SNX10‐mediated LPS sensing causes intestinal barrier dysfunction via a caspase‐5‐dependent signaling cascade

Article8 November 2021free access Source DataTransparent process SNX10-mediated LPS sensing causes intestinal barrier dysfunction via a caspase-5-dependent signaling cascade Xu Wang Xu Wang Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China These authors contributed equally to this work. Search for more papers by this author Jiahui Ni Jiahui Ni Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China The First Clinical Medical College of Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China These authors contributed equally to this work. Search for more papers by this author Yan You Yan You Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China These authors contributed equally to this work. Search for more papers by this author Guize Feng Guize Feng Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Sulin Zhang Sulin Zhang Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Weilian Bao Weilian Bao Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Hui Hou Hui Hou Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Haidong Li Haidong Li Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Lixin Liu Lixin Liu Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Mingyue Zheng Mingyue Zheng Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yirui Wang Yirui Wang Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Hua Zhou Corresponding Author Hua Zhou [email protected] orcid.org/0000-0002-7025-3690 Faculty of Chinese Medicine and State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macao, China Search for more papers by this author Weixing Shen Corresponding Author Weixing Shen [email protected] orcid.org/0000-0002-6454-3296 The First Clinical Medical College of Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China Search for more papers by this author Xiaoyan Shen Corresponding Author Xiaoyan Shen [email protected] orcid.org/0000-0003-4828-3268 Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Xu Wang Xu Wang Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China These authors contributed equally to this work. Search for more papers by this author Jiahui Ni Jiahui Ni Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China The First Clinical Medical College of Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China These authors contributed equally to this work. Search for more papers by this author Yan You Yan You Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China These authors contributed equally to this work. Search for more papers by this author Guize Feng Guize Feng Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Sulin Zhang Sulin Zhang Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Weilian Bao Weilian Bao Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Hui Hou Hui Hou Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Haidong Li Haidong Li Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Lixin Liu Lixin Liu Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Mingyue Zheng Mingyue Zheng Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yirui Wang Yirui Wang Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Hua Zhou Corresponding Author Hua Zhou [email protected] orcid.org/0000-0002-7025-3690 Faculty of Chinese Medicine and State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macao, China Search for more papers by this author Weixing Shen Corresponding Author Weixing Shen [email protected] orcid.org/0000-0002-6454-3296 The First Clinical Medical College of Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China Search for more papers by this author Xiaoyan Shen Corresponding Author Xiaoyan Shen [email protected] orcid.org/0000-0003-4828-3268 Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China Search for more papers by this author Author Information Xu Wang1, Jiahui Ni1,2, Yan You1, Guize Feng1, Sulin Zhang3, Weilian Bao1, Hui Hou3, Haidong Li1, Lixin Liu1, Mingyue Zheng3, Yirui Wang1, Hua Zhou *,4, Weixing Shen *,2 and Xiaoyan Shen *,1 1Department of Pharmacology & the Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, Shanghai, China 2The First Clinical Medical College of Nanjing University of Chinese Medicine, Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor, Nanjing, China 3Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China 4Faculty of Chinese Medicine and State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macao, China *Corresponding author. Tel: +853 88972458; E-mail: [email protected] *Corresponding author. Tel: +86 15150501221; E-mail: [email protected] *Corresponding author. Tel: +86 17317137196; E-mail: [email protected] The EMBO Journal (2021)40:e108080https://doi.org/10.15252/embj.2021108080 See also: MS Dickinson & J Coers (December 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Altered intestinal microbial composition promotes intestinal barrier dysfunction and triggers the initiation and recurrence of inflammatory bowel disease (IBD). Current treatments for IBD are focused on control of inflammation rather than on maintaining intestinal epithelial barrier function. Here, we show that the internalization of Gram-negative bacterial outer membrane vesicles (OMVs) in human intestinal epithelial cells promotes recruitment of caspase-5 and PIKfyve to early endosomal membranes via sorting nexin 10 (SNX10), resulting in LPS release from OMVs into the cytosol. Caspase-5 activated by cytosolic LPS leads to Lyn phosphorylation, which in turn promotes nuclear translocalization of Snail/Slug, downregulation of E-cadherin expression, and intestinal barrier dysfunction. SNX10 deletion or treatment with DC-SX029, a novel SNX10 inhibitor, rescues OMV-induced intestinal barrier dysfunction and ameliorates colitis in mice by blocking cytosolic LPS release, caspase-5 activation, and downstream signaling. Our results show that targeting SNX10 may be a new therapeutic approach for restoring intestinal epithelial barrier function and promising strategy for IBD treatment. SYNOPSIS Sorting nexin 10 (SNX10) mediates Gram-negative bacteria-induced intestinal epithelial barrier dysfunction via the PIKfyve-caspase-5-Lyn-Snail/Slug-E-cadherin signaling axis. Deletion or chemical inhibition of SNX10 restores intestinal epithelial barrier function in murine colitis models. SNX10 acts as an adaptor protein to assemble a complex containing PIKfyve, caspase-5 and Lyn for LPS release, sensing and signaling transduction. SNX10 promotes caspase-5 activation and Lyn phosphorylation to regulate nuclear trans-localization of Snail/Slug leading to downregulation of E-cadherin expression and impaired intestinal epithelial barrier function. SNX10 deletion or DC-SX029 treatment maintains intestinal epithelial barrier function and ameliorate colitis in mice. Introduction Alteration of intestinal microbial composition has a crucial impact on the intestinal barrier function. Impaired intestinal mucosal barrier function is the trigger for the initiation and recurrence of IBD (Plichta et al, 2019). Loss of an integrated gut mucosa barrier results in inappropriate translocation of intestinal luminal contents, commensal microbiota, and pathogenic microbes into the gut lamina propria, and ultimately induces intestinal inflammation (Graham et al, 2019). Current IBD treatment focuses on inflammation control rather than restoring intestinal epithelial barrier function. Accumulated proportions of Gram-negative bacteria in the inflamed lesion of intestinal epithelium have been detected in the patients of intestinal inflammatory diseases (Seksik et al, 2003; Martin et al, 2005), indicating the elevated LPS from the augmentative Gram-negative bacteria engages into the initiation and aggravation of intestinal inflammation. The OMVs derived from Gram-negative bacteria are regarded as a carrier that releases LPS into the cytosol for activating the caspase-11-dependent signaling pathway in vivo and in vitro (Vanaja et al, 2016). However, the precise mechanism that triggers LPS release from OMVs and its impact on intestinal epithelial barrier function are largely unknown. SNX10 is a member of sorting nexin (SNX) family proteins. Our previous studies found it acted as an adaptor protein and played crucial roles in endosome/lysosome homeostasis and function maintenance. Since endosomal events are related to OMV internalization and LPS release (Vanaja et al, 2016), in the present study, we explored the role of SNX10 in the cytosolic release of LPS from OMVs and its impact on intestinal epithelial barrier function. Results SNX10 involves in OMV-induced downregulation of E-cadherin in intestinal epithelial cells After LPS quantification, OMVs from Escherichia coli BL21 were used to treat Caco-2, HT-29, and NCM460 cells, no significant cytotoxicity was found at the concentration below 100 μg/ml (including 100 μg/ml, Fig EV1A–D). In bone marrow-derived macrophages (BMDMs), IL-1β and IL-18 secretion was significantly increased and the cell viability was reduced by OMV (100 μg/ml) induction, while undetectable IL-1β and slightly increased IL-18 secretion were found in Caco-2, HT-29, or NCM460 cells (Fig EV1E and F, Appendix Fig S1A). The OMV-induced IL-18 secretion in intestinal epithelial cells was consistent with the results of the previous study (Knodler et al, 2014), and we also found that SNX10 deficiency prominently inhibited OMV-induced IL-18 secretion in Caco-2 cells (Appendix Fig S2). These results suggest OMV treatment at 100 μg/ml might trigger a different signaling rather than inflammasome activation and pyroptosis in intestinal epithelial cells. Click here to expand this figure. Figure EV1. Effects of OMV treatment on the viability of intestinal epithelial cells A. LPS levels in purified OMVs were determined by LAL assay (n = 6 independent experiments). B–D. The cell viability of Caco-2 (B), HT-29 (C), and NCM460 (D) cells treated with indicated doses of OMVs for 24 h was detected by Cell Counting Kit-8 (CCK-8; n = 6 independent experiments). E. IL-1β secretion by indicated cell types stimulated with OMVs (100 μg/ml) for 24 h was detected by ELISA (n = 6 independent experiments). F. IL-18 secretion by indicated cell types stimulated with OMVs (100 μg/ml) for 24 h was detected by ELISA (n = 6 independent experiments). Data information: Data are means ± SD. Two-tailed unpaired t-test between two groups and one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons were utilized for statistical analyses. ***P < 0.001. Download figure Download PowerPoint Alterations in tight junctions (TJs) and adherens junctions (AJs) may result in disturbance of paracellular permeability. As shown in Fig 1A–C, OMV treatment at 10 or 100 μg/ml for 24 h could reduce the protein and mRNA (CDH1) levels of E-cadherin in a concentration-dependent manner. Surprisingly, SNX10 deletion by CRISPR/Cas9 technique prohibited the E-cadherin reduction caused by 10 or 100 μg/ml OMV treatment in Caco-2 cells (Figs 1A–C and EV2A). These results were also confirmed by time course of OMV treatment (100 μg/ml) for 6, 12, and 24 h in both Caco-2 and HT-29 cells (Figs 1D–F and EV2A–D). Conversely, OMV treatment did not affect the expression of TJ proteins (Fig EV2E). Confocal images of WT Caco-2 cell monolayers revealed that E-cadherin was distributed regularly along with the cell boundaries, exhibiting a characteristic ‘chicken wire’ markup type (Fig 1G). In accordance with the dramatical reduction in protein and mRNA levels, incubation of Caco-2 cells with OMV (100 μg/ml) treatment for 6, 12, and 24 h obviously impaired the structures of E-cadherin by breaking successive band pattern and disturbing the distribution of E-cadherin (Fig 1G and H). However, SNX10 deficiency reversed the OMV-induced variations (Fig 1G and H). The results from intestinal barrier permeability assay in Caco-2 and HT-29 cells showed that SNX10 deficiency substantially prevented the OMV-induced decline in transepithelial electrical resistance (TEER) and strikingly restrained OMV-induced leakage of FITC-dextran into the bottom chambers (Figs 1I and J, and EV2F and G). Based on these findings, we propose that SNX10 deficiency potentially maintains the intestinal barrier integrity through suppressing OMV-induced downregulation of the adherens junction. Figure 1. SNX10 involves in OMV-induced downregulation of E-cadherin in intestinal epithelial cells A–C. WT and SNX10 KO Caco-2 cells were treated with indicated doses of OMVs for 24 h. Protein expression of E-cadherin and SNX10 was determined by immunoblots (A) and quantified by ImageJ software (B) (n = 3 independent experiments). The fold changes in mRNA levels of CDH1 (encoding E-cadherin) and SNX10 in Caco-2 cells were determined by RT–qPCR (C) (n = 6 independent experiments). D–F. WT and SNX10 KO Caco-2 cells were treated with OMVs (100 μg/ml) for the indicated time. Protein expression of E-cadherin and SNX10 was determined by immunoblots (D) and quantified by ImageJ software (E) (n = 3 independent experiments). The fold changes in mRNA levels of CDH1 and SNX10 were determined by RT–qPCR (F) (n = 6 independent experiments). G, H. Confocal images of E-cadherin staining in Caco-2 cells treated with OMVs (100 μg/ml) for the indicated time were captured (G), and the fluorescence intensity of E-cadherin staining was quantified by ImageJ software (H) (n = 3 independent experiments, n = 18 fields analyzed). Scale bar: 20 μm. I. TEER value of Caco-2 cell monolayers incubated with or without OMVs (100 μg/ml) for 24 h was analyzed (n = 9 independent experiments). J. Caco-2 cell monolayers on transwell membranes were treated with or without OMVs (100 μg/ml) for 24 h. FITC-dextran was added to these cells (top of the membrane). After 2 h, FITC-dextran levels in the bottom chamber wells were measured (n = 9 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyses. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 1 [embj2021108080-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. SNX10 deficiency maintains the E-cadherin expression and intestinal epithelial barrier function in HT-29 cells treated with OMVs SNX10 was deleted in Caco-2 cells by CRISPR/Cas9 technology, and the efficiency was confirmed by immunoblots and quantified by ImageJ software (n = 6 independent experiments). SNX10 was deleted in HT-29 cells by CRISPR/Cas9 technology, and the efficiency was confirmed by immunoblots and quantified by ImageJ software (n = 6 independent experiments). Protein levels of E-cadherin in HT-29 cells were measured by western blots and quantified by ImageJ software (n = 3 independent experiments). The relative mRNA levels of CDH1 (encoding E-cadherin) in HT-29 cells were determined by RT–qPCR method (n = 6 independent experiments). The expression of tight junction proteins in Caco-2 cells treated with OMVs (100 μg/ml) was determined by immunoblots. TEER value of HT-29 cell monolayers incubated with or without OMVs (100 μg/ml) for 24 h was measured (n = 9 independent experiments). HT-29 cell monolayers on transwell membranes were treated with or without OMVs (100 μg/ml) for 24 h. FITC-dextran was added to these cells (top of the membrane). After 2 h, FITC-dextran levels in the bottom chamber wells were detected (n = 9 independent experiments). Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/ml OMVs for 24 h were analyzed by immunoblots. Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull-down assay with anti-Flag antibody-conjugated agarose, followed by immunoblots with indicated antibodies. Data information: Data are means ± SD. Two-tailed unpaired t-test between two groups (A and B), and one-way ANOVA followed by Bonferroni post hoc test for multiple comparisons were utilized for statistical analyses (C, D, F and G). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Download figure Download PowerPoint Blockage of OMV-induced E-cadherin reduction in SNX10-deficient cells is due to the inhibited nuclear localization of Snail/Slug proteins RT–qPCR showed OMV (100 μg/ml) treatment for 24 h did not affect the mRNA levels of SNAI1 (encoding Snail) or SNAI2 (encoding Slug; Fig 2A and B), but dramatically induced the nuclear localization of Snail and Slug (Fig 2C–F). SNX10 deficiency observably inhibited OMV-induced nuclear localization of Snail and Slug (Fig 2C–F). Consistently, overexpression of SNX10 by infecting Caco-2 cells with Ad-SNX10 promoted OMV-induced nuclear localization of Snail and Slug accompanied by more severe downregulation of E-cadherin (Fig 2G). Furthermore, reintroduction of SNX10 reversed the reduction of nuclear Snail and Slug caused by SNX10 deficiency, resulting in the decreased protein expression of E-cadherin (Fig 2H). These results suggest that SNX10 is involved in the maintenance of E-cadherin expression by regulating nuclear localization of Snail and Slug. Figure 2. Blockage of OMV-induced E-cadherin reduction in SNX10-deficient cells is due to the inhibited nuclear localization of Snail/Slug proteins A, B. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/ml) for 24 h. The fold changes in mRNA levels of SNAI1 (encoding Snail) (A) and SNAI2 (encoding Slug) (B) in Caco-2 cells were determined by RT–qPCR. n = 6 independent experiments. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyses. ns, not significant. C. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/ml) for 24 h. Cytoplasmic and nuclear proteins were extracted for Snail and Slug determination by immunoblots. D–F. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/ml) for 24 h. Snail and Slug were assessed by immunofluorescence staining (D) and quantified as the percentage of Snail (E) or Slug (F)-positive nuclei, respectively. Scale bar: 20 μm. n = 3 independent experiments, n = 18 fields analyzed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyses. ***P < 0.001. G. Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/ml OMVs for 24 h. Proteins from nucleus, cytoplasm, and total cell lysates were subjected to immunoblots with the indicated antibodies. H. WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without 100 μg/ml OMVs for 24 h. Proteins from nucleus, cytoplasm, and total cell lysates were subjected to immunoblots with the indicated antibodies. Source data are available online for this figure. Source Data for Figure 2 [embj2021108080-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint SNX10 deficiency impairs Lyn-mediated nuclear localization of Snail/Slug Lyn has emerged as an important upstream regulator of Snail and Slug (Thaper et al, 2017). The pull-down experiment revealed a strong interaction of SNX10-Flag with Lyn (Fig 3A), which was confirmed by co-localization of SNX10-Flag and Lyn (Fig 3B). OMV treatment increased the phosphorylated Lyn (p-Lyn), which was prevented by SNX10 deficiency along with a sustained E-cadherin expression (Fig 3C and D). Overexpression of SNX10 facilitated Lyn phosphorylation induced by OMVs together with a more distinct downregulation of E-cadherin (Fig 3E and F). Rescue of SNX10 eliminated the reduction of Lyn phosphorylation and sustentation of E-cadherin expression in SNX10-deleted Caco-2 cells with OMV treatment (Fig 3G and H). Tolimidone (Toli), a Lyn agonist, prominently increased the level of p-Lyn accompanied by increased nuclear Snail/Slug proteins and decreased E-cadherin in both WT and SNX10 KO cells (Fig 3I). Consistently, Tolimidone treatment also enhanced OMV-induced decrease of TEER and leakage of FITC-dextran into the bottom chambers (Fig 3J and K). These results indicate that the persistence of E-cadherin expression in SNX10-deficient cells resulted from the impaired Lyn-mediated nuclear localization of Snail/Slug. Figure 3. SNX10 deficiency impairs Lyn-mediated nuclear localization of Snail/Slug A. Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull-down assay with anti-Flag antibody-conjugated agarose, followed by immunoblots with the indicated antibodies. B. Representative images of co-staining of SNX10-Flag and Lyn in Caco-2 cells expressing SNX10-Flag. Scale bar: 20 μm. C, D. Levels of p-Lyn, Lyn, E-cadherin, and SNX10 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/ml) for 24 h were determined by immunoblots (C) and quantified by ImageJ software (D) (n = 3 independent experiments). E, F. Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/ml) for 24 h. Lysates were extracted and subjected to immunoblots with the indicated antibodies (E) and quantified by ImageJ software (F) (n = 3 independent experiments). G, H. WT and SNX10 KO Caco-2 cells were transfected with Ad-vector or Ad-SNX10, followed by treatment with or without OMVs (100 μg/ml) for 24 h. Total cell lysates were subjected to immunoblots with the indicated antibodies (G) and quantified by ImageJ software (H) (n = 3 independent experiments). I. Cytoplasmic and nuclear proteins were extracted from WT and SNX10 KO Caco-2 cells treated with or without Tolimidone (10 μM), and the indicated proteins were analyzed by immunoblots. J. TEER value of Caco-2 cell monolayers after incubation with or without Tolimidone (10 μM) for 24 h was analyzed (n = 9 independent experiments). K. Caco-2 cell monolayers on transwell membranes were treated with or without Tolimidone (10 μM) for 24 h. FITC dextran was added to these cells (top of the membrane). After 2 h, FITC-dextran levels in the bottom chamber wells were measured (n = 9 independent experiments). Data information: Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyses. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure. Source Data for Figure 3 [embj2021108080-sup-0006-SDataFig3.pdf] Download figure Download PowerPoint Interaction of SNX10 and caspase-5 is essential for OMV-induced Lyn phosphorylation We then explored whether OMV-induced Lyn phosphorylation was related to caspase-4 or −5 (caspase-11 in mice). Neither OMV treatment nor SNX10 deficiency had significant effects on the expression of caspase-4 or caspase-5 in Caco-2 cells (Fig 4A). Interestingly, caspase-5 rather than caspase-4 was pulled down by Flag-tagged SNX10 (Fig 4B). Co-localization of SNX10-Flag and caspase-5 was further confirmed (Fig 4C). Immunoprecipitation revealed that OMV treatment dramatically induced the interaction between caspase-5 and Lyn, which was blocked by SNX10 deficiency (Fig 4D). Consistently, the co-localization of caspase-5 and Lyn was obviously increased by OMV stimulation, and this was reversed by SNX10 deficiency (Fig 4E and F). Interference of caspase-5 rather than caspase-4 could inhibit OMV-induced Lyn phosphorylation, Snail/Slug nuclear localization, and E-cadherin reduction (Fig 4G). In addition, OMV treatment did not affect the expression of Toll-like receptor 4 (TLR4), and no interaction was found between TLR4 and SNX10 (Fig EV2H and I). These results indicate that interaction of caspase-5 with SNX10 is required for OMV-induced Lyn phosphorylation. To investigate which region is responsible for the interaction of caspase-5 and SNX10, deletion mutagenesis was done as described in our previous study (Zhang et al, 2020). Deletion of the PX domain, but not the C or N terminus region of SNX10 abolished the interaction of caspase-5 and SNX10 (Fig 4H) and inhibited Lyn phosphorylation induced by OMVs (Fig 4I). These results further confirmed the essential role of the interaction of caspase-5 and SNX10 in OMV-induced Lyn phosphorylation. Figure 4. Interaction of SNX10 and caspase-5 is essential for OMV-induced Lyn phosphorylation Lysates from WT and SNX10 KO Caco-2 cells treated with or without 100 μg/ml OMVs for 24 h were analyzed by immunoblots with the indicated antibodies. Lysates from Caco-2 cells transfected with SNX10-Flag were subjected to pull-down assay with anti-Flag antibody-conjugated agarose, followed by immunoblots with the indicated antibodies. Representative images of co-staining of SNX10-Flag and caspase-5 in Caco-2 cells expressing SNX10-Flag. Scale bar: 20 μm. WT and SNX10 KO Caco-2 cells were treated with or without OMVs (100 μg/ml) for 24 h and then subjected to IP with anti-caspase-5 antibody. Representative images of costaining of Lyn and caspase-5 in WT and SNX10 KO Caco-2 cells treated with or without OMVs (100 μg/ml) for 24 h. Scale bar: 20 μm. The co-localization of Lyn and caspase-5 was quantified by ImageJ software. n = 3 independent experiments, n = 18 fields analyzed. Data are means ± SD. One-way ANOVA followed by Bonferroni post hoc test was used for statistical analyses. ***P < 0.001. Cytoplasmic and nuclear proteins were extracted from Caco-2 cells transfected with control siRNA, CASP4 siRNA, or CASP5 siRNA and the indicated proteins were detected by immunoblots. Lysates of Flag-tagged full-length SNX10 or its different truncated mutants transfected with Caco-2 cells were subjected to immunoprecipitation. Caco-2 cells were transfected with Flag-tagged full-l