Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Article25 March 2021Open Access Source DataTransparent process Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner Anthony Lagnado Anthony Lagnado Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Jack Leslie Jack Leslie Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Marie-Helene Ruchaud-Sparagano Marie-Helene Ruchaud-Sparagano Translational Research Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Stella Victorelli Stella Victorelli Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Petra Hirsova Petra Hirsova Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Mikolaj Ogrodnik Mikolaj Ogrodnik Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Amy L Collins Amy L Collins Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Maria Grazia Vizioli Maria Grazia Vizioli Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Leena Habiballa Leena Habiballa Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Gabriele Saretzki Gabriele Saretzki Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Shane A Evans Shane A Evans Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Hanna Salmonowicz Hanna Salmonowicz Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Adam Hruby Adam Hruby Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Daniel Geh Daniel Geh Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Kevin D Pavelko Kevin D Pavelko Department of Immunology and Immune Monitoring Core, Mayo Clinic, Rochester, MN, USA Search for more papers by this author David Dolan David Dolan Computational Biology Unit, University of Bergen, Bergen, Norway Search for more papers by this author Helen L Reeves Helen L Reeves Newcastle University Translational and Clinical Research Institute, Liver Unit, Newcastle upon Tyne NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Sushma Grellscheid Sushma Grellscheid Computational Biology Unit, University of Bergen, Bergen, Norway Department of Biosciences, Durham University, Durham, UK Search for more papers by this author Colin H Wilson Colin H Wilson Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Sanjay Pandanaboyana Sanjay Pandanaboyana Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Madison Doolittle Madison Doolittle Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Thomas von Zglinicki Thomas von Zglinicki Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Fiona Oakley Fiona Oakley Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Suchira Gallage Suchira Gallage Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Caroline L Wilson Caroline L Wilson Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Jodie Birch Jodie Birch orcid.org/0000-0003-1727-1125 Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Bernadette Carroll Bernadette Carroll School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author James Chapman James Chapman Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Mathias Heikenwalder Mathias Heikenwalder Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Nicola Neretti Nicola Neretti Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Sundeep Khosla Sundeep Khosla Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Claudio Akio Masuda Claudio Akio Masuda Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Search for more papers by this author Tamar Tchkonia Tamar Tchkonia orcid.org/0000-0003-4623-7145 Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author James L Kirkland James L Kirkland Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Diana Jurk Diana Jurk Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Derek A Mann Corresponding Author Derek A Mann [email protected] orcid.org/0000-0003-0950-243X Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author João F Passos Corresponding Author João F Passos [email protected] orcid.org/0000-0001-8765-1890 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Anthony Lagnado Anthony Lagnado Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Jack Leslie Jack Leslie Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Marie-Helene Ruchaud-Sparagano Marie-Helene Ruchaud-Sparagano Translational Research Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Stella Victorelli Stella Victorelli Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Petra Hirsova Petra Hirsova Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Mikolaj Ogrodnik Mikolaj Ogrodnik Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Amy L Collins Amy L Collins Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Maria Grazia Vizioli Maria Grazia Vizioli Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Leena Habiballa Leena Habiballa Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Gabriele Saretzki Gabriele Saretzki Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Shane A Evans Shane A Evans Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Hanna Salmonowicz Hanna Salmonowicz Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Adam Hruby Adam Hruby Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Daniel Geh Daniel Geh Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Kevin D Pavelko Kevin D Pavelko Department of Immunology and Immune Monitoring Core, Mayo Clinic, Rochester, MN, USA Search for more papers by this author David Dolan David Dolan Computational Biology Unit, University of Bergen, Bergen, Norway Search for more papers by this author Helen L Reeves Helen L Reeves Newcastle University Translational and Clinical Research Institute, Liver Unit, Newcastle upon Tyne NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Sushma Grellscheid Sushma Grellscheid Computational Biology Unit, University of Bergen, Bergen, Norway Department of Biosciences, Durham University, Durham, UK Search for more papers by this author Colin H Wilson Colin H Wilson Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Sanjay Pandanaboyana Sanjay Pandanaboyana Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK Search for more papers by this author Madison Doolittle Madison Doolittle Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Thomas von Zglinicki Thomas von Zglinicki Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Fiona Oakley Fiona Oakley Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Suchira Gallage Suchira Gallage Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Caroline L Wilson Caroline L Wilson Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Jodie Birch Jodie Birch orcid.org/0000-0003-1727-1125 Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Bernadette Carroll Bernadette Carroll School of Biochemistry, University of Bristol, Bristol, UK Search for more papers by this author James Chapman James Chapman Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author Mathias Heikenwalder Mathias Heikenwalder Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Nicola Neretti Nicola Neretti Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA Search for more papers by this author Sundeep Khosla Sundeep Khosla Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Claudio Akio Masuda Claudio Akio Masuda Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil Search for more papers by this author Tamar Tchkonia Tamar Tchkonia orcid.org/0000-0003-4623-7145 Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author James L Kirkland James L Kirkland Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Diana Jurk Diana Jurk Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Derek A Mann Corresponding Author Derek A Mann [email protected] orcid.org/0000-0003-0950-243X Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK Search for more papers by this author João F Passos Corresponding Author João F Passos [email protected] orcid.org/0000-0001-8765-1890 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA Search for more papers by this author Author Information Anthony Lagnado1,2, Jack Leslie3, Marie-Helene Ruchaud-Sparagano4, Stella Victorelli1,2, Petra Hirsova5, Mikolaj Ogrodnik1,2, Amy L Collins3, Maria Grazia Vizioli1,2, Leena Habiballa1,2,6, Gabriele Saretzki7, Shane A Evans8, Hanna Salmonowicz1,2,7, Adam Hruby1,2, Daniel Geh3, Kevin D Pavelko9, David Dolan10, Helen L Reeves11, Sushma Grellscheid10,12, Colin H Wilson13, Sanjay Pandanaboyana13, Madison Doolittle2, Thomas Zglinicki7, Fiona Oakley3, Suchira Gallage14, Caroline L Wilson3, Jodie Birch7, Bernadette Carroll15, James Chapman7, Mathias Heikenwalder14, Nicola Neretti8, Sundeep Khosla2, Claudio Akio Masuda1,2,16, Tamar Tchkonia2, James L Kirkland2, Diana Jurk1,2, Derek A Mann *,3 and João F Passos *,1,2 1Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA 2Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA 3Newcastle Fibrosis Research Group, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK 4Translational Research Institute, Newcastle University, Newcastle upon Tyne, UK 5Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA 6NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK 7Ageing Research Laboratories, Faculty of Medical Sciences, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK 8Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA 9Department of Immunology and Immune Monitoring Core, Mayo Clinic, Rochester, MN, USA 10Computational Biology Unit, University of Bergen, Bergen, Norway 11Newcastle University Translational and Clinical Research Institute, Liver Unit, Newcastle upon Tyne NHS Foundation Trust, Newcastle upon Tyne, UK 12Department of Biosciences, Durham University, Durham, UK 13Department of Hepatobiliary Surgery, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK 14Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany 15School of Biochemistry, University of Bristol, Bristol, UK 16Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil *Corresponding author. Tel: +44 191 208351; E-mail: [email protected] *Corresponding author. Tel: +1 507 293 9785; E-mail: [email protected] The EMBO Journal (2021)40:e106048https://doi.org/10.15252/embj.2020106048 See also: MSJ Burbano et al (May 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 ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease. SYNOPSIS Whether infiltrating immune cells impact on tissue viability during inflammatory response is poorly characterized. Here, activated neutrophils are shown to induce ROS-dependent acute senescence in adjacent naïve cells, raising the possibility of intercellular senescence crosstalk during ageing and age-related pathologies. Neutrophils induce paracrine senescence in neighbouring cells via ROS-dependent telomere dysfunction. Neutrophil clearance prevents senescence and telomere dysfunction in a model of acute liver injury. Senescent cells recruit neutrophils via the senescence-associated secretory phenotype (SASP) in the aged liver. Introduction Neutrophils play a critical role in the process of microbial killing, which they achieve through the actions of a variety of neutrophil-expressed microbicidal factors including proteolytic enzymes and reactive oxygen species (ROS). In addition, neutrophils are rapidly recruited to sites of tissue damage in response to damage-associated molecular patterns (DAMPs) and their induction of neutrophil chemoattractants which include C-X-C chemokines, calprotectins, and lipid mediators important in the sterile immune response to tissue damage (Peiseler & Kubes, 2019). In acute resolving injuries, neutrophils stimulate wound repair by removing injured cells, clearing cellular remnants, and releasing growth factors and pro-angiogenic molecules. In this context, neutrophils are important for resolution of inflammation and enabling the subsequent regenerative response. Where damage is chronic, neutrophils fail to effectively resolve inflammation and can instead exacerbate cellular stress and injury through their release of matrix metalloproteinases and ROS. Less well understood is the degree to which neutrophilic inflammation may induce changes in tissues that impact on longer-term health and susceptibility to disease. Cellular senescence refers to an irreversible cell cycle arrest, which can be triggered by a number of stressors, including oxidative stress and telomere dysfunction among many others (Gorgoulis et al, 2019). Senescence is associated with a number of distinctive phenotypes, including the development of a pro-inflammatory response, commonly known as the senescence-associated secretory phenotype (SASP). The SASP is thought to communicate with the immune system and facilitate immune surveillance, stimulating the clearance of senescent or pre-malignant cells (Ovadya et al, 2018). However, chronic exposure to the SASP can lead to age-associated tissue dysfunction (Jurk et al, 2014; Xu et al, 2018). Consistent with this idea, senescent cells have been shown to accumulate in many tissues with age and at etiological sites in multiple chronic diseases (Ogrodnik et al, 2019a). Importantly, clearance of senescent cells either genetically or by using "senolytic" drugs has been shown to alleviate the onset of a number of pathologies during aging and disease (Zhu et al, 2015; Baker et al, 2016; Farr et al, 2017; Ogrodnik et al, 2017; Bussian et al, 2018; Musi et al, 2018; Anderson et al, 2019; Ogrodnik et al, 2019b). Senescent cells have been shown to accumulate during aging in murine liver (Wang et al, 2009; Hewitt et al, 2012) and in the context of age-related liver disease in humans (Ogrodnik et al, 2017). The pathophysiological impacts of senescence are exemplified by the aging liver where regeneration and capacity to overcome injuries wanes in older animals and humans. Consequently, aging is associated with a higher incidence of acute liver failure and chronic liver disease. Consistent with a role for senescent cells in liver pathology, it has been shown that clearance of senescent cells reduces liver steatosis in aged, obese, and diabetic mice (Ogrodnik et al, 2017). However, the underlying mechanisms driving senescence in the liver are not completely understood. Here, we investigated the hypothesis that neutrophils recruited from the circulation in response to damage can act as drivers of cellular senescence and as such contribute to organ dysfunction during aging and disease. We first observed that short-term co-culture of neutrophils with human primary fibroblasts accelerates the rate of telomere shortening and induces premature replicative senescence in the latter, a process which is dependent on ROS and requires cell-to-cell contact. Importantly, we found that ectopic expression of the catalytic subunit of telomerase (hTERT) prevents neutrophil-induced telomere shortening and premature senescence. Ex vivo studies involving precision-cut liver slices (PCLS) obtained from human subjects confirm that hepatic infiltration by neutrophils induces telomere dysfunction and senescence markers in liver hepatocytes. Further investigation involving mouse PCLS showed that neutrophils from transgenic mice overexpressing human catalase in mitochondria (MCAT) did not induce paracrine telomere dysfunction or expression of p21 as opposed to wild-type neutrophils. In vivo, we found that depletion of neutrophils reduces telomere dysfunction and senescence in a model of acute liver injury. Finally, we observed that during liver aging, neutrophils accumulate in the hepatic parenchyma and are found in close proximity to hepatocytes containing dysfunctional telomeres. Genetic clearance of p16Ink4a-positive cells in aged INK-ATTAC mice reduces the number of neutrophil infiltrates. In contrast, stimulation of neutrophil recruitment to the liver enhances hepatocyte senescence. Altogether, our findings suggest that neutrophils are both recruited by senescent cells and can be drivers of senescence. Therapies which counteract neutrophil-induced senescence may be beneficial during aging and age-related disease. Results 1-Neutrophils induce paracrine senescence In order to test the hypothesis that neutrophils contribute to cellular senescence, we began by co-culturing human MRC5 fibroblasts for 3 days with freshly isolated neutrophils from similarly aged healthy donors (Figs 1A, and EV1A and B). To avoid the confounding factor of neutrophil cell-death (which we observed to occur after 24 h), we replaced neutrophils daily and cultured them at 3% O2. In order to test the role of ROS as a mediator of neutrophil-induced senescence, neutrophils and fibroblasts were co-cultured in the presence or absence of extracellular recombinant catalase for 3 days. After 3 days of co-culture, neutrophils were removed (as well as the catalase) and MRC5 fibroblasts were passaged until they reached replicative senescence (Fig 1A). Figure 1. Neutrophils induce paracrine senescence A. Schematic depicting experimental design. B. Effect of neutrophils and recombinant catalase on population doublings (data are from three independent cultures are shown). Blue arrow indicates time point in which neutrophils were added to culture for consecutive 3 days. C. % Δ Population Doublings (PD) following neutrophil co-culture. D. Representative images of Ki67 and p16INK4A immunofluorescence and SA-β-Gal staining. E. % of Ki67-positive cells (28 days after co-culture). F. % of p16INK4A-positive cells (28 days after co-culture). G. % of SA-β-Gal-positive cells (28 days after co-culture). H. Western blotting for p21 and quantification. I. MitoSOX fluorescence. J. Heat map depicting expression of 48 human cytokines in conditioned media at 8 and 28 days following co-culture. Data information: Data are mean ± SEM of three independent experiments. Statistical analysis was performed using one-way ANOVA (Holm–Sidak method) for multiple comparisons and two-tailed t-test for single comparisons. **P < 0.01; *P < 0.04. Source data are available online for this figure. Source Data for Figure 1 [embj2020106048-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Neutrophils induce paracrine senescence A. Age of neutrophil healthy donors used in independent co-culture experiments. Data are mean ± SEM of n = 3. B. Table summarizing gender and donor age as well as % of neutrophil purity. C. Growth curves comparing population doublings in controls and MRC5 co-cultured with non-primed neutrophils. Blue arrow indicates time point in which neutrophils were added to culture for consecutive 3 days. D. Superoxide anion levels in neutrophils following treatment with different concentrations of LPS. The highest increase was observed after 1 h treatment with 100 ng/ml. Data are mean ± SEM of n = 5 independent experiments. E. Representative Western blot of p15 and tubulin expression 8 and 28 days after neutrophil co-culture. F. Quantification of p15 expression relative to tubulin. Data are mean ± SEM of three independent experiments. G. Expression of selected secreted factors (from cytokine array depicted in Figure 1j) at 8 and 28 days after neutrophil co-culture. Data are mean ± SEM of three independent experiments. Data information: Statistical analysis was performed using one-way ANOVA (Holm–Sidak method) for multiple comparisons and two-tailed t-test for single comparisons. **P < 0.01; *P < 0.04. Download figure Download PowerPoint Growth curves revealed that co-culture with neutrophils did not acutely affect the proliferation rate of MRC5 fibroblasts; however, instead it led to a premature growth arrest at later stages in culture, which could be prevented by pre-incubation with catalase (Fig 1B and C). We also observed that when neutrophils were LPS-primed the effects on replicative lifespan were more pronounced (Figs 1C and EV1C), which may be due to neutrophil activation and an associated stimulation of ROS (Fig EV1D). Analysis of the proliferation marker Ki67 after 28 days in culture confirmed that fibroblasts co-cultured with neutrophils displayed reduced proliferation (Fig 1D and E). In contrast, senescent markers p16INK4A (Fig 1D and F) and SA-β-Gal (Fig 1D and G) were elevated in co-cultured fibroblasts at 28 days and these effects were prevented by treatment with catalase. p21 expression showed a trend for an increase with activated neutrophils and reduction with catalase treatment (Fig 1H). We observed in one experiment that neutrophil co-culture led to an increase in p15 expression at the 28-day time point and a reduction with catalase (Fig EV1E) but this finding was not reproducible in independent experiments (Fig EV1F). Enhanced mitochondrial ROS generation is both a characteristic of senescent cells (Correia-Melo et al, 2016), but also can be a driver of senescence (Passos et al, 2007). We observed that fibroblasts that had been co-cultured with neutrophils displayed increased MitoSOX fluorescence, which was significantly reduced in catalase-treated