Cyclic nucleotide signalling in innate immunity

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences


Immune function in animals counters the possible effects of infection. In insects, an ancient form of immunity exists, which forms the template for human and animal immune systems. Studies conducted in the tiny fruit fly, Drosophila, using the insect analogue of the kidney (Malpighian tubule), have shown that this tissue itself can sense an immune attack, and can confer immune status on the whole animal. This suggests that peripheral tissue such as kidney may be more important than previously thought in immune surveillance for the whole body. How does the tubule mount an immune defense? We have evidence that a simple molecule, cyclic GMP, regulates gene expression of a key set of anti-microbial defence peptides in the tubule. This can affect the immune status of the entire animal. The studies described in the application will allow us to work out how cGMP can set the level of the immune state. As cGMP also exists and works in a similar way in humans as in the fly, it is possible that we may uncover new modulators of immunity by working out how such molecules work in Drosophila. This may have important consequences for both insect pest control strategies (many insects are major vectors of human disease) and also in the assessment of potential new therapeutic drugs, that act to control levels of these small molecules in various disease conditions.

Technical Summary

Innate immunity is an ancient defence against microbial attack, which in insects is thought to originate mainly in the fat body. We have recently shown that the fluid-transporting Malpighian (renal) tubule of D. melanogaster is an autonomous immune-sensing tissue, which uses the nitric oxide (NO) signalling pathway (MeGettigan et al., Ins Biochem. Mol. Biol. 2005). More recently, we have identified a novel role for cyclic nucleotides in the immune response: our unpublished tubule microarray dataset, validated by Q-PCR, show that cGMP modulates the expression of antimicrobial peptides (AMPs). We have thus uncovered a novel and powerful role of tubules in immune sensing and animal survival via a mechanism that involves cGMP. We also show that differential regulation of AMPs occurs via both cGMP and cAMP. We aim to unravel the potential role(s) of cyclic nucleotides on epithelial immune gene expression; and the role of such modulation of immune function on the whole animal by: 1: fully characterising cGMP/cAMP modulation of the Imd and Toll pathways; 2: understanding the contribution of other significant immune tissue to the modulation of immune status by cGMP ; 3: delineation of a novel range of transcriptional targets for cGMP and 4. defining the role of downstream effectors of cGMP ie., cGMP-dependent protein kinases on immune status. Techniques used include: Q-PCR, survival studies, generation of transgenic flies; use of reporter gene assays both in cultured cells and in transgenic flies.
Description Conserved innate immune pathways first identified in the tiny fruit fly, Drosophila, are critically important in vertebrates and are instrumental in the modulation of adaptive immunity in humans. The components of innate immune pathways are extremely well-mapped mapped but much less is known about potential modulation of innate immune function by small molecule second messengers. Moreover, epithelial innate immunity has emerged as a key area of investigation in relation to human disease. During the course of this grant we have made exciting discoveries in all these key areas by using a combination of molecular genetics, biochemistry, survival assays and computational methods:
I. Using the fly equivalent of our liver and kidney, the epithelial Malpighian tubule, we have identified the cyclic nucleotide, cGMP, and its two downstream cGMP-dependent kinases (cGKs) DG1 and DG2, which act as novel, direct 'on' or 'off' switches for innate immune gene regulation. Essentially, DG1 is activating, whilst DG2 is inhibitory, each acting to increase (DG1) or decrease (DG2) activity of a key transcription factor, Relish, a small protein which controls gene expression. Relish is a close relative of human NF-kB, which has roles in immune and stress signalling. This new mechanism of innate immune gene regulation also impacts on another key epithelial tissue, the gut, and also determines whole organism survival to infection. Thus we have shown that in mounting an immune response, tissue-tissue communication occurs in the organism. The Drosophila cGKs are very similar to the mammalian enzymes, and as we have shown that they modulate activity of a similar transcription factor to human Nf-kB, we have sought to extend this work to the mammalian system. We now have preliminary evidence that such a modulatory pathway of Nf-kB activity may occur in mammalian cells, using cell lines and also transgenic flies which express mammalian cGK in key tissues like the Malpighian tubule.
II. We have also shown that the regulation of immune genes is not necessarily part of the immune response in tissues and the whole organism to infection; but rather can also provide an early 'warning' for stress sensing and response. For example, under salt stress, we have shown that immune genes are up-regulated. To further this work, we have used computational methods to model stress networks and have discovered key network 'hubs' which act to deliver specific organismal responses to different stressors including immune, neuroendocrine and salt stress. This work will really uncover novel aspects of stress sensing and response, from the level of the molecule to the organism. Also, as signalling molecules are so well conserved across species, we may find similar pathway regulation in mammals. These will be key findings for modulation of mammalian innate immunity.
III. We have identified a novel regulatory peptide which modulates stress responses in the Malpighian tubule, and acts via cGMP signalling. In doing so have also 'de-orphaned' a plasma membrane receptor for which until now, there has been no known ligand. This work also supports our data described in section II, as although this peptide/receptor interaction up-regulates immune genes, this is not translated by the fly into a response to infection. Rather, this peptide-induced up - regulation of immune genes is a signal to resist stress, in particular, salt stress.
In addition to the work outlined above, we have also developed work on detoxification mechanisms, which has been of value to our industrial partners.
The grant award has also allowed us to collaborate with groups in the USA, Japan and Europe on different aspects of stress (eg., response to low oxygen), immunity and most recently, novel roles of cGKs in neurodegeneration (Parkinson's).
We have published 4 papers directly associated with this grant (a further one to be submitted in 2015), and a further 4 (one submitted to Science) with collaborators, as described above.

1: Overend G, Cabrero P, Guo AX, Sebastian S, Cundall M, Armstrong H, Mertens I,
Schoofs L, Dow JA, Davies SA. The receptor guanylate cyclase Gyc76C and a peptide
ligand, NPLP1-VQQ, modulate the innate immune IMD pathway in response to salt
stress. Peptides. 2012 Mar;34(1):209-18. doi: 10.1016/j.peptides.2011.08.019.
Epub 2011 Aug 27. PubMed PMID: 21893139.

2: Stergiopoulos K, Cabrero P, Davies SA, Dow JA. Salty dog, an SLC5 symporter,
modulates Drosophila response to salt stress. Physiol Genomics. 2009 Mar
3;37(1):1-11. doi: 10.1152/physiolgenomics.90360.2008. Epub 2008 Nov 18. PubMed
PMID: 19018044; PubMed Central PMCID: PMC2661102.

3: Davies SA, Dow JA. Modulation of epithelial innate immunity by autocrine
production of nitric oxide. Gen Comp Endocrinol. 2009 May 15;162(1):113-21. doi:
10.1016/j.ygcen.2008.09.012. Epub 2008 Oct 10. Review. PubMed PMID: 18952086.

4: Kanao T, Sawada T, Davies SA, Ichinose H, Hasegawa K, Takahashi R, Hattori N,
Imai Y. The nitric oxide-cyclic GMP pathway regulates FoxO and alters
dopaminergic neuron survival in Drosophila. PLoS One. 2012;7(2):e30958. doi:
10.1371/journal.pone.0030958. Epub 2012 Feb 29. PubMed PMID: 22393355; PubMed
Central PMCID: PMC3290610.

5: Patel U, Davies SA, Myat MM. Receptor-type guanylyl cyclase Gyc76C is required
for development of the Drosophila embryonic somatic muscle. Biol Open. 2012 Jun
15;1(6):507-15. doi: 10.1242/bio.2012943. Epub 2012 Apr 16. PubMed PMID:
23213443; PubMed Central PMCID: PMC3509439.

6: Scheunemann L, Jost E, Richlitzki A, Day JP, Sebastian S, Thum AS, Efetova M,
Davies SA, Schwärzel M. Consolidated and labile odor memory are separately
encoded within the Drosophila brain. J Neurosci. 2012 Nov 28;32(48):17163-71.
doi: 10.1523/JNEUROSCI.3286-12.2012. PubMed PMID: 23197709.

7: McGettigan J, McLennan RK, Broderick KE, Kean L, Allan AK, Cabrero P, Regulski
MR, Pollock VP, Gould GW, Davies SA, Dow JA. Insect renal tubules constitute a
cell-autonomous immune system that protects the organism against bacterial
infection. Insect Biochem Mol Biol. 2005 Jul;35(7):741-54. Epub 2005 Mar 31.
PubMed PMID: 15894191.
Exploitation Route The cGMP networks/xenobiotic work may be taken forward and co-developed by industry.
Sectors Agriculture, Food and Drink

Description In progress (see key findings).