HIF1A regulates xenophagic degradation of adherent and invasive <i>Escherichia coli</i> (AIEC)

<p>The hypoxia inducible transcription factor HIF1 activates autophagy, a general catabolic pathway involved in the maintenance of cellular homeostasis. Dysfunction in both autophagy and HIF1 has been implicated in an increasing number of human diseases, including inflammatory bowel disease (IBD), such as Crohn disease (CD). Adherent invasive <i>E. coli</i> (AIEC) colonize ileal mucosa of CD patients and strongly promote gastrointestinal inflammatory disorders by activation of HIF-dependent responses. Here, we aim to characterize the contribution of HIF1 in xenophagy, a specialized form of autophagy involved in the degradation of intracellular bacteria. Our results showed that endogenous HIF1A knockdown increased AIEC survival in intestinal epithelial cells. We demonstrate that the increase in survival rate correlates with a dramatic impairment of the autophagic flux at the autolysosomal maturation step. Furthermore, we show that AIEC remained within single-membrane LC3-II-positive vesicles and that they were unable to induce the phosphorylation of ULK1. These results suggested that, in the absence of HIF1A, AIEC were found within LC3-associated phagosomes. Using blocking antibodies against TLR5 and CEACAM6, the 2 well-known AIEC-bound receptors, we showed that downstream receptor signaling was necessary to mediate ULK1 phosphorylation. Finally, we provide evidence that HIF1 mediates CEACAM6 expression and that CEACAM6 is necessary to recruit ULK1 in a bacteria-containing signaling hub. Collectively, these results identify a new function for HIF1 in AIEC-dedicated xenophagy, and suggest that coactivation of autophagy and HIF1A expression may be a potential new therapy to resolve AIEC infection in CD patients.</p

PMNs undergo autophagic death and NETosis on infection with AIEC LF82.

<p>Differentiated PLB-985 cells or PMN were infected (MOI 50) with K12 or AIEC LF82 bacteria for 1 h and gentamycin was added for the following 5 h, cells were then analysed. (<b>A</b>) Control or K12- or AIEC LF82-infected PLB-985 cells, treated or not with 3-methyladenine (3-MA, 5 mM, autophagy inhibitor) or Zvad (5 µM, a pancaspase inhibitor), were incubated with propidium iodide (PI) (5 µg/ml) and cell death was analysed by flow cytometric analysis as described in the materials and methods section. The average ± S.D. is shown for three independent experiments, *p<0.003. Inset: immunoblot analysis of LC3-II testifying to the induction of autophagy in infected cells. (<b>B,</b> left panel) PMNs were infected with AIEC LF82 and cell death was assayed as described in A. Maximal cell death (+) was obtained after treatment with etoposide phosphate (100 µg/ml). To test whether cell death was due to apoptosis, a caspase-3 activity test was performed as described in the materials and methods section (right panel). Maximal cell apoptosis (+) was obtained by treatment with staurosporine (10 µM). (<b>C</b>) Cleavage of PARP and caspase-3 in non-infected or K12- and AIEC LF82-infected differentiated PLB-985 cells were analysed by immunoblot analyses. Etoposide phosphate (100 µg/ml) was used as a positive control. LC3-II was detected in LF82-infected cells and compared to uninfected or K12-infected cells (1+5 h, MOI 50). Actin was used as a loading control. (<b>D</b>) Survival of AIEC LF82 and K12 in differentiated PLB-985 cells was analysed with the gentamycin assay (panel one). To compare the ultrastructural morphology of vesicles and bacteria in PLB-985 cells transmission electron microscopy was performed. Ultrastructural analysis of K12-infected differentiated PLB-985 cells shows bacterial sequestration and degradation in phagocytosis vacuoles (panel two). In contrast, accumulation of autophagic vesicles and bacteria (arrowheads) inside the vacuoles was observed in PLB-985 cells (panel three) and PMNs (panel four). Scale bar = 1 µm. (<b>E</b>) Differentiated PLB-985 were seeded on glass coverslips coated with poly D lysine (5 µg/cm²) and allowed to settle for 1 h. Cells were then infected with AIEC-LF82 (MOI 50) for 4 h (1 h+3 h) and 6 h (1 h+5 h), fixed with 3% paraformaldehyde and stained with Hoechst 33342 (0.5 µg/ml) to visualize DNA. Cells were examined with an epifluorescence Axiophot microscope (Zeiss). Early apoptosis and necrosis were assayed by measuring Annexin-V-fluos (Roche) and propidium iodide (PI). Differentiated non-infected PLB-985 cells or cells infected with AIEC LF82 (MOI 50) for 1 h+5 h were incubated with Annexin-V-fluos and PI and fluorescence was detected on a FACS Calibure.</p

Inhibition of autophagic flux by AIEC LF82 infection.

<p>Human neutrophils or neutrophil-like PLB-985 cells were infected with AIEC LF82 at a MOI of 50 for 1 h than gentamicin (100 µg/ml) was added for 3 h. Cells were and processed for immunoblotting (<b>A,</b> left panel, <b>B</b>), quantitative RT-PCR (<b>A</b> rigth panel), immunofluorescence (<b>C</b>, <b>D</b>) and ultrastructural TEM analysis (<b>E</b>). (<b>A</b>) Time-dependent accumulation (1–8 h) of LC3-II and p62 in infected human neutrophils or neutrophil-like PLB-985 cells compared to uninfected cells analyzed at the end time point. Longer exposure detects the LC3-I band. We checked that AIEC infection did not affect p62 mRNA levels by qRT-PCR analysis (<b>A</b> right panel). Data are means ± SEM of three experiments. ** p<0.001. (<b>B</b>) Autophagic flux was analysed by immunoblot analysis in differentiated PLB-985 cells infected for 3 h with AIEC LF82 bacteria (MOI 50) in the absence or in the presence of E64d/PEPS. Actin was used as a loading control. Control unstimulated cells were analysed at the end time point. (<b>C</b>) Representative confocal images of control (0) or infected cells (LF82) (3 h post infection) showed the colocalization of bacteria with LC3-II and LAMP-1 proteins as indicated by yellow punctiform staining. Insets highlight individual staining of bacteria (DNA staining, blue), LC3-II (Alexa 488, green) and LAMP-1 (Alexa 594, red). (<b>D</b>) Representative confocal micrographs of control (0) and LF82 infected cells (LF82) showing the co-localization of bacteria (DNA staining, blue) within autophagic (LC3-II positive, Alexa 594, red) but not acidic compartments (LysoTracker negative, green). (<b>E</b>) Representative TEM images showing bacteria within endosomes (asterisk), autophagosomes (arrowheads) or free in the cytosol (arrow) in LF82-infected cells (3 h post infection). Bar = 2 µm.</p

Resistance to sunitinib in renal clear cell carcinoma results from sequestration in lysosomes and inhibition of the autophagic flux

<p>Metastatic renal cell carcinomas (mRCC) are highly vascularized tumors that are a paradigm for the treatment with antiangiogenesis drugs targeting the vascular endothelial growth factor (VEGF) pathway. The available drugs increase the time to progression but are not curative and the patients eventually relapse. In this study we have focused our attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives only suboptimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neoadjuvant setting, we exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which downregulated the activity of the lysosomal protease CTSB (cathepsin B) and led to incomplete autophagic flux. Amino acid deprivation initiates autophagy enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib stimulated the expression of ABCB1 (ATP-binding cassette, sub-family B [MDR/TAP], member 1), which participates in the accumulation of the drug in autolysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar or the permeabilization of lysosome membranes with Leu-Leu-O-methyl (LLOM) resensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50. Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on our results we propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.</p