Molecular characterization of nitric oxide stimulatory molecules of Brugia malayi parasite

Abstract

Lymphatic filariasis (LF), one of the major causes of chronic disability in the developing countries produces a considerable economic burden. Globally, around 1100 million people in 80 countries are at risk and 118 million people show clinical manifestations. It is caused by the nematode parasites Wuchereria bancrofti, Brugia malayi and B. timori. Current estimate shows that in India about 553 million people live in endemic areas with approximately 48 million have either circulating Microfilariae (Mf) or overt disease like hydrocele, lymphoedema and elephantiasis [1, 2]. Around 50% of the infected persons suffer from acute episodic attacks of adenolymphangitis (ADL) and fever. A million individuals have cryptic infections resulting in conditions such as tropical pulmonary eosinophilia (TPE). The infection is transmitted by mosquitoes (W. bancrofti by Culex quinquefasciatus and B. malayi by Mansonia sp.). The infection is initiated by introduction of third stage infective larvae (L3) of the parasite into the host by the bites of L3-bearing mosquitoes. Adults parasites reside into the lymphatics and Mf circulate and available in the peripheral blood. The parasites survive for several years in mammalian hosts, often without causing overt clinical manifestations. At initial stage the disease is characterized by acute episodes of adenolymphangitis, fever and associated constitutional symptoms. The recurrent episodic bouts of acute manifestations occurring over a period of time lead to the development of chronic disease manifestations such as elephantiasis, lymphedema and/ or hydrocoele. It is believed that the presence of adult worms in lymphatics results in dilation of the vessels, and this is considered to be the primary reason that predisposes to disease manifestations because of chronic exposure of inflammatory elements. Filarial parasites present a diverse array of antigens possessing both suppressive and stimulatory type and therefore the host response against these antigens is expected to be complex and variable. However, precise identity of the molecules and how the molecules are involved in the host-parasite interactions and their outcome in relation to protection of host from infection or protection for the parasites or infection-induced manifestations is unresolved. The pre-requisite to achieve this is the identification of parasite molecules that are responsible for initiating such reaction. The next logical step is delineation of the immune mediated responses to the parasite molecules. Proinflammatory mediators, like proinflammatory cytokines, nitric oxide (NO) etc are known to have a direct implication in the protection and pathology of LF. Nitric oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology including as a toxic defense molecule against infectious organisms. It is a highly reactive and pervasive biological mediator produced by mammalian cells, and its physiological actions are broad. NO mediated functions fall into three categories: (1) smooth muscle relaxation [3, 4], (2) neurotransmission [5] and (3) cell mediated immune response [6, 7]. It also regulates the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells [8]. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation like filariasis is poorly understood. NO does not act through a receptor—its target cell specificity depends on its concentration, its chemical reactivity, the vicinity of target cells and the way that target cells are programmed to respond. At high concentration as generated by NOS-2, NO is rapidly oxidized to reactive nitrogen oxide species (RNOS) that mediate most of the immunological effects. RNOS can S-nitrosate thiols to modify key signaling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy. A combination of these interactions may lead to the multiple actions of NO in the regulation of immune and inflammatory cells. Because the immune system is activated in response to infection, any associated NO response would develop in parallel, over days or weeks rather than within a fraction of a second as in physiological responses. Also, for NO to be effective as a toxic or immune regulatory mediator it needs to be generated at high levels for a sustained period of time. NO exerts immunosuppressive effects in vivo also since in NOS-2 gene knockout mice Th1 responses have been shown be enhanced to Leishmania infection [9] and implicated in exacerbated severity of autoimmune encephalitis [10]. In addition, NO regulates death of immune cells, either through induction or inhibition of apoptosis, or by necrosis. In some cell types NO can promote apoptosis, whereas in other cells NO inhibits apoptosis. High NO concentrations lead to the formation of toxic reaction products like dinitrogen trioxide or peroxynitrite that induce cell death, if not by apoptosis, then by necrosis. Long-term exposure to nitric oxide in certain conditions like chronic inflammatory states may predispose cells to tumorigenesis through DNA damage, inhibition of DNA repair, alteration in programmed cell death, or activation of proliferative signaling pathways. In filariasis these phenomenon is not clear. Cytokines are potent messenger molecules at sites of inflammation. Parasiteinduced cytokines, such as IFN-γ, TNF-α, IL-1β, and pathogen products such as glycosylphosphatidylinositols, can stimulate iNOS expression in infected hosts [11-13] (Figure). In other words, agents that induce iNOS expression include various proinflammatory cytokines (IL-1β, TNF-α- and IFN-γ), endotoxin lipopolysaccharide (LPS) and other agents [14]. The specific relationship among IL-1β, TNF-α- and IFN-γ on iNOS induction and NO synthesis in cells has not been reported in filariasis. In addition synergistic action between NO and cytokines is not clear in filariasis. Earlier study carried out in this laboratory revealed that level of IFN-γ and NO increased after L3 exposure to animals immunized with predominantly proinflammatory cytokine stimulating fraction of B. malayi which was found implicated in suppressing L3-induced filarial infection [15]. Thus, these reports encourage us to study synergistic or direct or indirect effects of proinflammatory cytokines on the NO expression. In nature, both endemic normals and chronic filarial patients, who are considered immune to L3 invasion or adult worms, have shown to have higher IFN-γ levels [16, 17]. Further, investigators have also demonstrated increased level of IFN-γ and IL-2 in endemic normal individuals and suggested that type 1 inflammatory responses may be involved in killing of L3 stage of the parasite and could be the mechanism by which endemic normals remain infection free. Das et al. (1996) [18] have reported that proinflammatory cytokines particularly TNF-α was raised during acute episodic attacks in filariasis but whether NO is also raised during this period and relationship between the two is not clear. In filariasis, NO mediated mechanisms have been shown to be capable of killing Mf in vitro and L3 in vivo and protect the host through Type 1 responses and IFN-γ stimulated toxic mediator’s release [19-21]. iNOS gene expression is regulated by complex mechanisms. Studies carried out in this laboratory have shown that BmAFII, a Sephadex G-200 eluted fraction of B. malayi adult worm extract, with molecules ranging from 21 to 84kDa, protected the host against B. malayi in M. coucha [15] and cross protected Leishmania donovani infection in hamsters [22] by stimulating predominantly proinflammatory responses. Further, SDS - PAGE fractions of the parasite corresponding to the molecular weight range of BmAFII were able to stimulate pro- and anti-inflammatory cytokines release in vitro [23]. Of these F6 (67.8-54.35 kDa) revealed that it protected the host from the parasite via Th1/Th2 type responses. A ~62kDa molecule of B. malayi suppressed the establishment of L3-induced infection in M. coucha and this correlated well with enhanced IFN-γ release, DNA damage in lymphocytes and downregulation of IL-10. These findings have provided leads for exploring whether some of the parasite molecules may also stimulate NO. The present study was therefore aimed for identifying and characterizing NO-stimulating molecules and to determine their role in protecting the host from infection. The present study was therefore undertaken with the following Objectives: 1. Identification, isolation and purification of NO stimulating fractions of B. malayi adult worm. 2. To investigate effect of the identified fraction(s) on the development and establishment of B. malayi L3 induced infection in M. coucha. 3. To investigate immunogenicity of the fraction(s). 4. To resolve proteins of the fraction(s) by 2DE and identify them by MALDI-TOF-MS. 5. To investigate role of identified fraction(s) in apoptosis. 6. Expression of NO genes in response to identified fraction(s) under Th1 /Th2 cytokine milieu.