Modulation of innate immunity by the cGMP signalling pathway in the drosophila malpighian tubule.
PhD thesis, University of Glasgow.
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The Drosophila innate immune system is one of the most widely characterised of all metozoan defense systems, and shares many similar characteristics to the innate immune systems of higher organisms. As such, Drosophila has become the model organism of choice for many researchers with regards to the study of the general mechanisms and regulatory elements of innate immunity. There are a number of mechanisms that Drosophila employ in order to combat infection, and these include both humoral and cellular responses. However, perhaps the most widely characterised of these mechanisms is the systemic production of anti-micorobial peptides (AMPs) via the activation of two specific immune signalling pathways – Toll and Imd (Lemaitre et al. 1995a; Belvin and Anderson 1996).
In Drosophila, a number of recent studies have identified a role for the diffusible second messenger nitric oxide (NO) in the positive regulation of the Imd pathway, a pathway that is fundamental to host defence against Gram-negative bacteria (Lemaitre et al. 1995a; Nappi et al. 2000; Foley and O'Farrell 2003; McGettigan et al. 2005). To date, the exact mechanism by which NO is mediating its effects on the Imd pathway has not yet been determined. However, it can be suggested that this effect is mediated through activation of the cGMP signalling pathway, via interaction with one of its upstream components, soluble guanylate cyclase (sGC), the main intracellular target for NO (Marletta and Spiering 2003).
Therefore, the aim of this study was to determine the potential role of the cGMP signalling pathway on regulation of the Drosophila Imd immune pathway. To do this, the Drosophila Malpighian (renal) tubule was used as a model system. The Malpighian tubule is a very well characterised, extensively studied epithelial tissue and for a number of years has comprised the model system of choice with regards to the study of the epithelial roles of signalling and transport genes (Dow and Davies 2001). The suitability of this tissue as a model system for this study is two-fold: Firstly, for many years, the NO/cGMP signalling pathway has been deemed as critical to tubule function (Dow et al. 1994a). Secondly, a recent study has identified the tubule as an important autonomous immune-sensing tissue where, upon immune challenge with Gram-negative bacteria, Imd pathway-associated AMPs are systemically produced in the tubule principle cells. Importantly, it has been demonstrated that activation of the Imd pathway in the principle cells is regulated via the autocrine production of NO (McGettigan et al. 2005).
Data obtained from this study has demonstrated a completely novel role for cGMP signalling in the tubule. Expression analysis has revealed that cGMP acts to modulate the expression of Imd pathway-associated AMPs in a dose-dependent manner; whereby low nanomolar concentrations are shown to stimulate diptericin expression and higher micromolar concentrations of cGMP are shown to inhibit it. This effect does not appear to extend to the fat body, the canonical tissue involved in the systemic induction of AMPs, thus suggesting a completely tissue-specific mechanism.
Importantly, it is shown here that the cognate cGMP-dependent protein kinases (cGKs), DG1 and DG2 (MacPherson et al. 2004a; 2004b), mediate differential effects on AMP production in the tubule. Targeted modulation of the expression of these kinases to the principle cells of the tubule using the GAL4/UAS system demonstrates that activation of DG1 mediates positive modulation of diptericin expression in the tubule. By contrast, negative modulation of diptericin expression is shown to occur following the activation of the two main isoforms of DG2, DG2P1 and DG2P2. These data therefore describe a completely novel role for each of these kinases. Significantly, the effects of these kinases on diptericin expression in the tubule are sufficient to impact on survival of the whole fly in response to septic infection with Gram-negative bacteria, as well as contribute significantly to bacterial clearance in the gut following natural infection with E.coli. This study has therefore revealed a critical novel role for both the tubule and cGKs in the regulation of defence mechanisms in response to both septic and natural infection in the adult fly.
Interestingly, Q-PCR has revealed that DG1 mediates its effects downstream of Imd. Additionally, studies have revealed that both DG1 and DG2 act to regulate the Imd pathway via modulation of Relish activation, the NFκB transcription factor responsible for the induction of AMPs following activation of the Imd pathway (Hedengren et al. 1999). Translocation assays have demonstrated that targeted over-expression of dg1 to the principal cells of the tubule results in enhanced translocation of activated Relish into the nucleus, whereas targeted knock-down of this kinase by RNAi results inhibition of Relish activation. In contrast to DG1, overexpression of either dg2P1 or dg2P2 to the principal cells of the tubule results in inhibition of Relish activation, even in the presence of immune challenge. However, this study has not revealed the exact mechanism by which these kinases mediate their effects on Relish activation, and therefore it is not clear whether DG1 and/or DG2 are acting directly on Relish, or indirectly via phosphorylation of an, as of yet, unidentified substrate(s). Despite this, a completely novel function for each of these kinases is described here for the first time.
Importantly, data described in this study also identifies that, with regards to Imd pathway regulation, DG1 and DG2 may be activated via different sources of cGMP within the cell. Data shows that stimulation of the Imd pathway in the tubule is facilitated by the activation of sGC via interaction with NO. Alternatively, inhibition of the Imd pathway in the tubule is shown to be facilitated by the activation of a receptor guanylate cyclase (rGC). Additionally, it is demonstrated by this study that cGMP-mediated inhibition of the Imd pathway in the tubule is regulated by the dual-specificity, tubule-enriched phosphodiesterase (PDE), PDE11 (Day et al. 2005), thus describing a functional role for this regulatory enzyme for the first time in Drosophila.
In conclusion, this study further validates the role of the tubule as a critical immune-sensing tissue in Drosophila melanogaster. In addition, a completely novel role for the cGMP signalling pathway, as a differential regulator of Imd pathway activation in the tubule, is described here for the first time. In particular, an important novel functional role for each of the Drosophila cGKs, DG1 and DG2, is revealed. The data shown in this study therefore contributes to fuller understanding of not only Imd pathway regulation in Drosophila, but also provides a significant advance in the understanding of the complexities of cGMP signalling and its regulation of tubule function.
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