Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

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Abstract

Extracellular bacteria, such as Pseudomonas aeruginosa and Klebsiella pneumoniae, have been reported to induce autophagy; however, the role and machinery of infection-induced autophagy remain elusive. We show that the pleiotropic Src kinase Lyn mediates phagocytosis and autophagosome maturation in alveolar macrophages (AM), which facilitates eventual bacterial eradication. We report that Lyn is required for bacterial infection-induced recruitment of autophagic components to pathogen-containing phagosomes. When we blocked autophagy with 3-methyladenine (3-MA) or by depleting Lyn, we observed less phagocytosis and subsequent bacterial clearance by AM. Both morphological and biological evidence demonstrated that Lyn delivered bacteria to lysosomes through xenophagy. TLR2 initiated the phagocytic process and activated Lyn following infection. Cytoskeletal trafficking proteins, such as Rab5 and Rab7, critically facilitated early phagosome formation, autophagosome maturation, and eventual autophagy-mediated bacterial degradation. These findings reveal that Lyn, TLR2 and Rab modulate autophagy related phagocytosis and augment bactericidal activity, which may offer insight into novel therapeutic strategies to control lung infection.

Author Summary

It is vital to establish the mechanistic basis for initiation of host defenses and immune responses that are required to eliminate bacterial infection. This line of inquiry will increase knowledge of bacterial pathogenesis and uncover new insights that can enhance design and effectiveness of novel therapeutics. We demonstrate that TLR-2 is required for inducing Lyn activity in host defense against Pa infection through assistance in autophagosome maturation, and may link autophagy to phagocytosis in a TLR-2-Lyn-dependent manner. Thus, these results may further help to alleviate human acute lung injury/adult respiratory distress syndrome (ALI/ARDS) caused by Gram-negative bacteria.

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Most cited references 34

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Guidelines for the use and interpretation of assays for monitoring autophagy.

C N Estevez, S M Lee, Qing Zhong … (2012)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

Tatsuya Saitoh, Naonobu Fujita, Myoung Ho Jang … (2008)

Systems for protein degradation are essential for tight control of the inflammatory immune response. Autophagy, a bulk degradation system that delivers cytoplasmic constituents into autolysosomes, controls degradation of long-lived proteins, insoluble protein aggregates and invading microbes, and is suggested to be involved in the regulation of inflammation. However, the mechanism underlying the regulation of inflammatory response by autophagy is poorly understood. Here we show that Atg16L1 (autophagy-related 16-like 1), which is implicated in Crohn's disease, regulates endotoxin-induced inflammasome activation in mice. Atg16L1-deficiency disrupts the recruitment of the Atg12-Atg5 conjugate to the isolation membrane, resulting in a loss of microtubule-associated protein 1 light chain 3 (LC3) conjugation to phosphatidylethanolamine. Consequently, both autophagosome formation and degradation of long-lived proteins are severely impaired in Atg16L1-deficient cells. Following stimulation with lipopolysaccharide, a ligand for Toll-like receptor 4 (refs 8, 9), Atg16L1-deficient macrophages produce high amounts of the inflammatory cytokines IL-1beta and IL-18. In lipopolysaccharide-stimulated macrophages, Atg16L1-deficiency causes Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-dependent activation of caspase-1, leading to increased production of IL-1beta. Mice lacking Atg16L1 in haematopoietic cells are highly susceptible to dextran sulphate sodium-induced acute colitis, which is alleviated by injection of anti-IL-1beta and IL-18 antibodies, indicating the importance of Atg16L1 in the suppression of intestinal inflammation. These results demonstrate that Atg16L1 is an essential component of the autophagic machinery responsible for control of the endotoxin-induced inflammatory immune response.

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Conditional gene targeting in macrophages and granulocytes using LysMcre mice.

B. Clausen, C Burkhardt, W. Reith … (1999)

Conditional mutagenesis in mice has recently been made possible through the combination of gene targeting techniques and site-directed mutagenesis, using the bacteriophage P1-derived Cre/loxP recombination system. The versatility of this approach depends on the availability of mouse mutants in which the recombinase Cre is expressed in the appropriate cell lineages or tissues. Here we report the generation of mice that express Cre in myeloid cells due to targeted insertion of the cre cDNA into their endogenous M lysozyme locus. In double mutant mice harboring both the LysMcre allele and one of two different loxP-flanked target genes tested, a deletion efficiency of 83-98% was determined in mature macrophages and near 100% in granulocytes. Partial deletion (16%) could be detected in CD11c+ splenic dendritic cells which are closely related to the monocyte/macrophage lineage. In contrast, no significant deletion was observed in tail DNA or purified T and B cells. Taken together, LysMcre mice allow for both specific and highly efficient Cre-mediated deletion of loxP-flanked target genes in myeloid cells.

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Author and article information

Contributors

Mary O'Riordan: Role: Editor

Journal

Journal ID (nlm-ta): PLoS Pathog

Journal ID (iso-abbrev): PLoS Pathog

Journal ID (publisher-id): plos

Journal ID (pmc): plospath

Title: PLoS Pathogens

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Print): 1553-7366

ISSN (Electronic): 1553-7374

Publication date (Electronic): 6 January 2016

Publication date Collection: January 2016

Volume: 12

Issue: 1

Electronic Location Identifier: e1005363

Affiliations

[1 ]State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, P.R. China

[2 ]Department of Biomedical Sciences, University of North Dakota, Grand Forks, North Dakota, United States of America

[3 ]Department of Thoracic Oncology, West China Hospital, Sichuan University, Chengdu, Sichuan, P.R. China

[4 ]Inflammations & Allergic Diseases Research Unit, Affiliated Hospital of Luzhou Medical College, Luzhou, Sichuan, P.R. China

University of Michigan Medical School, UNITED STATES

Author notes

The authors have declared that no competing interests exist.

Conceived and designed the experiments: XL SH GL CH MW. Performed the experiments: XL SH XZ YY ST SZ RL MY MCJ AH. Analyzed the data: XL SH MCJ AH YW MW. Contributed reagents/materials/analysis tools: YW GL CH MW. Wrote the paper: XL SH CH MW.

* E-mail: lzlgp@ 123456163.com (GL); hcanhua@ 123456hotmail.com (CH); min.wu@ 123456med.und.edu (MW)

Article

Publisher ID: PPATHOGENS-D-15-01739

DOI: 10.1371/journal.ppat.1005363

PMC ID: 4703367

PubMed ID: 26735693

SO-VID: 1366a704-14cb-4ec7-bd99-00377f7d47e6

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

History

Date received : 27 July 2015

Date accepted : 4 December 2015

Page count

Figures: 7, Tables: 0, Pages: 23

Funding

This work was supported by grants from the National 973 Basic Research Program of China (2013CB911300 and 2012CB518900), the Chinese NSFC (81225015 and 81430071), and Sichuan Science-Technology Innovative Research Team for Young Scientist (2013TD0001); this work was also supported by Flight Attendant Medical Research Institute (FAMRI, Grant #103007), National Institute of Health AI109317-01A1, AI101973-01, and AI097532-01A1 to MW. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Data Availability All relevant data are within the paper and its Supporting Information files.

Lyn Delivers Bacteria to Lysosomes for Eradication through TLR2-Initiated Autophagy Related Phagocytosis

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Most cited references 34

Guidelines for the use and interpretation of assays for monitoring autophagy.

Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.

Conditional gene targeting in macrophages and granulocytes using LysMcre mice.

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