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... expression and reduced H2S biosynthesis in the models of high fat fed and endotoxin tolerant mice These results give us an insight into the roles of H2S during the early stages of atherosclerosis and. .. from the H2S concentration at the site of inflammation, another factor which may play a part in determining the involvement of this gas in inflammation, is the duration and perhaps also the intensity... platelets and triggering of the clotting cascade Depending on the location of the clot, the thrombotic event may be serious or life-threatening H2S performs several roles during inflammation These include
ROLE OF HYDROGEN SULFIDE IN ACUTE AND CHRONIC INFLAMMATION DAVID NG SHEN WEN (B.Sc. (Hons.), NATIONAL UNIVERSITY OF SINGAPORE) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ______________________ David Ng Shen Wen 20 August 2014 I ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to my supervisor, Prof. Philip Keith Moore for giving me the opportunity to work on this research project and his guidance. I would also like to thank my co-supervisor, A/Prof. Low Chian Ming for his advice. I would like to thank Dr. Mohamed Shirhan Bin Mohamed Atan, Dr. Li Ling, Dr. Tsai Chin-Yi, Ms. Peh Meng Teng, Mr. Huang Weihao Caleb and Ms. Ng Li Theng, for their help over the course of the project. Finally, I would also like to thank my family for their support throughout my studies. II TABLE OF CONTENTS DECLARATION…………………………………………………………………………………I ACKNOWLEDGEMENTS…………………………………………………………………….II TABLE OF CONTENTS………………………………………………………………………III SUMMARY……………………………………………………………………………………VII LIST OF FIGURES………………………………………………………………………….VIII ABBREVIATIONS…………………………………………………………………………….IX CHAPTER 1: Introduction and Overview……………………………………………………..1 1 Introduction……………………………………………………………………………….2 1.1 Hydrogen Sulfide – overview and historical perspective…………………………2 1.2 Physical and chemical properties of H2S………………………………………….2 1.3 Biosynthesis of H2S……………………………………………………………….3 1.4 Biological effects and physiological functions of H2S……………………………4 1.5 Research Objectives……………………………………………………………….8 CHAPTER 2: Effects of administration of a high fat diet on H2S metabolism in mice……..9 2 Introduction………………………………………………………………………………10 2.1 Materials and methods…………………………………………………………...13 2.1.1 Animals and diet…………………………………………………………13 III 2.2 2.1.2 Measurement of tissue H2S synthesizing enzyme activity………………13 2.1.3 Measurement of plasma H2S concentration……………………………...14 2.1.4 Western blotting for H2S synthesizing enzymes…………………………15 2.1.5 Measurement of plasma cytokine and chemokine levels………………...16 2.1.6 Measurement of plasma SAA and CRP levels…………………………..16 2.1.7 Immunohistochemistry staining for CSE, CBS and 3-MST……………..16 2.1.8 Oil red O staining………………………………………………………...17 2.1.9 Statistics………………………………………………………………….17 Results……………………………………………………………………………18 2.2.1 Effect of a high fat diet on plasma H2S concentration…………………...18 2.2.2 Effect of a high fat diet on CSE, CBS and 3-MST expression…………..19 2.2.3 Effect of a high fat diet on tissue H2S synthesising activity……………..21 2.2.4 Effect of a high fat diet on vascular H2S synthesizing enzyme localisation……………………………………………………………….23 2.3 2.2.5 Effect of a high fat diet on plasma cytokines and chemokines…………..25 2.2.6 Effect of a high fat diet on plasma SAA and CRP levels………………..28 2.2.7 Effect of a high fat diet on lipid deposition in aorta……………………..29 Discussion………………………………………………………………………..31 IV CHAPTER 3: Effects of endotoxin tolerance on H2S metabolism in mice………………….35 3 Introduction………………………………………………………………………………36 3.1 3.2 Materials and methods…………………………………………………………...39 3.1.1 Animals and treatment groups…………………………………………...39 3.1.2 Measurement of plasma IL-1β, IL-6 and TNF-α levels………………….40 3.1.3 Measurement of myeloperoxidase activity in tissues……………………40 3.1.4 Haematoxylin and eosin staining………………………………………...41 3.1.5 Measurement of plasma H2S concentration……………………………...41 3.1.6 Western blotting for H2S synthesizing enzymes…………………………41 3.1.7 Measurement of tissue H2S synthesizing enzyme activity………………41 3.1.8 Statistics………………………………………………………………….42 Results……………………………………………………………………………43 3.2.1 Effect of endotoxin tolerance on inflammatory cytokines in plasma……43 3.2.2 Effect of endotoxin tolerance on myeloperoxidase activity in tissues…...45 3.2.3 Effect of endotoxin tolerance on cell infiltration and tissue remodelling..46 3.2.4 Effect of endotoxin tolerance on plasma H2S concentration…………….48 3.2.5 Effect of endotoxin tolerance on CBS, CSE and 3-MST expression……48 3.2.6 Effect of endotoxin tolerance on tissue H2S synthesising activity………51 V 3.3 Discussion………………………………………………………………………..53 CHAPTER 4: Conclusion……………………………………………………………………...55 4 Conclusion……………………………………………………………………………….56 BIBLIOGRAPHY………………………………………………………………………………58 VI SUMMARY H2S been reported to exhibit both pro-inflammatory and anti-inflammatory properties. In this study, we evaluated the contribution of the H2S system in two different models of inflammation. In the first model, we fed mice a high fat diet over a period of 16 weeks to induce a chronic low grade inflammatory model. We observed that the CSE expression and H2S biosynthesis in high fat fed mice was reduced by approximately 50% compared to normal diet mice. As these changes occurred prior to the development of atherosclerosis, these results suggest that H2S deficiency may predispose mice to atherosclerosis. In the second model, we injected mice with LPS to induce an acute inflammatory state. Mice re-exposed to LPS developed a state of endotoxin tolerance. CSE expression and H2S biosynthesis in endotoxin tolerant mice was observed to be reduced by approximately 25% compared to endotoxic shock mice. We observed down-regulated CSE expression and reduced H2S biosynthesis in the models of high fat fed and endotoxin tolerant mice. These results give us an insight into the roles of H2S during the early stages of atherosclerosis and endotoxin tolerance. The results also opens up the possibility of using the H2S system as a therapeutic target in early atherosclerosis and endotoxin tolerance. VII LIST OF FIGURES Figure 1. The biosynthesis of H2S within mammalian tissues…………………………………….4 Figure 2. Therapeutic targets for H2S……………………………………………………………..8 Figure 3. Plasma H2S concentration……………………………………………………………..18 Figure 4. Expression of CBS, CSE and 3-MST………………………………………………….20 Figure 5. Biosynthesis of H2S……………………………………………………………………22 Figure 6. Representative photographs of IHC staining in aorta………………………………….24 Figure 7. Plasma concentration of cytokines and chemokines…………………………………..26 Figure 8. Plasma concentrations of biochemical markers of atherosclerosis……………………28 Figure 9. Representative photographs showing Oil red O staining for lipid in aorta……………30 Figure 10. Plasma concentration of inflammatory cytokines……………………………………44 Figure 11. Myeloperoxidase activity…………………………………………………………….46 Figure 12. Representative photographs of histological changes within liver and lung………….47 Figure 13. Plasma H2S concentration……………………………………………………………48 Figure 14. Expression of CBS, CSE and 3-MST………………………………………………...50 Figure 15. Biosynthesis of H2S…………………………………………………………………..52 Figure 16. Experimental flowchart……..………………………………………………………..57 VIII ABBREVIATIONS Symbols Full name 3MP 3-mercaptopyruvate 3-MST 3-mercaptopyruvate sulfurtransferase AP-1 Activator protein 1 ApoE-/- Apolipoprotein E-deficient ATB-346 2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoylphenyl ester BSA Bovine serum albumin C19H42BrN Hexadecyltrimethylammonium bromide cAMP Cyclic adenosine monophosphate CARS Compensatory anti-inflammatory response syndrome CAT Cysteine aminotransferase CBS Cystathionine β synthase CD11b/CD18 β2 integrin Mac-1 CD36 Cluster of differentiation 36 CL Cysteine lyase CO Carbon monoxide COPD Chronic obstructive pulmonary disease IX CRP C-reactive protein CSE Cystathionine γ lyase DTT Dithiothreitol ERK Extracellular signal-regulated kinases FeCl3 Iron (III) chloride G-CSF Granulocyte-colony stimulating factor GMP Guanosine monophosphate GYY4137 Morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate H2S Hydrogen sulfide HCl Hydrochloric acid HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A HPLC High-performance liquid chromatography IACUC Institutional Advisory Care and Use Committee ICAM-1 Intracellular adhesion molecule 1 IFN-γ Interferon gamma IHC Immunohistochemistry IKK IκB kinase IL Interleukin X KATP ATP-sensitive potassium KC Keratinocyte chemoattractant LL LPS+LPS LPS Lipopolysaccharide MAPK Mitogen-activated protein kinase MBB Monobromobimane MCP-1 Monocyte chemotactic protein 1 MIP Macrophage inflammatory protein MPO Myeloperoxidase MyD88 Myeloid differentiation primary response gene 88 NaHS Sodium hydrosulfide NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NMDA N-methyl-D-aspartic acid NO Nitric oxide NUS National University of Singapore OxLDL Oxidised low density lipoprotein P5P Pyridoxal 5’-phosphate PAMPs Pathogen-associated molecular patterns XI PBS Phosphate buffered saline PRR Pattern recognition receptors RANTES Regulated on activation, normal T expressed and secreted ROS Reactive oxygen species SAA Plasma serum amyloid A SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of mean SL Saline+LPS SRA Scavenger receptor class A SS Saline+saline TBS Tris-buffered saline Th1 T helper 1 Th17 T helper type 17 TIR Toll-interleukin-1 receptor TLR4 Toll-like receptor 4 TNF-α Tumor necrosis factor alpha VCAM-1 Vascular cell adhesion molecule 1 XII Chapter 1 Introduction and Overview 1 1 Introduction 1.1 Hydrogen Sulfide – overview and historical perspective Hydrogen sulfide (H2S) is a colourless gas with the distinctive odor of rotten eggs [1]. Until recently, H2S was widely regarded as a noxious gas. H2S is naturally occurring within volcanoes, sulfur springs and swamps. H2S is also produced by the petrochemical industrial activities and by tanneries. H2S has been known for many years to inhibit cytochrome c oxidase, an essential enzyme for mitochondrial respiration [2]. Even with the characteristic odour of H2S, individuals may be unaware of its presence in the air as the sense of smell is severely compromised by H2S concentrations above 150 ppm [3]. Exposure to H2S concentrations of above 500 ppm leads to respiratory paralysis, unconsciousness and may consequently result in death [4]. In the past two decades, the view that H2S is purely an environmental pollutant has been increasingly challenged and is no longer tenable. Indeed, H2S is now seen as an endogenous biologically active molecule with a range of significant functions inside the body. In this regard, H2S influences numerous signaling processes within cells. H2S is now classified alongside nitric oxide (NO) and carbon monoxide (CO) as gasotransmitters. As the newest member of the gasotransmitter family there has been considerable interest in the biology of H2S 1.2 Physical and chemical properties of H2S H2S is able to diffuse readily through plasma membranes without the help of membrane transporters. H2S dissolves freely in water to form a weak acid which dissociates as follows: H2S 2 ↔ HS- + H+. Dissolved H2S under physiological conditions of pH 7.4 at 37oC, will exist predominantly as the hydrosulfide anion (HS-, 81.5%) compared to 18.5% as H2S [5]. H2S is a powerful reducing agent as it is easily oxidized. 1.3 Biosynthesis of H2S H2S production within mammalian tissues occurs via both enzymatic and non-enzymatic means [6; 7; 8; 9]. Generation of H2S via non-enzymatic means occurs through the reduction of thiols. Using L-cysteine as a starting material, H2S can be synthesized by at least four distinct routes enzymatically [6]. These four routes are: (i) cystathionine γ lyase (CSE) produces thiocysteine from cystine, which then reorganizes to produce H2S; (ii) cystathionine β synthase (CBS) works on L-cysteine to create H2S and L-serine; (iii) cysteine aminotransferase (CAT) working in concert with 3-mercaptopyruvate sulfurtransferase (3-MST) to produce H2S with 3mercaptopyruvate as the intermediary product; (iv) cysteine lyase (CL) catalyzes L-cysteine and sulfite to H2S and L-cysteate. These enzymes occur in mammalian tissues and are differentially expressed. CBS is abundantly expressed in brain whereas CSE is the predominant enzyme in liver, kidney and smooth muscle cells. CAT and 3-MST are primarily localized within mitochondria [10]. CSE, CBS, CAT and CL need pyridoxal 5’-phosphate (P5P) as a cofactor. 3MST requires zinc as cofactor. H2S can also be synthesized from homocysteine in a reaction catalyzed by CBS. The four enzymatic pathways that catalyze H2S production are shown in Figure 1. 3 Figure 1. The biosynthesis of H2S within mammalian tissues. Figure taken from Li et al., (2011) [6]. 1.4 Biological effects and physiological functions of H2S H2S, which has been long viewed purely as an environmental pollutant, has in recent years increasingly come to be recognized as an influential biological molecule with important physiological roles. Recently, there has been an abundance of reports illustrating the role of H2S in diverse fields such as cardiovascular, reproductive, neurobiology and inflammation [5; 6; 11; 12; 13; 14]. These studies have not only considerably increased our understanding of H2S but have additionally illuminated the complexity of this seemingly chemically simple gas. 4 Within the cardiovascular system, H2S acts as a physiological vasodilator, modulator of blood flow within the microcirculation and regulator of blood pressure through the opening of ATPsensitive potassium (KATP) channels within vascular smooth muscle cells [15; 16; 17]. In contrast, lack of endogenous H2S may contribute to the development of hypertension [18]. H2S promotes angiogenesis and is anti-atherosclerotic in nature [19; 20]. During atherosclerosis, there is oxidative stress due to increased formation of reactive oxygen species (ROS). Oxidative stress promotes proliferation of vascular smooth muscle cells and increased foam cell formation which are key steps in atherosclerosis. H2S can directly quench ROS with its strong reducing properties and inhibit ROS production [21; 22]. Within atherosclerotic lesions, there is massive proliferation of vascular smooth muscle cells. H2S inhibits vascular smooth muscle cells proliferation and also induces apoptosis of vascular smooth muscle cells [23]. Foam cells formation from macrophages by OxLDL is critical for the initiation and progression of atherosclerotic lesions. H2S was shown to inhibit formation of foam cells [24]. In CSE knockout mice, a deficiency of endogenous H2S has been associated with accelerated atherosclerosis [25]. In reproductive biology, H2S relaxes both the corpus cavernosum and vaginal smooth muscles again through the modulation of KATP channels. These data suggest that sexual function may, at least partly, be controlled by H2S [11; 12]. In neurobiology, H2S has been reported to alter activity of potassium channels and curb synaptic potentials in dorsal raphe serotonergic neurons [26]. In neurons, H2S enhances N-methyl-Daspartic acid (NMDA) receptor-mediated responses and modifies the induction of hippocampal long-term potentiation [27]. Additionally, sensitizing of NMDA receptor by H2S been reported to 5 have a role in pain sensation [28]. H2S also been shown to confer protection on neurons from oxidative stress and prevent neurodegeneration [29; 30]. In inflammation research, H2S exhibits both pro-inflammatory and anti-inflammatory properties. H2S been reported to be an endogenous potentiator of T cell activation [14]. T cells interact with many cell types and recognize a large number of pathogens. T cell activation leads to either production of antibodies, activation of phagocytic cells or direct cell killing which are essential components of host defense. However, uncontrolled T cell activation might result in autoimmune destruction of cells and tissue [14]. During inflammation, H2S increases leukocyte adhesion via activation of the β2 integrin Mac-1 (CD11b/CD18) [31]. Remarkably, this gas also has opposing roles on the effect of administration of lipopolysaccharide (LPS). In a pro-inflammatory role, elevated H2S level and myeloperoxidase (MPO) activity, which serves as a marker for neutrophil infiltration, were observed after either administration of LPS or treatment with the H2S donor sodium hydrosulfide (NaHS) [32]. On the other hand, H2S donors (NaHS and S-diclofenac) demonstrated anti-inflammatory effects through decreased activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and reduced production of inflammatory cytokines [33; 34]. In a burn injury model, H2S caused markedly increased inflammatory damage [35]. Another study reported a negative association between endogenous H2S levels and the severity of chronic obstructive pulmonary disease (COPD) [36]. It has been proposed that the pro-inflammatory properties of H2S might be mediated through increased release of the neuropeptide, substance P, leading to the resultant activation of extracellular signal-regulated kinases (ERK) and NF-κB to generate inflammatory 6 mediators such as interleukin (IL)-1β, tumor necrosis factor α (TNF-α) [37]. H2S has been shown to ameliorate emphysema in a murine smoke exposure model [38]. H2S donors have also been reported to possess anti-inflammatory properties in murine models of ischemia-reperfusion injury of small intestine and colitis [39; 40]. The anti-inflammatory properties of H2S may be partly due to inhibition of phosphodiesterase activity resulting in an increased cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (GMP) levels within aortic cells [41]. Recently, H2S was reported to function as an endogenous mediator of the resolution phase of inflammation [42]. H2S donors that release H2S rapidly over a short period (e.g. NaHS) are typically pro-inflammatory [32; 43]. On the other hand, H2S donors releasing H2S slowly over a long period (e.g. morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) and 2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester (ATB-346)) are usually anti-inflammatory [44; 45]. As such, the overall effect of H2S in inflammation most likely depends on the actual experimental conditions and the concentration of H2S at the site of inflammation [6]. Apart from the H2S concentration at the site of inflammation, another factor which may play a part in determining the involvement of this gas in inflammation, is the duration and perhaps also the intensity of the inflammatory insult [6]. In addition to the topics discussed above, H2S been additionally implicated in other disease states including cancer, organ transplant and metabolic syndromes [46; 47; 48]. The above-mentioned examples serve to illustrate the complexity and wide ranging roles of H2S across different physiological and pathophysiological states. Consequently, H2S, and its donor drugs, are increasingly being considered as potential therapeutic agents for a number of diseases [49; 50; 51]. An illustration of the various potential therapeutic targets for H2S is provided in Figure 2. 7 Figure 2. Therapeutic targets for H2S. Figure taken from Predmore et al., (2012) [51]. 1.5 Research Objectives H2S performs multiple roles in inflammation. Therefore, understanding the factors which govern the overall effects of this gas during inflammation is essential. The present study aims therefore to evaluate the contribution of the H2S system in two different models of inflammation. The first such model is a chronic low grade inflammatory model produced by feeding mice a high fat diet over an interval of up to 16 weeks whilst the second is an acute inflammatory state of endotoxic shock following injection of mice with E. coli LPS. 8 Chapter 2 Effects of administration of a high fat diet on H2S metabolism in mice 9 2 Introduction Cardiovascular disease is currently one of the chief causes of mortality within developed countries and is a leading health concern worldwide [52]. High cholesterol levels increases the risk of cardiovascular diseases [53]. One third of heart disease cases worldwide can be attributed to high blood cholesterol levels which is especially obvious within the developed world [52]. One of the biggest influences on high blood cholesterol levels is diet high in saturated fats [54]. Atherosclerosis is the principal cause of cardiovascular disease. Atherosclerosis is characterized by accumulation of lipids, fibrous elements, smooth muscle cells, endothelial cells, leukocytes and foam cells in large arteries [55]. Atherosclerosis is increasingly being recognized as an inflammatory disease. Over the past decades, the involvement of immune cells and inflammatory mechanisms in the development of atherosclerosis has become clear [56; 57]. During atherogenesis, a process in which atherosclerotic plaques are formed, monocyte recruitment to sites of lesion in large arteries occurs as an early event [56]. Circulating oxidised low density lipoprotein (OxLDL) leads to activation of endothelial cells. The attachment of monocytes to the activated endothelial cells is facilitated by various adhesion molecules. The two main molecules are vascular cell adhesion molecule 1 (VCAM-1) and intracellular adhesion molecule 1 (ICAM1). The recruited monocytes undergo maturation into macrophages and releases several mediators to further drive atherogenesis [57]. The macrophages migrate into the intima of vasculature and increase expression of surface scavenger receptors such as scavenger receptor class A (SRA) and cluster of differentiation 36 (CD36). These receptors bind and internalize lipoprotein with the cells. The lipids accumulate in the macrophages to form foam cells. Foam cells release pro-inflammatory cytokines and ROS which augments the inflammation within the lesion. Foam cells also augment smooth muscle cell migration into the intima which drives the 10 progression of the atherosclerosis [58]. These processes result in the formation of fatty steaks. Over time, these fatty streaks evolve into atherosclerotic plaques. The initiation and progression of atherosclerotic plaques generally take place over many years without any symptoms. In the event of a plaque rupture, acute thrombosis occurs by activation of platelets and triggering of the clotting cascade. Depending on the location of the clot, the thrombotic event may be serious or life-threatening. H2S performs several roles during inflammation. These include dilation of blood vessels and regulation of leukocyte adhesion [15; 31]. The role of H2S in atherosclerosis has been well documented [59]. It has been reported that apolipoprotein E-deficient (ApoE-/-) mice have lowered plasma H2S levels with increased plasma ICAM-1 concentrations [20]. This is interesting since ApoE-/- mice are known to develop atherosclerosis spontaneously even when fed a normal diet. Proliferation of vascular smooth muscle cells is one of the key features of atherosclerosis. H2S been described to induce apoptosis and suppress the proliferation of vascular smooth muscle cells [23]. These suggest that H2S may be protective against atherosclerosis [60]. During the progression of atherosclerosis, the increased ROS formation within the lesion results in oxidative stress which plays an important function in driving the atherosclerotic process. H2S can directly quench ROS with its strong reducing properties [21]. Additionally, H2S also been shown to inhibit ROS production [22]. The H2S donor, NaHS inhibited the formation of foam cells from macrophages by OxLDL [24]. H2S also reduced the extent of aortic lesions in ApoE-/- mice [20; 61]. CSE knockout mice, which suffer from deficiency of endogenous H2S have accelerated development of atherosclerosis [25]. Taken together, a strong case can be made that deficiency of H2S is linked with the development of 11 atherosclerosis. Whether there are any changes to the metabolism of H2S in mice before the development of atherosclerotic lesions in animals or in man is not clear. Moreover, whether such changes, if they occur, would be good predictors of future development of atherosclerosis is also unclear. In this work, I examine whether administration of a high fat diet to mice affects tissue H2S biosynthesis and synthesising enzymes as well as plasma markers of both inflammation and atherosclerosis. 12 2.1 Materials and methods 2.1.1 Animals and diet Male C57/Bl6 mice (23–25 g) were maintained in Comparative Medicine at National University of Singapore (NUS) in an environment with regulated temperature (21-24oC) and lighting (12:12 h light-darkness cycle). Drinking water was provided ad libitum. A period of at least three days was allowed for animals to acclimatize before any experimental manipulations were undertaken. Thereafter, mice were fed either a high fat (16% fat, 12.5% cholesterol and 5% sodium cholic acid) or normal diet (Research Diets Inc., NJ, USA) for up to 16 weeks. At the end of 8, 12 or 16 weeks, groups of animals were anaesthetised with a mixture of ketamine (75 mg/kg, i.p.) and medetomidine (1 mg/kg, i.p.) and blood obtained by cardiac puncture, anticoagulated with heparin (100 U/ml). Blood was then centrifuged at 10000g for 3 min at 4oC to prepare plasma which was then stored at -80oC. Livers, kidneys and lungs were rapidly excised and immediately snap frozen in liquid nitrogen prior to biochemical analyses. Aortae were also removed and placed in 4% v/v paraformaldehyde in phosphate buffer for 24 hours and then embedded in paraffin for subsequent histology. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of NUS. 2.1.2 Measurement of tissue H2S synthesizing enzyme activity Tissue H2S synthesizing enzyme activity was determined as previously described [32]. Lung, kidney and liver were homogenized in ice-cold potassium phosphate buffer (100 mM, pH 7.4). 13 The homogenates were centrifuged at 900g for 10 min at 4oC and the resulting supernatant were collected. The assay mixture (500 µl) contained tissue homogenate (430 µl), L-cysteine (10 mM; 20 µl), P5P (2 mM; 20 µl) (Sigma–Aldrich Ltd., MO, USA), and saline (30 µl). For the detection of H2S synthesizing activity by 3-MST, the protocol was modified from a previous paper [62]. All reagents in the assay mixture remained the same, except that L-cysteine was replaced with 3mercaptopyruvate (3MP) (0.1 mM, 40 µl), P5P was omitted and dithiothreitol (DTT) (1 mM) (Sigma–Aldrich Ltd., MO, USA) was added. The assay mixtures were incubated for 30 min at 37oC in parafilm-sealed microcentrifuge tubes. Thereafter, zinc acetate (1% w/v, 250 µl) was injected to trap H2S followed by trichloroacetic acid (10% w/v, 250 µl) to stop the reaction. Subsequently, N,N-dimethyl-p-phenylenediamine sulfate (20 mM; 133 µl) dissolved in 7.2 M hydrochloric acid (HCl) was added followed immediately by iron (III) chloride (FeCl3) (30 µM; 133 µl) dissolved in 1.2 M HCl. Absorbance of samples (300 µl) was determined at an absorbance wavelength of 670 nm using a 96-well microplate reader (Tecan Systems Inc., CA, USA). All standards and samples were assayed in duplicate. The H2S concentration of each sample was calculated against a calibration curve of NaHS standards (3.125–250 µM) and results are expressed as nm H2S formed per mg soluble protein as determined using the Bradford assay (Bio-Rad Ltd., CA, USA). 2.1.3 Measurement of plasma H2S concentration Plasma H2S was measured by a high-performance liquid chromatography (HPLC) method as described [63]. Mouse plasma (15 µl) and NaHS standards were derivatised for 30 min at room temperature with the fluorescent probe monobromobimane (MBB, 2 mM) in the dark. 14 Thereafter, a standard curve of NaHS (0.018–1.5 µM) was prepared from the derivatised NaHS stock solution. HPLC analysis of derivatized plasma or NaHS solution was carried out on a C18 column using an Agilent 1100 Series HPLC System (Agilent Technologies., CA, USA) with mobile phases comprising of 10% v/v methanol and 0.25% v/v acetic acid and 90% v/v methanol and 0.25% v/v acetic acid. Excitation and emission wavelengths were 385 nm and 475 nm respectively. Derivatised H2S has a retention time in this system of 24.4 ± 0.01 min. 2.1.4 Western blotting for H2S synthesizing enzymes Lung, kidney and liver were homogenized (1:12.5 w/v) in ice cold lysis buffer comprising EDTA (5 mM) containing protease and phosphatase inhibitors (HaltTM Protease Inhibitor Cocktail and Halt™ Phosphatase Inhibitor Cocktail) and 1% v/v Triton-X 100 in phosphate buffered saline (PBS). The homogenates were incubated on ice for one hour before being centrifuged at 16,000g for 10 min at 4oC and the resulting supernatant were collected. Protein concentration was quantified using the Bradford assay (Bio-Rad Ltd., CA, USA). Samples were resolved on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) gel and transferred onto nitrocellulose membranes (Bio-Rad Ltd., CA, USA). The membranes were incubated for one hour at room temperature with blocking buffer (PBS, containing 5% v/v skimmed milk and 0.1% v/v Tween-20). Membranes were then incubated overnight at 4oC with primary antibodies directed against CBS, CSE (Abcam Ltd., USA) or 3-MST or actin (Sigma–Aldrich Ltd., MO, USA). After overnight incubation, primary antibodies were removed, membranes were washed three times with PBS containing 0.1% v/v Tween-20 and thereafter incubated for one hour at room temperature with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse 15 IgG and goat anti-rabbit IgG) (Thermo Fisher., MA, USA). The immunoreactive bands were visualized using LuminataTM Crescendo Western HRP Substrate (Merck Millipore Ltd., MA, USA) and exposed to X-ray film. Resulting blots were scanned and quantified using ImageJ software. 2.1.5 Measurement of plasma cytokine and chemokine levels A range of cytokines and chemokines were assayed in mouse plasma using a Bio-Plex Pro™ Mouse Cytokine 23-plex Assay (Bio-Rad Ltd., CA, USA) according to the manufacturer’s instructions. Fluorescence was measured using the Luminex 100 system and results analysed using Bio-plex Manager™ software (Bio-Rad Ltd., CA, USA). 2.1.6 Measurement of plasma SAA and CRP levels Plasma serum amyloid A (SAA) and C-reactive protein (CRP) levels were measured using ELISA kits (USCN Life Science Inc., HB, China and GenWay Biotech Inc., CA, USA) respectively. Experiments were performed according to the manufacturers’ instructions. 2.1.7 Immunohistochemistry (IHC) staining for CSE, CBS and 3-MST Fixed tissue samples were sliced into 5 µm sections using microtome (Leica AG., HE, Germany). The sections were deparaffinised, rehydrated before antigen recall was performed. Sections were blocked with blocking buffer (tris-buffered saline (TBS) containing 1% v/v bovine 16 serum albumin (BSA)) for one hour at room temperature. Sections were then incubated overnight at 4oC with primary antibodies directed against CBS, CSE or 3-MST. After overnight incubation, primary antibodies were removed; sections were washed with PBS containing 0.1% v/v Tween20 and thereafter incubated for one hour at room temperature with horseradish peroxidaseconjugated secondary antibodies (goat anti-mouse IgG and goat anti-rabbit IgG). Photo-images were captured in a light microscope equipped with a digital camera (Olympus., TK, Japan). 2.1.8 Oil red O staining Fixed tissue samples were sliced into 5 µm sections using microtome. The sections were deparaffinised and rehydrated. Sections were then rinsed with 60% v/v isopropanol and stained for 15 min with Oil Red O solution (Sigma–Aldrich Ltd., MO, USA). Sections were rinsed with PBS, counterstained with haematoxylin (Sigma–Aldrich Ltd., MO, USA) and rinsed again with PBS. Photo-images were captured in a light microscope equipped with a digital camera. 2.1.9 Statistics Data is expressed as mean ± standard error of mean (SEM) with the number of observations shown in parenthesis. Analysis was carried out using Student's t-test and statistical significance was set at p < 0.05. 17 2.2 Results 2.2.1 Effect of a high fat diet on plasma H2S concentration Plasma H2S concentration in control animals prior to administration of a high fat diet was 310.1 ± 16.1 nM (n = 6) as determined by HPLC. No significant change in plasma H2S concentration was detected in either control or fat fed animals at 8, 12 or 16 weeks (Fig. 3). Figure 3. Plasma H2S concentration. Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks. Results show H2S concentration (nM) and are mean ± SEM, n = 6– 7. Hatched column is mice at 0 weeks. 18 2.2.2 Effect of a high fat diet on CSE, CBS and 3-MST expression The expression of the three H2S synthesising enzymes in liver, kidney and lung from control and high fat fed animals were determined by Western blotting. The effect of a high fat diet on expression of these enzymes was tissue-dependent. In the liver, both CSE and 3-MST were down-regulated after 8, 12 or 16 weeks of high fat diet. In contrast, an up-regulation of CBS in liver was observed at the 8 and 16 week time points (Fig. 4A and D). Perhaps this up-regulation may be compensatory in nature. In the kidney, the expression of CBS was only up-regulated at 8 weeks and not at the other two time points. There was no significant effect on the expression of either CSE or 3-MST after high fat diet treatment (Fig. 4B and D). In the lung, CSE expression was reduced at 8 and 16 weeks but not 12 weeks after feeding a high fat diet. There was no significant effect on the expression of either CBS or 3-MST after high fat diet treatment (Fig. 4C and D). 19 Figure 4. Expression of CBS, CSE and 3-MST. (A) Liver, (B) Kidney, (C) Lung. Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks. Results show expression of each protein (c.f. actin) and are mean ± SEM, n = 4–6, *p < 0.05. (D) Representative blots. 20 2.2.3 Effect of a high fat diet on tissue H2S synthesising activity Experiments were performed to monitor tissue H2S synthesising activity ex vivo using liver, kidney and lung samples. Either cysteine or 3MP was used as substrate to monitor enzyme activity of CSE/CBS or 3-MST respectively. In the liver, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of high fat diet (Fig. 5A). In the lung, H2S biosynthesis using cysteine was significantly reduced after 8 and 12 weeks of high fat diet. Interestingly, no significant different was observed at 16 weeks (Fig. 5B). In the kidney, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of high fat diet (Fig. 5C). In the liver, H2S biosynthesis using 3MP was significantly reduced at all three time points after administration of high fat diet (Fig. 5D). In the kidney, there was no significant effect on H2S biosynthesis using 3MP at all three time points after administration of high fat diet (Fig. 5E). The interpretation of the liver biosynthesis data may be complicated by the observed increased expression of CBS in livers of animals fed a high fat diet. This increased expression of CBS might compensate for the decreased CSE expression during H2S biosynthesis. Interestingly, H2S biosynthesis from cysteine was reduced in kidney even though there were no significant changes in kidney CSE, CBS or 3-MST expression. Whether this reflects synthesis of H2S from another source or perhaps enhanced catabolism or quenching of the synthesised gas is not clear. 21 Figure 5. Biosynthesis of H2S. (A) Liver, (B) Lung (B), (C) Kidney from cysteine with P5P, (D) Liver and (E) Kidney from 3MP with DTT. Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks. Results show H2S synthesis from starting substrate expressed as nmol/mg protein and are mean ± SEM, n = 10–14, *p < 0.05. 22 2.2.4 Effect of a high fat diet on vascular H2S synthesizing enzyme localisation In aortic sections of control mice after 16 weeks, IHC staining for CSE revealed that the localisation of CSE was in the endothelial cell layer and not in vascular smooth muscle cells (Fig. 6A). In aortic sections of high fat fed mice after 16 weeks, CSE was greatly reduced or absent in the endothelial cell layer (Fig. 6B). IHC staining for CBS and 3-MST revealed that neither enzyme was present in aorta of either control nor mice fed a high fat diet for up to 16 weeks (Fig. 6C-E). 23 Figure 6. Representative photographs of IHC staining in aorta. Representative sections from 3 animals in each group. (A) control diet mice CSE, (B) high fat fed mice CSE, (C) control diet mice CBS, (D) high fat fed mice CBS (E) control diet mice 3-MST, (F) high fat fed mice 3MST. Arrows highlight areas of brown CSE staining. Scale shows dimension (20 µm). 24 2.2.5 Effect of a high fat diet on plasma cytokines and chemokines Plasma of mice at the time points of 8, 12 or 16 weeks were screened for changes in profile of various inflammatory cytokines and chemokines. No significant difference in plasma concentrations between control and high fat mice was detected for IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-10, IL-13, IL-17, interferon gamma (IFN-γ), monocyte chemotactic protein 1 (MCP-1), regulated on activation, normal T expressed and secreted (RANTES) or TNF-α (Fig. 7G-R). Significant differences were observed at some time points for IL-6, granulocyte-colony stimulating factor (G-CSF), keratinocyte chemoattractant (KC), IL-12p40, macrophage inflammatory protein (MIP)-1β and IL-5. Plasma concentrations of IL-6 and G-CSF were significantly increased in the high fat diet mice at both 12 and 16 weeks. (Fig. 7A and C). At all three time points, plasma concentration of IL-12p40 was increased for high fat fed mice (7B). Plasma concentration of KC was only significant increased at 16 weeks for high fat fed mice (7D). Plasma MIP-1β levels were significantly lower at 8 weeks but become significantly increased at 12 weeks for high fat fed mice (Fig. 7E). Plasma concentration of IL-5 was decreased at 8 weeks but not at other time points for high fat fed mice (Fig. 7F). 25 26 Figure 7. Plasma concentration of cytokines and chemokines. (A) IL-6, (B) IL-12p40, (C) GCSF, (D) KC (E) MIP-1β, (F) IL-5, (G) IL-1α, (H) IL-1β, (I) IL-2, (J) IL-3, (K) IL-4, (L) IL-10, (M) IL-13, (N) IL-17, (O) IFN-γ, (P) MIP-1α, (Q) RANTES and (R) TNF-α. Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks. Results show concentration of cytokine/chemokine in pg/ml and are mean ± SEM, n = 6–7, *p < 0.05. 27 2.2.6 Effect of a high fat diet on plasma SAA and CRP levels Plasma of mice at the time points of 8, 12 or 16 weeks were tested for changes SAA and CRP levels. No significant difference in plasma concentrations between control and high fat mice was observed for SAA and CRP across all three time points. Figure 8. Plasma concentrations of biochemical markers of atherosclerosis. (A) SAA and (B) CRP. Control (open columns) and high fat fed (black columns) mice at 8, 12 and 16 weeks. Results show concentration of SAA in µg/ml and CRP in ng/ml. Values are mean ± SEM, n = 6– 7 28 2.2.7 Effect of a high fat diet on lipid deposition in aorta Oil red O stained sections of aorta from both control and high fat fed mice at 16 weeks showed no lipid deposition. The sections had thin intima lined by endothelium and a thick media with no evidence of fatty streaks (Fig. 9A and B). These indicate that there was no atherosclerosis in either set of animals. 29 Figure 9. Representative photographs showing Oil red O staining for lipid in aorta. Representative sections from 3 animals in each group. (A) control diet mice and (B) high fat fed mice at 16 weeks. Scale shows dimension (20 µm). 30 2.3 Discussion A diet high in saturated fats have been shown to raise the risk of developing coronary heart disease whilst a reduction in intake of saturated fats corresponds to lower risk of the disease [64; 65]. Currently, atherosclerosis is managed through a combination of lifestyle changes and pharmacotherapy. The most widely used drugs for atherosclerosis are angiotensin-converting enzyme inhibitors, statins and antiplatelet agents. Statins, the gold standard in treatment of atherosclerosis, reduces blood cholesterol level through inhibition of 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase [66]. In addition to their better known cholesterol lowering effects, statins also show anti-inflammatory activity through suppression of ROS and inhibition of NF-κB [67; 68]. Recently, there is increased interest in the development of anti-inflammatory drugs to treat atherosclerosis [69]. It is well established that atherosclerotic animals suffer from H2S deficiency [25; 59; 70]. However, it is unclear whether there are changes in the H2S system during chronic low grade inflammation which occurs prior to the development of atherosclerosis. It has been reported that 12 weeks of high fat diet is enough to generate atherosclerosis [71]. In this project, we observed no signs of atherosclerosis even at the 16 weeks. This is probably be due to different experimental conditions. This absence of atherosclerosis allowed us to study the changes to the H2S system during the chronic low grade inflammation phase before the development of atherosclerosis. In this project, changes to the H2S system as a result of chronic low grade inflammation were investigated. In mice fed a high fat diet, changes in expression of H2S synthesising enzymes could be detected from 8 weeks onwards and were tissue specific. In the liver, expression of CSE and 3-MST in 31 high fat fed mice were down-regulated from 8 weeks onwards. This change was accompanied by up-regulation of CBS in high fat fed mice which might perhaps be a compensatory response to the down-regulation of CSE and 3-MST. Liver H2S biosynthesis using either L-cysteine or 3MP was also reduced in high fat fed mice which correlated with the observed down-regulation of CSE and 3-MST. H2S biosynthesis was also reduced in kidney and lungs of high fat mice. These observations suggest that administration of a high fat diet results in reduced tissue H2S biosynthesis and CSE expression. Furthermore, CSE expression within aortic endothelial cells was down-regulated after 16 weeks of a high fat diet. We observed that the decrease of liver and kidney H2S synthesis in high fat mice is more at 16 weeks as compared to those in 8 and 12 weeks (Fig. 5A and C). The observed smaller decrease in H2S synthesis in high fat mice at 16 weeks could be a compensatory effect as the H2S synthesis in the liver and kidney drops off sharply at 16 weeks. A sensitive HPLC assay was used to determine plasma H2S levels. However, no difference could be detected between the control and high fat fed animals. This may be due to metabolism of H2S by enzymes away from the affected tissues or rapid oxidation of H2S into thiosulfate [6]. This suggests that there is no correlation between plasma H2S concentrations and any alterations in enzyme expression/activity in tissues from high fat fed mice. There was no evidence of lipid deposition within the aorta of high fat fed animals even after 16 weeks of feeding a high fat diet. No difference could be detected in plasma concentrations of either SAA or CRP which are regarded as early markers of atherosclerosis between the normal and high fat fed mice at any time point [72; 73]. These data strongly suggest that atherosclerosis had not developed in high fat fed mice in this study. Thus, we surmise that H2S deficiency occurs 32 prior to the development of atherosclerosis as seen by both histological and biochemical assays [74]. We propose that, H2S deficiency may predispose mice to atherosclerosis. Pro-inflammatory cytokines such as IL-1α, IL-1β, IFN-γ and TNF-α are known to perform significant functions in the initiation and progression of atherosclerosis [75]. No differences was observed in the plasma concentrations of these cytokines between the normal and high fat fed mice even after 16 weeks which suggests atherosclerosis has not commenced in the high fat mice. Plasma concentrations of IL-5, IL-6, IL-12p40, MIP-1β, KC and G-CSF were elevated in the high fat fed mice. The plasma concentrations of these cytokines were up-regulated at different time points. As such, these cytokines could be useful potential biomarkers for the chronic, low grade inflammation which occurs prior to the development of full blown atherosclerosis. Of these, plasma levels of IL-6, IL-12p40 and G-CSF were consistently elevated in high fat fed mice. IL-6 possess both pro- and anti-inflammatory effects and exerts a wide range of effects on immune cells. It has been proposed as a possible biomarker for coronary heart disease in man [76; 77]. IL-12p40 functions as a subunit of both IL-12 and IL-23. IL-12 been shown to be critical for the development of a T helper 1 (Th1) response and for differentiation into T cell phenotype. IL-23 regulates the differentiation of T cells into T helper type 17 (Th17) cells. Together, IL-12 and IL-23 are able to influence cell-mediated immune responses and Th1 inflammatory reactions [78; 79]. Interestingly, human atherosclerotic plaque was found to be richly laden with IL-12 p40 mRNA [80]. Some of the functions of G-CSF include regulating the survival and proliferation of neutrophils. In an atherosclerotic mice model, G-CSF was reported to increase the size of lesions [81]. 33 We observed a down-regulation of CSE expression and deficiency of H2S biosynthesis in tissues from high fat fed mice. It is increasingly recognized that H2S has multiple anti-atherosclerotic effects which include inhibition of vascular smooth muscle cell proliferation, promotion of endothelial cell proliferation, reduction of oxidative stress, reduction of inflammation and inhibition of foam cell formation. As such, the deficiency of H2S in high fat fed mice would result in a reduction of protective anti-atherosclerotic benefits of H2S. The observed H2S deficiency would lead to early development and progression of atherosclerosis in the high fat fed mice. It has been shown that CSE knockout mice have accelerated development of atherosclerosis. We propose that endogenous H2S inhibits the chronic low grade inflammation during the early atherosclerosis. Administration of a high fat diet over a period of 16 week resulted in chronic low grade inflammation. Using histological and biochemical assays, no signs of atherosclerosis within the aorta or plasma was observed. As such, we were able to utilize our high fat diet model to study the H2S system during the period prior to the development of atherosclerosis. A down-regulation of CSE expression and deficiency of H2S biosynthesis in tissues from high fat fed mice was observed. As the changes in H2S metabolism and rise in inflammatory cytokine levels happened at the same time after administration of a high fat diet, it still remains unclear whether the deficiency of H2S precedes the induction of chronic low grade inflammation. As such, more research is needed to discover whether a deficiency of H2S can be used as a predictive biomarker prior to development of atherosclerosis. 34 Chapter 3 Effects of endotoxin tolerance on H2S metabolism in mice 35 3 Introduction Inflammation is a host immune response triggered by stimuli such as damaged cells, foreign bodies or pathogens [82]. During inflammation, various cell signaling pathways are activated resulting in the secretion of inflammatory mediators and activation of leukocytes. Pathogens such as bacteria and viruses express distinctive structural motifs known as pathogen-associated molecular patterns (PAMPs) [83]. Different PAMPs are recognized by its corresponding pattern recognition receptors (PRR) expressed on surfaces of immune cells. Toll-like receptor 4 (TLR4) is the PRR responsible for sensing LPS, a major constituent of the outer membrane of Gramnegative bacteria [84; 85]. Upon detection of LPS, TLR4 undergoes dimerization, followed by recruitment of its downstream adaptors via its Toll-interleukin-1 receptor (TIR) domains. TLR4 signal transduction is mediated through myeloid differentiation primary response gene 88 (MyD88), activating the downstream IκB kinase (IKK) and mitogen-activated protein kinase (MAPK) pathways [84]. Consequently, NF-κB and activator protein 1 (AP-1) are activated and up-regulate the expression of various pro-inflammatory cytokines such as IL-1β, IL-6 and TNFα. This inflammatory response is an essential part of host defence against invading pathogens. The inflammatory response is a tightly regulated process as uncontrolled inflammation results in widespread damage to tissues and may lead to serious and chronic problems such as cancer and autoimmune diseases [85; 86]. High doses of LPS can induce severe systemic inflammation and sepsis [87]. Sepsis occurs when the excessive inflammatory cytokines are released into the bloodstream as a ‘cytokine storm’ during an infection, causing extensive inflammation throughout the body [88]. This systemic inflammation may potentially lead to multiple organ 36 failure. Sepsis may progress to septic shock if the patient’s blood pressure plunges and remains low despite medical intervention [88]. Sepsis is a major healthcare problem with millions of cases diagnosed worldwide yearly [89]. Currently, sepsis patients are administered an aggressive treatment of intravenous broad-spectrum antibiotics. Additionally, intravenous fluids and vasopressor agents are also given to septic shock patients [88; 89]. Even with the advances in medical care, the prognosis of sepsis still remains poor with a mortality rate of approximately 30% [90]. Patients recovering from sepsis often exhibit features of immunosuppression, also known as compensatory anti-inflammatory response syndrome (CARS) [91]. These patients are vulnerable to acquiring second infections from normal avirulent microorganisms. Endotoxin tolerance is a phenomenon whereby cells or organisms previously challenged with LPS exhibit hyporesponsiveness to subsequent challenges with LPS [92; 93]. Monocytes from patients recovering from sepsis exhibit blunted expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α upon re-exposure to LPS [94]. Endotoxin tolerance is caused by inhibition of the MyD88 mediated signaling pathway of TLR4 during subsequent LPS exposure resulting in decreased production of pro-inflammatory cytokines [95; 96; 97]. Tissue H2S synthesizing activity and CSE expression were reported to be increased in an endotoxic shock model in mice which suggests a pro-inflammatory role for H2S in endotoxic shock [32]. On the other hand, a H2S-releasing derivative of diclofenac and the slow-releasing H2S donor GYY 4137 been reported to be anti-inflammatory during endotoxic shock [34; 44]. Thus, the exact role of H2S during endotoxic shock probably depends on the experimental 37 conditions. Whether there are any changes to the H2S system in mice during endotoxin tolerance is not clear. In this work, I examine whether endotoxin tolerance affects tissue H2S biosynthesis and H2S synthesizing enzymes. 38 3.1 Materials and methods 3.1.1 Animals and treatment groups Male C57/Bl6 mice (23–25 g) were maintained in Comparative Medicine at NUS in an environment with regulated temperature (21-24oC) and lighting (12:12 h light-darkness cycle). Drinking water was provided ad libitum. A period of at least three days was allowed for animals to acclimatize before any experimental manipulations were undertaken. Mice were divided into three treatment groups; saline+saline (SS), saline+LPS (SL) and LPS+LPS (LL). Mice in the SS or negative control group received a dose of saline (10 mg/kg, i.p.) followed by another dose of saline (10 mg/kg, i.p.) after 24 hours. Mice in the SL or septic group received a dose of saline (10 mg/kg, i.p.) followed by LPS (5 mg/kg, i.p.) after 24 hours. Mice in the LL or endotoxin tolerant group received LPS (0.5 mg/kg, i.p.) followed by LPS (5 mg/kg, i.p.) after 24 hours. 6 hours after the last treatment, the mice were anaesthetised with a mixture of ketamine (75 mg/kg, i.p.) and medetomidine (1 mg/kg, i.p.) and blood obtained by cardiac puncture and anticoagulated with heparin (100 U/ml). Blood was then centrifuged at 10000g for 3 min at 4oC to prepare plasma which was stored at -80oC. Livers, kidneys and lungs were rapidly excised and immediately snap frozen in liquid nitrogen prior to biochemical analyses. Sections of liver and lungs were placed in 4% v/v paraformaldehyde in phosphate buffer for 24 hours and then embedded in paraffin for subsequent histology. All animal experiments were approved by the IACUC of NUS. 39 3.1.2 Measurement of plasma IL-1β, IL-6 and TNF-α levels IL-1β, IL-6 and TNF-α levels in mouse plasma were measured using a Bio-Plex kit (Bio-Rad Ltd., CA, USA) according to the manufacturer’s instructions. Fluorescence was measured using the Luminex 100 system and results analysed using Bio-plex Manager™ software (Bio-Rad Ltd., CA, USA). Plasma TNF-α levels was measured using a BD ELISA kit (BD., NY, USA) 3.1.3 Measurement of myeloperoxidase activity in tissues Measurement of MPO activity, a marker for neutrophil infiltration was determined as previously described [32]. Liver and lung samples were homogenized in ice cold phosphate buffer (20 mM) before being centrifuged at 450 g for 10 min at 4°C and the resulting supernatant were collected. 100 µl of the supernatant was collected for DNA quantification and the remaining supernatant was re-centrifuged at 20,000 g for 15 min at 4°C. The resulting pellet was resuspended in phosphate buffer (50 mM) containing 0.5% v/v hexadecyltrimethylammonium bromide (C19H42BrN) (Sigma–Aldrich Ltd., MO, USA). The suspension was subjected to four cycles of freeze and thaw and additionally disrupted using sonication. Samples were then centrifuged at 18000 g for 5 min at 4°C and the resulting supernatant was used for MPO assay. SureBlueTM TMB substrate (KPL., MD, USA) was added to the supernatant and the reaction stopped with sulfuric acid (2 mM). Absorbance of samples was determined at an absorbance wavelength of 450 nm using a 96-well microplate reader (Tecan Systems Inc., CA, USA). Tissue MPO activity was corrected for DNA concentration, which was determined spectrofluorimetrically using Nanodrop (Thermo Fisher., MA, USA). 40 3.1.4 Haematoxylin and eosin staining Fixed tissue samples were sliced into 5 µm sections using microtome. The sections were deparaffinised and rehydrated. Sections were stained with haematoxylin (Sigma–Aldrich Ltd., MO, USA) for 5 min, washed with water, differentiated in 1% acid alcohol (70% ethanol containing 1% v/v HCl), washed again with water and counterstained with eosin (Sigma–Aldrich Ltd., MO, USA). Photo-images were captured using a light microscope equipped with a digital camera. 3.1.5 Measurement of plasma H2S concentration Plasma H2S was measured by a HPLC method as described in section 2.1.3. 3.1.6 Western blotting for H2S synthesizing enzymes Western blotting for H2S synthesizing enzymes was performed as described in section 2.1.4. 3.1.7 Measurement of tissue H2S synthesizing enzyme activity Tissue H2S synthesizing enzyme activity was determined as previously described in section 2.1.2. 41 3.1.8 Statistics Data is expressed as mean ± SEM with the number of observations shown in parenthesis. Analysis was carried out using one-way ANOVA with post hoc Turkey test and statistical significance was set at p < 0.05. 42 3.2 Results 3.2.1 Effect of endotoxin tolerance on inflammatory cytokines in plasma The concentration of inflammatory cytokines was measured in the plasma of SS, SL and LL mice. A significant increase in plasma concentration of IL-1β, IL-6 and TNF-α was detected in SS cf. SL mice (Fig. 10A, B and C). A significant decrease in plasma concentration of IL-6 and TNF-α but not IL-1β was noted in SL compared to LL animals (Fig. 10A, B and C). 43 Figure 10. Plasma concentration of inflammatory cytokines. (A) IL-1β, (B) IL-6, (C) TNF-α. SS (control), SL (septic) and LL (endotoxin tolerant). Results show concentration of cytokine in pg/ml and are mean ± SEM, n = 5–15, *p < 0.05 (cf. SS), #p < 0.05 (cf. SL). 44 3.2.2 Effect of endotoxin tolerance on myeloperoxidase activity in tissues MPO activity, a marker for neutrophil infiltration, was measured in liver and lung. A significant increase in MPO activity in SL mice compared to SS animals was observed in both liver and lung. A significant decrease in MPO activity between SL and LL animals was observed in both liver and lung. 45 Figure 11. Myeloperoxidase activity. (A) Liver, (B) Lung. SS (control), SL (septic) and LL (endotoxin tolerant). Results show percentage increase in MPO activity over control and are mean ± SEM, n = 5–8, *p < 0.05 (cf. SS), #p < 0.05 (cf. SL). 3.2.3 Effect of endotoxin tolerance on cell infiltration and tissue remodelling Inflammatory cells such as macrophages and neutrophils are activated and migrate into tissues in response to LPS [98]. Visualization of inflammatory cell infiltration and tissue remodelling was determined using H&E staining. Infiltration of inflammatory cells into the liver of SL and LL mice as compared to SS mice were detected (Fig. 12). SL animals showed infiltration of cells 46 into the lung, alveolar wall thickening and interstitial edema as compared to the SS animals. The LL mice had reduced infiltration of inflammatory cells into the lung, alveolar wall thickening and interstitial edema as compared to the SL mice (Fig. 12). Figure 12. Representative photographs of histological changes within liver and lung. SS (control), SL (septic) and LL (endotoxin tolerant). Arrows highlight areas of cell infiltration. Scale shows dimension (100 µm). 47 3.2.4 Effect of endotoxin tolerance on plasma H2S concentration The plasma H2S concentration of SS, SL and LL mice were determined using HPLC. No significant change in plasma H2S concentration in the SS, SL and LL groups of mice was observed (Fig.13). Figure 13. Plasma H2S concentration. SS (control), SL (septic) and LL (endotoxin tolerant). Results show H2S concentration (µM) and are mean ± SEM, n = 6–7. 3.2.5 Effect of endotoxin tolerance on CBS, CSE and 3-MST expression The expression of the three H2S synthesising enzymes in liver and kidney from SS, SL and LL animals were determined by Western blotting. No significant change was seen in expressions of CBS and 3-MST in liver of SS, SL and LL animals. In contrast, CSE expression in liver of SL animals was increased as compared to SS animals. There was decreased liver CSE expression in 48 LL animals as compared to SL mice (Fig. 14A and C). In kidney, there was no significant change to CBS and 3-MST between the SS, SL and LL mice. CSE expression in kidney of SL animals was increased as compared to between SS animals, however there was no significant change to CSE expression in kidney between SL and LL animals (Fig. 14B and C). 49 Figure 14. Expression of CBS, CSE and 3-MST. (A) Liver, (B) Kidney, (C) Representative blots. SS (control), SL (septic) and LL (endotoxin tolerant). Results show expression of each protein (cf. actin) and are mean ± SEM, n = 3-4, *p < 0.05 (cf. SS), #p < 0.05 (cf. SL). 50 3.2.6 Effect of endotoxin tolerance on tissue H2S synthesising activity Experiments to monitor tissue H2S synthesising activity ex vivo using liver, kidney and lung homogenates was performed. Either cysteine or 3MP was used as substrate to monitor enzyme activity of CSE/CBS or 3-MST respectively. In the liver, H2S biosynthesis from cysteine was significantly increased in SL as compared to SS mice. However, LL animals had significantly decreased H2S biosynthesis with cysteine as substrate as compared to SL animals (Fig. 15A). In lung, H2S biosynthesis using cysteine was significantly increased in SL mice as compared to SS mice. LL mice had significantly decreased H2S biosynthesis using cysteine as compared to SL mice (Fig. 15B). In kidney, H2S biosynthesis using cysteine was significantly increased in SL mice as compared to SS mice. In contrast, LL mice had significantly decreased H2S biosynthesis using cysteine as compared to SL mice (Fig. 15C). In the liver, lung and kidney, there was no significant change in H2S biosynthesis using 3MP between SS, SL and LL mice (Fig. 15D, E and F). 51 Figure 15. Biosynthesis of H2S. (A) Liver, (B) Lung (B), (C) Kidney from cysteine with P5P, (D) Liver, (F) Lung and (E) Kidney from 3MP with DTT. SS (control), SL (septic) and LL (endotoxin tolerant). Results show H2S synthesis from starting substrate expressed as nmol/mg protein and are mean ± SEM, n = 5-8, *p < 0.05 (cf. SS), #p < 0.05 (cf. SL). 52 3.3 Discussion Sepsis is a major healthcare problem despite the advances in its medical care. The increasing frequent utilization of invasive medical procedures, immunosuppressive drugs and the growing levels of microbial resistance may contribute towards a rising incidence and poor medical outcome of sepsis [99]. After surviving the inflammatory phase of sepsis, patients still have to get through a period of general immunosuppression where they are especially vulnerable to iatrogenic infections [100]. Therefore, it is crucial to comprehend the mechanisms regulating endotoxin tolerance to improve outcome during the immunosuppression period. It is not known if there are changes in the H2S system during endotoxin tolerance. In the event where changes are observed, it opens up the possibility of using the H2S system as a therapeutic target in endotoxin tolerance. In this project, changes to the H2S system as a result of endotoxin tolerance were investigated. The plasma concentration of the inflammatory cytokines IL-1β, IL-6 and TNF-α was diminished in the LL mice compared to SL mice. On top of its pro-inflammatory activity, IL-1β also contributes to neutrophil recruitment [101]. TNF-α also regulates the production of IL-1β and IL6, thus the reduced concentrations of IL-1β and IL-6 in LL mice is partly due to decreased TNFα concentrations [102]. Neutrophils are essential part of the host inflammatory response and perform both benefit and harmful roles during sepsis. In the early stages, neutrophils promote clearance of bacteria. Subsequently, neutrophils cause tissue injury through the release of proinflammatory mediators, MPO, and proteases [103]. Decreased MPO activity which is a marker of neutrophil infiltration was seen in LL mice as compared to SL mice which has been reported 53 [104]. This may explain the decreased amount of inflammatory cytokines in LL mice. Every parameter above serves as a marker of an inflammatory state and these were all reduced in the LL mice. These data strongly suggest that the endotoxin tolerance model has been established successfully in these animals. Thereafter, LL mice were examined for changes in the H2S system. No differences in plasma H2S levels were detected between SL and LL animals. This may be due to metabolism of H2S by enzymes away from the affected tissues or rapid oxidation of H2S into thiosulfate [6]. Next, the effect of endotoxin tolerance on expression of H2S synthesising enzymes and H2S biosynthesis were examined. A down-regulation of CSE expression in tissues of LL mice was observed when compared to SL mice. LPS-induced inflammation in SL mice was linked with increased CSE expression while decreased inflammation in LL mice was linked with decreased CSE expression. CSE expression was reported to be increased in mice exposed to LPS which suggests a proinflammatory role for H2S in endotoxic shock [32]. Decreased H2S biosynthesis was also observed in LL mice which correlates with reduced CSE expression. From these results, H2S plays a pro-inflammatory role during sepsis which is similar to other studies [32; 105]. The correlation of decreased inflammation with lowered CSE expression and H2S biosynthesis in LL mice suggests that H2S may function as a pro-inflammatory mediator during endotoxin tolerance. In this study, mice previously challenged with LPS exhibit endotoxin tolerance upon re-exposure to LPS. Decreased CSE expression and deficiency of H2S biosynthesis in tissues from LL mice was observed. Whether the deficiency of H2S precedes endotoxin tolerance is still not clear. There is also a need to discover if administration of H2S donors can reverse endotoxin tolerance. 54 Chapter 4 Conclusion 55 4 Conclusion In this study, the contribution of the H2S system in two different models of inflammation was evaluated (Fig. 16). In a chronic low grade inflammatory model produced by feeding mice a high fat diet, tissues of high fat fed mice were observed to have reduced CSE expression and H2S biosynthesis. Whether this deficiency of H2S can be used as a predictive biomarker for atherosclerosis requires more research. The second model was an acute inflammatory model of endotoxin tolerance following injection of mice with LPS. In this model, the LL or endotoxin tolerant mice was observed to have lowered CSE expression and H2S biosynthesis. Whether H2S donors can be used to reverse endotoxin tolerance requires more study. We observed that changes to H2S system during endotoxin tolerance and during the chronic low grade inflammation before atherosclerosis. 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Bhatia, Endogenous hydrogen sulfide regulates inflammatory response by activating the ERK pathway in polymicrobial sepsis, J Immunol 181 (2008) 4320-31. 63 [...]... anti-inflammatory [44; 45] As such, the overall effect of H2S in inflammation most likely depends on the actual experimental conditions and the concentration of H2S at the site of inflammation [6] Apart from the H2S concentration at the site of inflammation, another factor which may play a part in determining the involvement of this gas in inflammation, is the duration and perhaps also the intensity of. .. ameliorate emphysema in a murine smoke exposure model [38] H2S donors have also been reported to possess anti-inflammatory properties in murine models of ischemia-reperfusion injury of small intestine and colitis [39; 40] The anti-inflammatory properties of H2S may be partly due to inhibition of phosphodiesterase activity resulting in an increased cyclic adenosine monophosphate (cAMP) and guanosine monophosphate... effects of this gas during inflammation is essential The present study aims therefore to evaluate the contribution of the H2S system in two different models of inflammation The first such model is a chronic low grade inflammatory model produced by feeding mice a high fat diet over an interval of up to 16 weeks whilst the second is an acute inflammatory state of endotoxic shock following injection of mice... platelets and triggering of the clotting cascade Depending on the location of the clot, the thrombotic event may be serious or life-threatening H2S performs several roles during inflammation These include dilation of blood vessels and regulation of leukocyte adhesion [15; 31] The role of H2S in atherosclerosis has been well documented [59] It has been reported that apolipoprotein E-deficient (ApoE-/-)... respectively In the liver, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of high fat diet (Fig 5A) In the lung, H2S biosynthesis using cysteine was significantly reduced after 8 and 12 weeks of high fat diet Interestingly, no significant different was observed at 16 weeks (Fig 5B) In the kidney, H2S biosynthesis using cysteine was significantly reduced after 12 and 16 weeks of. .. other hand, H2S donors (NaHS and S-diclofenac) demonstrated anti-inflammatory effects through decreased activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and reduced production of inflammatory cytokines [33; 34] In a burn injury model, H2S caused markedly increased inflammatory damage [35] Another study reported a negative association between endogenous H2S levels and. .. produces thiocysteine from cystine, which then reorganizes to produce H2S; (ii) cystathionine β synthase (CBS) works on L-cysteine to create H2S and L-serine; (iii) cysteine aminotransferase (CAT) working in concert with 3-mercaptopyruvate sulfurtransferase (3-MST) to produce H2S with 3mercaptopyruvate as the intermediary product; (iv) cysteine lyase (CL) catalyzes L-cysteine and sulfite to H2S and L-cysteate... min 2.1.4 Western blotting for H2S synthesizing enzymes Lung, kidney and liver were homogenized (1:12.5 w/v) in ice cold lysis buffer comprising EDTA (5 mM) containing protease and phosphatase inhibitors (HaltTM Protease Inhibitor Cocktail and Halt™ Phosphatase Inhibitor Cocktail) and 1% v/v Triton-X 100 in phosphate buffered saline (PBS) The homogenates were incubated on ice for one hour before being... 15 IgG and goat anti-rabbit IgG) (Thermo Fisher., MA, USA) The immunoreactive bands were visualized using LuminataTM Crescendo Western HRP Substrate (Merck Millipore Ltd., MA, USA) and exposed to X-ray film Resulting blots were scanned and quantified using ImageJ software 2.1.5 Measurement of plasma cytokine and chemokine levels A range of cytokines and chemokines were assayed in mouse plasma using a... there has been an abundance of reports illustrating the role of H2S in diverse fields such as cardiovascular, reproductive, neurobiology and inflammation [5; 6; 11; 12; 13; 14] These studies have not only considerably increased our understanding of H2S but have additionally illuminated the complexity of this seemingly chemically simple gas 4 Within the cardiovascular system, H2S acts as a physiological