## 6 NP Immunomodulators
### Fungal Metabolites  
The natural product cyclosporin A (CyA) is a small lipophilic cyclic peptide produced by the entomopathogenic fungus _Tolypocladium inflatum_, which functions to promote mycotic growth by severely compromising its insect host's immune system. CyA has similar effects on the mammalian immune system by inhibiting antigen receptor signal-transduction pathways conserved in both lymphocytes and haemocytes [62] and by blocking transcription of pro-inflammatory mammalian cytokines [63]. CyA also inhibits apoptosis in human macrophage/monocyte populations and inhibits neutrophil functions in vitro. It has a long clinical history of use as a potent immunosuppressiveant in humans to prevent organ transplant rejection and is also used to suppress autoimmune hypersensitivity, such as rheumatoid arthritis [64]. The insecticidal effect of CyA is not observed in _G. mellonella_[65], making this an ideal model to study this immunodulator. In vivo treatment of _G. mellonella_ larvae (7 th instar; \(n=10\)) with CyA elicited an increase in humoral defence systems in the haemolymph (anti-microbial lysozyme release (numerical data not published) but inhibited cellular immune processes in isolated haemocyte (in vitro phagocytic activity) in a dose-dependent manner [65]. The conflicting results can be attributed either to (i) the injection process triggering an inflammatory response [66], (ii) the difference in treatment regimens (in vivo work involved injection of CyA whereas \"in vitro\" studies involved ex vivo incubation with naive haemocytes), or (iii) the different CyA doses employed (in vivo = 10-30 ug; in vitro = 100 ug). In another study, larvae (7 th instar; \(n=\) unpublished) inoculated with bacterial-derived LPS (_Pseudomonas aeruginosa_; 39 ng/larvae) induced high levels of humoral anti-bacterial activity which was completely abrogated by the co-administration of CyA [67]. This immunosuppressive effect of the insect humoral defences correlated with a loss in protective immunity (30%) directed against _E. coli_ challenge, resulting in a higher mortality rate in CyA-treated bacterially challenged insects compared to the negative controls. The authors then compared the effect of CyA (22.5 ug/larvae) on LPS-inoculated pupae (2-3 days post-cocoon; \(n=\) unpublished) in the initial phase (CyA + LPS together) and in the effector phase (LPS + CyA 18 h later) of the _G. mellonella_ immune response. CyA was more effective in inhibiting LPS-induced haemolymph lysosomal activity (40% reduction) and reducing bacterial (_E. coli_) infection load (50% reduction) in the initial phase. CyA administration in the effector phase was lesseffective, causing only 20% reduction in lysosomal activity and a 24% in bacterial load compared to LPS-inoculated controls [68]. Taken together, these studies demonstrate that the well-characterised immunosuppressionent effects of CyA fungal derivative in humans can be replicated in _G. mellonella_ immunity and entomopathogenic infection models and that these properties are used by the fungi to disable insect immunity to facilitate host-pathogen interactions. Although CyA has been shown to exert its immunosuppressiveent effect by inhibiting human and insect phagocytes, the very real possibility exists that the insecticidal activity of CyA in insects (postulated to be via blocking of insect detoxification P-glycoprotein efflux pumps) is via a different mechanism than described in humans (calcineurin inhibition). CyA has a very successful clinical history but, at high doses, is associated with severe adverse effects, so there is an urgent need to a find new, less toxic NP immunosuppressiveent. The entomopathogenic fungal-derived cyclic lipophilic amino acid metabolite myriocin (ISP-1) has demonstrated more potent immunosuppressive effects than CyA in vitro (myriocin IC50 = 0.0056 vs. 0.03 \(\upmu\)g/mL CyA) and in vivo (myriocin 10-fold \(>\) CyA in lymphoproliferation in mice) [69]. A comprehensive study was undertaken using the _G.