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Pseudomonas Aeruginosa-Derived Pyocyanin Reduces Adipocyte Differentiation, Body Weight, and Fat Mass As Mechanisms Contributing to T Septic Cachexia

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Pseudomonas Aeruginosa-Derived Pyocyanin Reduces Adipocyte Differentiation, Body Weight, and Fat Mass As Mechanisms Contributing to T Septic Cachexia Food and Chemical Toxicology 130 (2019) 219–230 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Pseudomonas aeruginosa-derived pyocyanin reduces adipocyte differentiation, body weight, and fat mass as mechanisms contributing to T septic cachexia Nika Lariana, Mark Ensora, Sean E. Thatchera, Victoria Englisha, Andrew J. Morrisb, ∗ Arnold Strombergc, Lisa A. Cassisa, a Department of Pharmacology and Nutritional Sciences, University of Kentucky, Lexington, KY, USA b Department of Internal Medicine,University of Kentucky, Lexington, KY, USA c Department of Statistics, University of Kentucky, Lexington, KY, USA ARTICLE INFO ABSTRACT Keywords: Pseudomonas aeruginosa, a leading cause of sepsis, produces pyocyanin, a blue-pigmented virulence factor. Sepsis Pyocyanin is associated with cachexia, but mechanisms are unknown and conventional nutrition approaches are not ef- Pseudomonas aeruginosa fective treatments. Pyocyanin has affinity for the aryl hydrocarbon receptor (AhR), which is expressed on adi- Adipocyte pocytes and regulates adipocyte differentiation. The purpose of this study was to define in vitro and in vivo effects Sepsis of pyocyanin on adipocyte differentiation and body weight regulation as relates to septic cachexia. In 3T3-L1 Cachexia preadipocytes, pyocyanin activated AhR and its downstream marker CYP1a1, and reduced differentiation. Aryl hydrocarbon receptor Administration of pyocyanin to male C57BL/6J mice acutely reduced body temperature with altered locomotion, but caused sustained weight loss. Chronic pyocyanin administration to male and female C57BL/6J mice resulted in sustained reductions in body weight and fat mass, with adipose-specific AhR activation. Pyocyanin-treated male mice had decreased energy expenditure and physical activity, and increased adipose explant lipolysis. In females, pyocyanin caused robust reductions in body weight, adipose-specific AhR activation, and increased expression of inflammatory cytokines in differentiated adipocytes. These results demonstrate that pyocyanin reduces adipocyte differentiation and decreases body weight and fat mass in male and female mice, suggesting that pyocyanin may play a role in septic cachexia. 1. Introduction Furthermore, there is currently no FDA-approved medication that spe- cifically targets sepsis (Kaneki, 2017). Anti-inflammatory strategies are Sepsis is a life-threatening condition characterized by dysregulated used to manage acute sepsis; however, no therapeutic approaches im- host response to infection and multiple organ dysfunction (Singer et al., prove septic cachexia, and wasting of adipose tissue in sepsis has been 2016). Sepsis pathogenesis includes metabolic alterations such as hy- largely unexplored (Callahan and Supinski, 2009; Schefold et al., 2010; percatabolism, muscle wasting, and lipoatrophy (Kaneki, 2017), Crowell et al., 2017). leading to cachexia characterized by decreased body weight, lean body Although adipose cachexia in sepsis is an understudied area, white mass, and fat mass as well as metabolic disruption and systemic in- adipose tissue (WAT) is known to be an important regulator of cachexia flammation (Evans et al., 2008; Argiles et al., 2010; Marques et al., associated with cancer, with increases in WAT apoptosis, lipolysis, 2013). Sepsis remains a leading cause of death in critically ill patients, NLRP3 inflammasome activation, inflammatory cytokine expression, with global estimates of 31.5 million cases resulting in 5.3 million and decreases in adipogenesis and adipocyte size (Neves et al., 2016; deaths annually (Fleischmann et al., 2016). While advances in critical Batista et al., 2013; Franco et al., 2017). Similarly, a recent study found care have increased survival from acute sepsis, a growing number of that septic mice lost more weight than controls and failed to replete patients progress to chronic critical illness that includes septic cachexia, their fat mass even when body weight returned to baseline (Crowell a condition for which nutritional therapies have limited efficacy. et al., 2017). This state of septic adipose cachexia was associated with ∗ Corresponding author. Department of Pharmacology & Nutritional Sciences, Vice President for Research, University of Kentucky, Room 521b, Wethington Building, 900 S. Limestone, Lexington, KY, 40536-0200, USA. E-mail address: [email protected] (L.A. Cassis). https://doi.org/10.1016/j.fct.2019.05.012 Received 25 March 2019; Received in revised form 7 May 2019; Accepted 8 May 2019 Available online 09 May 2019 0278-6915/ © 2019 Published by Elsevier Ltd. N. Larian, et al. Food and Chemical Toxicology 130 (2019) 219–230 increases in inflammation, apoptosis, and lipolysis, and decreases in penicillin/streptomycin, 0.2 μM insulin, 0.5 mM IBMX and 1.0 μM lipogenesis of WAT (Crowell et al., 2017). These results suggest that dexamethasone. Forty-eight hours later (day 2), media was changed to adipose tissue is a site where cachexia from sepsis is manifest, but adipocyte maintenance media containing 90% DMEM, 10% FBS, 1% mechanisms for adipose tissue wasting during acute or chronic sepsis penicillin/streptomycin, and 0.2 μM insulin. Following day 2, media are not well defined. was changed every 48 h (day 4 and day 6) using maintenance media Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bac- containing 90% DMEM, 10% FBS, and 1% penicillin/streptomycin. On terium with a high propensity for antibiotic resistance that commonly day 8, cells were considered fully differentiated adipocytes. colonizes the lung and gut and is a leading cause of sepsis. P. aeruginosa produces pyocyanin, a blue-pigmented toxin that is a major virulence factor for this organism. The toxic and inflammatory properties of 2.2. Pyocyanin treatment in 3T3-L1 cells pyocyanin are due, in part, to its ability to increase production of re- active oxygen species, induce neutrophil apoptosis, and facilitate the Pyocyanin was purchased from Sigma-Aldrich (catalogue number formation of biofilms (Singer et al., 2016; Caldwell et al., 2009; Hall P0046, Sigma-Aldrich, St. Louis, MO) and dissolved in DMSO. For ff fl et al., 2016; Jayaseelan et al., 2014). Pyocyanin possesses both anti- treatment during di erentiation, 3T3-L1 cells were grown to con uence biotic and antifungal properties, which allow it to kill off competitors of in preadipocyte expansion medium in 6-well plates. Two days after fl P. aeruginosa. The majority of studies on pyocyanin have focused on its reaching con uence, cells were treated with 0 (DMSO, 0.02%), 10, 50, μ ability to facilitate the virulence of P. aeruginosa. or 100 M pyocyanin, with fresh media containing vehicle or pyocyanin Recent studies demonstrated that pyocyanin binds the aryl hydro- added on days 0, 2, 4, and 6. On day 8, cells were harvested for RNA carbon receptor (AhR) and results in AhR activation in macrophages extraction and gene expression analysis, or Oil Red O (ORO) staining as ff ff and pneumocytes (Moura-Alves et al., 2014). Pyocyanin has structural a marker of adipocyte di erentiation. For post-di erentiation treatment ff similarity to 2,3,7,8-tetrachlorodibenzodioxin (TCDD), a prototypical of mature adipocytes, fully di erentiated 3T3-L1 adipocytes (day μ ligand of AhR, and similar to TCDD, easily crosses the cell membrane as 8 cells) were treated with 0, 10, 50, or 100 M pyocyanin for 24 h and a low molecular weight zwitterion (Singer et al., 2016). AhR-deficient then harvested for RNA extraction or ORO staining. Cells were scraped μ ff mice are more susceptible to P. aeruginosa lung infection than wild type using 175 L of RNA lysis bu er (Maxwell, catalogue # MC501C, − mice, indicating a role of AhR in resistance against P. aeruginosa that is Promega, Madison, WI), and stored at 80 °C. Oil Red O staining was potentially related to clearance of pyocyanin (Moura-Alves et al., performed on separate plates on day 8 or 9 using the ORO Staining Kit 2014). In its classical role in drug and xenobiotic metabolism, AhR from Lifeline Cell Technology (catalogue number LL-0052, Frederick, binds to xenobiotic response elements in the promoter regions of genes MD). Stained cells were imaged at 100x. After imaging, water was re- μ such as cytochrome P450 CYP1a1, CYP1a2, and CYP1b1 (Fujii- moved from the stained cells, 500 L of isopropanol was added to each μ Kuriyama and Mimura, 2005). AhR exhibits promiscuous ligand well for 1 min to extract ORO from the cells, and 200 L was removed binding to many drug and xenobiotic ligands, including persistent or- from each well and absorbance measured in a 96-well plate at 510 nm ganic pollutants (POPs), such as TCDD and coplanar polychlorinated in a spectrophotometer. biphenyls (PCBs) (Singer et al., 2016). AhR has been implicated in the ff ff di erentiation and functional regulation of several di erent cell types, 2.3. Animal treatments and sample collection including adipocytes. The functions of adipocyte AhR are of particular interest due to the lipophilicity of AhR ligands, including POPs, which All experiments met the approval of the Animal Care and Use results in their accumulation in adipose tissue in close proximity to Committee of the University of Kentucky. Mice were housed in micro- adipocyte AhR (La Merrill et al., 2013). isolator, polystyrene cages with a 14 h light/10 h dark cycle. Room Pyocyanin has been detected in sputum, wounds, and urine in temperature was at a range of 20–21 °C, and humidity ranged from 30 μ concentrations up to 130 M(Wilson et al., 1988; Cruickshank and to 70%. Male and female mice C57BL/6J mice were purchased from Lowbury, 1953). While there are several studies demonstrating pyo- The Jackson Laboratories (Bar Harbor, MA) and aged 6 months to have cyanin promotes the virulence of P. aeruginosa, levels of pyocyanin sufficient fat mass to assess adipose cachexia. All mice were fed stan- released by the bacteria may also regulate the function of mammalian dard murine diet (Harlan Teklad 2918 Global Rodent Diet, irradiated; cells, including activation of AhR in adipocytes, a cell type potentially Harlan Laboratories, Indianapolis, IN) ad libitum for the duration of the fi ff related to cachexia of chronic sepsis.
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