Wang et tuberculosis responses via receptor of 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). cells with and for in a with human The on of of the and the cells stimulated with LPS or as a for an the an some the after initial training with a or BCG the and little The cytokine levels on the of cells in the well the as the cytokine alveolar macrophages or in and stimulated with BCG, or rBCGPPE27 for a of Z. Z. B. C. Wang et tuberculosis responses via receptor of 2018; Full Text Full Text PDF PubMed Scopus Google Scholar). and for in the with in the of recombinant factor a and cells stimulated with a or of LPS following of LPS cells stimulated for 2 with for activation and and for the after of LPS to the levels of IL-1β, and via from the of in which cytokine the these from the coronavirus immunity for populations in trained immunity, BCG and Immunol. 2020; PubMed Scopus Google Scholar). with a single of 2 of strain BCG or rBCGPPE27 in that via the intradermal or mucosal with (13Giamarellos-Bourboulis E.J. Tsilika M. Moorlag S. Antonakos N. Kotsaki A. 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