Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes

Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn’s disease. Crohn’s disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn’s disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn’s Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn’s disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn’s Disease due to defects in autophagy. Introduction NOD2 receptor due to its ubiquitylation activity towards RIPK2 IAPs are ubiquitin ligases that regulate the activity of . TNF Super Family Receptors (TNFSFR), TLRs and NOD Genetic loss of cIAP1, cIAP2 and XIAP results in severe receptors. By attaching ubiquitin onto substrates such as systemic inflammation characterised by massive increases RIPK1 they regulate the activation of NF-κB and deter- in many cytokines including TNF and IL-1β . IAP mine the outcome of signals from these receptors. Their antagonist drugs are also able to trigger activation of inhibition results in skewing of signals towards death and NLRP3 inflammasomes in LPS primed macrophages .In 1–4 also production of an inflammatory cytokine response . both cases there appears to be an important role for XIAP, The three best characterised and functionally related IAP in addition to cIAP1 and cIAP2, in suppressing this members are cIAP1, cIAP2 and XIAP. cIAP1 and cIAP2 inflammatory cascade suggesting some redundancy in the 5–7 act together in complex with TRAF2 and TRAF3 . Loss function of IAPs. of cIAP1 leads to defective NF-κB signals from partner Humans with mutations in XIAP often develop Crohn’s receptors such as TNFR1 and additional activation of disease (CD) but may also suffer from X-linked lympho- 1 9–12 non-canonical NF-κB . Less is known about XIAP and its proliferative disease 2 (XLP-2) . NOD2 is also com- 13–16 regulation other than it is required for NF-κB signals from monly mutated in CD patients . Although mutations in XIAP affect the activation of NOD2, the molecular mechanisms behind CD in these mutations are still not clear. Another gene commonly mutated in CD is the Correspondence: Ian E. Gentle (ian.gentle@uniklinik-freiburg.de) 1 17 Institute of Medical Microbiology and Hygiene, University Medical Center autophagy gene ATG16L1 . NOD2 and Atg16L1 are also Freiburg, Freiburg, Germany functionally linked with NOD2 being required for Faculty of Medicine, University of Freiburg, Freiburg, Germany recruiting ATG16L1 to internalised bacteria such as Full list of author information is available at the end of the article. Edited by G. Raschellà © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Gradzka et al. Cell Death and Disease (2018) 9:529 Page 2 of 15 Fig. 1 IAP inhibition triggers autophagosome accumulation. a Wild type MEFs expressing mcherry-GFP-LC3b were treated with the indicated doses of LCL161or thapsigargin for 6 h. DNA was stained using Hoechst and live Cells were imaged to visualise mCherry, GFP and Hoechst. The number of GFP+ puncta and mCherry+ puncta were then counted per cell and the ratio of GFP + /mCherry + puncta calculated. Graphs represent the mean and error bars show SEM of at least 3 experiments. b IAP antagonism does not block lysosomal acidification. Wild type MEFs were treated with either LCL161 (5 μM), thapsigargin (3 μM) or concanamycin A (2 nM) for 6 h. Cells were stained with lysotracker red and visualised by microscopy or lysotracker intensity measured via flow cytometry. Graphs represent the mean of the geometric mean fluorescence intensity measured by flow cytometry and error bars show SEM of at least 3 independent experiments. P values were calculated using T-test in Prism software Salmonella during xenophagy, the targeted autophagy of autophagy is a key pathway linking NOD2 and ATG16L1 invading bacteria . CD associated mutations in NOD2 in the development of Crohn’s disease and hint that XIAP and Atg16L1 were also shown to block autophagy may also have some role in autophagy regulation. induction by NOD2 and reduce the xenophagy of invad- Autophagy is a highly conserved pathway for recycling ing Salmonella . Defects in Atg16L1 or NOD2 also cellular components in times of nutrient limitation. increased the replication of adherent-invasive Escherichia Autophagosomes are formed around cellular components coli (AIEC) in macrophages, resulting in enhanced cyto- such as bulk cytoplasm (macroautophagy), but also spe- kine production. Conversely, induction of autophagy in cific targets including mitochondria (mitophagy), invasive −/− NOD2 macrophages reduced survival of AIEC and bacteria (xenophagy) and aggregated proteins (aggre- cytokine production . Together these data suggest that phagy). In each case phagophores, fuse to make Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 3 of 15 autophagosomes that fuse with lysosomes, releasing their autophagosomes or an increase in activation of autop- 21– contents to be degraded and recycled (reviewed in refs. hagy. Defects in autophagosome turnover occur by a ). Autophagy is also linked to many other functions in number of mechanisms. One possibility is a failure to animals, including regulation of the immune system at acidify the autolysosome, resulting in defective lysosomal various levels such as xenophagy, MHCII presentation, enzyme function. Such an effect is seen upon the addition limiting cytokine production in response to infection, and of drugs like concanamycin A, an inhibitor of this later stage in autophagy that blocks the activity of ATPase in protein folding diseases such as Alzheimer’s disease 24–27 (AD) (reviewed in refs. ). proton pumps in the lysosome, preventing acidification We here show that both cIAP2 and XIAP promote and blocking degradation of lysosomal cargo. autophagosome–lysosome fusion and that their loss or To rule out an effect of IAP antagonists on lysosomal inhibition results in accumulation of autophagosomes and acidification, MEFs were treated with IAP antagonists or lysosomes and defects in mitophagy and xenophagy. concanamycin A to block acidification, or thapsigargin to block autophagosomal fusion with lysosomes. MEFs were Results then stained with Lysotracker Red, which only fluoresces IAP antagonism causes accumulation of autophagosomes when lysosomes are acidified. Cells were analysed by To determine if there was any effect of IAP antagonists microscopy and flow cytometry (Fig. 1b). IAP antagonist on autophagy, Mouse Embryonic Fibroblasts (MEFs) were treatment did not reduce lysotracker fluorescence infected with pBabe-mCherry-EGFP-LC3b. GFP fluores- whereas Concanamycin A did (Fig. 1b). Both thapsigargin cence is sensitive to pH and decreases in the acidic treatment and higher doses of LCL161 enhanced lyso- environment of autolysosomes while mCherry retains its tracker staining (Fig. 1b), suggesting a possible accumu- fluorescence. Due to this property of GFP, this reporter lation of lysosomes in addition to autophagosomes. IAP can be used to assess the rate at which autophagosomes antagonists do not therefore affect the acidification of are synthesised and degraded by lysosomes. lysosomes, but do lead to a build-up of mCherry + /GFP Since there is continuous fusion with lysosomes at + positive puncta in a fashion similar to thapsigargin. steady state, there are more single mCherry + puncta in Electron Microscopy (EM) was performed on LCL161 cells expressing GFP-mCherry-LC3b than GFP + treated MEFs, as well as thapsigargin treated MEFs and mCherry + puncta, and the ratio of GFP + and mCherry untreated controls. LCL161 treated cells showed a clear + puncta can be used to illustrate this spontaneous increase in vesicular structures containing cellular debris fusion. The Cells were treated with the IAP antagonist suggesting they are autophagosomes (Fig. 2). The same LCL-161 . Upon treatment of MEFs with LCL161, there was seen for thapsigargin treatment. Of note is that was a clear accumulation of GFP + puncta at doses as low electron dense lysosomes cluster in regions adjacent to as 500 nM (Fig. 1a). A slight accumulation of mCherry + autophagosomes, but do not appear to be fused with the puncta could also be detected at this dose but the ratio of autophagosomes (Fig. 2). Immunofluoresence was also GFP/mCherry increased substantially. Treatment with 5 perfomed on wt MEFs treated with LCL161 or birinapant μM LCL161 resulted in more mCherry + GFP + puncta to confirm that endogenous LC3 also shows a similar and a ratio of GFP + /mCherry + close to one (Fig. 1a). accumulation after IAP antagonism (Fig. 3). There is a Similar results were seen with an unrelated IAP antago- striking accumulation of autophagosomes, in the peri- nist, birinapant that also lead to significant blockage of nuclear region. Surprisingly these autophagosomes appear fusion events, but only at doses of 50 µM or above to surround lysosomes (LAMP2 structures) as opposed (Supplementary Fig. 1). Thapsigargin, an irreversible to co-localising with them which is similar to the non- inhibitor of the SERCA calcium pumps of the ER, has fused, but associated, autophagosomes and lysosomes been shown to specifically inhibit the fusion of autophagic seen by EM (Fig. 2) and is indicative of recruitment of vesicles with lysosomes, resulting in build up of GFP + autophagosomes to lysosomes, but failure to fuse. The mCherry + puncta . As a positive control, MEFs were same effect was seen with birinapant but to a lesser extent also treated with thapsigargin, which triggered a build up than with LCL161. of GFP + mCherry + puncta as expected. The ratio of GFP + /mCherry + puncta was close to one, reflecting its IAP antagonists block autophagic flux function in blocking fusion of autophagosomes with Blocking fusion of autophagosomes with lysosomes lysosomes and similar to the results seen for treatment hinders autophagic flux. Autophagic flux can be analysed with IAP antagonists (Fig. 1a). IAP-antagonism therefore by preventing formation of new autophagosomes and causes a build-up of GFP + mCherry + LC3 positive chasing preformed autophagosomes through to their vesicles in MEFs. fusion with lysosomes. Inhibition of PI3K activity blocks This build-up of GFP + mCherry + LC3 positive vesi- the early stages of autophagosome formation, but allows cles could be a result of either a deficiency in turnover of existing autophagosomes to continue through autophagic Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 4 of 15 Fig. 2 Electron microscopy of IAP antagonist treated cells shows autophagosome and lysosome accumulation. MEFs were treated with thapsigargin (3 μM) or LCL161 (0.5 μM) for 6 h. Cells were fixed and analysed by EM. a Overview of whole cells showing increased vesicularization in LCL161 and thapsigargin treated cells. b Magnification of LCL161 (I–II) or thapsigargin (IV–VI) treated cells showing I+IV. Accumulation of lysosomes, II+V. Lysosome associated but not fused to autophagosome, III+VI. Autophagosomes with cellular debris inside Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 5 of 15 Fig. 3 Endogenous LC3 accumulates around lysosomes. Wild type MEFs were treated with LCL161 (5 µM) or birinapant (50 µM) or DMSO as a control for 6 h. Cells were fixed and stained with antibodies against LAMP2 (red channel) and LC3 (green channel). Cells were imaged by confocal microscopy flux, and be degraded by lysosomes. In cells expressing These data show that LCL and thapsigargin can both mCherry-GFP-LC3b this leads to the loss in the GFP block the turnover of existing autophagosomes that have signal . been triggered to form by starvation. These experiments To analyse if IAP antagonists can block fusion with were also performed with 0.5 μM LCL161 with similar lysosomes, cells were first starved in HBSS and treated results although reduced in degree (data not shown). with different combinations of PI3K inhibitor and/or IAP Autophagic flux was further analysed by measuring antagonist or thapsigargin. When cells were first starved induction of LC3-II in response to concanamycin A by incubation in HBSS for 2 h, there was, as expected, a treatment. Concanamycin A, as described above, blocks substantial increase in the number of GFP + puncta, while the last stages of autophagy by blocking acidification of the number of mCherry + puncta remained grossly lysosomes. Addition of concanamycin A therefor leads to similar (Fig. 4a). Addition of LY294002 (LY), a PI3K increased LC3-II levels when autophagy is induced for inhibitor known to block autophagy, for 1 h caused the example by starvation, but shows no increase in LC3-II number of GFP + puncta to return to control levels, levels if the late stages of autophagy are blocked, due to showing that the existing autophagosomes fused with the redundancy in effect. We therefore treated MEFs lysosomes and lost their GFP fluorescence. Addition of expressing mCherry-GFP-LC3 with either HBSS to LCL161 with LY reversed this GFP + puncta loss, as did induce starvation, or with LCL161. Cells were treated with addition of thapsigargin. Conversely, addition of LY to concanamycin A and analysed by Western blot for levels cells pre-treated with LCL161 or thapsigargin failed to of mCherry-GFP-LC3-II. There was a clear concanamycin reduce the number of autophagosomes which had accu- A induced increase in LC3-II in both control and HBSS mulated (Fig. 4a) confirming that pre-existing autopha- treated cells, however concanamycin A failed to increase gosomes did not chase through to fusion with lysosomes. LC3-II in LCL161 treated cells (Fig. 4). Similar results Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 6 of 15 Fig. 4 (See legend on next page.) Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 7 of 15 (see figure on previous page) Fig. 4 IAP antagonism blocks autophagic flux. a MEFs expressing mcherry-gfp-lc3b were treated as shown in the timeline by either starving cells in HBSS for 1 h or treating with thapsigragin (3 μM) or LCL161 (5 μM) for 5 h. LY294002 (20 μM) was then added for 1 h either alone or, in the HBSS starved conditions, thapsigargin or LCL161 were also added together with LY294002. Cells were imaged and GFP+ and mCherry+ puncta /cell counted. Graphs show means error bars show SEM of at least 3 experiments. b MEFs expressing mCherry-GFP-LC3b were either left untreated, starved in HBSS for 2 h or treated with LCL161 (0.5 μM) for 6 h. In each condition cells were also treated with or without concanamycin A (2 nM) for the final hour. Cells were lysed and levels of mCherry-GFP-LC3B detected by western blot. For each condition the amount of mCherry-GFP-LC3BII was normalised to actin levels in the non concanamycin A treated sample. The graph shows the mean fold change in mCherry-GFP-LC3BII from at least 3 independent experiments and error bars show SEM. P values were calculated using T-test in Prism software fl/fl −/− −/− were seen using birinapant (data not shown). These less so for cIAP1 cIAP2 XIAP cells, indicating a results again argue that IAP antagonism blocks autophagy reduced rate of flux (Fig. 5c). Immunofluorescence against autophagosome–lysosome fusion stage. Lamp2 and LC3 showed dramatic accumulation of LC3 in fl/fl −/− −/− the starved cIAP1 cIAP2 XIAP cells compared cIAP2 and XIAP but not cIAP1 regulate autophagic flux to the wild type (Fig. 5d). At steady state, while there is LCL161 and birinapant both target cIAP1 and cIAP2 some accumulation of autophagosomes, the difference is with higher affinity than XIAP. The increase in severity of not as clear, suggesting adaptation to loss of cIAP2 and the phenotype in response to higher doses of XIAP (Fig. 5d). Loss of both cIAP2 and XIAP together LCL161 suggests that XIAP may play a role. To identify therefore inhibits autophagosome–lysosome fusion caus- which IAPs regulate autophagy and to confirm that this ing reduced flux through the autophagy pathway phenotype was specific to IAP antagonism and not an off target effect of the IAP antagonist drugs, siRNA knock- IAP antagonists block turnover of long-lived proteins down was used to silence expression of cIAP1, cIAP2 or Basal autophagic activity is responsible for degradation XIAP. Efficient silencing was achieved for each gene of long-lived proteins that are not normally turned over (Fig. 5a). cIAP2 loss was confirmed using qPCR, cIAP1 by proteasomal degradation. To determine if IAP inhibi- and XIAP loss were determined by western blot. Loss of tion could block turnover of long-lived proteins, wild type −/− cIAP2 or XIAP increased GFP + /mCherry + ratio closer and Atg5 MEFs were labelled using click-iT chemistry to 1, similar to IAP antagonist treatment at lower doses with L-Azidohomoalanine (AHA). AHA is incorporated (0.5 µM) (Fig. 5). Surprisingly however, siRNA against in place of methionine when cells are cultured in cIAP1 had no significant effect on the GFP + /mCherry + methionine free media. The degradation of long-lived ratio suggesting that build-up of GFP + autophagosomes proteins can then be monitored by labelling with a due to IAP antagonism is likely a result of cIAP2 and fluorophore and monitoring fluorescence by flow XIAP antagonism. We were unable to suppress expres- cytometry . sion of both cIAP2 and XIAP simultaneously using siRNA Starvation in HBSS for three hours resulted in a in MEFs; at the concentrations required the transfection decrease in fluorescence indicating a reduction of existing −/− reagents alone induced build-up of autophagosomes, stained proteins (Fig. 6). The same treatment in ATG5 something that has previously been reported . However, MEFs showed no significant reduction in fluorescence given the higher affinity of birinapant and LCL161 for confirming the reduction observed is specific to autop- cIAP1 and cIAP2 over XIAP, the effect seen at low doses hagy mediated degradation. Co-incubation of wild type is likely mostly due to cIAP2 inhibition. cells with HBSS and thapsigargin also completely blocked To determine if loss of both cIAP2 and XIAP together the decrease in labelled protein, showing that blocking could replicate inhibition of autophagosome–lysosome fusion of autophagosomes and lysosomes also blocks fusion seen with higher doses of IAP antagonist drugs, protein turnover. Co-incubation with HBSS and LCL161 fl/fl −/− −/− cIAP1 cIAP2 XIAP dermal fibroblasts were or birinapant also blocked turnover of long-lived proteins infected with lentivirus expressing mCherry-GFP-LC3, although incompletely, presumably because at these lower and the ratio of GFP + /mCherry + vesicles quantified. doses XIAP is not targeted and only the loss of cIAP2- Genotyping of the cells confirmed knockout of cIAP2 and function was observed (Fig. 6). fl/fl −/− XIAP (Supplementary Fig. 2). The cIAP1 cIAP2 −/− XIAP cells showed a GFP + /mCherry + ratio close to IAPs do not regulate endocytosis fl/fl 1 without any treatment (Fig. 5b). Additionally, cIAP1 Autophagosomes use some of the same machinery for −/− −/− cIAP2 XIAP cells have increased endogenous LC3 lysosomal fusion as the endocytic trafficking system levels (Fig. 5c). Starvation reduced LC3-II levels in wild including SNAREs, Rab7 and the class C/HOPS tethering 29, 32, 33 type cells, probably due to a high rate of flux, but this was complex . Blockage of autophagosome–lysosome Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 8 of 15 Fig. 5 cIAP2 and XIAP regulate autophagosome fusion, but not cIAP1. a MEFs expressing mCherry-GFP-LC3b were transfected with siRNA against either cIAP1, cIAP2 or XIAP. Cells were analysed with the microscope and the number of mCherry+, and GFP+ puncta/cell were calculated and the ratio of GFP+ /mCherry+ puncta is indicated. Shown are the means and the error bars represent the SEM of at least three independent experiments. Westerns show efficient knockdown of cIAP1 and XIAP expression. cIAP2 siRNA efficiency was determined by real time PCR as shown in −/− −/− the graph below the westerns. b Wild type and cIAP2 XIAP dermal fibroblasts expressing mCherry-GFP-LC3b were treated analysed on the −/− microscope and mCherry+, and GFP+ puncta/cell were calculated. The ratio of GFP+/mCherry+ puncta is indicated. c Wild type and cIAP2 −/− XIAP dermal fibroblasts were left in complete media (CM) or starved for 2 h in EBSS. Cells were lysed and proteins analysed by western blot for −/− XIAP, LC3, and Actin. cIAP2 was confirmed by PCR due to lack of effective antibodies for mouse cIAP2 (see supplemental Fig. 2). d −/− −/− −/− −/− Immunofluoresence showing accumulation of LC3 in starved cIAP2 XIAP dermal fibroblasts. Wild type and cIAP2 XIAP dermal fibroblasts were incubated in complete media or starved in EBSS for 2 h, then fixed and stained for LC3 (green channel) and LAMP2 (red channel). Nuclei are stained blue with Hoechst. Shown in upper panels are overviews. Lower panels show zoomed in regions indicated in the upper panels fusion may represent a more general defect in endocytic endocytosis using uptake of fluorescently labelled dextran membrane fusion caused by IAP antagonists. To test this, and trafficking of labelled endosomes to lysosomes by co- we monitored internalisation and degradation of EGFR, staining with lysotracker. No difference could be detected which upon activation is degraded in lysosomes after in the amount of dextran-labelled endosomes co- endosomal trafficking. There was no change in EGFR localising with lysosomes in treated and control cells degradation rate in LCL161 treated cells while con- (Fig. 7b). This was confirmed by Pearson’s correlation canamycin A treated cells showed a near complete loss of coefficient analysis, which also clearly showed that the degradation (Fig. 7a). Additionally we analysed fluid phase degree of co-localisation is not altered significantly by IAP Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 9 of 15 Oligomycin and Antimycin A to induce mitochondrial damage either with or without LCL161 or birinapant for 16 h and examined the levels of cytochrome C as a marker of mitochondria. There was a decrease in cytochrome C levels with the oligomycin and Antimycin A treatment only in the Parkin over-expressing cells (Fig. 8c). Addition of LCL161 restored the levels of cytochrome C to some extent, however not completely (Fig. 8c). MEFs were also induced to over-express Parkin and treated in the same way, but in our hands with all mitochondrial proteins −/− tested we also saw degradation in ATG5 MEFs and also in non-Parkin over-expressing cells (data not shown), suggesting that the degradation is not mitophagy in the MEFs. Similar results have been published previously indicating that there are few reliable markers for mito- phagy in MEFs . Therefor, not only is mitophagy inhib- ited in IAP antagonist treated cells, but IAP antagonism also blocks or at least slows the degradation of mito- chondria by mitophagy in human cells too. Xenophagy, which is conceptually similar to mitophagy as it involves the tagging of intracellular bacteria with ubiquitin followed by engulfment by autophagosomes and Fig. 6 IAP antagonism blocks turnover of long lived proteins. degradation, is an early step in the recognition of a −/− Wild type or Atg5 MEFs were labelled overnight with AHA as number of pathogenic bacteria. Salmonella have been described in methods. Cells were starved in HBSS or treated with HBSS used extensively as a model to study xenophagy and there and LCL161 (0.5 μM), birinapant (0.5 μM) or thapsigargin (3 μM) for is significantly more bacterial survival in autophagy defi- three hours. Click it chemistry was used to label proteins and the cient cells . A block in mitophagy suggests loss of XIAP geometric mean fluorescence intensity was measured for each condition. Shown are the means of at least three independent could also lead to a deficiency in xenophagy. To test this, experiments and error bars show SEM XIAP was knocked out in MEFs using CRISPR-Cas9. Cells infected under these conditions were fixed and stained using an antibody against S. Typhimurium LPS and the antagonism (Fig. 7b). Together these results clearly show number of bacteria in each cell counted, revealing more that IAP antagonism has no effect on the endosomal salmonella in the XIAP CRISPR cells than in the wild type pathway but instead specifically affects the autophagoso- (Fig. 8d). This was then confirmed by lysing cells and mal system. calculating the colony forming units (CFU) (Fig. 8e). Most studies analysing xenophagy have used treatments or Mitophagy and xenophagy are blocked in IAP antagonist- mutants that block the early stages of autophagosome treated cells formation. To the best of our knowledge little is known One consequence defective autophagy is the accumu- about the effect of blocking fusion of autophagosomes lation of mitochondria due to failure loss of mitophagy. with lysosomes on xenophagy. As a model for blockage of We examined the amount of mitochondria in MEFs the fusion of autophagosomes with lysosomes, cells were treated with LCL161 or thapsigargin by staining with also treated with thapsigargin. Following 5 h of infection, −/− Mitotracker Green (MG). High dose (5 µM) LCL161 and there is consistently more surviving bacteria in ATG5 thapsigargin treated cells showed a clear increase in MG cells than in wild type as previously reported. Thapsi- signal, while low doses (0.5 μM) showed no significant gargin treatment also resulted in accumulation of viable S. increase (Fig. 8a, b). Birinapant also showed an increase in Typhimurium, as did knockout of XIAP in the XIAP- mitochondrial mass only at the high doses that fully block CRISPR cells (Fig. 8e). Both thapsigargin and XIAP defi- fusion (Fig. 8a, b). To confirm that mitophagy is impaired ciency result in increased S. Typhimurium survival to a we turned to HeLa cells overexpressing Parkin as a well- similar extent to Atg5 deficiency, supporting that established model for inducing mitophagy. HeLa cells do Autophagy inhibition is involved in all conditions. not express endogenous Parkin, a ubiquitin ligase that ubiquitylates damaged mitochondria and induces their Discussion clearance by mitophagy . We treated HeLa cells without While we have demonstrated that cIAP2 and XIAP Parkin and cells overexpressing mCherry-Parkin with promote autophagosome-lysosome fusion, the Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 10 of 15 Fig. 7 IAP antagonism does not block endocytosis. a Wild type MEFs were grown overnight in 0.5% serum. Cells were either left untreated as a control or treated with LCL161 (0.5 μM), birinapant (0.5 μM) for 4 h, or concanamycin A (2 nM) for 1 h before treatment with 100 ng/ml EGF in the presence of 10 μg/ml cycloheximide for 0, 1 or 2 h. Cells were lysed and levels of EGFR were analysed by western blot. b Wild type MEFs were treated with LCL161 (0.5 μM) or not for 4 h. Alexafluor 647-dextran (100 μg/mL) was added for 30 mins and then cells were washed and grown in normal media for a further 2 h. The final 30 min cells were stained with lysotracker (100 nM). Cells were fixed and analysed on the fluorescence microscope. Shown are representative images and line profile of lysotracker and dextran Alexafluor 647. Experiments were performed at least 3 times. Pearsons correlation coefficients are shown in the right panel. Error bars represent the SEM from at least three experiments mechanism is still not clear. Ubiquitylation regulates One interesting aspect of these results is that we have many steps in vesicle trafficking and it is likely that identified cIAP2 and XIAP as playing redundant roles in cIAP2 and XIAP are regulating specific components of regulating autophagy in MEFs. XIAP depletion alone in −/− the autophagosome–lysosome fusion machinery via their humans leads to disease but XIAP mice are in most ubiquitin ligase activity. The observation that endocytosis aspects normal, although they can be induced to develop is also not affected by IAP inhibition suggests ubiquity- disease similar to XLP-2 in humans by infection with a lation of some target on the autophagosomes themselves herpes virus, MHV-68 . One might expect that if there is likely, however we cannot at this stage rule out that was an absolute requirement for XIAP or cIAP2 in some other function such as scaffolding may also be autophagy that mice deficient for either alone would playing a role. present with phenotypes associated with defects in Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 11 of 15 Fig. 8 IAP antagonism blocks mitophagy and xenophagy. Wild type MEFs were treated overnight with the indicated concentrations of LCL161, birinapant or thapsigargin. Cells were stained with mitotracker green for 30 min and fluorescence intensity quantified using flow cytometry. a representative histograms showing increases in mitotracker signal in IAP antagonist treated cells. b quantification of mitotracker staining. Shown are the means of the geometric mean fluorescence intensity. Error bars are SEM from at least 3 independent experiments. P values were calculated using t tests in Prism software. c IAP antagonism can block mitophagy. Wild type HeLa cells or HeLa cells expressing mCherry-Parkin were treated with either LCL161 (5 µM), birinapant (50 µM) or oligomycin (1 µM) and Antimycin A (1 µM) (O/A) alone or in combination as indicated. LCL161 or birinapant were added 4 h prior to Oligomycin and Antimycin A. Cells were incubated for 16 h before cells were harvested and lysates run on SDS- PAGE and levels of cytochrome C and β-Actin analysed by western. d Immunofluoresence and quantification of Salmonella Typhimurium infection in wild type and XIAP CRISPR MEFs. MEFs of the indicated genotypes were infected as described in methods, fixed and stained for Salmonella Typhimurium LPS. Shown are representative images. The mean number of Salmonella Typhimurium /cell is shown from two experiments. e −/− −/− Xenophagy of Salmonella Typhimurium is impaired in XIAP deficient cells. Wild type, Atg5 and XIAP -CRISPR cells were infected with Salmonella Typhimurium and incubated for 5 h. CFU/mL of internalised bacteria was then calculated. Thapsigargin was used where indicted to block autophagosome–lysosome fusion by treating with 3 µM thapsigargin for 1 h before and then throughout the infection protocol. Error bars are SEM from at least 3 independent experiments. P values were calculated using T tests in Prism software Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 12 of 15 autophagy, but loss of both seems to be required before Many questions remain including dissection of the spontaneous inflammation is seen . This may be partly mechanism behind cIAP2 and XIAPs regulation of explained by the overlapping functions seen for cIAP2 autophagosome–lysosome fusion, as well as the degree to and XIAP in this study and may explain why XIAP defi- which autophagy defects contribute to inflammation cient mice do not develop disease like humans do. induced by IAP loss. It will be interesting to see if IAPs or Relatively little is known about the substrates of XIAP, their substrates are also involved in other autophagy related diseases such as protein misfolding diseases and or however ubiquitylation of RIPK2 at NOD2 receptors was shown to be required for NOD2 activation of NF-κB . lysosomal storage disorders. Loss of NF-κB signalling results in a failure to up- regulate cytokines in response to NOD2 signalling and Methods this link to NOD2 signalling is thought to be the cause of Reagents Crohn’s disease in XIAP deficient patients. While this LCL161 and birinapant were purchased from ApexBio. molecular connection between NOD2 and XIAP may The following antibodies were used; EGFR (PA1-1110— contribute to disease progression, NOD2 mutation does Thermo fisher), XIAP (MAB822—R&D Systems), cIAP1 not lead to the multiple other conditions that XIAP (was a kind gift from John Silke—WEHI Melbourne deficiency causes , suggesting that the story is not so Australia), LC3b (nb100–2220—Novus), LAMP2 simple. There are numerous genes that are involved in (ab13524—Abcam). EGF was purchased from Biolegend autophagy, including ATG16L1, NDP52, IRGM, and (catalogue No. 713108). Alexafluor 647 Dextran LRRK2 that have been found to be mutated in Crohn’s MW10,000 (catalogue No. D-22914), Lysotracker Red Disease patients; a number of these mutations also DND-99 (catalogue No. L7528) and Mitotracker Green manifest with multiple clinical presentations. Recently, (catalogue No. M7514) were purchased from Thermo Niemann–Pick disease type C (NPC), a lysosomal sto- Fisher Scientific. Concanamycin A (catalogue No. Cay- rage disorder in which the NPC1 gene coding for a 11050-25) was purchased from Cayman Chemical. lysosomal protein involved in lipid transport is mutated, was also linked to early onset Crohn’s disease. Loss of Cell culture NPC1 function was also shown to be linked to defective All cells were grown in DMEM High glucose (Gibco) autophagy resulting in increased survival of intracellular with 10% FCS and 1% Pen/Strep in 5% CO at 37 °C Salmonella and adherent-invasive E. coli . Importantly, unless otherwise stated. this study showed that NOD2 induced NF-κB signalling is normal in NPC1 mutated cells and enforced autophagy Fluorescence microscopy of LC3 induction could bypass the xenophagy defect and reduce MEFs of the indicated genotypes were infected with intracellular bacterial survival. Similarly impaired xeno- pBABE-puro-mCherry-EGFP-LC3B (pBABE-puro- phagy has also been seen in macrophages from patients mCherry-EGFP-LC3B was a gift from Jayanta Debnath with NOD2 and XIAP mutations. We observed defects in (Addgene plasmid # 22418)). Cells were selected in Pur- xenophagy of S. Typhimurium in XIAP deficient fibro- omycin 4 μg/ml. The selected cells were seeded at 50,000 blasts. Such defects in xenophagy may also contribute to cells/well of an 8 chamber μ-slide (Ibidi) and allowed to accumulation of invasive bacteria in XIAP deficient plate down overnight. Cells were typically treated for 6 h humans, again increasing the burden of bacteria that with the indicated concentrations of LCL161, birinapant needs to be dealt with by the immune system and or thapsigargin (3 μM). Cells were treated for 30 min with resulting in overactive inflammation. Additionally we Hoechst to stain nuclei. Cells were imaged with a Keyence observed an accumulation of mitochondria in cells BZ9000 fluorescence microscope taking Z-stacks to and treated with IAP antagonists at doses high enough to using the “full focus” function in the analysis software to block XIAP, as well as cIAP1 and cIAP2, suggesting that make a single layer image from full focused features in the loss of IAPs also causes defects in mitophagy. each layer of the Z-stack. These images were used to Mitophagy is important for clearing old or damaged count the number of GFP + and mCherry + puncta per mitochondria and defects in mitophagy have been linked cell in image J using the analyse particles function to to excessive ROS production and excessive inflamma- count nuclei and find maxima function to count puncta. some activation . Mitophagy is in many ways mechan- The ratio of mCherry + /GFP + puncta/cell was calcu- istically analogous to xenophagy and defects therein may lated from these measurements. also contribute to inflammation in XIAP deficient For Immunofluorescence of LC3 and LAMP2, Wild patients. This suggests that the driving mechanism type MEFs were seeded in 8 well µ-slides (Ibidi) at 50,000 behind multiple genetic mutations causing Crohn’s cells/well and incubated overnight. Cells were treated Disease may be a loss of autophagy, and supports a role with IAP antagonists as indicated and cells were washed for XIAP in regulating this process. twice in warm PBS. Cell were fixed in 4% PFA in PBS for Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 13 of 15 10 min followed by washing three times in PBS. Cells were (0.5 μM), thapsigargin (3 μM), or birinapant (0.5 μM) were subsequently permeabilized in PBS with 50 µg/ml Digi- added at this stage. Cells were washed in PBS and then tonin for 5 min followed by washing three times in PBS cultured for a further 3 h either in full media or HBSS then blocking with 3% BSA in PBS for 30 min. Antibodies containing the indicated drugs. Cells were washed 2× in against LC3 and Lamp2 were added 1:200 for 1 h in BSA PBS and then harvested and fixed in 4% formaldehyde in 3% in a humidified chamber at 37 C. Cells were washed PBS for 15 min at room temperature. After fixation, the cells are washed twice with 3% BSA in PBS. Cells were three times in PBS followed by incubating with appro- priate secondary antibodies at 1:500 in PBS 3% BSA for 1 permeabilized with 0.5% Triton X-100 in PBS for 20 min h followed by washing 5 times in PBS. Images were taken at room temperature. Cells were resuspended in PBS and using a Zeiss LSM 880 with an Airyscan confocal store at 4 °C for detection of the corresponding alkyne- microscope at a 63 × magnification (oil immersion) and tagged detection molecule. analysed with Zen (Zeiss) software. Cells from the last step above were washed with 3% BSA in PBS. 100 μL Click-iT® reaction cocktail was added to siRNA treatment each sample. Cells were incubated for 30 min at room MEFs were seeded at 150,000 cells/well of a 6 well plate. temperature in the dark. Cells were washed once with 3% The next day cells were washed with PBS and media BSA in PBS before being analysed using flow cytometry. without antibiotics was added. Cells were transfected with siRNA’s complexed with Lipofectamine RNAiMAX EGFR degradation assay according to manufacturers instructions. Briefly, 7.5 µL of A total of 500,000 cells/well were seeded in the morning 20 µM stock stealth siRNA oligo was added to 150 µL of and let to plate down. In the evening, media was optimem. Nine microlitre of RNAiMAX was added to exchanged for DMEM 0.5% FCS and cells incubated another 150 µL of optimum. The tubes were mixed overnight. The next day, cells were pre-treated for 4 h with together before incubating at room temperature for 5 min. IAP antagonists birinapant (0.5 μM) or LCL161 (0.5 μM) 250 µL of this was then added to the cells and incubated or for 1 h with concanamycin A (2 nM). Media was then overnight. Cells were seeded at 50,000 cells/well of an 8 exchanged with serum free media alone or containing the chamber μ-slide (Ibidi) and allowed to plate down over- drugs. Cells were treated with EGF (100 ng/ml) and night before microscopy was performed as described cycloheximide (10 μg/ml) for 0, 1 or 2 h. Cells were lysed above. The following stealth siRNAs were used from on ice in RIPA buffer and 50 µg/ml protein per lane was Thermo Fisher Scientific; cIAP1 (MSS273215), cIAP2 run on 10% SDS-PAGE before transferring to nitrocellu- (MSS202113) and XIAP (MSS202115). Activity of each lose membranes and western blotting for EGFR siRNA was confirmed using qPCR with SYBR Select Master Mix (applied biosystems # 4472908) and the fol- Assay of endocytosis and fusion with lysosomes lowing primers: cIAP1 (Forward 5′-GAAGAAAATGC 30,000 cells/well were seeded in 8 well microscope TGACCCTACAGA-3′, Reverse 5′-CATGACGACATC culture slides (Ibidi) in 300–500 µL media. The next day TTCCGA-3′), cIAP2 (Forward 5′- TCGATGCAGAA cells were treated with DMSO, LCL161 0.5 μM or bir- GACGAGA-3′ Reverse 5′-TTTGTTCTTCCGGATTAG inapant (0.5 μM) for 6 h. 2.5 h before end of drug treat- TGC-3′, XIAP (Forward 5′-GCTTGCAAGAGCTGGA ments, Dextran Alexafluor 647 was added to a final of TTTT-3′, Reverse 5′-TGGCTTCCAATCCGTGAG-3′). concentration of 100 µg/ml. Cells were incubated for 30 Actin was used as a reference gene. PCR was done in 384 min at 37 °C. Cells were washed 2× in warm PBS. Fresh well plates in a 7900HT Fast Real-Time PCR System. media containing LCL161 or birinapant was added and cells were incubated for a further 2 h. Lysotracker Red Long-lived protein degradation assay (100 nM) was added for the final 30 min. Cells were Cells were grown to ~70–80% confluency in a 6-well washed 2× in warm PBS before fixation in 4% paraf- plate then washed with warm PBS and cultured in L- ormaldehyde (PFA) for 20 min at room temperature. Cells methionine-free DMEM (cat. no. 21013-024, Gibco) for were washed 2× in PBS and stored at 4 °C until images 30–60 min to deplete the intracellular methionine were taken on a Keyence BZ9000 fluorescence micro- reserves. Following methionine depletion, the cells were scope. Hoechst was added 30 min prior to imaging. labelled with 25 μM AHA in 10% dialysed FBS DMEM Pearsons correlation coefficients were calculated using the (methionine-free) for 18 h. Dialysed FBS was made by FIJI distribution of ImageJ and the Coloc2 plugin. dialyzing against PBS with Slide-A-Lyzer mini dialysis devices 3.5 k MWCO (Pierce—cat. No. 88403) overnight. Electron microscopy After labelling, the cells were washed with PBS and cul- MEFs were cultured on glass cover slips and fixed for tured in regular DMEM containing 10 × L-methionine (2 20 min in 4% PFA plus 1% glutaraldehyde (Roth, Ger- mM) for 2 h to chase out short-lived proteins. LCL161 many) in PBS. After contrastation in 0.5% Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 14 of 15 Osmiumtetroxide (30 min at RT) cells were dehydrated BSA for 30 min followed by staining with anti mouse and embedded in epoxy resin (Durcupan, Sigma-Aldrich, IgG-Cy3 (1:500) in PBS 3% BSA. Nuceli were stained Gillingham, UK). Images were taken using a Philips CM with Hoechst. Cells were imaged and the number of 100 transmission electron microscope. bacteria per cell was counted from at least 50 cells for each genotype per experiment. Mitophagy assays Acknowledgements For mitotracker Green staining, Wild type MEFs were −/− The authors thank Barbara Joch for her excellent technical assistance. Atg5 +/+ +/− treated overnight with the indicated concentrations of and Atg5 MEFs were made from ATG5 mice, which were a kind gift from Prof. Dr. Tobias Huber (University Medical Center Freiburg). mCherry- LCL161, birinapant or thapsigargin. Cells were incubated Parkin expressing lentiviral constructs were a kind gift from Dr. Florian with 100 nM mitotracker Green for 30 min in complete Steinberg (Center for Biological Systems Analysis, University of Freiburg). media then washed in PBS and trypsinized then analysed for fluorescence intensity using flow cytometry on a FACS Author details Institute of Medical Microbiology and Hygiene, University Medical Center Calibur then analysed using FlowJo software. Freiburg, Freiburg, Germany. Faculty of Medicine, University of Freiburg, For analysis of Parkin dependent mitophagy, HeLa cells 3 Freiburg, Germany. Renal Division, University Medical Center Freiburg, were infected with a lentivirus (pXLG3) expressing Freiburg, Germany. Department of Neuroanatomy, University Freiburg, Freiburg, Germany. Department of Medicine, University Medical Center mCherry-Parkin (human) or left uninfected. These Hamburg-Eppendorf, Hamburg, Germany. Institute of Experimental 5 5 HeLas were then seeded at 4 × 10 for wild type and 6 × 10 Immunology, University of Zurich, Zurich, Switzerland cells/well in 6 well plates, left overnight and then incubated Conflict of interest for 4 h with 5 µM LCL161 or 50 µM birinapant followed by The authors declare that they have no conflict of interest. addition of Oligomycin (1 µM) (Sigma Aldrich—O4876) and Antimycin A (1 µM) (Sigma Aldrich—A8674) for 16 h. Cells were harvested and lysed in DISC lysis buffer fol- Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in lowed by detection of Cytochrome C by western blot. Actin published maps and institutional affiliations. was used as a loading control. Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-0508-y). Xenophagy assays S. Typhimurium used in all analysis was a patient Received: 23 November 2017 Revised: 8 March 2018 Accepted: 13 March derived strain identified through sera agglutination. Salmonella were cultured overnight in LB at 37 °C. On the same day 100,000 cells/well of MEFs were seeded in 12 well plates. The next day, Salmonella were diluted 1:33 and incubate without shaking for 3 h at 37 °C. Sal- References 1. Vince, J. E. et al. IAP antagonists target cIAP1 to induce TNFalpha-dependent monella were harvested by centrifugation and washed apoptosis. Cell 131,682–693 (2007). with PBS two times before re-suspending in DMEM 2. Wong,W.W.-L. et al.cIAPs andXIAP regulate myelopoiesis through cytokine without any antibiotics. The cells were adjusted to have production in an RIPK1- and RIPK3-dependent manner. Blood 123,2562–2572 (2014). an OD600 of 0.5 by diluting with DMEM. One hundred 3. Vince, J. E. et al. 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Inhibitor of apoptosis proteins are required for effective fusion of autophagosomes with lysosomes

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Abstract

Inhibitor of Apoptosis Proteins act as E3 ubiquitin ligases to regulate NF-κB signalling from multiple pattern recognition receptors including NOD2, as well as TNF Receptor Superfamily members. Loss of XIAP in humans causes X-linked Lymphoproliferative disease type 2 (XLP-2) and is often associated with Crohn’s disease. Crohn’s disease is also caused by mutations in the gene encoding NOD2 but the mechanisms behind Crohn’s disease development in XIAP and NOD2 deficient-patients are still unknown. Numerous other mutations causing Crohn’s Disease occur in genes controlling various aspects of autophagy, suggesting a strong involvement of autophagy in preventing Crohn’s disease. Here we show that the IAP proteins cIAP2 and XIAP are required for efficient fusion of lysosomes with autophagosomes. IAP inhibition or loss of both cIAP2 and XIAP resulted in a strong blockage in autophagic flux and mitophagy, suggesting that XIAP deficiency may also drive Crohn’s Disease due to defects in autophagy. Introduction NOD2 receptor due to its ubiquitylation activity towards RIPK2 IAPs are ubiquitin ligases that regulate the activity of . TNF Super Family Receptors (TNFSFR), TLRs and NOD Genetic loss of cIAP1, cIAP2 and XIAP results in severe receptors. By attaching ubiquitin onto substrates such as systemic inflammation characterised by massive increases RIPK1 they regulate the activation of NF-κB and deter- in many cytokines including TNF and IL-1β . IAP mine the outcome of signals from these receptors. Their antagonist drugs are also able to trigger activation of inhibition results in skewing of signals towards death and NLRP3 inflammasomes in LPS primed macrophages .In 1–4 also production of an inflammatory cytokine response . both cases there appears to be an important role for XIAP, The three best characterised and functionally related IAP in addition to cIAP1 and cIAP2, in suppressing this members are cIAP1, cIAP2 and XIAP. cIAP1 and cIAP2 inflammatory cascade suggesting some redundancy in the 5–7 act together in complex with TRAF2 and TRAF3 . Loss function of IAPs. of cIAP1 leads to defective NF-κB signals from partner Humans with mutations in XIAP often develop Crohn’s receptors such as TNFR1 and additional activation of disease (CD) but may also suffer from X-linked lympho- 1 9–12 non-canonical NF-κB . Less is known about XIAP and its proliferative disease 2 (XLP-2) . NOD2 is also com- 13–16 regulation other than it is required for NF-κB signals from monly mutated in CD patients . Although mutations in XIAP affect the activation of NOD2, the molecular mechanisms behind CD in these mutations are still not clear. Another gene commonly mutated in CD is the Correspondence: Ian E. Gentle (ian.gentle@uniklinik-freiburg.de) 1 17 Institute of Medical Microbiology and Hygiene, University Medical Center autophagy gene ATG16L1 . NOD2 and Atg16L1 are also Freiburg, Freiburg, Germany functionally linked with NOD2 being required for Faculty of Medicine, University of Freiburg, Freiburg, Germany recruiting ATG16L1 to internalised bacteria such as Full list of author information is available at the end of the article. Edited by G. Raschellà © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; Gradzka et al. Cell Death and Disease (2018) 9:529 Page 2 of 15 Fig. 1 IAP inhibition triggers autophagosome accumulation. a Wild type MEFs expressing mcherry-GFP-LC3b were treated with the indicated doses of LCL161or thapsigargin for 6 h. DNA was stained using Hoechst and live Cells were imaged to visualise mCherry, GFP and Hoechst. The number of GFP+ puncta and mCherry+ puncta were then counted per cell and the ratio of GFP + /mCherry + puncta calculated. Graphs represent the mean and error bars show SEM of at least 3 experiments. b IAP antagonism does not block lysosomal acidification. Wild type MEFs were treated with either LCL161 (5 μM), thapsigargin (3 μM) or concanamycin A (2 nM) for 6 h. Cells were stained with lysotracker red and visualised by microscopy or lysotracker intensity measured via flow cytometry. Graphs represent the mean of the geometric mean fluorescence intensity measured by flow cytometry and error bars show SEM of at least 3 independent experiments. P values were calculated using T-test in Prism software Salmonella during xenophagy, the targeted autophagy of autophagy is a key pathway linking NOD2 and ATG16L1 invading bacteria . CD associated mutations in NOD2 in the development of Crohn’s disease and hint that XIAP and Atg16L1 were also shown to block autophagy may also have some role in autophagy regulation. induction by NOD2 and reduce the xenophagy of invad- Autophagy is a highly conserved pathway for recycling ing Salmonella . Defects in Atg16L1 or NOD2 also cellular components in times of nutrient limitation. increased the replication of adherent-invasive Escherichia Autophagosomes are formed around cellular components coli (AIEC) in macrophages, resulting in enhanced cyto- such as bulk cytoplasm (macroautophagy), but also spe- kine production. Conversely, induction of autophagy in cific targets including mitochondria (mitophagy), invasive −/− NOD2 macrophages reduced survival of AIEC and bacteria (xenophagy) and aggregated proteins (aggre- cytokine production . Together these data suggest that phagy). In each case phagophores, fuse to make Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 3 of 15 autophagosomes that fuse with lysosomes, releasing their autophagosomes or an increase in activation of autop- 21– contents to be degraded and recycled (reviewed in refs. hagy. Defects in autophagosome turnover occur by a ). Autophagy is also linked to many other functions in number of mechanisms. One possibility is a failure to animals, including regulation of the immune system at acidify the autolysosome, resulting in defective lysosomal various levels such as xenophagy, MHCII presentation, enzyme function. Such an effect is seen upon the addition limiting cytokine production in response to infection, and of drugs like concanamycin A, an inhibitor of this later stage in autophagy that blocks the activity of ATPase in protein folding diseases such as Alzheimer’s disease 24–27 (AD) (reviewed in refs. ). proton pumps in the lysosome, preventing acidification We here show that both cIAP2 and XIAP promote and blocking degradation of lysosomal cargo. autophagosome–lysosome fusion and that their loss or To rule out an effect of IAP antagonists on lysosomal inhibition results in accumulation of autophagosomes and acidification, MEFs were treated with IAP antagonists or lysosomes and defects in mitophagy and xenophagy. concanamycin A to block acidification, or thapsigargin to block autophagosomal fusion with lysosomes. MEFs were Results then stained with Lysotracker Red, which only fluoresces IAP antagonism causes accumulation of autophagosomes when lysosomes are acidified. Cells were analysed by To determine if there was any effect of IAP antagonists microscopy and flow cytometry (Fig. 1b). IAP antagonist on autophagy, Mouse Embryonic Fibroblasts (MEFs) were treatment did not reduce lysotracker fluorescence infected with pBabe-mCherry-EGFP-LC3b. GFP fluores- whereas Concanamycin A did (Fig. 1b). Both thapsigargin cence is sensitive to pH and decreases in the acidic treatment and higher doses of LCL161 enhanced lyso- environment of autolysosomes while mCherry retains its tracker staining (Fig. 1b), suggesting a possible accumu- fluorescence. Due to this property of GFP, this reporter lation of lysosomes in addition to autophagosomes. IAP can be used to assess the rate at which autophagosomes antagonists do not therefore affect the acidification of are synthesised and degraded by lysosomes. lysosomes, but do lead to a build-up of mCherry + /GFP Since there is continuous fusion with lysosomes at + positive puncta in a fashion similar to thapsigargin. steady state, there are more single mCherry + puncta in Electron Microscopy (EM) was performed on LCL161 cells expressing GFP-mCherry-LC3b than GFP + treated MEFs, as well as thapsigargin treated MEFs and mCherry + puncta, and the ratio of GFP + and mCherry untreated controls. LCL161 treated cells showed a clear + puncta can be used to illustrate this spontaneous increase in vesicular structures containing cellular debris fusion. The Cells were treated with the IAP antagonist suggesting they are autophagosomes (Fig. 2). The same LCL-161 . Upon treatment of MEFs with LCL161, there was seen for thapsigargin treatment. Of note is that was a clear accumulation of GFP + puncta at doses as low electron dense lysosomes cluster in regions adjacent to as 500 nM (Fig. 1a). A slight accumulation of mCherry + autophagosomes, but do not appear to be fused with the puncta could also be detected at this dose but the ratio of autophagosomes (Fig. 2). Immunofluoresence was also GFP/mCherry increased substantially. Treatment with 5 perfomed on wt MEFs treated with LCL161 or birinapant μM LCL161 resulted in more mCherry + GFP + puncta to confirm that endogenous LC3 also shows a similar and a ratio of GFP + /mCherry + close to one (Fig. 1a). accumulation after IAP antagonism (Fig. 3). There is a Similar results were seen with an unrelated IAP antago- striking accumulation of autophagosomes, in the peri- nist, birinapant that also lead to significant blockage of nuclear region. Surprisingly these autophagosomes appear fusion events, but only at doses of 50 µM or above to surround lysosomes (LAMP2 structures) as opposed (Supplementary Fig. 1). Thapsigargin, an irreversible to co-localising with them which is similar to the non- inhibitor of the SERCA calcium pumps of the ER, has fused, but associated, autophagosomes and lysosomes been shown to specifically inhibit the fusion of autophagic seen by EM (Fig. 2) and is indicative of recruitment of vesicles with lysosomes, resulting in build up of GFP + autophagosomes to lysosomes, but failure to fuse. The mCherry + puncta . As a positive control, MEFs were same effect was seen with birinapant but to a lesser extent also treated with thapsigargin, which triggered a build up than with LCL161. of GFP + mCherry + puncta as expected. The ratio of GFP + /mCherry + puncta was close to one, reflecting its IAP antagonists block autophagic flux function in blocking fusion of autophagosomes with Blocking fusion of autophagosomes with lysosomes lysosomes and similar to the results seen for treatment hinders autophagic flux. Autophagic flux can be analysed with IAP antagonists (Fig. 1a). IAP-antagonism therefore by preventing formation of new autophagosomes and causes a build-up of GFP + mCherry + LC3 positive chasing preformed autophagosomes through to their vesicles in MEFs. fusion with lysosomes. Inhibition of PI3K activity blocks This build-up of GFP + mCherry + LC3 positive vesi- the early stages of autophagosome formation, but allows cles could be a result of either a deficiency in turnover of existing autophagosomes to continue through autophagic Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 4 of 15 Fig. 2 Electron microscopy of IAP antagonist treated cells shows autophagosome and lysosome accumulation. MEFs were treated with thapsigargin (3 μM) or LCL161 (0.5 μM) for 6 h. Cells were fixed and analysed by EM. a Overview of whole cells showing increased vesicularization in LCL161 and thapsigargin treated cells. b Magnification of LCL161 (I–II) or thapsigargin (IV–VI) treated cells showing I+IV. Accumulation of lysosomes, II+V. Lysosome associated but not fused to autophagosome, III+VI. Autophagosomes with cellular debris inside Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 5 of 15 Fig. 3 Endogenous LC3 accumulates around lysosomes. Wild type MEFs were treated with LCL161 (5 µM) or birinapant (50 µM) or DMSO as a control for 6 h. Cells were fixed and stained with antibodies against LAMP2 (red channel) and LC3 (green channel). Cells were imaged by confocal microscopy flux, and be degraded by lysosomes. In cells expressing These data show that LCL and thapsigargin can both mCherry-GFP-LC3b this leads to the loss in the GFP block the turnover of existing autophagosomes that have signal . been triggered to form by starvation. These experiments To analyse if IAP antagonists can block fusion with were also performed with 0.5 μM LCL161 with similar lysosomes, cells were first starved in HBSS and treated results although reduced in degree (data not shown). with different combinations of PI3K inhibitor and/or IAP Autophagic flux was further analysed by measuring antagonist or thapsigargin. When cells were first starved induction of LC3-II in response to concanamycin A by incubation in HBSS for 2 h, there was, as expected, a treatment. Concanamycin A, as described above, blocks substantial increase in the number of GFP + puncta, while the last stages of autophagy by blocking acidification of the number of mCherry + puncta remained grossly lysosomes. Addition of concanamycin A therefor leads to similar (Fig. 4a). Addition of LY294002 (LY), a PI3K increased LC3-II levels when autophagy is induced for inhibitor known to block autophagy, for 1 h caused the example by starvation, but shows no increase in LC3-II number of GFP + puncta to return to control levels, levels if the late stages of autophagy are blocked, due to showing that the existing autophagosomes fused with the redundancy in effect. We therefore treated MEFs lysosomes and lost their GFP fluorescence. Addition of expressing mCherry-GFP-LC3 with either HBSS to LCL161 with LY reversed this GFP + puncta loss, as did induce starvation, or with LCL161. Cells were treated with addition of thapsigargin. Conversely, addition of LY to concanamycin A and analysed by Western blot for levels cells pre-treated with LCL161 or thapsigargin failed to of mCherry-GFP-LC3-II. There was a clear concanamycin reduce the number of autophagosomes which had accu- A induced increase in LC3-II in both control and HBSS mulated (Fig. 4a) confirming that pre-existing autopha- treated cells, however concanamycin A failed to increase gosomes did not chase through to fusion with lysosomes. LC3-II in LCL161 treated cells (Fig. 4). Similar results Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 6 of 15 Fig. 4 (See legend on next page.) Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 7 of 15 (see figure on previous page) Fig. 4 IAP antagonism blocks autophagic flux. a MEFs expressing mcherry-gfp-lc3b were treated as shown in the timeline by either starving cells in HBSS for 1 h or treating with thapsigragin (3 μM) or LCL161 (5 μM) for 5 h. LY294002 (20 μM) was then added for 1 h either alone or, in the HBSS starved conditions, thapsigargin or LCL161 were also added together with LY294002. Cells were imaged and GFP+ and mCherry+ puncta /cell counted. Graphs show means error bars show SEM of at least 3 experiments. b MEFs expressing mCherry-GFP-LC3b were either left untreated, starved in HBSS for 2 h or treated with LCL161 (0.5 μM) for 6 h. In each condition cells were also treated with or without concanamycin A (2 nM) for the final hour. Cells were lysed and levels of mCherry-GFP-LC3B detected by western blot. For each condition the amount of mCherry-GFP-LC3BII was normalised to actin levels in the non concanamycin A treated sample. The graph shows the mean fold change in mCherry-GFP-LC3BII from at least 3 independent experiments and error bars show SEM. P values were calculated using T-test in Prism software fl/fl −/− −/− were seen using birinapant (data not shown). These less so for cIAP1 cIAP2 XIAP cells, indicating a results again argue that IAP antagonism blocks autophagy reduced rate of flux (Fig. 5c). Immunofluorescence against autophagosome–lysosome fusion stage. Lamp2 and LC3 showed dramatic accumulation of LC3 in fl/fl −/− −/− the starved cIAP1 cIAP2 XIAP cells compared cIAP2 and XIAP but not cIAP1 regulate autophagic flux to the wild type (Fig. 5d). At steady state, while there is LCL161 and birinapant both target cIAP1 and cIAP2 some accumulation of autophagosomes, the difference is with higher affinity than XIAP. The increase in severity of not as clear, suggesting adaptation to loss of cIAP2 and the phenotype in response to higher doses of XIAP (Fig. 5d). Loss of both cIAP2 and XIAP together LCL161 suggests that XIAP may play a role. To identify therefore inhibits autophagosome–lysosome fusion caus- which IAPs regulate autophagy and to confirm that this ing reduced flux through the autophagy pathway phenotype was specific to IAP antagonism and not an off target effect of the IAP antagonist drugs, siRNA knock- IAP antagonists block turnover of long-lived proteins down was used to silence expression of cIAP1, cIAP2 or Basal autophagic activity is responsible for degradation XIAP. Efficient silencing was achieved for each gene of long-lived proteins that are not normally turned over (Fig. 5a). cIAP2 loss was confirmed using qPCR, cIAP1 by proteasomal degradation. To determine if IAP inhibi- and XIAP loss were determined by western blot. Loss of tion could block turnover of long-lived proteins, wild type −/− cIAP2 or XIAP increased GFP + /mCherry + ratio closer and Atg5 MEFs were labelled using click-iT chemistry to 1, similar to IAP antagonist treatment at lower doses with L-Azidohomoalanine (AHA). AHA is incorporated (0.5 µM) (Fig. 5). Surprisingly however, siRNA against in place of methionine when cells are cultured in cIAP1 had no significant effect on the GFP + /mCherry + methionine free media. The degradation of long-lived ratio suggesting that build-up of GFP + autophagosomes proteins can then be monitored by labelling with a due to IAP antagonism is likely a result of cIAP2 and fluorophore and monitoring fluorescence by flow XIAP antagonism. We were unable to suppress expres- cytometry . sion of both cIAP2 and XIAP simultaneously using siRNA Starvation in HBSS for three hours resulted in a in MEFs; at the concentrations required the transfection decrease in fluorescence indicating a reduction of existing −/− reagents alone induced build-up of autophagosomes, stained proteins (Fig. 6). The same treatment in ATG5 something that has previously been reported . However, MEFs showed no significant reduction in fluorescence given the higher affinity of birinapant and LCL161 for confirming the reduction observed is specific to autop- cIAP1 and cIAP2 over XIAP, the effect seen at low doses hagy mediated degradation. Co-incubation of wild type is likely mostly due to cIAP2 inhibition. cells with HBSS and thapsigargin also completely blocked To determine if loss of both cIAP2 and XIAP together the decrease in labelled protein, showing that blocking could replicate inhibition of autophagosome–lysosome fusion of autophagosomes and lysosomes also blocks fusion seen with higher doses of IAP antagonist drugs, protein turnover. Co-incubation with HBSS and LCL161 fl/fl −/− −/− cIAP1 cIAP2 XIAP dermal fibroblasts were or birinapant also blocked turnover of long-lived proteins infected with lentivirus expressing mCherry-GFP-LC3, although incompletely, presumably because at these lower and the ratio of GFP + /mCherry + vesicles quantified. doses XIAP is not targeted and only the loss of cIAP2- Genotyping of the cells confirmed knockout of cIAP2 and function was observed (Fig. 6). fl/fl −/− XIAP (Supplementary Fig. 2). The cIAP1 cIAP2 −/− XIAP cells showed a GFP + /mCherry + ratio close to IAPs do not regulate endocytosis fl/fl 1 without any treatment (Fig. 5b). Additionally, cIAP1 Autophagosomes use some of the same machinery for −/− −/− cIAP2 XIAP cells have increased endogenous LC3 lysosomal fusion as the endocytic trafficking system levels (Fig. 5c). Starvation reduced LC3-II levels in wild including SNAREs, Rab7 and the class C/HOPS tethering 29, 32, 33 type cells, probably due to a high rate of flux, but this was complex . Blockage of autophagosome–lysosome Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 8 of 15 Fig. 5 cIAP2 and XIAP regulate autophagosome fusion, but not cIAP1. a MEFs expressing mCherry-GFP-LC3b were transfected with siRNA against either cIAP1, cIAP2 or XIAP. Cells were analysed with the microscope and the number of mCherry+, and GFP+ puncta/cell were calculated and the ratio of GFP+ /mCherry+ puncta is indicated. Shown are the means and the error bars represent the SEM of at least three independent experiments. Westerns show efficient knockdown of cIAP1 and XIAP expression. cIAP2 siRNA efficiency was determined by real time PCR as shown in −/− −/− the graph below the westerns. b Wild type and cIAP2 XIAP dermal fibroblasts expressing mCherry-GFP-LC3b were treated analysed on the −/− microscope and mCherry+, and GFP+ puncta/cell were calculated. The ratio of GFP+/mCherry+ puncta is indicated. c Wild type and cIAP2 −/− XIAP dermal fibroblasts were left in complete media (CM) or starved for 2 h in EBSS. Cells were lysed and proteins analysed by western blot for −/− XIAP, LC3, and Actin. cIAP2 was confirmed by PCR due to lack of effective antibodies for mouse cIAP2 (see supplemental Fig. 2). d −/− −/− −/− −/− Immunofluoresence showing accumulation of LC3 in starved cIAP2 XIAP dermal fibroblasts. Wild type and cIAP2 XIAP dermal fibroblasts were incubated in complete media or starved in EBSS for 2 h, then fixed and stained for LC3 (green channel) and LAMP2 (red channel). Nuclei are stained blue with Hoechst. Shown in upper panels are overviews. Lower panels show zoomed in regions indicated in the upper panels fusion may represent a more general defect in endocytic endocytosis using uptake of fluorescently labelled dextran membrane fusion caused by IAP antagonists. To test this, and trafficking of labelled endosomes to lysosomes by co- we monitored internalisation and degradation of EGFR, staining with lysotracker. No difference could be detected which upon activation is degraded in lysosomes after in the amount of dextran-labelled endosomes co- endosomal trafficking. There was no change in EGFR localising with lysosomes in treated and control cells degradation rate in LCL161 treated cells while con- (Fig. 7b). This was confirmed by Pearson’s correlation canamycin A treated cells showed a near complete loss of coefficient analysis, which also clearly showed that the degradation (Fig. 7a). Additionally we analysed fluid phase degree of co-localisation is not altered significantly by IAP Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 9 of 15 Oligomycin and Antimycin A to induce mitochondrial damage either with or without LCL161 or birinapant for 16 h and examined the levels of cytochrome C as a marker of mitochondria. There was a decrease in cytochrome C levels with the oligomycin and Antimycin A treatment only in the Parkin over-expressing cells (Fig. 8c). Addition of LCL161 restored the levels of cytochrome C to some extent, however not completely (Fig. 8c). MEFs were also induced to over-express Parkin and treated in the same way, but in our hands with all mitochondrial proteins −/− tested we also saw degradation in ATG5 MEFs and also in non-Parkin over-expressing cells (data not shown), suggesting that the degradation is not mitophagy in the MEFs. Similar results have been published previously indicating that there are few reliable markers for mito- phagy in MEFs . Therefor, not only is mitophagy inhib- ited in IAP antagonist treated cells, but IAP antagonism also blocks or at least slows the degradation of mito- chondria by mitophagy in human cells too. Xenophagy, which is conceptually similar to mitophagy as it involves the tagging of intracellular bacteria with ubiquitin followed by engulfment by autophagosomes and Fig. 6 IAP antagonism blocks turnover of long lived proteins. degradation, is an early step in the recognition of a −/− Wild type or Atg5 MEFs were labelled overnight with AHA as number of pathogenic bacteria. Salmonella have been described in methods. Cells were starved in HBSS or treated with HBSS used extensively as a model to study xenophagy and there and LCL161 (0.5 μM), birinapant (0.5 μM) or thapsigargin (3 μM) for is significantly more bacterial survival in autophagy defi- three hours. Click it chemistry was used to label proteins and the cient cells . A block in mitophagy suggests loss of XIAP geometric mean fluorescence intensity was measured for each condition. Shown are the means of at least three independent could also lead to a deficiency in xenophagy. To test this, experiments and error bars show SEM XIAP was knocked out in MEFs using CRISPR-Cas9. Cells infected under these conditions were fixed and stained using an antibody against S. Typhimurium LPS and the antagonism (Fig. 7b). Together these results clearly show number of bacteria in each cell counted, revealing more that IAP antagonism has no effect on the endosomal salmonella in the XIAP CRISPR cells than in the wild type pathway but instead specifically affects the autophagoso- (Fig. 8d). This was then confirmed by lysing cells and mal system. calculating the colony forming units (CFU) (Fig. 8e). Most studies analysing xenophagy have used treatments or Mitophagy and xenophagy are blocked in IAP antagonist- mutants that block the early stages of autophagosome treated cells formation. To the best of our knowledge little is known One consequence defective autophagy is the accumu- about the effect of blocking fusion of autophagosomes lation of mitochondria due to failure loss of mitophagy. with lysosomes on xenophagy. As a model for blockage of We examined the amount of mitochondria in MEFs the fusion of autophagosomes with lysosomes, cells were treated with LCL161 or thapsigargin by staining with also treated with thapsigargin. Following 5 h of infection, −/− Mitotracker Green (MG). High dose (5 µM) LCL161 and there is consistently more surviving bacteria in ATG5 thapsigargin treated cells showed a clear increase in MG cells than in wild type as previously reported. Thapsi- signal, while low doses (0.5 μM) showed no significant gargin treatment also resulted in accumulation of viable S. increase (Fig. 8a, b). Birinapant also showed an increase in Typhimurium, as did knockout of XIAP in the XIAP- mitochondrial mass only at the high doses that fully block CRISPR cells (Fig. 8e). Both thapsigargin and XIAP defi- fusion (Fig. 8a, b). To confirm that mitophagy is impaired ciency result in increased S. Typhimurium survival to a we turned to HeLa cells overexpressing Parkin as a well- similar extent to Atg5 deficiency, supporting that established model for inducing mitophagy. HeLa cells do Autophagy inhibition is involved in all conditions. not express endogenous Parkin, a ubiquitin ligase that ubiquitylates damaged mitochondria and induces their Discussion clearance by mitophagy . We treated HeLa cells without While we have demonstrated that cIAP2 and XIAP Parkin and cells overexpressing mCherry-Parkin with promote autophagosome-lysosome fusion, the Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 10 of 15 Fig. 7 IAP antagonism does not block endocytosis. a Wild type MEFs were grown overnight in 0.5% serum. Cells were either left untreated as a control or treated with LCL161 (0.5 μM), birinapant (0.5 μM) for 4 h, or concanamycin A (2 nM) for 1 h before treatment with 100 ng/ml EGF in the presence of 10 μg/ml cycloheximide for 0, 1 or 2 h. Cells were lysed and levels of EGFR were analysed by western blot. b Wild type MEFs were treated with LCL161 (0.5 μM) or not for 4 h. Alexafluor 647-dextran (100 μg/mL) was added for 30 mins and then cells were washed and grown in normal media for a further 2 h. The final 30 min cells were stained with lysotracker (100 nM). Cells were fixed and analysed on the fluorescence microscope. Shown are representative images and line profile of lysotracker and dextran Alexafluor 647. Experiments were performed at least 3 times. Pearsons correlation coefficients are shown in the right panel. Error bars represent the SEM from at least three experiments mechanism is still not clear. Ubiquitylation regulates One interesting aspect of these results is that we have many steps in vesicle trafficking and it is likely that identified cIAP2 and XIAP as playing redundant roles in cIAP2 and XIAP are regulating specific components of regulating autophagy in MEFs. XIAP depletion alone in −/− the autophagosome–lysosome fusion machinery via their humans leads to disease but XIAP mice are in most ubiquitin ligase activity. The observation that endocytosis aspects normal, although they can be induced to develop is also not affected by IAP inhibition suggests ubiquity- disease similar to XLP-2 in humans by infection with a lation of some target on the autophagosomes themselves herpes virus, MHV-68 . One might expect that if there is likely, however we cannot at this stage rule out that was an absolute requirement for XIAP or cIAP2 in some other function such as scaffolding may also be autophagy that mice deficient for either alone would playing a role. present with phenotypes associated with defects in Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 11 of 15 Fig. 8 IAP antagonism blocks mitophagy and xenophagy. Wild type MEFs were treated overnight with the indicated concentrations of LCL161, birinapant or thapsigargin. Cells were stained with mitotracker green for 30 min and fluorescence intensity quantified using flow cytometry. a representative histograms showing increases in mitotracker signal in IAP antagonist treated cells. b quantification of mitotracker staining. Shown are the means of the geometric mean fluorescence intensity. Error bars are SEM from at least 3 independent experiments. P values were calculated using t tests in Prism software. c IAP antagonism can block mitophagy. Wild type HeLa cells or HeLa cells expressing mCherry-Parkin were treated with either LCL161 (5 µM), birinapant (50 µM) or oligomycin (1 µM) and Antimycin A (1 µM) (O/A) alone or in combination as indicated. LCL161 or birinapant were added 4 h prior to Oligomycin and Antimycin A. Cells were incubated for 16 h before cells were harvested and lysates run on SDS- PAGE and levels of cytochrome C and β-Actin analysed by western. d Immunofluoresence and quantification of Salmonella Typhimurium infection in wild type and XIAP CRISPR MEFs. MEFs of the indicated genotypes were infected as described in methods, fixed and stained for Salmonella Typhimurium LPS. Shown are representative images. The mean number of Salmonella Typhimurium /cell is shown from two experiments. e −/− −/− Xenophagy of Salmonella Typhimurium is impaired in XIAP deficient cells. Wild type, Atg5 and XIAP -CRISPR cells were infected with Salmonella Typhimurium and incubated for 5 h. CFU/mL of internalised bacteria was then calculated. Thapsigargin was used where indicted to block autophagosome–lysosome fusion by treating with 3 µM thapsigargin for 1 h before and then throughout the infection protocol. Error bars are SEM from at least 3 independent experiments. P values were calculated using T tests in Prism software Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 12 of 15 autophagy, but loss of both seems to be required before Many questions remain including dissection of the spontaneous inflammation is seen . This may be partly mechanism behind cIAP2 and XIAPs regulation of explained by the overlapping functions seen for cIAP2 autophagosome–lysosome fusion, as well as the degree to and XIAP in this study and may explain why XIAP defi- which autophagy defects contribute to inflammation cient mice do not develop disease like humans do. induced by IAP loss. It will be interesting to see if IAPs or Relatively little is known about the substrates of XIAP, their substrates are also involved in other autophagy related diseases such as protein misfolding diseases and or however ubiquitylation of RIPK2 at NOD2 receptors was shown to be required for NOD2 activation of NF-κB . lysosomal storage disorders. Loss of NF-κB signalling results in a failure to up- regulate cytokines in response to NOD2 signalling and Methods this link to NOD2 signalling is thought to be the cause of Reagents Crohn’s disease in XIAP deficient patients. While this LCL161 and birinapant were purchased from ApexBio. molecular connection between NOD2 and XIAP may The following antibodies were used; EGFR (PA1-1110— contribute to disease progression, NOD2 mutation does Thermo fisher), XIAP (MAB822—R&D Systems), cIAP1 not lead to the multiple other conditions that XIAP (was a kind gift from John Silke—WEHI Melbourne deficiency causes , suggesting that the story is not so Australia), LC3b (nb100–2220—Novus), LAMP2 simple. There are numerous genes that are involved in (ab13524—Abcam). EGF was purchased from Biolegend autophagy, including ATG16L1, NDP52, IRGM, and (catalogue No. 713108). Alexafluor 647 Dextran LRRK2 that have been found to be mutated in Crohn’s MW10,000 (catalogue No. D-22914), Lysotracker Red Disease patients; a number of these mutations also DND-99 (catalogue No. L7528) and Mitotracker Green manifest with multiple clinical presentations. Recently, (catalogue No. M7514) were purchased from Thermo Niemann–Pick disease type C (NPC), a lysosomal sto- Fisher Scientific. Concanamycin A (catalogue No. Cay- rage disorder in which the NPC1 gene coding for a 11050-25) was purchased from Cayman Chemical. lysosomal protein involved in lipid transport is mutated, was also linked to early onset Crohn’s disease. Loss of Cell culture NPC1 function was also shown to be linked to defective All cells were grown in DMEM High glucose (Gibco) autophagy resulting in increased survival of intracellular with 10% FCS and 1% Pen/Strep in 5% CO at 37 °C Salmonella and adherent-invasive E. coli . Importantly, unless otherwise stated. this study showed that NOD2 induced NF-κB signalling is normal in NPC1 mutated cells and enforced autophagy Fluorescence microscopy of LC3 induction could bypass the xenophagy defect and reduce MEFs of the indicated genotypes were infected with intracellular bacterial survival. Similarly impaired xeno- pBABE-puro-mCherry-EGFP-LC3B (pBABE-puro- phagy has also been seen in macrophages from patients mCherry-EGFP-LC3B was a gift from Jayanta Debnath with NOD2 and XIAP mutations. We observed defects in (Addgene plasmid # 22418)). Cells were selected in Pur- xenophagy of S. Typhimurium in XIAP deficient fibro- omycin 4 μg/ml. The selected cells were seeded at 50,000 blasts. Such defects in xenophagy may also contribute to cells/well of an 8 chamber μ-slide (Ibidi) and allowed to accumulation of invasive bacteria in XIAP deficient plate down overnight. Cells were typically treated for 6 h humans, again increasing the burden of bacteria that with the indicated concentrations of LCL161, birinapant needs to be dealt with by the immune system and or thapsigargin (3 μM). Cells were treated for 30 min with resulting in overactive inflammation. Additionally we Hoechst to stain nuclei. Cells were imaged with a Keyence observed an accumulation of mitochondria in cells BZ9000 fluorescence microscope taking Z-stacks to and treated with IAP antagonists at doses high enough to using the “full focus” function in the analysis software to block XIAP, as well as cIAP1 and cIAP2, suggesting that make a single layer image from full focused features in the loss of IAPs also causes defects in mitophagy. each layer of the Z-stack. These images were used to Mitophagy is important for clearing old or damaged count the number of GFP + and mCherry + puncta per mitochondria and defects in mitophagy have been linked cell in image J using the analyse particles function to to excessive ROS production and excessive inflamma- count nuclei and find maxima function to count puncta. some activation . Mitophagy is in many ways mechan- The ratio of mCherry + /GFP + puncta/cell was calcu- istically analogous to xenophagy and defects therein may lated from these measurements. also contribute to inflammation in XIAP deficient For Immunofluorescence of LC3 and LAMP2, Wild patients. This suggests that the driving mechanism type MEFs were seeded in 8 well µ-slides (Ibidi) at 50,000 behind multiple genetic mutations causing Crohn’s cells/well and incubated overnight. Cells were treated Disease may be a loss of autophagy, and supports a role with IAP antagonists as indicated and cells were washed for XIAP in regulating this process. twice in warm PBS. Cell were fixed in 4% PFA in PBS for Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 13 of 15 10 min followed by washing three times in PBS. Cells were (0.5 μM), thapsigargin (3 μM), or birinapant (0.5 μM) were subsequently permeabilized in PBS with 50 µg/ml Digi- added at this stage. Cells were washed in PBS and then tonin for 5 min followed by washing three times in PBS cultured for a further 3 h either in full media or HBSS then blocking with 3% BSA in PBS for 30 min. Antibodies containing the indicated drugs. Cells were washed 2× in against LC3 and Lamp2 were added 1:200 for 1 h in BSA PBS and then harvested and fixed in 4% formaldehyde in 3% in a humidified chamber at 37 C. Cells were washed PBS for 15 min at room temperature. After fixation, the cells are washed twice with 3% BSA in PBS. Cells were three times in PBS followed by incubating with appro- priate secondary antibodies at 1:500 in PBS 3% BSA for 1 permeabilized with 0.5% Triton X-100 in PBS for 20 min h followed by washing 5 times in PBS. Images were taken at room temperature. Cells were resuspended in PBS and using a Zeiss LSM 880 with an Airyscan confocal store at 4 °C for detection of the corresponding alkyne- microscope at a 63 × magnification (oil immersion) and tagged detection molecule. analysed with Zen (Zeiss) software. Cells from the last step above were washed with 3% BSA in PBS. 100 μL Click-iT® reaction cocktail was added to siRNA treatment each sample. Cells were incubated for 30 min at room MEFs were seeded at 150,000 cells/well of a 6 well plate. temperature in the dark. Cells were washed once with 3% The next day cells were washed with PBS and media BSA in PBS before being analysed using flow cytometry. without antibiotics was added. Cells were transfected with siRNA’s complexed with Lipofectamine RNAiMAX EGFR degradation assay according to manufacturers instructions. Briefly, 7.5 µL of A total of 500,000 cells/well were seeded in the morning 20 µM stock stealth siRNA oligo was added to 150 µL of and let to plate down. In the evening, media was optimem. Nine microlitre of RNAiMAX was added to exchanged for DMEM 0.5% FCS and cells incubated another 150 µL of optimum. The tubes were mixed overnight. The next day, cells were pre-treated for 4 h with together before incubating at room temperature for 5 min. IAP antagonists birinapant (0.5 μM) or LCL161 (0.5 μM) 250 µL of this was then added to the cells and incubated or for 1 h with concanamycin A (2 nM). Media was then overnight. Cells were seeded at 50,000 cells/well of an 8 exchanged with serum free media alone or containing the chamber μ-slide (Ibidi) and allowed to plate down over- drugs. Cells were treated with EGF (100 ng/ml) and night before microscopy was performed as described cycloheximide (10 μg/ml) for 0, 1 or 2 h. Cells were lysed above. The following stealth siRNAs were used from on ice in RIPA buffer and 50 µg/ml protein per lane was Thermo Fisher Scientific; cIAP1 (MSS273215), cIAP2 run on 10% SDS-PAGE before transferring to nitrocellu- (MSS202113) and XIAP (MSS202115). Activity of each lose membranes and western blotting for EGFR siRNA was confirmed using qPCR with SYBR Select Master Mix (applied biosystems # 4472908) and the fol- Assay of endocytosis and fusion with lysosomes lowing primers: cIAP1 (Forward 5′-GAAGAAAATGC 30,000 cells/well were seeded in 8 well microscope TGACCCTACAGA-3′, Reverse 5′-CATGACGACATC culture slides (Ibidi) in 300–500 µL media. The next day TTCCGA-3′), cIAP2 (Forward 5′- TCGATGCAGAA cells were treated with DMSO, LCL161 0.5 μM or bir- GACGAGA-3′ Reverse 5′-TTTGTTCTTCCGGATTAG inapant (0.5 μM) for 6 h. 2.5 h before end of drug treat- TGC-3′, XIAP (Forward 5′-GCTTGCAAGAGCTGGA ments, Dextran Alexafluor 647 was added to a final of TTTT-3′, Reverse 5′-TGGCTTCCAATCCGTGAG-3′). concentration of 100 µg/ml. Cells were incubated for 30 Actin was used as a reference gene. PCR was done in 384 min at 37 °C. Cells were washed 2× in warm PBS. Fresh well plates in a 7900HT Fast Real-Time PCR System. media containing LCL161 or birinapant was added and cells were incubated for a further 2 h. Lysotracker Red Long-lived protein degradation assay (100 nM) was added for the final 30 min. Cells were Cells were grown to ~70–80% confluency in a 6-well washed 2× in warm PBS before fixation in 4% paraf- plate then washed with warm PBS and cultured in L- ormaldehyde (PFA) for 20 min at room temperature. Cells methionine-free DMEM (cat. no. 21013-024, Gibco) for were washed 2× in PBS and stored at 4 °C until images 30–60 min to deplete the intracellular methionine were taken on a Keyence BZ9000 fluorescence micro- reserves. Following methionine depletion, the cells were scope. Hoechst was added 30 min prior to imaging. labelled with 25 μM AHA in 10% dialysed FBS DMEM Pearsons correlation coefficients were calculated using the (methionine-free) for 18 h. Dialysed FBS was made by FIJI distribution of ImageJ and the Coloc2 plugin. dialyzing against PBS with Slide-A-Lyzer mini dialysis devices 3.5 k MWCO (Pierce—cat. No. 88403) overnight. Electron microscopy After labelling, the cells were washed with PBS and cul- MEFs were cultured on glass cover slips and fixed for tured in regular DMEM containing 10 × L-methionine (2 20 min in 4% PFA plus 1% glutaraldehyde (Roth, Ger- mM) for 2 h to chase out short-lived proteins. LCL161 many) in PBS. After contrastation in 0.5% Official journal of the Cell Death Differentiation Association Gradzka et al. Cell Death and Disease (2018) 9:529 Page 14 of 15 Osmiumtetroxide (30 min at RT) cells were dehydrated BSA for 30 min followed by staining with anti mouse and embedded in epoxy resin (Durcupan, Sigma-Aldrich, IgG-Cy3 (1:500) in PBS 3% BSA. Nuceli were stained Gillingham, UK). Images were taken using a Philips CM with Hoechst. Cells were imaged and the number of 100 transmission electron microscope. bacteria per cell was counted from at least 50 cells for each genotype per experiment. Mitophagy assays Acknowledgements For mitotracker Green staining, Wild type MEFs were −/− The authors thank Barbara Joch for her excellent technical assistance. Atg5 +/+ +/− treated overnight with the indicated concentrations of and Atg5 MEFs were made from ATG5 mice, which were a kind gift from Prof. Dr. Tobias Huber (University Medical Center Freiburg). mCherry- LCL161, birinapant or thapsigargin. Cells were incubated Parkin expressing lentiviral constructs were a kind gift from Dr. Florian with 100 nM mitotracker Green for 30 min in complete Steinberg (Center for Biological Systems Analysis, University of Freiburg). media then washed in PBS and trypsinized then analysed for fluorescence intensity using flow cytometry on a FACS Author details Institute of Medical Microbiology and Hygiene, University Medical Center Calibur then analysed using FlowJo software. Freiburg, Freiburg, Germany. Faculty of Medicine, University of Freiburg, For analysis of Parkin dependent mitophagy, HeLa cells 3 Freiburg, Germany. Renal Division, University Medical Center Freiburg, were infected with a lentivirus (pXLG3) expressing Freiburg, Germany. Department of Neuroanatomy, University Freiburg, Freiburg, Germany. Department of Medicine, University Medical Center mCherry-Parkin (human) or left uninfected. These Hamburg-Eppendorf, Hamburg, Germany. Institute of Experimental 5 5 HeLas were then seeded at 4 × 10 for wild type and 6 × 10 Immunology, University of Zurich, Zurich, Switzerland cells/well in 6 well plates, left overnight and then incubated Conflict of interest for 4 h with 5 µM LCL161 or 50 µM birinapant followed by The authors declare that they have no conflict of interest. addition of Oligomycin (1 µM) (Sigma Aldrich—O4876) and Antimycin A (1 µM) (Sigma Aldrich—A8674) for 16 h. Cells were harvested and lysed in DISC lysis buffer fol- Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in lowed by detection of Cytochrome C by western blot. Actin published maps and institutional affiliations. was used as a loading control. Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41419-018-0508-y). Xenophagy assays S. 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Cell Death & DiseaseSpringer Journals

Published: May 9, 2018

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