ABSTRACT
Toll like receptors are components of the innate immune system. They are immune system mediators which act as signaling receptors. The purpose of this study was to assess the levels of TLR4 in pediatrics Typhoid fever of different demographics and identify similarities or differences that could affect its levels. 14 plasma samples obtained from blood of individuals aged 0 – 16 within the city of Abak, Akwa Ibom, Nigeria. TLR4 levels was estimated was using the Enzyme Linked Immunosorbent Assay (ELISA). However, there was no significant difference in the concentration between the different age groups. Individuals between 1_4year had mean concentration of 2.7ng/ml, 5-8 age group, 77.55ng/m l9-12 age group had 36.59ng/ml and 13-59 age group had 26.73ngml as their mean concentration. Individual between 1-4year had the lowest mean concentration in their blood plasma and individual between the age of 6-8 years had the highest mean concentration in their plasma. This study shows that the concentration of TLR4 in pediatrics typhoid fever patients decreases with age. The result showed that all the patients have TLR4 in their serum plasma.
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Toll-like receptors (TLRs) are transmembrane proteins. They form a part of the Toll/interleukin-1 (TIR) super family that includes the interleukin-1 receptors (IL-1Rs) because of the shared homology of their cytoplasmic domains. However, the extracellular domain of IL-1Rs consist of an immunoglobulin G (IgG) domain while TLR extracellular domains are made up of tandem repeats of leucine-rich regions termed leucine-rich repeats. Macrophages initiate the innate immune response by recognizing pathogens, phagocytizing them and secreting inflammatory mediators. An effective immune response requires that macrophages recognize pathogen-associated molecular patterns (PAMPs) that distinguish the infectious agents from ‘‘self’’ and in addition discriminate among pathogens. A novel family of receptors, the Toll-like receptors (TLR), has been demonstrated to identify pathogen-associated molecular patterns (PAMPs) on infectious agents and discriminate between pathogens (Janeway and Medzhitov, 2002).
Central to this role is the broad specificity with which they can detect pathogen-associated patterns and danger associated patterns via the pattern recognition receptors (PRRs) they express. Several families of PRRs have been identified including Toll-like receptors (TLRs), C-type lectin-like receptors, retinoic acid-inducible gene-like receptors and nucleotide-binding oligomerization domain–like receptors. TLRs are one of the most largely studied families of PRRs. The binding of ligands to TLRs on antigen presenting cells (APCs) mainly dendritic cells leads to APC maturation, induction of inflammatory cytokines and the priming of naive T cells to drive acquired immunity (Janeway and Medzhitov, 2002). Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors usually expressed on sentinel cells such as macrophages and dendritic cells, which recognize structurally conserved molecules derived from microbes (Janeway, 1989). Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs, which activate immune cell responses. Toll-like receptors are a family of type I transmembrane pattern recognition receptors (PRRs) that sense invading pathogens or endogenous damage signals and initiate the innate and adaptive immune response. Furthermore, Toll-like receptors ligation triggers several adapter proteins and downstream kinases, leading to the induction of key pro-inflammatory mediators but also anti-inflammatory and anti-tumor cytokines (Beck and Habicht,1996).The result of this activation goes beyond innate immunity to shape the adaptive responses against pathogens and tumor cells, and maintains host homeostasis via cell debris utilization (Beck and Habicht,1996).
The TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, though the latter three are not found in humans. Of the human TLRs, TLR1, 2, 4, 5, 6, and 10 are expressed on the cell surface and primarily recognize microbial membrane and/or cell wall components, while TLR3, 7, 8, and 9 are expressed in the membranes of endolysosomal compartments and recognize nucleic acids.TLR2 is one of the toll-like receptors and plays a role in the immune system. TLR2 is a membrane protein, a receptor, which is expressed on the surface of certain cells and recognizes foreign substances and passes on appropriate signals to the cells of the immune system. TLR2 recognizes the largest number of microbial ligands, including various fungal, gram-positive and mycobacterial components such as peptidoglycans, lipoarabinomannan and bacterial lipoproteins (Bell et al; 2003).
The immediate or innate immune response is the first line of defense against diverse microbial pathogens and it requires the expression of sensor molecules such as toll-like receptors (TLRs). TLR4, one of the TLRs serves as a specific receptor for lipopolysaccharide (LPS) and is localized on the surface of a subset of mammalian cells (Lehnardt et al., 2002). Toll-like receptors (TLRs) play key roles in detecting pathogens and initiating inflammatory responses which subsequently elicit specific adaptive responses (O’Neill et al., 2006). Several mechanisms control TLR activity to avoid excessive inflammation and consequent immunopathology, including the anti-inflammatory cytokine IL-10 (Rossato, 2012). Recently, several TLR-responsive microRNAs (miRs) have also been proposed as potential regulators of this signaling pathway but their functional role during the inflammatory response is not completely understood (Graziella et al., 2013).
Toll-like receptor (TLR) 4 is essential for the defense against infection with gram-negative pathogens but reduced TLR4 expression has not been linked to altered disease susceptibility in humans (Ragnarsdo´ttir et al., 2007). In mice, Tlr4 controls the mucosal response to Escherichia coli urinary tract infections. Inactivation of mouse Tlr4 causes an asymptomatic carrier state resembling asymptomatic bacteriuria (ABU). Altered TLR4 and adaptor protein expression might impair TLR4 signalling and explain the weak mucosal response to urinary tract infection in patient who develop ABU rather than symptomatic disease (Ragnarsdo ‘ttir et. al., 2007).
It is therefore important that TLR signaling pathways are tightly regulated. One of the most effective suppressor of TLR-induced inflammatory cytokine production is IL-10, which displays powerful inhibitory actions on innate immune cells (Murthy et al., 2000 3; Siewe, 2006), not only by direct inhibition of cytokine transcription (Zhou et al., 2004) but also by destabilizing their coding RNA and blocking their translation (Knödler, 2009).
1.2 TYPHOID FEVER
Enteric fever is a systemic infection caused by the human adapted pathogens Salmonella enterica serotype Typhi (S. Typhi) and S. enterica serotpe Paratyphi (S. Paratyphi) A, B, and C. These organisms are important causes of febrile illness in crowded and impoverished populations with inadequate sanitation that are exposed to unsafe water and food and also pose a risk to travellers visiting countries of endemicity (Whitaker et al., 2009).
In 2000, typhoid fever caused an estimated 21.7 million illnesses and 217,000 deaths, and paratyphoid fever caused an estimated 5.4 million illnesses worldwide (Luby et al., 2004). Infants, children, and adolescents in south-central and Southeastern Asia experience the greatest burden of illness (Luby et al., 2004). Typhoid and paratyphoid fever most often present as clinically similar acute febrile illnesses and accurate diagnosis relies on laboratory confirmation (Youssef et al., 2003).
1.3 STATEMENT OF PROBLEM
Efforts to develop serologic methods for the diagnosis of typhoid fever that improve on the poor performance of the Widal test still suffer from substantial limitations of both sensitivity and specificity (Olsen et al., 2004). Serological approaches to the diagnosis of S. Paratyphi A, B, and C have been developed but have not been evaluated or adapted for field use (Chart et al., 2007). However, most enteric fever occurs in low- and middle income countries (Chart et al., 2007). The most robust approach to the measurement of incidence of typhoid and paratyphoid fever is by regular, community-wide household visits to identify persons with febrile illness from whom blood samples for culture confirmation may be obtained. Alternatively, the results of surveys of health-seeking behavior and sentinel health care facility–based surveillance may be combined to estimate incidence (Youssef et al., 2003).
1.4 Significance of the Study
Toll-like receptors (TLRs) are a family of proteins that play a key role in the innate immune response to infectious agents through their ability to discriminate conserved microbial structures, known as pathogen-associated molecular patterns (PAMPs), from self (Akira et al., 2006). TLR recognition of PAMPs such as lipopolysaccharide (LPS), teichoic acid, and surface lipoproteins, initiates signal transduction through the NF-kB pathway (Akira et al., 2006). Nuclear trans-location of NF-kB induces transcription of pro-inflammatory cytokine genes essential to mounting a protective immune response and host defense (Tapping et al., 2000).
Different TLRs recognize distinct classes of products synthesized by pathogens. For example, TLR4 is a receptor for LPS made by Gram-negative bacteria (Tapping et al., 2000).. Consistent with this role, it has been reported that amino acid polymorphisms in the extracellular domain of TLR4 conferred altered responsiveness to inhaled LPS in humans (Akira et al., 2006).
Among the human TLR family members described to date, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, and TLR9 have been implicated in the recognition of bacterial components (Akira et al., 2006).. Associations between TLR polymorphisms and susceptibility to infectious agents have been reported (Smirnova et al., 2003; Hawn et al., 2003; Schroder et al., 2005 and Johnson et al., 2007). However, the conclusions that can be drawn from these studies are limited because, in general, relatively few polymorphic sites have been analyzed, small patient populations have been studied, and the analyses have largely been confined to a single TLR gene (Xin et al., 2007).
TLRs have been reported to participate in the interaction of pathogenic mycobacteria or their extracellular products with mice and humans. In particular, TLR2 in association with TLR1 and TLR6, and TLR4 have been implicated as receptors involved in the recognition of mycobacterial antigens and activation of macrophages and dendritic cells (DCs) (Abel et al., 2002; Reiling et al., 2002; Lopez et al., 2003; Drennan et al., 2004; Roura-Mir et al., 2005; Branger et al., 2004; Chang et al., 2006; Schumann et al., 2007; Omueti et al., 2007 and Hawn et al., 2007).
1.5 Aims and Objectives
- To determine the level of positivity for typhoid fever infection
- To measure the level of TLR4 in patient
- To assess association of TLR4 levels with disease