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As evolution takes its course, the human body has proven its ability to adapt to numerous physiological changes, hence strengthening its ability to survive any harsh environment that it faces. This magnificent adaptability also termed acclimatisation relies heavily on our complex homeostatic system which holds and maintains the balance among our internal and external environment. Besides that, the immune system which represents one of the key cores of our homeostatic system plays an undoubtedly important role in maintaining and restoring the well being and normal function of the host. Generally, the immune system functions by providing the host with an array of protection from foreign treats such as infections and even injuries by first eliciting an inflammatory response. This inflammatory response would then trigger the expression and activation of various pro-inflammatory cytokines and chemokines. They are essentially responsible for recruiting immune cells to the site of infection/injury. In general, inflammation can be broadly divided into either acute inflammation or chronic inflammation. While acute inflammatory response is beneficial to the host (it triggers the self-healing nature of the human body), chronic inflammation has been linked to various detrimental diseases such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD) to name a few. Therefore, to invent a novel treatment for chronic inflammation, we first have to understand the underlying mechanism involving inflammation.
Lipopolysaccharide (LPS) which always been associated with the inducement of inflammation, consists of both polysaccharides and lipids which form the constituent of the outer-membrane of all Gram-negative bacteria. This core structure is fundamentally made up of three distinctive components: O-antigen, Core oligosaccharide (inner region and outer region) and Lipid A. The O-antigen or also known as the O-polysaccharide or the O-chain usually represents a saccharide subunit that is arranged in an orderly and repeated manner (Rittig et al., 2003). However, such structure orientation might not persist in certain strains of bacteria (i.e. Brucella s.p) where a disordered and truncated orientation of the O-antigen is displayed in their LPS (Rittig et al., 2003). It has been suggested that with such orientation in the O-antigen, LPS would be rendered non-virulent. In some literature, this type of LPS is commonly termed as the rough LPS (Rittig et al., 2003). This probably explains why O-antigen is commonly regarded as the most diverse component of the LPS that can be documented across different types of bacterial species. In addition to that, in some infectious pathogens especially in those enteric Gram-Negative organisms, it was found that the orientation of its unique elongated, hydrophilic and neutral O-chain serves as a protective mechanism by preventing it from being solubilised by both intestinal enzyme and bile acid (Stewart, Schluter and Shaw, 2006). On the other hand, Lipid A which is an acylated glycolipid manifests itself as the most important constituent of the LPS as it represents the endotoxic constituent of the LPS (Stewart, Schluter and Shaw, 2006). Hence, any divergence from the normal nature and position of the acyl group and hydrophilic backbone would lead to a reduction or even a full disruption of normal biological activity. This therefore enhances the fact that it is necessary for Lipid A to contain at least one secondary acyl chain for it to elicit its endotoxic property (Stewart, Schluter and Shaw, 2006). Apart from that, a study which was conducted by Schromm et al. (2000) have also documented that it is necessary for Lipid A to adopt a specific conformation (conical shape conformation) in order for it to display its endotoxic characteristic. Only Lipid A with such conformational shape would be capable of inducing mechanical stress on associated signalling proteins. Lipid A with different types of conformation such as the cylindrical shape conformation would usually hinder the LPS ability to activate signalling proteins (Schromm et al., 2000).
In general, when LPS binds to a specific receptor on certain types of proteins such as CD14, LPS binding protein and Toll-like receptor 4 (TLR4), an array of host-mediated response would be induced to combat the invasion of this toxic substance. With monocytes and macrophages being the component of our innate immunity system, they serve as our first line of defence and therefore are the first to accumulate at the site of infection/injury. Platelets and neutrophils are eventually recruited as well to elicit their designated purposes. In addition to that, an array of endogenous mediators such as the Platelet-Activating Factor (PAF), Arachidonic Acid (AA) metabolites, Nitric Oxide (NO) and cytokines would also be released by inflammatory cells (Stewart, Schluter and Shaw, 2006). The cytokines that are involved includes Tumour Necrosis Factor-alpha (TNF-Î±), Interleukin-1 (IL-1) and Interleukin-6 (IL-6), all of which plays a pivotal role in inflammation.
Pathogen Recognition Receptors (PRRs) are commonly recognised as the receptors being associated with LPS-induced signalling pathways. It functions by recognising the Pathogen Associated Molecular Patterns (PAMPs) which represent sets of specific molecular structures unique to that individual pathogen (Pasare and Medzhitov, 2005). One of the most vital subgroup of the PRRs would be the Toll-like receptors (TLRs) which is essentially a primary focus of this study.
Toll-like receptors (TLRs)
Fundamentally, the Toll-like receptor (TLR) family can be subdivided into 13 distinctive members with their own unique gene targets and ligand specificities. Among them, TLR2 is understood to be sensitive to the peptidoglycan constituent of the gram-positive bacteria (Takeuchi et al., 1999). On the other hand, TLR3 is known to target double-stranded Ribonucleic Acid (dsRNA) from both negative stranded and double stranded viruses (Alexpoulou et al., 2001). In addition to that, the well-documented TLR4 is known to be LPS specific. Besides that, Heil et al., (2004) documented that both TLR7 and TLR8 specifically recognises the RNA component of single stranded viruses while Hemmi et al., (2000) documented that TLR9 is involved in the recognition of unmethylated CpG site of the Deoxyribonucleic Acid (DNA) found in DNA viruses and prokaryotes.
The TLR family inevitably plays a heavy role in the induction of host adaptive immunity. It was found that the maturation of dendritic cells (DCs) through the induction of TLR is a prerequisite to activate naÃ¯ve T cells (Pasare and Medzhitov, 2005). This finding was supported with experimental studies which concluded that transgenic mice which lack Myeloid Differentiation Primary Response Gene 88 (MyD88) were unable to activate the T cells as well as interferon-gama associated with immune response (Lenschow, Walunas and Bluestone, 1996). MyD88 represents a type of adapter protein that is intrinsically linked to TLR functionality which fundamentally activates the Nuclear Factor kappa B (NF-ÎºB). Although the dendritic cells were able to undergo maturation, they were found to be unable to produce cytokines associated with inflammation. In conclusive, the mutation or deletion of this adapter protein would disrupt the TLR signal tranduction pathway (Pasare and Medzhitov, 2005).
As mentioned, TLR4 has long been associated with the induction of the LPS-induced intracellular signalling pathway. In depth, the activation of this signalling pathway requires the adhesion of the CD14-bound LPS to the TLR4-myeloid differentiation protein (MD)-2 complex which manifests itself at cellular membrane (Seki et al., 2010). In response to the activation, several adaptor proteins would be first recruited by the TLR4, namely the TRIF-Related Adaptor Molecule (TRAM) and the Toll/IL-1 Receptor-Associated Protein (TIRAP). TRIF which undergoes a subsequent activation commonly represents the Toll/IL-1 Receptor-domain that expresses adaptor proteins responsible for inducing IFN-c (Seki et al., 2010). In addition to that, the MyD88, IL-1 Receptor Associated Kinases (IRAKs) and Tumour Necrosis Factor Receptor-Activated Factor 6 (TRAF6) which forms the downstream signalling pathway would also undergo similar activation. With the sequential activation cascade involving all the mentioned proteins, NF-ÎºB expression and activation would then be increased, leading to the upregulation of pro-inflammatory chemokines and cytokines production and expression. To provide a better representation of the TLR pathway, a schematic diagram is being illustrated below:
Figure 1. A schematic of the TLR pathway in inducing NF-ÎºB (Akira and Hemmi, 2003).
Nuclear Factor-kappa B (NF-ÎºB)
The Nuclear Factor kappa B (NF-ÎºB) proteins represent collectively one of the most important transcription factors families that regulate numerous cellular responses, including cell proliferation and inflammation. Basically, the NF-ÎºB complexes consist of five distinctive components which are fundamentally the constitutively expressed p50, p52, p65(also known as RelA), RelB and c-Rel, all of which contains a Rel-Homology Domain (RHD) which essentially functions as a DNA binding domain (Simmonds and Foxwell, 2008). Nevertheless, an interesting discovery was documented in which it was noticed that only p65, RelB and c-Rel are capable of interacting with common co-activators and transcription factors as they represent the only component of the NF-ÎºB family that expresses a Transactivation Domain (TAD) which is vital for gene transcription (Simmonds and Foxwell, 2008). Such distinguishing characteristic would render either an activator or repressor property on that particular dimer such as the one observed with p50 heterodimers which function as repressor dimers during the transcriptional process. It elicits its competitive inhibition effect by binding to the p50/p65 binding site on the NF-ÎºB consensus sequence in gene promotion which regulates the process of gene transcription (Li, et al., 2005).
In general, the NF-ÎºB proteins function as dimers; either as heterodimers or homodimers in regulating the transcription process of NF-ÎºB (Simmonds and Foxwell, 2008). When no cellular stimuli are perceived, the NF-ÎºB transcription factors which is commonly present in the cells' cytosol would remain in an inactivated state due to a ÎºB molecule inhibitor (IÎºB). They essentially exist as an IÎºBÎ± and IÎºBÎ² subunit which works by impeding NF-ÎºB entry into the nucleus through the masking of p50/p65 heterodimer nuclear localisation sequence (Simmonds and Foxwell, 2008). However, interestingly the IÎºBs (both IÎºBÎ± and IÎºBÎ² subunit) would undergo degradation whenever they are targeted by the IÎºB kinase( IKK) complex following the activation of the ubiquitin/proteasome pathway. This ultimately results in the increase in NF-ÎºB transcription factor activity (Simmonds and Foxwell, 2008). To date, three subunits of IKK complex have been identified and they are specifically known as IKK1, IKK2 and NF-ÎºB Essential Modulator (NEMO) whilst in some various studies, they are also commonly termed as IKKÎ±, IKKÎ² and IKKÎ³ respectively (Simmonds and Foxwell, 2008). Although it has been implicated that both IKKÎ± and IKKÎ² are involved in IÎºBÎ± phosphorylation activities, there are growing evidences indicating that IKKÎ± might serve as a "negative-regulator" in regulating the activity of NF-ÎºB in order to maintain cellular balance (Lawrence, et al., 2005).
Mitogen Activated Protein Kinases (MAPK)
Representing another important and well documented protein family to be involved in signal transduction would be the Mitogen Activated Protein Kinase (MAPK). It generally encompasses a few different subfamilies such as the p38 isoforms (p38s), the Extracellular Signal-Regulated Kinases (ERKs) and the c-Jun NH2-terminal Kinases (JNKs). Whenever these subfamilies were to activate the Early Growth Response Factor 1 (EGR-1) and the transcription factor protein (AP-1), it has been documented that the expression of pro-inflammatory genes would be upregulated to cope with inflammatory response (Huang, Han and Hui, 2010). In general, MAPK kinase (MAPKK or MEK) is responsible for activating MAPK by phosphorylating both the threonine and tyrosine residue on MAPK. On the other hand, the MAPKK itself is activated by MAPKK kinase such as Raf-1 (a type of proto-oncogene) due to the phophorylation process involving the serine residue on MAPKK. In conclusion, this sequential phosphorylation process which takes place generally makes up the MAPK cascade which basically forms the basic signalling pathway in regulating cell proliferation and differentiation (Huang, Han and Hui, 2010).
If we look into individual subfamilies of the MAPK, we can notice that p38 represents a fundamental protein kinase that has been intrinsically linked with a number of inflammatory pathologies such as rheumatoid arthritis (RA), psoriasis and inflammatory bowel disease (IBD). It has been experimentally established that both p38 isoforms, p38Î± and p38Î² are primarily presented in inflammatory cells (Huang, Han and Hui, 2010). Being one of the most widely expressed isoform in most tissues, p38Î± fundamentally manifests itself as a serine/theorine kinase that predominantly responds to stress stimuli such as heat shock and LPS. The extensive role that p38Î± plays in inflammation has been substantiated with experiments which studies inflammatory response in p38Î±-deficit macrophages. Interestingly, it was found in these macrophages that the expression and activation of various LPS-induced proinflammatory cytokines such as IL-18, IL-12 and TNF-Î± was suppressed (Kang, et al., 2008). Apart from that, it has also been proposed that p38Î± isoform might be the representation for p38 that is responsible for playing the crucial role in cell proliferation and cell differentiation (Huang, Han and Hui, 2010). On the other hand, JNK genes are well-known to regulate both the expression and activation of inflammatory mediators, namely TNF-Î±, E-selectin, IL-2 and matrix metalloproteinases (Huang, Han and Hui, 2010). Such understanding derives from experiments which examine the extensive inflammation in the intestinal tract in patients with IBD where higher JNK phosphorylation activities are displayed. The inhibition of JNK activity with a pan-JNK inhibitor which resulted in the reduction of TNF-Î± & IL-6 expression substantiates the role that JNK genes play in inflammation (Assi et al., 2006). Conversely, the ERKs generally induce the upregulation of inflammatory cytokines when stimulated, where a higher expression of IL-8 activity is visible.
Phosphatidyl-inositol-3 kinase (PI3K)
In addition to above mentioned LPS-induced signalling pathways (through the action of TLR4), another important signalling pathway that is worth mentioning in this study would be the Phosphatidyl-Inositol-3 Kinase (PI3K) pathway. The PI3K family which are made up of four distinguishing subfamilies, namely IA, IB, II and III also commonly plays an important role in regulating cell differentiation and gene expression (Schabbauer et al., 2004). Structure wise, PI3K consists of two domains: one essentially for its regulatory functions whilst the other for its catalytic properties (Schabbauer et al., 2004). Generally, when PI3K are activated, its associated downstream kinases such as Akt/PKB and PDK-1 would then undergo a similar activation. It has been documented in some studies that when PI3K-Akt/PKB are activated, the expression and activity of TNF-Î± would be suppressed in human monocytic cells through a negative modulation mechanism (Schabbauer et al., 2004). Nevertheless, the most interesting characteristic of PI3K would be its ability to induce different effects on NF-ÎºB in different cells. In monocytic cells, it was found that the expression and activation of NF-ÎºB would be increased whenever PI3K activity is suppressed/inhibited; while in the human endothelial cells, the activation of NF-ÎºB is dependent on the activation of the PI3K pathway (Ojaniemi et al., 2003).
One particular enzyme that has always been heavily associated with inflammation would be the Cyclooxygenase enzyme, famously abbreviated as COX. Being an isoform, COX can generally be subdivided into COX-1 or COX-2 which essentially stimulates the production of PGH2. PGH2 functions as a precursor to produce prostacyclin, prostaglandins and thromboxanes, all of which plays a specific role in inflammatory response (Chang et al., 2009). In general, the rate limiting step in PGH2 production would be the conversion rate of Arachidonic Acid (AA) to PGH2 by which the presence of oxygen is essential for such conversion process to take place. In terms of functionality, the abundantly expressed COX-1 is by and large responsible for maintaining the homeostatic balance of cells while the meagrely expressed COX-2 is mainly inflammatory-sensitive. Therefore, during an inflammatory response, we can notice an upregulation in COX-2 expression and production, leading to a higher COX-2 activity. Interestingly, different levels of activity were observed from individual isoform of COX in relation to the type of inflammation being induced (acute inflammation or chronic inflammation). In acute inflammatory response, it has been documented that the activity of COX-1 supersedes the activity of COX-2 whilst the opposite occurs during chronic inflammation (Mitchell et al., 1995). Such finding might have an interesting implication for potential treatment for diseases associated with chronic inflammation.
Generally, the converted products of PGH2 itself play a pivotal role in inflammatory response. For instance, the vasodilator effect induced by both prostaglandins E2 and I2 would promote leukocyte extravasations. On the other hand, the vasoconstriction effect induced by thromboxane would promote platelet aggregation (Mitchell et al., 1995). In addition to that, another fascinating discovery with regards to prostanoid was that it portrays a localised yet different biological action, depending on the site of action (Mitchell et al., 1995). The classic example would be prostaglandin D2 which is able to induce a vasoconstrictor effect in mesenteric area and yet a vasodilator effect at the kidney area.
Nitric Oxide (NO)
Nitric oxide (NO) which usually serves as a vessel tone regulator, have also a crucial role in host immunity, specifically in terms of inflammatory response. In general, Nitric Oxide Synthase (NOS) are understood to be responsible in activating the oxidation process of L-arginine by which would ultimately produce NO. Basically, NOS are also available as an isoform, which can be generally broken down to either cNOS or iNOS (Clancy et al., 1998). While the calcium-dependent cNOS represents the isoform that is widely expressed in cells, the calcium-independent iNOS on the other hand represents the type of isoform that are only normally induced in response to a stress stimulus such as inflammatory cytokines (LPS, TNF-Î± or IL-1) (Clancy et al., 1998). An important characteristic to note with regards to NO would be its diverse effect that it is able to induce of which is dependent to its concentration at target site as well as the site of action. For example, low concentration of NO at the vascular endothelium would promote the relaxation of the associated smooth muscles while high concentration of NO in response to cytokine stimulation would elicit a cytotoxic effect of which would mostly target invading pathogens (but host cells as well) to provide the host with a proper yet a non-specific immunity (Clancy et al., 1998).
Cyclooxygenase (COX)/Nitric Oxide (NO) Relationship
If we view both COX and NO pathway in unison, we are able to establish a rather interesting yet not completely understood relationship between these two pathways. Interestingly, it was observed during inflammation in some cell types (i.e murine macrophages, rat vascular smooth muscle) that a coexpression/coinduction of COX-2 and iNOS incidence would occur (Mitchell et al., 1995). Such findings were exciting as some studies have demonstrated that endogenously-released NOs exhibit both the inhibition and activation properties on COX-2. The possible explanation for its inhibition properties probably lies with its capability in reducing COX from its ferric-active form to its non-ferric active form (Mitchell et al., 1995). Apart from that, another possible explanation would be the involvement of the S-nitrosylation and nitration process involving some the vital amino acid in which would also justify the inhibition properties portrayed by NOs (Prieto, 2010).
Therefore, the aim of this study is to investigate various compounds including plant-derived products, natural products and some synthetic small molecules for their potential anti-inflammatory properties. Their potential inhibitory action on the LPS-induced signalling pathways would primarily be assessed using a murine marcophage cell model, by which we would investigate the changes in iNOS expression and activity.