Autophagy: A new strategy for host-directed therapy of tuberculosis

Figures & data

Figure 1. Overview of macroautophagy.

Macroautophagy is one of autophagic process in which is induced by stimuli or stress such as starvation and infection. Autophagy sequesters aggregated proteins, damaged organelles, and microbes from cytoplasm through formation of double-membraned structure (autophagosome). These autophagosomes fuse with lysosome or endosome that contains endocytic compartments to form amphisome or autolysosome. Finally, cargos are degraded in autolysosome for the maintenance of cellular homeostasis.

Figure 2. Xenophagy versus LAP during Mtb infection.

(Left) During mycobacterial infection with Mtb, xenophagy is triggered by the cytoplasmic release of bacteria via its ESX-1 system. Next, the recognition of extracellular bacterial DNA by the STING-dependent pathway allows for the ubiquitination of bacteria. In addition, ubiquitin ligases such as Parkin and Smurf1 allow for the ubiquitination of different chain linkages (K63 and K48, respectively). TRIM16 cooperates with Galectin-3, ATG16L1, ULK1, and BECN1 for the subsequent ubiquitination of bacteria and autophagy activation. These ubiquitin chains recruit autophagy adaptors such as NDP52 and p62, which link to LC3 of the autophagosomal membranes. The exact function of IFN-γ-dependent IRGM1 (LRG47) in the regulation of selective autophagy requires further clarification.

(Right) LAP is an LC3-conjugation process onto the single-membrane phagosome (LAPosome). It is triggered by pathogenic microbes, such as Mtb, through numerous receptors, including toll-like receptor signals. During the LAP process, recruitment of the class III PI3-kinase complex (composed of VPS34, Beclin-1, UVRAG, and Rubicon) increases the production of phosphatidylinositol-3-phosphate (PI3P), which is needed to stabilize the NOX2 complex for the production of reactive oxygen species and the recruitment of autophagic proteins (ATG5, ATG12, ATG16L, ATG7, and ATG3) for the conjugation of lipidated LC3-II to the LAPosomal membrane. Mtb protein CpsA is required for bacteria to block NADPH oxidase activity in order to evade killing by LAP.

Table 1. Potential adjunctive anti-TB therapeutics targeting autophagy.

Candidates	Mechanisms	References
Vitamin D receptor signaling
1,25-dihydroxyvitamin D3	Induction of cathelicidin, autophagy gene transcription IL1R signaling and DEFB4/HBD2 induction	[54, 57]
Mycobacterial LpqH antigen	Functional VDR activation through TLR2/1/CD14-Ca^2+-AMPK-p38 MAPK signaling	[55]
4-phenylbutyrate	LL-37 expression, P2RX7 receptor signaling, intracellular calcium influx and AMPK pathways	[56]
IFN-γ	Crosstalk with vitamin D-dependent pathway	[58,59]
Nuclear receptors and agonists
NR1D1	Increase of MAPL1LC3-II and LAMP1	[60]
PPAR-α	Upregulation of autophagy and lysosomal genes	[61]
ESRRA	Transcriptional and post-tranaslational regulation of autophagy genes/protein	[62]
AMPK pathway
Calcium-mobilizing agents	Ca(2+)/calmodulin-dependent kinase kinase-β (CaMKKβ)-mediated AMPK activation	[63]
AICAR	AMPK-PGC1α-mediated autophagy-related gene transcription	[64]
Cyclic peptides Ohmyungsamycins A/B	AMPK-mediated autophagy activation and regulation of inflammation	[65]
Phytochemicals; steroid glycoside chemicals	AMPK-ULK1-dependent pathway	[66]
Resveratrol	Activation of AMPK and sirtuin 1	[67,68]
Small molecules/Chemicals
Gefitinib	Inhibition of p38 MAPK pathway; activation of autophagy	[69]
Fluoxetine	Increased secretion of TNF-α; induction of autophagy	[69]
Baicalin	Modulation of PI3K/Akt/mTOR pathway	[70]
Loperamide, Verapamil	Induction of autophagy; mechanisms are unknown	[71]
Src kinase inhibitors	Activation of autophagy; mechanisms are unknown	[72]
Carbamazepine	Inositol triphosphate (IP3) depletion; mTOR-independent mechanisms	[73]
Antibiotics
Isoniazid, Pyrazinamide	Bacterial hydroxyl radicals and cellular ROS-dependent mechanisms	[74]
Thiostrepton	ER stress-mediated autophagy activation	[75]
Ambroxol	Induction of autophagy; mechanisms are unknown	[76]

Table 2. Clinical studies of autophagy-adjunctive therapeutics for tuberculosis.

Study name		Phase	Results	Clinical trials. gov Identifier*	References
Vitamin D replacement
Trial of Adjunctive Vitamin D in TB Treatment	III		Vitamin D did not significantly affect time to sputum culture conversion	NCT 419068	[87]
A Clinical Trial to Study the Effect of Vitamin D to Treatment in New Pulmonary TB Patients	II		Vitamin D supplementation did not reduce time to sputum culture conversion	NCT 366470	[88]
Impact of Vitamin D supplementation on Host Immunity to Mtb and Response to Treatment	II		A high-dose vitamin D3 regimen safely corrected vitamin D deficiency but did not improve the rate of sputum Mtb clearance over 16 weeks in this pulmonary TB cohort	NCT 918086	[89]
Replacement of Vitamin D in Patients With Active TB	II		High doses of vitamin D accelerated clinical, radiographic improvement in all TB patients and increased immune activation in patients with deficient serum vitamin D levels	NCT 1130311	[90,91]
Role of Vitamin D in Innate Immunity to TB	II		Vitamin D supplementation for 6 months had significant favorable effects on serum 25(OH) D concentrations and on growth in stature	NCT 1244204	[92]
Effects of Vitamin D Supplementation on Antimycobacterial Immunity	II		A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro	NCT 00157066	[93]
Vitamin D + other replacement
L-arginine and Vitamin D Adjunctive Therapy in Pulmonary TB	III		Neither vitamin D nor L-arginine supplementation affected TB outcomes	NCT 677339	[94]
Clinical Trial of PBA and Vitamin D in TB	II		Adjunct therapy with PBA+vitamin D3 or vitamin D3 or PBA to standard short-course therapy demonstrated beneficial effects towards clinical recovery	NCT 01580007	[95]
Drug repositioning
HDT-TB (Everolimus, Auranofin)	II		Enrolling	NCT 2968927
Doxycycline in human pulmonary TB	II		Enroll: completed	NCT 2774993	[96]

BCG, Bacille Calmette-Guérin * Further details for trial with NCT numbers can be accessed at http://clinicaltrials.gov.

Yuk JM, Shin DM, Lee HM, et al. Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host Microbe. 2009 Sep 17;6(3):231–243.

 Google Scholar

Verway M, Bouttier M, Wang TT, et al. Vitamin D induces interleukin-1beta expression: paracrine macrophage epithelial signaling controls M. tuberculosis infection. PLoS Pathog. 2013;9(6):e1003407.

 Google Scholar

Shin DM, Yuk JM, Lee HM, et al. Mycobacterial lipoprotein activates autophagy via TLR2/1/CD14 and a functional vitamin D receptor signalling. Cell Microbiol. 2010 Nov;12(11):1648–1665.

 Google Scholar

Rekha RS, Rao Muvva SS, Wan M, et al. Phenylbutyrate induces LL-37-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human macrophages. Autophagy. 2015;11(9):1688–1699.

 Google Scholar

Edfeldt K, Liu PT, Chun R, et al. T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism. Proc Natl Acad Sci U S A. 2010 Dec 28;107(52):22593–22598.

 Google Scholar

Fabri M, Stenger S, Shin DM, et al. Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages. Sci Transl Med. 2011 Oct 12;3(104):104ra102.

 Google Scholar

Chandra V, Bhagyaraj E, Nanduri R, et al. NR1D1 ameliorates Mycobacterium tuberculosis clearance through regulation of autophagy. Autophagy. 2015 Nov 2;11(11):1987–1997.

 Google Scholar

Kim YS, Lee HM, Kim JK, et al. PPAR-alpha activation mediates innate host defense through induction of TFEB and lipid catabolism. J Immunol. 2017 Apr 15;198(8):3283–3295.

 Google Scholar

Kim SY, Yang CS, Lee HM, et al. ESRRA (estrogen-related receptor alpha) is a key coordinator of transcriptional and post-translational activation of autophagy to promote innate host defense. Autophagy. 2018;14(1):152–168.

 Google Scholar

Hoyer-Hansen M, Bastholm L, Szyniarowski P, et al. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell. 2007 Jan 26;25(2):193–205.

 Google Scholar

Yang CS, Kim JJ, Lee HM, et al. The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy. Autophagy. 2014 May;10(5):785–802.

 Google Scholar

Kim TS, Shin YH, Lee HM, et al. Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway. Sci Rep. 2017 Jun 13;7(1):3431.

 Google Scholar

Fan Y, Wang N, Rocchi A, et al. Identification of natural products with neuronal and metabolic benefits through autophagy induction. Autophagy. 2017 Jan 2;13(1):41–56.

 Google Scholar

Vingtdeux V, Giliberto L, Zhao H, et al. AMP-activated protein kinase signaling activation by resveratrol modulates amyloid-beta peptide metabolism. J Biol Chem. 2010 Mar 19;285(12):9100–9113.

 Google Scholar

Vingtdeux V, Chandakkar P, Zhao H, et al. Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-beta peptide degradation. FASEB J. 2011 Jan;25(1):219–231.

 Google Scholar

Stanley SA, Barczak AK, Silvis MR, et al. Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog. 2014 Feb;10(2):e1003946.

 Google Scholar

Zhang Q, Sun J, Wang Y, et al. Antimycobacterial and anti-inflammatory mechanisms of Baicalin via induced autophagy in macrophages Infected with Mycobacterium tuberculosis. Front Microbiol. 2017;8:2142.

 Google Scholar

Juarez E, Carranza C, Sanchez G, et al. Loperamide restricts intracellular growth of Mycobacterium tuberculosis in lung macrophages. Am J Respir Cell Mol Biol. 2016 Dec;55(6):837–847.

 Google Scholar

Chandra P, Rajmani RS, Verma G, et al. Targeting drug-sensitive and -resistant strains of Mycobacterium tuberculosis by inhibition of Src family kinases lowers disease burden and pathology. mSphere. 2016 Mar–Apr;1(2):e00043-15.

 Google Scholar

Schiebler M, Brown K, Hegyi K, et al. Functional drug screening reveals anticonvulsants as enhancers of mTOR-independent autophagic killing of Mycobacterium tuberculosis through inositol depletion. EMBO Mol Med. 2015 Feb;7(2):127–139.

 Google Scholar

Kim JJ, Lee HM, Shin DM, et al. Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action. Cell Host Microbe. 2012 May 17;11(5):457–468.

 Google Scholar

Zheng Q, Wang Q, Wang S, et al. Thiopeptide antibiotics exhibit a dual mode of action against intracellular pathogens by affecting both host and microbe. Chem Biol. 2015 Aug 20;22(8):1002–1007.

 Google Scholar

Choi SW, Gu Y, Peters RS, et al. Ambroxol induces autophagy and potentiates rifampin antimycobacterial activity. Antimicrob Agents Chemother. 2018 Sep;62(9):e01019-18.

 Google Scholar

Martineau AR, Timms PM, Bothamley GH, et al. High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lancet. 2011 Jan 15;377(9761):242–250.

 Google Scholar

Daley P, Jagannathan V, John KR, et al. Adjunctive vitamin D for treatment of active tuberculosis in India: a randomised, double-blind, placebo-controlled trial. Lancet Infect Dis. 2015 May;15(5):528–534.

 Google Scholar

Tukvadze N, Sanikidze E, Kipiani M, et al. High-dose vitamin D3 in adults with pulmonary tuberculosis: a double-blind randomized controlled trial. Am J Clin Nutr. 2015 Nov;102(5):1059–1069.

 Google Scholar

Hasan Z, Salahuddin N, Rao N, et al. Change in serum CXCL10 levels during anti-tuberculosis treatment depends on vitamin D status [Short Communication]. Int J Tuberc Lung Dis. 2014 Apr;18(4):466–469.

 Google Scholar

Salahuddin N, Ali F, Hasan Z, et al. Vitamin D accelerates clinical recovery from tuberculosis: results of the SUCCINCT Study [Supplementary Cholecalciferol in recovery from tuberculosis]. A randomized, placebo-controlled, clinical trial of vitamin D supplementation in patients with pulmonary tuberculosis’. BMC Infect Dis. 2013 Jan 19;13:22.

 Google Scholar

Ganmaa D, Giovannucci E, Bloom BR, et al. Vitamin D, tuberculin skin test conversion, and latent tuberculosis in Mongolian school-age children: a randomized, double-blind, placebo-controlled feasibility trial. Am J Clin Nutr. 2012 Aug;96(2):391–396.

 Google Scholar

Martineau AR, Wilkinson RJ, Wilkinson KA, et al. A single dose of vitamin D enhances immunity to mycobacteria. Am J Respir Crit Care Med. 2007 Jul 15;176(2):208–213.

 Google Scholar

Ralph AP, Waramori G, Pontororing GJ, et al. L-arginine and vitamin D adjunctive therapies in pulmonary tuberculosis: a randomised, double-blind, placebo-controlled trial. PLoS One. 2013;8(8):e70032.

 Google Scholar

Mily A, Rekha RS, Kamal SM, et al. Significant effects of oral phenylbutyrate and vitamin D3 adjunctive therapy in pulmonary tuberculosis: A randomized controlled trial. PLoS One. 2015;10(9):e0138340.

 Google Scholar

Xing Y, Liqi Z, Jian L, et al. Doxycycline induces mitophagy and suppresses production of interferon-beta in IPEC-J2 cells. Front Cell Infect Microbiol. 2017;7:21.

 Google Scholar