Zinc inhibits lethal inflammatory shock by preventing microbe‐induced interferon signature in intestinal epithelium
2020; Springer Nature; Volume: 12; Issue: 10 Linguagem: Inglês
10.15252/emmm.201911917
ISSN1757-4684
AutoresJolien Souffriau, Steven Timmermans, Tineke Vanderhaeghen, Charlotte Wallaeys, Kelly Van Looveren, Lindsy Aelbrecht, Sylviane Dewaele, Jolien Vandewalle, Evy Goossens, Serge Verbanck, Filip Boyen, Melanie Eggermont, Lindsey De Commer, Riet De Rycke, Michiel De Bruyne, Raúl Y. Tito, Marlies Ballegeer, Sofie Vandevyver, Tiago R. Velho, Luís F. Moita, Tino Hochepied, Karolien De Bosscher, Jeroen Raes, Filip Van Immerseel, Rudi Beyaert, Claude Libert,
Tópico(s)Immune responses and vaccinations
ResumoArticle11 September 2020Open Access Transparent process Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium Jolien Souffriau Jolien Souffriau Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Steven Timmermans Steven Timmermans Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tineke Vanderhaeghen Tineke Vanderhaeghen Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Charlotte Wallaeys Charlotte Wallaeys Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Kelly Van Looveren Kelly Van Looveren Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsy Aelbrecht Lindsy Aelbrecht Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sylviane Dewaele Sylviane Dewaele Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Vandewalle Jolien Vandewalle orcid.org/0000-0003-1844-6476 Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Evy Goossens Evy Goossens Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Serge Verbanck Serge Verbanck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Filip Boyen Filip Boyen Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Melanie Eggermont Melanie Eggermont Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsey De Commer Lindsey De Commer Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Michiel De Bruyne Michiel De Bruyne orcid.org/0000-0002-1276-1857 Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Raul Tito Raul Tito Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Marlies Ballegeer Marlies Ballegeer Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sofie Vandevyver Sofie Vandevyver Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tiago Velho Tiago Velho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Luis Ferreira Moita Luis Ferreira Moita Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Tino Hochepied Tino Hochepied Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Karolien De Bosscher Karolien De Bosscher VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Filip Van Immerseel Filip Van Immerseel Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Rudi Beyaert Rudi Beyaert orcid.org/0000-0002-5704-582X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Corresponding Author Claude Libert [email protected] orcid.org/0000-0001-6408-036X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Souffriau Jolien Souffriau Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Steven Timmermans Steven Timmermans Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tineke Vanderhaeghen Tineke Vanderhaeghen Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Charlotte Wallaeys Charlotte Wallaeys Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Kelly Van Looveren Kelly Van Looveren Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsy Aelbrecht Lindsy Aelbrecht Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sylviane Dewaele Sylviane Dewaele Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Vandewalle Jolien Vandewalle orcid.org/0000-0003-1844-6476 Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Evy Goossens Evy Goossens Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Serge Verbanck Serge Verbanck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Filip Boyen Filip Boyen Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Melanie Eggermont Melanie Eggermont Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsey De Commer Lindsey De Commer Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Michiel De Bruyne Michiel De Bruyne orcid.org/0000-0002-1276-1857 Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Raul Tito Raul Tito Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Marlies Ballegeer Marlies Ballegeer Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sofie Vandevyver Sofie Vandevyver Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tiago Velho Tiago Velho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Luis Ferreira Moita Luis Ferreira Moita Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Tino Hochepied Tino Hochepied Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Karolien De Bosscher Karolien De Bosscher VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Filip Van Immerseel Filip Van Immerseel Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Rudi Beyaert Rudi Beyaert orcid.org/0000-0002-5704-582X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Corresponding Author Claude Libert [email protected] orcid.org/0000-0001-6408-036X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Author Information Jolien Souffriau1,2,‡, Steven Timmermans1,2,‡, Tineke Vanderhaeghen1,2, Charlotte Wallaeys1,2, Kelly Van Looveren1,2, Lindsy Aelbrecht1,2, Sylviane Dewaele1,2, Jolien Vandewalle1,2, Evy Goossens3, Serge Verbanck3, Filip Boyen3, Melanie Eggermont1,2, Lindsey De Commer4,5, Riet De Rycke6,7, Michiel De Bruyne6,7, Raul Tito4,5, Marlies Ballegeer1,2, Sofie Vandevyver1,2, Tiago Velho8, Luis Ferreira Moita8, Tino Hochepied1,2, Karolien De Bosscher9,10, Jeroen Raes4,5, Filip Van Immerseel3, Rudi Beyaert1,2 and Claude Libert *,1,2 1Center for Inflammation Research, VIB, Ghent, Belgium 2Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 3Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 4Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium 5VIB-KU Leuven Center for Microbiology, Leuven, Belgium 6Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium 7VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium 8Instituto Gulbenkian de Ciência, Oeiras, Portugal 9VIB Center for Medical Biotechnology, Ghent, Belgium 10Department of Biochemistry, Ghent University, Ghent, Belgium ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +32 9 3313700; E-mail: [email protected] EMBO Mol Med (2020)12:e11917https://doi.org/10.15252/emmm.201911917 The authors want to dedicate this paper to the memory of Prof. Walter Fiers (1931–2019), who passed away on 31 July 2019, and who was mentor and strong supporter of this project and research team. 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 The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system. Synopsis This study provides genetic and biochemical evidence that the intestinal health promoting effects of zinc supplementation is based on a specific, direct effect on members of the microbial community in the ileum. The cytokine TNF has dramatic effects on the viability of ileal crypt Paneth cells The TNF effect depends on expression of Interferon Stimulated Response Element (ISRE) and Interferon Regulatory Factor (IRF) genes which are induced by gut microbes By killing bacteria such as several Staphylococci species, zinc reduces this ISRE/IRF expression, preventing Paneth cell death during inflammation Since glucocorticoids, like dexamethasone, reduce ISRE/IRF on the transcriptional level, zinc works in concert with such steroids The paper explained Problem Zinc has therapeutic effects, mainly in intestinal infections and diarrhea. The mechanism of this zinc effect is poorly known. Zinc also protects against intestinal effects induced by the cytokine tumor necrosis factor (TNF), which plays a key role in Crohn's disease. Since zinc was previously shown to protect in the TNF model in mice, we applied this model to investigate zinc's mode of action. Results By applying RNA-seq in ileum biopsies in mice treated with water or ZnSO4 in the drinking water, we found that zinc causes strong downregulation of a large group of genes, known as ISRE/IRF genes. These are thought to (confirmed here) be induced in intestinal epithelium by gut microbes. Zinc lowers these genes, by actively and directly modulating the composition of the gut microbes, and our data suggest that mainly Staphylococci would be targeted by zinc. The reduction in ISRE/IRF genes is important, because several of these genes play an important role in killing certain epithelial cells (known as Paneth cells), when TNF is present. Because of the effects of zinc on bacteria and ISRE/IRF genes in these cells, they resist killing by TNF. How this leads to survival of the animals is not yet clear, but since zinc also reduces the escape of bacteria from the gut to other organs, the impact of zinc on the gut microbes or on the Paneth cell's death appears responsible as well. Mice with extremely high ISRE/IRF gene expression (e.g., because they have no adrenals) or extremely low ISRE/IRF genes, because they have no gut microbes, are not susceptible to the beneficial effects of zinc. Impact Now that the mechanism of zinc is unfolded, more precise zinc targeting or zinc replacing strategies in intestinal diseases where TNF is implicated (Crohn's disease) can be considered. Introduction The cytokine TNF is a central player in inflammatory diseases and infections. TNFR1 (Puimege et al, 2014) is the main pathogenic TNF receptor and is strongly stimulated by acute and chronic expressed TNF. The systemic effects of TNF have been studied in detail. It is striking that the intestinal epithelium is sensitive to TNF-induced damage: Transgenic TNF overexpression, via a myriad of mechanisms, clearly leads to inflammatory bowel disease and/or arthritis, and injection of TNF in mammals has shown that intestinal toxicity is a major dose-limiting issue (Piguet et al, 1998; Kontoyiannis et al, 1999). Cell death of IECs as well as reduced expression of tight junctions appears essential in the increase in gut permeability induced by TNF. This reduced barrier between the homeostatic body and the gut luminal content is crucial (Van Hauwermeiren et al, 2015). In the lumen, the microbiota, consisting of many billions of microbes, predominantly bacteria, is present (Vrancken et al, 2019). Most of these are harmless if they remain in the gut lumen. TNF has been shown to lead to outflow of bacteria from the lumen and colonization of draining mesenteric lymph nodes (MLNs) and spleen (Van Hauwermeiren et al, 2015). Hence, broad-spectrum antibiotics confer significant protection against TNF-induced lethal systemic inflammatory response syndrome (SIRS; Van Hauwermeiren et al, 2015). Since IEC-specific depletion of TNFR1 leads to a similar protective effect as an antibiotic treatment (Van Hauwermeiren et al, 2013), it appears conceivable that TNF, via TNFR1, induces IEC damage, followed by a bacterial contamination of organs. The damage that is caused by TNF to IECs is incompletely understood, but is of utmost importance (Van Assche et al, 2010), as it may play a role in Crohn's disease. TNF induces cell death of IECs, shrinkage of villi and erosion (Piguet et al, 1998), and death of goblet cells as well as Paneth cells (Van Hauwermeiren et al, 2015). Glucocorticoids (GCs; e.g., the synthetic dexamethasone (Dex)), which function by binding to the glucocorticoid receptor (GR), confer protection against Crohn's diseases (Van Assche et al, 2010) and against TNF-induced intestinal damage and lethal shock, when given prior to TNF. From a therapeutic viewpoint, this preventive therapy is not relevant, but this effect can help elucidating essential mechanisms. It was also shown that removal of adrenals which produce endogenous GCs drastically sensitizes to TNF and that GR mutant mice have sensitized IECs toward TNF (Ballegeer et al, 2018). The mechanism by which GCs/GR dampens TNF effects in the gut is thought to relate to the gut commensal flora: The microbes were recently shown to chronically induce, in the ileum, interferon-stimulated response element (ISRE) genes, and interferon regulatory factor (IRF) genes, some of which are involved in necroptosis (Ripk3, Mlkl, and Zbp1; Ballegeer et al, 2018). When GCs or GR was absent, the IECs lost control over these genes, and hence, these genes underwent high expression. Under such conditions, low doses of TNF sufficed to cause induction of necroptosis followed by death of the animals. Interestingly, this necroptosis was confined to crypt cells known as Paneth cells (Ballegeer et al, 2018). In humans, 25% of the global mortalities are caused by microbial infections. Many of these problems are due to the development of resistance against antibiotics. Investigation of interesting alternative antimicrobial strategies has become mandatory. Zinc has a huge potential in this respect, since zinc has been shown to increase the resistance of vulnerable patients (children, elderly, underfed) against diarrhea and gastrointestinal infections (Souffriau & Libert, 2018). We have previously published that a pretreatment of mice with ZnSO4 in the drinking water for 1 week leads to strong protection against TNF-induced SIRS (Waelput et al, 2001; Van Molle et al, 2007), but the mechanism of protection remained unclear. Zinc is essential for a healthy intestinal homeostasis and is known to be essential for Paneth cell function and survival (Jouppila et al, 1976; Podany et al, 2016). In these cells, zinc is actively adsorbed by transporters and shuttled into the secretory vesicles, which contain anti-bacterial defensins, some of which are activated by zinc-dependent proteases, the most important of which being matrix metalloproteinase-7 (MMP7; Wilson et al, 1999). Zinc has been shown to activate a specific transcription factor, metal transcription factor 1 (MTF1) by direct binding (Gunther et al, 2012), leading to numerous transcriptional changes, e.g., induction of a zinc transporter known as ZnT2 and coded by Slc30a2. ZnT2 is of importance in the intracellular zinc transport in Paneth cells (Podany et al, 2016). Zn has also been shown to have profound effects on the composition of the gut microbiota (Li et al, 2016; Zackular et al, 2016), but whether this is via direct anti-bacterial effects, or via Paneth cells or other mechanisms is not clear. In this paper, we studied the mechanism of protection of zinc against TNF-induced lethal SIRS. We studied the cross-talk of zinc with GCs/GR and found that zinc is unable to protect against TNF when GR is mutated or absent in the IECs or when corticosterone is absent. Zinc appeared to reduce the ISRE/IRF-dependent genes in the IECs, thereby protecting these cells (particularly Paneth cells) against TNF-induced necroptosis and bacterial influx from the gut lumen into the system. The mechanism by which zinc reduces ISRE/IRF genes was investigated and appears to relate to direct anti-bacterial effects of zinc against certain bacterial taxa, such as Staphylococcus sciuri and Staphylococcus nepalensis. The elucidation of the action mechanism of zinc may lead to better understanding of zinc's effects in the treatment of intestinal infections and diarrhea in humans and farm animals. Results Glucocorticoids and GR dimers play an essential role in the zinc protection against TNF-induced lethality Endogenous and synthetic GCs and their receptor GR are crucial for the survival of mice against TNF-induced SIRS (Vandevyver et al, 2012; Ballegeer et al, 2018). We investigated whether GCs are involved in the zinc-induced protection against TNF. We used adrenalectomized (Adx) mice, which are unable to produce corticosterone. Adx mice were pretreated for 7 days with 25 mM ZnSO4 via the drinking water, a protocol that was published to confer optimal protection in the TNF model in normal mice (Waelput et al, 2001). In contrast to naïve C57BL/6J mice, Adx mice were not protected by ZnSO4 against a lethal intraperitoneal (i.p.) injection of TNF (Fig 1A). Serum corticosterone levels were indeed very low in Adx mice and were not increased upon 1-week treatment of mice with ZnSO4 (Fig 1B). GRDim mice, which express a point-mutant GR protein that forms less efficient GR homodimers and DNA interaction, were previously found to be extremely sensitive for TNF-induced SIRS (Reichardt et al, 1998; Van Bogaert et al, 2011; Vandevyver et al, 2012; Ballegeer et al, 2018). ZnSO4 was unable to confer protection against TNF in these mice (Fig 1C). Increasing the zinc dose from 25 to 75 mM (the maximal tolerated dose for mice) for a week had no protective effect in GRDim mice either. Therefore, we conclude that the zinc protection against TNF requires the presence of corticosterone and functional GR dimers, i.e., involves GR-mediated gene regulation. Figure 1. ZnSO4 fails to protect against a lethal TNF injection in Adx, GRDim, and GRVillKO mice A. Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 μg (naïve) or 2 μg (Adx) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of three experiments. B. Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N = 6 per group). C. Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 50 μg (GRWT) or 12.5 μg (GRDim) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 50 h after TNF injection. Results represent combined data of two experiments. D, E. Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL/6J naïve and Adx mice (D, N = 3–6), or GRWT and GRDim mice (E, N = 6–9). F. Agtpbp1 gene expression measured with RT–qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water (N = 3 per group). G. Survival curves of GRfl/fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 35 μg TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 150 h after TNF injection. Results represent combined data of six experiments. H. Mouse Nr3c1 gene expression measured with RT–qPCR in the intestinal epithelial cells (IECs) of GRfl/fl and GRVillKO mice (N = 3–6). Hprt and Villin were used as housekeeping genes. I. Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl/fl and GRVillKO mice (N = 9 per group). J. Number of genes differentially expressed (up and down) by zinc (LFC > 0.8 and P < 0.05) measured by RNA sequencing in the ileum and liver of C57BL/6J mice treated with 25 mM ZnSO4 in the drinking water for 7 days (N = 3). Data information: For the survival curves, P-values were analyzed with a chi-square test. In (B, D, E, F, H, I), the data are shown as mean ± SD. P-values were analyzed with Student's t-test (unpaired, two-tailed) on the log-transformed data in (B, H) and with a two-way ANOVA followed by a Tukey multiple comparisons test in (D, E, F, I). Significant expression of genes in the RNA sequencing was assessed with a Wald test with negative binomial distribution in DESEQ2. Download figure Download PowerPoint We further confirmed that the complete lack of protection of zinc in Adx and GRDim mice is not reflected in a lack of zinc uptake by these mice, since blood zinc levels 1 week after 25 mM ZnSO4 were equally increased in these as in control mice (Fig 1D and E). Blood zinc levels fluctuate in mammals between 75 and 125 μg/dl (Reyes, 1996). In our studies, 1 week of treatment increases these levels to 200–300 μg/dl. Zinc is able to upregulate the mRNA expression of several genes (see further Fig 2) in the ileum of mice, and several key control genes, e.g., Agtpbp1, were found to be perfectly induced by zinc in GRDim as in GRWT mice, illustrating that zinc in these GRDim mice does have biological activities (Fig 1F). Figure 2. Transcriptional effects of ZnSO4 in the ileum and the role of antibiotics and germ-free aspects A–C. RNA sequencing on ileum samples of C57BL/6J mice treated with water or 25 mM ZnSO4 water for 7 days and then injected i.p. with 200 μg Dex (Rapidexon) solved in 200 μl PBS, or with PBS only. 2 h after injection, the ileum was isolated (N = 3 per group). Overlap between genes (A) upregulated or (B) downregulated by ZnSO4 (Zn) or Dex (LFC > 0.8, P < 0.05). (C) Scheme of numbers of genes upregulated in ZnSO4, Dex, or ZnSO4+Dex-treated mice (LFC > 0.8, P < 0.05). Significant expression of genes in the RNA sequencing was assessed with a Wald test with negative binomial distribution in DESEQ2. D. Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC > 0.8, P < 0.05 are considered, while these genes with P < 0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient was calculated by GraphPad Prism. E. C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week of 25 mM ZnSO4 in the drinking water. During this week, AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 μg TNF solved in sterile PBS and survival recorded. Combined data of two experiments. F. Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N = 5–8). G,H. Mice housed in germ-free (GF) or specific pathogen-free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 μg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of two experiments. (H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N = 3/4 per group). I. Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week, and 750 μl of this IS was injected i.p. in C57BL/6J GF mice. Data information: For the survival curves, P-values were analyzed with a chi-square test. In (F) and (H), data are shown as mean ± SD and P-values were analyz
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