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The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal influenza A virus infection at epithelial surfaces

Yingxue Wang, Weijiao Zhang, Matthew Jefferson, Parul Sharma, Ben Bone, Anja Kipar, Janine L. Coombes, Timothy Pearson, Angela Man, Alex Zhekova, Yongping Bao, Ralph A Tripp, Simon R. Carding, Ulrike Mayer, Penny P. Powell, James P. Stewart, Thomas Wileman

Posted on: 9 March 2020

Preprint posted on 15 January 2020

Loss of non-canonical autophagy in mice increases sensitivity to influenza A virus infection by disrupting defences at the lung epithelial barrier, but not in phagocytic leukocytes.

Selected by Kirsty Hooper

Background 

Autophagy is a well-characterised pathway, in which intracellular components sequestered in a double membrane autophagosome are degraded upon fusion with a lysosome (Dikic et al., 2018). The degraded subunits can then be recycled by the cell to promote survival and maintain homeostasis (Dikic et al., 2018). Non-canonical autophagy, however, is the recruitment of the autophagy protein LC3, among other components of the autophagy machinery, to single membrane compartments to help facilitate degradation of engulfed material taken up via phagocytosis, endocytosis or micropinocytosis (Upadhyay et al., 2019). This pathway is independent of upstream autophagy regulators and has been linked to innate immune defences (Upadhyay et al., 2019), cancer (Cunha et al., 2018) and Alzeimer’s disease (Heckmann et al., 2019). Among the pathogens known to induce non-canonical autophagy is influenza A virus (IAV) (Fletcher et al., 2018). To date, however, most studies investigating non-canonical autophagy in innate immune defence have been limited to in vitro models. There are existing mouse models that can be used to study non-canonical autophagy, such as the Rubicon-/- mouse (Martinez et al., 2015). Although these mice display loss of non-canonical autophagy, Rubicon also has distinct functions in the immune system that are separate from non-canonical autophagy (Yang et al., 2012).   

 Experimental model  

ATG16L1 is a key protein in both canonical and non-canonical autophagy that facilitates LC3 lipidation to membranes (Dikic et al., 2018). This study used a mouse model with a truncation in ATG16L1 to remove the C-terminal WD and linker domains (δWD [ATG16L1δWD/δWD]), which enabled them to dissect these pathways by specifically disrupting the non-canonical pathway (Rai et al., 2019). The authors validated the mouse model by showing that cells from δWD mice were proficient in canonical autophagy but defective in non-canonical autophagy.  

 Key Findings  

Systemic loss of non-canonical autophagy increases sensitivity to IAV in mice  

Upon infection with a low pathogenicity strain of IAV, δWD mice had increased mortality compared to control mice, along with more severe weight loss, increased lung viral titres and evidence of fulminant viral pneumonia.  

 Non-canonical autophagy controls lung inflammation after IAV infection 

δWD mice also displayed prolonged increases in pro-inflammatory cytokine expression (IL-1β, TNF-α and CCL2) and increased neutrophil and macrophage/monocyte infiltration post-IAV infection, compared with control mice. Previous studies have identified plasmacytoid dendritic cells (pDCs) as key mediators of cytokine storms (Davidson et al., 2014). Depletion of pDCs in δWD mice decreased morbidity in response to IAV infection, suggesting that exacerbated cytokine responses via pDCs led to the increased severity of infection in δWD mice.  

Interestingly, there was no increase in IL-1β secretion, or frequencies of T cells, B cells and macrophages upon LPS treatment in δWD mice compared to littermate controls. This suggests that there was no alteration of inflammatory threshold or immune homeostasis in δWD mice, despite the amplified responses to IAV infection.  

 Non-canonical autophagy limits IAV infection independently of phagocytic cells  

The authors determined that the high sensitivity to IAV infection seen in δWD mice was not due to defects in myeloid cells. Bone marrow chimeras of δWD mice with WT leukocytes had the same phenotype as δWD mice, and furthermore, LysMcre generated mice, with truncated ATG16L1 protein only present in myeloid cells had the same phenotype as control mice.   

The activity of non-canonical autophagy in epithelial cells during IAV infection was then tested ex vivo, in precision cut lung slices and MEFs from δWD mice. This demonstrated that in the absence of phagocytic cells the exaggerated inflammatory responses and increased viral load in δWD mice persisted.  

 Why did I choose this paper?  

Non-canonical autophagy is a rapidly emerging field implicated in a growing number of pathogenicities. As highlighted by the authors, however, the study of this pathway in vivo has been limited. The creation of the δWD mouse that specifically dissects canonical and non-canonical autophagy with minimal off-target effects can allow great advancements in this field. By characterising this pathway in vivo, the authors can define its systemic role in response to infection with IAV. This can pave the way for the characterisation of non-canonical autophagy in many more infection models and disease settings, and help delineate its molecular mechanisms.   

 Questions to the author/future directions? 

  1. As stated by the authors, there are many studies showing the importance of non-canonical autophagy in innate immune responses in macrophages and dendritic cells. Would the authors predict that there would be a response more dependent on myeloid cells in this in vivo model when using an alternative pathogen that has more of a tropism for phagocytes, such as Aspergillus fumigatus or Listeria monocytogenes? 
  1. In cell culture it was shown that IAV stimulated non-canonical autophagy via a mechanism involving the viral protein M2 (Fletcher et al., 2018). Would the authors expect this mechanism to be more preferential in epithelial cells? And could this mechanism be delineated further using this mouse model? 

 References 

Cunha, L.D. et al. LC3-Associated Phagocytosis in Myeloid Cells Promotes Tumor Immune Tolerance. Cell. 4;175(2):429-441.e16. doi:10.1016/j.cell.2018.08.061 (2018) 

Davidson, S. et al. Pathogenic potential of interferon alphabeta in acute influenza infection. Nat Commun 5, 3864, doi:10.1038/ncomms4864 (2014).  

Dikic, I. and Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat Rev Mol Cell Biol. 19:349–364. (2018) 

Fletcher, K. et al. The WD40 domain of ATG16L1 is required for its noncanonical role in lipidation of LC3 at single membranes. EMBO J. 37, doi:10.15252/embj.201797840 (2018). 

Heckmann, B.L., et al. LC3-Associated Endocytosis Facilitates β-Amyloid Clearance and Mitigates Neurodegeneration in Murine Alzheimer’s Disease. Cell. 25;178(3):536-551.e14. doi:10.1016/j.cell.2019.05.056. (2019) 

Martinez, J. et al. Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat Cell Biol 17, 893-906, doi:10.1038/ncb3192 (2015). 

Rai, S. et al. The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis. Autophagy 15, 599-612, doi:10.1080/15548627.2018.1534507 (2019). 

Upadhyay, S. and Philips, J.A. LC3-associated phagocytosis: host defense and microbial response. Curr Opin Immunol. 60:81-90. doi:10.1016/j.coi.2019.04.012 (2019) 

Yang, C. S. et al. The autophagy regulator Rubicon is a feedback inhibitor of CARD9-mediated host innate immunity. Cell Host Microbe 11, 277-289, doi:10.1016/j.chom.2012.01.019 (2012). 

 

 

doi: https://doi.org/10.1242/prelights.17439

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Author's response

Professor Thomas Wileman shared

In response to your questions:

1. As stated by the authors, there are many studies showing the importance of non-canonical autophagy in innate immune responses in macrophages and dendritic cells. Would the authors predict that there would be a response more dependent on myeloid cells in this in vivo model when using an alternative pathogen that has more of a tropism for phagocytes, such as Aspergillus fumigatus or Listeria monocytogenes?

Yes that is a very good point. At the outset we thought that LAP in phagocytic cells would play a major role in defence against IAV. It was a surprise when we found that LAP in epithelial cells played the major role. I do however think that it will be different for pathogens with a tropism for phagocytes. This is something we would like to look at in the future.

2. In cell culture it was shown that IAV stimulated non-canonical autophagy via a mechanism involving the viral protein M2 (Fletcher et al., 2018). Would the authors expect this mechanism to be more preferential in epithelial cells? And could this mechanism be delineated further using this mouse model?

Another good point. It might be difficult to dissect the roles played by the M2 protein ‘in vivo‘. M2 has a positive role when it facilitates release of RNPs from viruses during endosome acidification, but would have an negative role when M2 activates LAP/non-canonical autophagy.

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