General and Comparative Endocrinology 162 (2009) 113–121

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General and Comparative Endocrinology

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Modulation of epithelial innate immunity by autocrine production of nitric oxide

Shireen-Anne Davies *, Julian A.T. DowFaculty of Biomedical and Life Sciences, Integrative and Systems Biology, University of Glasgow, Anderson Complex, 56 Dumbarton Road, Glasgow G12 8QQ, Scotland, UK

a r t i c l e i n f o

Article history:Received 13 May 2008Revised 5 September 2008Accepted 8 September 2008Available online 10 October 2008

Keywords:Innate immunityDrosophilaSecretory epitheliaMalpighian tubules autocrineNitric oxide

0016-6480/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.ygcen.2008.09.012

* Corresponding author. Fax: +44 141 330 4878.E-mail address: s.a.davies@bio.gla.ac.uk (S.-A. Dav

a b s t r a c t

Mechanisms of innate immunity especially with relevance to epithelial tissue, are currently the focus ofintense research, as epithelial immunity greatly impacts on health and disease. However, many findingsregarding innate immunity signalling pathways in vertebrates stems from research using the geneticmodel Drosophila melanogaster.

Here we discuss the central importance of epithelial tissues in innate immunity in Drosophila; the mod-ulation of the Imd pathway via autocrine production of nitric oxide (NO); and the central importance ofthe Malpighian (renal) tubule in immune function of the whole animal.

� 2008 Elsevier Inc. All rights reserved.

1. Introduction

Control of the mammalian immune response via mechanisms ofinnate immunity is currently the focus of intense research efforts.In mammals, adaptive immunity has traditionally been viewed asthe major form of immune resistance; however, more recently,the ancient and phylogenetically conserved innate immune sys-tem, which utilizes receptors to recognize microbial infection,has been elevated to equally important status. Innate immunitypathways in mammals are known to regulate defense againstinfections, confer protection against injury, mediate wound healingand tissue repair, and regulate inflammation (Stavitsky, 2007). In-nate immune pathways in mammals are therefore implicated inmajor disease conditions including obesity and inflammation-asso-ciated disorders which currently afflict developed countries (Tilgand Moschen, 2006); and with aging (Aw et al., 2007).

While understanding of the innate immune system in mammalshas come on apace, the original identification of some componentsof innate immune signaling pathways (Fig. 1) e.g., the Toll receptor,came from studies in the genetic model Drosophila melanogaster(Lemaitre et al., 1996). The Toll signaling pathway is highly con-served between Drosophila and humans; and the initial discoveryof Toll receptor signaling in Drosophila has led to significant find-ings in Toll biology in mammalian systems. Toll-like receptor(TLR) function is implicated in tumor cells (Huang et al., 2008),while polymorphisms in TLR are being investigated in the contextof disease susceptibility (Misch and Hawn, 2008). Targeted thera-

ll rights reserved.

ies).

peutic compounds are also being developed for endocrine media-tors of the innate immune system including TLR (Ulevitch, 2004).

Use of model organisms allows the determination of gene func-tion in vivo by use of transgenics and mutagenesis, coupled withphysiological analysis. D. melanogaster has excellent tools fortargeted expression and transgenesis; particularly the GAL4/UASG

binary expression system (Brand and Perrimon, 1993). A transpos-able DNA element (‘P’ element) is engineered to contain an expres-sion construct for the yeast transcription factor GAL4, and ismobilized around the genome (P-element screen, see (Sozenet al., 1997)). Subsequent expression of GAL4 is modulated bytissue- or cell-specific enhancers (enhancer trapping) and is usedto drive expression of transgenes cloned downstream of the Up-stream Activating Sequence (UASG) contained in distinct, trans-formed, fly lines, and tissue- or cell-specific expression of anygene of choice occurs in the progeny of a GAL4/UAS cross. Further-more, conditional GAL4 drivers (utilising temperature e.g., heat-shock, GAL80; and Tet-on/off; FLP recombinase/FRT (McGuireet al., 2004)) allow temporally-controlled targeted gene expressionto be achieved. The GAL4 system is also used to take advantage ofRNAi technology (Kennerdell and Carthew, 2000) allowing tar-geted gene silencing in vivo. Thus the utility of D. melanogasterand other invertebrate genetic models e.g., Caenorhabditis elegansin elucidating the fundamentals of innate immunity is still verypromising (Ferrandon et al., 2007; Fuchs and Mylonakis, 2006).

Activation of the innate immunity pathways in Drosophilaresults in the production of distinct anti-microbial peptides(AMPs), encoded by seven separate gene families which are ex-pressed by the fat body and/or by epithelial tissue, both in larvaeand adults. Diptericin, attacin, drosocin, and cecropin are effective

Fig. 1. The Drosophila Toll/Imd Immune Pathways. The Toll pathway (left panel) is activated in response to Gram-positive bacteria and fungi; while the Imd pathway (rightpanel) is activated by Gram-negative bacteria. These activated signaling pathways lead to the activation of a family of NFjB/REL transcription factors i.e., Dif or Dorsal(associated with Toll signaling) and Relish (associated with Imd signaling). These transcription factors translocate into the nucleus after processing, where they initiatetranscription of anti-microbial peptide genes e.g., drosomycin (Toll) and diptericin (Imd). Figure reproduced with permission from (Ferrandon et al., 2007).

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against Gram-negative bacteria; defensin acts against Gram-posi-tive bacteria; while metchnikowin and drosomycin act against fun-gi (Ferrandon et al., 2007). The peptidoglycan-recognition proteins(PGRPs) and the Gram-negative binding proteins (GNBPs) bindmicrobial ligands, activating either the Toll, or Imd pathways.PGRPs are evolutionarily conserved: Drosophila, has 13 membersof the PGRP family (Werner et al., 2000); whilst there are 4 PGRPfamily members in mammals (Dziarski, 2004). In Drosophila,PGRP-LC and PGRP-SD exist as transmembrane receptors; whilstPGRP-LE functions in two different ways: full-length PGRP-LE actsas an intracellular receptor, whilst a short form of PGRP-LE is anextracellular receptor which interacts with PGRP-LC to enhancerecognition of peptidoglycans at the cell surface (Kaneko et al.,2006; Takehana et al., 2004). By contrast, PGRP-SA, which activatesToll signaling, is a circulating hemolymph protein which binds tothe Micrococcus luteus peptidoglycan (Werner et al., 2000).

Downstream of the PGRPs and GNBPs, distinct receptors partic-ipate in activating immune defense, including cytokine receptors,and Toll and domeless (interleukin JAK/STAT) receptors. Toll,which contains the TIR (Toll/interleukin 1 receptor (IL-1R)) domainfound in mammalian TLRs (Lemaitre et al., 1996) is stimulated byproteolytic cleavage induced by Gram-positive bacterial activationof PGRP (-SA, -SD), and GNPB1. Toll is also activated by GNBP3,which detects fungal wall components, and by the Drosophila pro-tease Persephone, which is subject to proteolysis by fungal prote-ases (Gottar et al., 2006). The Imd pathway is activated byrecognition of DAP–PGN by PGRP-LE and -LC, which bind to areceptor-interacting protein homotypic interaction motif-like(RHIM) motif, but not Imd itself (Kaneko et al., 2006).

The fat body has been described as the canonical tissue for pro-duction of AMPs (Silverman and Maniatis, 2001); however, it is

now known that in both larvae and the adult fly, the epithelial tis-sues, especially tubules, play major roles in AMP production (Kane-ko et al., 2006; McGettigan et al., 2005; Takehana et al., 2004; Tzouet al., 2000), Fig. 2 and below.

2. Role of epithelia in innate immunity

2.1. Production of anti-microbial peptides

Epithelial tissue plays a significant role in immune defense inboth vertebrates and invertebrates. Indeed, in vertebrates and hu-mans, AMP production by epithelial tissues is a very important fea-ture of the immune response. Given that the mucosa of respiratory,intestinal and urogenital tract form the main sites of colonizationof pathogenic or commensal microorganisms, immune sensing bysuch epithelial tissues is critical to survival of the organism. Theseepithelial tissues combat infections via production of AMPs whichinclude human a-defensins, HD5 and HD6 (intestine, female geni-tal tract); human b-defensins, HBD-1, and -2 (respiratory airways,kidney, and female genital tract); human cathelicidin-derived pep-tide LL-37 (respiratory airways, male reproductive tract); and cryp-tidin defensin (intestine, male reproductive tract). Finally, the skin,the largest organ of the human body, produces LL-37, HBD-1, andHBD-2 (from (Tzou et al., 2000)). Recent work has revealed thatmammalian epithelial cells express PGRPs (Uehara et al., 2005);whilst Toll-like receptors (TLRs) including TLR-2, -3, -4, and -7;and NOD1 and NOD2 are expressed by epithelial cells from the oralcavity, tongue, salivary gland, pharyngeal, esophageal, intestinal,cervical, breast, lung, and kidney (Uehara et al., 2007), and produceAMPs upon activation in response to immune challenge. Thus, it isnow understood that deregulation of epithelial sensing and im-

Fig. 2. AMP production in epithelial tissues of the adult fly. The figure is based on (Hartenstein, 1993), with major tissues indicated. The AMPs produced by these tissues areindicated in the boxes. The trachea, which are important in first-line immune defense (Takehana et al., 2004) are not shown, but these produce drosomycin and drosocin.Adapted from Ferrandon et al. (2007).

Table 1Adult and larval tissues which express the highest levels of each of the anti-microbialpeptide genes. Data were collated from FlyAtlas. (flyatlas.org) ‘mRNA enrichment’ iscalculated from four independent microarray replicates and is compared to theexpression signal in whole fly (Chintapalli et al., 2007). FlyAtlas presently covers onlytwo larval tissues, the tubules and fat body, so these tissues will be over-representedin the list.

AMP Adult tissue Enrichment Larval tissue Enrichment

Diptericin Midgut 2.5 Fat body 2.3

AttacinA/B Head (fat body) 9.6 Fat body 5.0C Head (fat body) 8.2 Fat body 7.6D Midgut 4.4 Tubules 2.7Drosocin Head (fat body) 2.6 Fat body 2.1

CercropinB Head (fat body) 3.2 Fat body 4.7C Head (fat body) 9.6Defensin Head (fat body) 1.5Metchnikowin Head (fat body) 5.8 Tubules 7.8

DrosomycinDro-3 Midgut 5.5 Fat body 2.3Dro-1 Midgut 3.5 Fat body 1.7Dro-4 Carcass (fat body) 4.4 Fat body 1.6Dro-5 Head (fat body) 5.5 Fat body 3.6Dro-6 Head (fat body) 4.3 (Tubule) (1.5)

Tubule 4.2

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mune defense is linked to major disorders including inflammatorybowel disease (Xavier and Podolsky, 2007), Crohn’s Disease(Kobayashi et al., 2005) and asthma (Schleimer et al., 2007); andemphasises the importance of the study of epithelial immunity.The fly has been extremely useful in determining innate immunepathways and novel regulators of these pathways, (Aggarwal andSilverman, 2008), so study of epithelial immune signaling in Dro-sophila should be a fruitful research direction.

Epithelial tissues in Drosophila produce the full range of sevenAMPs (Tzou et al., 2000), Fig. 2. Given that most microorganismsgain entry to the animal via the digestive tract, it is perhaps notsurprising that the gut epithelia, in particular, are important in im-mune defense. Furthermore, the Malpighian (renal) tubules—which are present as a pair each of blind-ended, single-cell thickanterior and posterior tubules, which are connected to the hind-gut—perform the roles of vertebrate liver and kidney, includingosmoregulatory and detoxifying functions, which are critical tothe survival of the fly (Dow and Davies, 2006). For example, tu-bules are the site for insecticide metabolism for the whole animal,and express very high levels of many of the large cytochrome P450family, particularly those implicated in xenobiotic metabolism(Yang et al., 2007).

Adult tissues with the highest expression for the AMPs arelisted in Table 1, using data collated from FlyAtlas, a tissue-specificgene expression resource for Drosophila (Chintapalli et al., 2007). Itshould be noted that AMP gene expression e.g. diptericin, is gener-ally very low or barely detectable under non-induced conditions.However, a reduced but constitutive level of AMP is produced bytubules (see evidence for the tubule as an autonomous immunesensor, below).

In the adult, even under non-immune challenge, the highest le-vel of AMP gene expression occurs mainly in the fat body (con-tained in either the head or body of adult flies) or in epithelia,notably the midgut (see expression levels of diptericin, attacin D,Dro-1, and Dro-3 Table 1). Compare this to the larval state, whereexpression of AMPs occurs almost exclusively in the fat body, withthe exception of Attacin D, metchnikowin, and Dro-6, which aremost highly expressed in larval tubules.

A rather more significant picture of expression emerges whenlooking at receptors, the PGRPs, for either the Toll or Imd pathways(Table 2). With the exception of PGRP-SA, which is highly ex-pressed in fat body, the other PGRPs are most highly expressed

in gut epithelia, notably midgut. It is interesting that statisticallysignificant levels of PGRP-Sc1a, -SC2, and -LB expression abovethose seen in whole fly, either in the adult or larval state, only oc-cur in adult midgut. However, in the larvae, the PGRPs share distri-bution between the fat body and the tubules. In larvae, PGRP-SAexpression in the fat body is 10-fold that of tubule (Table 2); how-ever, given that the mass of fat body in the larva (the most signif-icant tissue) will greatly outweigh that of tubules, the expressionlevel of PGRP-SA in tubules is of some significance for immunedefense.

Thus, although the fat body is a major tissue for immune de-fense in the fly, the epithelial tissues are not merely ‘peripheral’for immune sensing but should be viewed as central, ‘early warn-ing’ immune-sensing tissues. This may also be the case for other in-sects: in Glossina morsitans, the vector for African sleeping sickness,the proventriculus (cardia), at the junction of the foregut/midgut

Table 2Adult and larval tissues which express the highest levels of each of the genesencoding Peptidoglycan Recognition Particles (PGRPs). Data were collated fromFlyAtlas (flyatlas.org) ‘mRNA enrichment’ is calculated from four independentmicroarray replicates and is compared to the expression signal in whole fly(Chintapalli et al., 2007).

PGRP Adult tissue Enrichment Larval tissue Enrichment

-SA Adult carcass (fat body) 5.7 Fat body 24.1Tubules 2.7 Tubules 2.3

-SC1a Midgut 4.2-SC2 Midgut 5.7-LB Midgut 10.0-LC Hindgut 3.1 Fat body 1.1

Crop 3.0-LE Midgut 3.4 Fat body 1.1

Tubule 1.1

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shows increased AMP expression upon infection (Hao et al., 2003)which is dependent on Imd pathway-activation; and which affectsboth the prevalence and intensity of midgut infections by trypan-osomes (Hu and Aksoy, 2006).

2.2. The Drosophila Malpighian tubule is an immune-sensing tissue

In support of a central role for epithelial tissue in immunesensing, previous data have demonstrated that tubules possesscell-autonomous, immune-sensing capabilities. In keeping withthe immune-sensing role of tubules, they express all componentsof the Imd pathway (McGettigan et al., 2005) and the Toll pathway(FlyAtlas.org). Acutely-dissected tubules are capable of binding andinternalizing lipopolysaccharide (LPS) (McGettigan et al., 2005), acomponent of the coat of Gram-negative bacteria. Furthermore,PGRP-LE, which binds diaminopimelic-acid (DAP) containing pep-tidoglycan from Gram-negative bacteria is localized to the plasmamembrane of tubules-this PGRP-LE is a truncated form, whichenhances PGRP-LC recognition of peptidoglycan on the membranesurface. By contrast, the full-length PGRP-LE is internalized in thetubules upon activation by DAP peptidoglycan, suggesting that likemammals, both extracellular and intracellular receptors are usedto modulate the immune signaling pathways (Kaneko et al.,2006). Excised tubules can mount a significant bacterial killing re-sponse, with less than 50% of an Escherichia coli population remain-ing after treatment of the bacterial cultures with the supernatantin which the excised tubules have been incubated, reflecting a sig-nificant constitutive production of anti-microbial peptides (McG-ettigan et al., 2005). Tubules have also been shown to produceseveral AMPs in response to an immune trigger-these include dip-tericin, cecropin, and metchnikowin (Fig. 2, (Tzou et al., 2000)).

LPS, used to induce an immune response, induces diptericinproduction in acutely-dissected, intact tubules (McGettigan et al.,2005). Although the use of LPS as an immune stimulant is stillwidespread in mammalian work (Freudenberg et al., 2008), forDrosophila, however, the stimulatory effects of LPS on immune sig-naling were shown to be due to peptidoglycan from Gram-negativebacteria (PGN�), a common contaminant of commercial LPS prep-arations (Kaneko et al., 2004). Therefore, although the use of LPS isnow discredited in Drosophila, we have shown robust effects of LPSon Imd-signaling (McGettigan et al., 2005), and our current work(Aitchison, in prep) supports the use of either LPS or PGN� as acti-vators of the Imd pathway in tubules.

2.3. Osmoregulation by the tubule is associated with the whole animalresponse to immune challenge

Is the osmoregulatory role of the tubules linked to immunesensing? The obvious primary task of the tubule is to clear waste

metabolites and toxins from the hemolymph, and to achieve thisthey transport fluid at very high rates relative to the hemolymphvolume (Dow et al., 1994a; Dow et al., 1998). Given this role, thetubule is one of the first tissues that would be exposed to key mol-ecules derived from bacterial coat, e.g., peptidoglycan (PGN), whichis associated with immune challenge.

In order to test the importance of hemolymph clearance andimmune sensing, mutant flies which show aberrant fluid transportby the tubules, due to defects in key ion transport pumps e.g., Na+,K+ ATPase, were tested for tubule AMP production in response toan immune challenge. The Na+, K+ ATPase is an ouabain-sensitivepump important in the detoxification process, and is located atthe basolateral membrane of principal cells of the tubule (Torrieet al., 2004).

Tubules from a mutant in the Na+, K+ ATPase, P{ATP{a}}(Schubiger et al., 1994) show reduced rates of basal and neuropep-tide-stimulated fluid transport even as a heterozygote, P{ATP{a}}/+(Torrie et al., 2004), Fig. 3A. Interestingly, when P{ATP{a}}/+ fliesare inoculated with E. coli, the tubules do not mount a robust a dip-tericin response as wild-type flies (Fig. 3B). Thus, a significantreduction in fluid transport by the tubules, which results in animpairment in hemolymph clearance, also significantly affects pro-duction of AMPs, at least via the Imd pathway. Is there any impactof the qualitative reduction in tubule AMP production on survival?If the P{ATP{a}}/+ flies are subject to bacterial challenge, they sur-vive less well at all time points tested after inoculation comparedto wild-type flies (Fig. 3C). Thus, it appears that for the Na+, K+ ATP-ase mutant at least, reduced fluid transport is associated with areduction in ability to survive bacterial challenge.

3. NO in immune signaling

Although the investigation of the individual components of im-mune signaling pathways and the regulation of Imd and Toll path-way components is an area of intense research activity, the impactof cell signaling molecules on the immune pathways is an importantarea and therefore, has long been of interest and is actively beinginvestigated, both in mammals and in model organisms. For exam-ple, the JAK/STAT pathway, which is intensively researched in thevertebrate arena, is known to be important in modulation of immu-nity in Drosophila (Arbouzova and Zeidler, 2006).

The gaseous second messenger, nitric oxide (NO), is critical forphysiological processes (Murad, 2006) and has been shown to beimportant in immune signaling in vertebrates (Nagy et al., 2007;Niedbala et al., 2006; Tripathi, 2007). NO, produced via nitric oxidesynthase (NOS), acts to activate soluble guanylate cyclase, sGC(Krumenacker et al., 2004; Zhao et al., 1999), which results in pro-duction of cyclic GMP (cGMP) and downstream activation of thecGMP signaling pathway. The state of the NO:sGC complex viainteraction of NO with different sGC subunits, in addition to allo-steric regulation of sGC by cyclic nucleotides, can influence thetemporal range of cGMP production (Cary et al., 2006), suggestingcomplex regulation of the NO/cGMP pathway by NO itself.

There are three NOS genes in vertebrates (Mungrue et al., 2003)but a single NOS gene in insects, including that in Drosophila, dNOS(Tully, 1994) and Anopheles mosquitoes, AsNOS (Luckhart et al.,1998). The single NOS genes in insects, including dNOS in D. mela-nogaster and AsNOS in Anopheles stephensi is subject to complexregulation via alternative splicing and multiple transcripts: thereare 10 transcripts from dNOS, which also encode dominant nega-tive isoforms (Stasiv et al., 2004, 2001); and 18–22 transcripts inthe case of AsNOS (Luckhart and Li, 2001). The different gene prod-ucts encoded by the mammalian NOS genes; and the complexity oftranscription, and thus isoforms encoded by the single insect NOSgenes, will also greatly impact on NO-mediated immunity.

Fig. 3. Disruption of the gene encoding the Na+, K+ ATPase results in a fluid transport phenotype and associated reduction in survival of the flies to bacterial challenge. (A)Fluid transport data (secretion rate in nl min�1 ± SEM, shown on y-axis) for tubules from wild-type Oregon R 7-day old adults (squares) and from P{ATP{a}} (diamonds) 7-dayadult flies. Tubules from the Na+, K+ ATPase mutant display significantly reduced neuropeptide-stimulate fluid transport rates compared to wild-type tubules (addition ofcapa-1 and Drosokinin (Terhzaz et al., 1999) for maximal rates of secretion, arrowed). Data from Torrie et al. (2004). (B) Diptericin expression in acutely-dissected tubulesfrom either Oregon R or P{ATP{a}} heterozygous 7-day old adults, which were either mock-inoculated (unshaded bars), or inoculated with a culture of E. coli (shaded bars).Data shown are expressed as diptericin expression normalized against mock-injected Oregon R flies (value of 1), ±SD. Data: Susie Wan. (C) Batches of thirty P{ATP{a}}heterozygous flies, aged 7-days were either mock-challenged (inoculated with needle dipped in Schneider’s Medium) or challenged with E. coli suspension (McGettigan et al.,2005). Survivors were counted for up to 84 h post-inoculation. The numbers of survivors for each line after mock or infective treatment were expressed as percentage of thestarting number of flies. Data are expressed as % survivors ± SD for P{ATP{a}} heterozygotes (grey). For comparison, results for wild-type flies (red) from a single experimentare shown; survival rates of the wild-type flies are comparable to previously published data for Oregon R adults (McGettigan et al., 2005). Data: Winnie Tay.

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Furthermore, NO can also have direct effects on protein func-tion, via S-nitrosylation (Foster et al., 2003), thus affecting immuneresponses. For example, in lung epithelial and in Jurkat T cells,inhibitory jB kinase (IKK), the enzyme complex which inducesNF-jB, is inhibited by S-nitrosothiols (SNO) in a dose-dependentmanner, due to S-nitrosylation of the IKK complex by either exog-enous or endogenous NO (Reynaert et al., 2004). Thus, a unifiedview of nitric oxide action in immunity, especially in mammals,has been slow to emerge (Guzik et al., 2003) due to the diverseexperimental systems and preparations in use; the very complexregulation of NO production, the concentration of NO produced un-der different cellular conditions, and spatio-temporal effects of NO,which are either cGMP-dependent or -independent.

In Drosophila, NO modulates immune function by acting onhemocytes (Nappi et al., 2000) and on fat body (Foley and O’Farrell,2003) to activate AMP (diptericin) expression. The Drosophila NOSenzyme, like vertebrate nNOS (NOS1) and eNOS (NOS3), is also cal-cium/calmodulin-regulated (Tully, 1994), thus allowing anothertier of regulation of NO production. Recent work has shown thatcalcinuerin mediates NO-induced AMP production in Drosophilalarval fat body (Dijkers and O’Farrell, 2007), demonstrating theimportance of calcium in the NO-regulated immune response.

However, in other insects, NO has a direct anti-parasitic killingrole killing role (Rivero, 2006). In Anopheles mosquitoes, inductionof NOS occurs in the midgut upon infection with Plasmodium(Luckhart et al., 1998) via Plasmodium falciparum glycosylphos-phatidylinositols, suggesting that the parasite-derived GPIs areimportant triggers for the immune response in the host (Limet al., 2005). Furthermore, infection by P. falciparum of the blood-fed mosquito midgut is limited by toxic metabolites of NO, the pro-duction of which are catalyzed by hemoglobin-thus demonstratinga direct killing role of NO and its metabolites (Peterson et al.,

2007). NOS also acts in defense of trypanosome infection in midgutof other insects: in G. morsitans, NOS activity is increased in theproventriculas upon immune challenge, thus restricting coloniza-tion by trypanosomes (Hao et al., 2003); in Rhodnius prolixus mid-gut, NOS activity and transcription is increased upon infection withtrypanosomes (Whitten et al., 2007). Therefore, in insects, NOdemonstrably modulates the immune response, either by produc-tion of AMPs (as in Drosophila) or via a direct killing role in the pre-vention of infection.

4. Regulation of Malpighian tubule physiology by autocrineproduction of NO

In many cells and tissues, NO is an intercellular signaling mole-cule, where it signals to a sGC-containing cell which is differentfrom the cell containing NOS. However, NO functions as an auto-crine molecule in some cell types, notably the macula densa of ver-tebrate kidney (Welch and Wilcox, 2002), and the principal cells ofthe Drosophila Malpighian tubule. The Drosophila tubule, like thetubules of Dipteran mosquito species, contains two main cell types,the principal cell and the stellate cells. NOS is expressed only inprincipal cells in tubules from Drosophila, as well as the mosquitospecies Aedes and Anopheles (Pollock et al., 2004). However, whenDrosophila NOS is conditionally expressed throughout the intacttubule via the GAL4/UASG system for transgene expression, NOproduction is elevated, as expected—but cGMP content is only in-creased in principal cells (Broderick et al., 2003). Thus, in thisin vivo situation, the endogenous localization of NOS to principalcells acts to stimulate an autocrine production of NO. Furthermore,this autocrine production of NO is modulated by the endocrineproduction of a neuropeptide, capa, which activates calciumsignaling and NO production in the principal cells, resulting in

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elevation of intracellular calcium and activation of the cGMP sig-naling pathway (Kean et al., 2002). The activation of the NO/cGMPsignaling pathway in tubules results in a dose-dependent modula-tion of fluid transport (Davies, 2007), and so is associated with epi-thelial osmoregulation.

The capa peptide family (which has three members in Drosophila,capa 1–3), are processed from a pre-pro-peptide encoded by the sin-gle capa gene (Kean et al., 2002). The capa gene is expressed in theneurosecretory cells of the suboesophageal ganglion, retrocerebralcomplex in the adult and the ring gland in the larva (Kean et al.,2002; Predel and Wegener, 2006). The capa precursor is cleaved toform different peptide products in either the suboesophagael gan-glion neurons or the Va neurons-two of the peptides derived fromthe precursor produced by the Va neurons are capa-1 and capa-2,which both activate the autocrine production of NO in tubule princi-pal cells (Predel and Wegener, 2006), via binding to the G-protein-coupled capa receptor (Iversen et al., 2002). As they signal throughcalcium, NO and cGMP, capa peptides are necessarily diuretic in Dro-sophila (Kean et al., 2002). Interestingly, the capa/NO/cGMP signal-ing pathway is conserved between Drosophila, Aedes, Anophelesand the tsetse fly, Glossina (Pollock et al., 2004) suggesting the funda-mental importance of this pathway in these insects.

The unusual autocrine production of NO in the tubule, the ef-fects of NO on fluid transport, and the involvement of NO in the im-mune response together suggested that NO may have a role forimmune sensing by the tubule. This was investigated in intact Mal-pighian tubules, using both pharmacological and transgenic tools.

5. Modulation of the Imd pathway via autocrine NO

5.1. Increased tubule nitric oxide synthase (NOS) activity in immune-challenged tubules

NADPH diaphorase activity is a reliable indicator of NOS activityin Drosophila tubules (Broderick et al., 2003; Dow et al., 1994b),

Fig. 4. Immune challenge activates autocrine production of NO in tubule principal cells;posterior tubule (Sozen et al., 1997) from Oregon R adults inoculated with E. coli for 6 hpower magnification of B to show staining only in principal cells. Note unstained stellate(McGettigan et al., 2005). (C) Resting intact tubule from 7-day old diptericin-GFP adulttubule as C but challenged with LPS for 6 h. The tubule diameter can be taken as 35 lm

and can be effectively assayed in acutely-dissected tubules. Whenadult wild-type flies are challenged with E. coli, increased NADPHdiaphorase staining is observed in tubule principal cells in excisedtissue (Fig. 4). The figure shows the NADPH response after 6 h ofinfection; however, a less potent response is also observed after3 h of infection.

Quantification of NADPH diaphorase activity in the absence andpresence of an inhibitor of NOS, L-NAME, showed that the LPS-in-duced NADPH activity is wholly attributable to NOS activity (McG-ettigan et al., 2005). Interestingly, however, after 12–24 h post-infection, there is also a small increase activity of NADPH diapho-rase which is not associated with NOS: this may reflect increasedactivity of cytochrome P450s, several of which are highly abundantin tubule (Wang et al., 2004).

Increased activation of NOS via immune challenge results in en-hanced diptericin expression, both in LPS-treated excised tubules,and in tubules excised from flies inoculated with E. coli. In tubules,diptericin is produced only in principal cells, as transgenic fliesbearing a diptericin promoter:GFP construct show increased fluo-rescence in only principal cells in intact excised tubules upon LPStreatment (McGettigan et al., 2005), Fig. 4D. Thus, immune activa-tion of the tubule results in activation of the autocrine productionof NO, which in turn triggers cell-specific production of diptericin.Interestingly, LPS treatment of intact, excised tubules also resultsin a qualitative increase in basal and stimulated fluid transportrates (McGettigan et al., 2005), which also suggests that increasedhemolymph clearance may occur upon immune challenge; andsupports the data shown in Fig. 3, which demonstrates a link be-tween fluid transport and immune sensing.

5.2. Activation of NO signaling is directly linked to the AMP response intubules

While the data shown above are highly suggestive of a directlink between immune challenge, NO and the activation of AMP

and principal-cell specific diptericin expression. (A) Main segment of excised, intact, then stained for NADPH diaphorase activity in the presence of substrate. (B) Highcells (arrowed). Anterior tubules also respond in a similar way to immune challengeflies (Tzou et al., 2000) imaged for fluorescence using confocal imaging. (D) Intactin all cases. Data from McGettigan et al. (2005).

Fig. 5. The NO-induced diptericin response occurs via NO-activated solubleguanylate cyclase. Expression of a dNOS transgene (dN1-8, (McGettigan et al.,2005)) using a principal-cell specific GAL4 driver (UO-GAL4) increases diptericinproduction in tubules excised from E. coli-challenged UO-GAL4/UAS-dN1-8 flies.Diptericin expression (quantified by real-time quantitative-PCR) is normalizedagainst expression in E. coli-challenged 7-day old parental UAS-dN1-8 flies. ForODQ-treated samples, tubules were excised from UO-GAL4/UAS-dN1-8 flies 2 hpost-inoculation, and treated with 1 lM ODQ for 1 h prior to preparation of samplesfor Q-PCR of diptericin gene expression. Results expressed as ± SEM, N = 4,significance indicated by � (p < 0.05). Data: Susie Wan.

S.-A. Davies, J.A.T. Dow / General and Comparative Endocrinology 162 (2009) 113–121 119

production in tubule principal cells, direct testing of this hypothe-sis comes from the availability of elegant genetic tools for Drosoph-ila research. The GAL4/UASG binary system of expression allowsthe targeted expression of transgenes of choice to cells and tissues.Of relevance to the tubule is the availability of GAL4 ‘driver’ lineswhich drive expression in either segments of the tubule, in princi-pal, or in stellate cells (Sozen et al., 1997). A principal-cell specificGAL4 driver, c42 (Broderick et al., 2004; McGettigan et al., 2005;Sozen et al., 1997), was used to drive a NOS-encoding transgenein tubules.

Such an approach showed that over-expression of the NOStransgene only in principal cells in the adult tubule results in in-creased NO production compared to tubules from parental con-trols. This rise in NO in principal cells is directly associated withincreased diptericin production in either excised, LPS-treated tu-bules, or tubules which were acutely-dissected from flies of thec42/UAS-dNOS genotype, which were immune-challenged withE. coli (McGettigan et al., 2005).

The NO-associated diptericin response is due to the activationof NO-responsive soluble guanylate cyclase, as inhibition of thesoluble guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxa-lin-1-one (ODQ) abolishes the NO-induced diptericin response(Fig. 5). In this experiment, the NOS transgene was driven by a no-vel principal-cell specific driver (Terhzaz, Kean, Davies and Dow, inpreparation), UO-GAL4, based on the gene encoding urate oxidase,which is expressed uniquely in these cells in the entire fly, both atthe larval and adult stage (flyatlas.org). Use of this driver thus ne-gates any potential ‘off-target’ effects of other GAL drivers duringdevelopment.

5.3. Autocrine production of NO only in principal cells in adult fliesprotects the whole organism from infection

The effect of AMP production in only tubule principal cells uponactivation of autocrine NO production is dramatic. Significantimprovement in survival rates of the whole animal upon immunechallenge is observed (Fig. 6). These survival rates are significantlyimproved at all time points sampled after immune challenge, com-pared to parental transgenic lines and wild-type flies.

That no improvement in survival is observed in the c42/UAS-dN1-8 flies compared to parental lines upon mock-injection sug-gests that principal-cell NO signaling is directly linked to NO-acti-vated immune responses and is not associated with effects ofwounding, nor general with stress effects. Thus, by extension, pro-duction of diptericin in only tubule principal cells, numbering �77per tubule, is sufficient to protect the whole organism from im-mune challenge.

Fig. 6. Targeted over-expression of a dNOS transgene in tubule principal cells results inparental c42, UAS-dN1-8) were either mock-challenged (A) or challenged with E. coli suspthereafter (days 1–6). The numbers of survivors for each line after mock or infective tresurvivors ± SEM (N = 4) for c42/UAS-dN1-8 (black); as well as parental lines, c42 (gredifferent from E. coli-infected c42 or UAS-dN1-8. Data for mock-injected flies were simishowed similar survival to immune-challenged wild-type flies (McGettigan et al., 2005)

6. Conclusion

The roles of NO in biology are wide-ranging, and affect almostall known physiological functions, including regulation of the im-mune response (Bian et al., 2008).

In Drosophila tubules, the effect of NO on immune function isnot merely incidental to that of other canonical immune tissues(for example the fat body), but is critical to survival of the wholeanimal under conditions of stress. This important central role of tu-bules may not be insect-specific: recent work in vertebrates hasshown that renal tubular epithelial cells (TEC) respond to pro-inflammatory cytokines by producing NO (Du et al., 2006). This re-sponse determines the susceptibility of TEC to apoptosis, whichinfluences maintenance of overall kidney function; and thereforeis critical for survival. Thus, while NO is known to have wide-rang-ing roles in vertebrate kidney function, especially in relation tosalt-water balance (Kone, 2004), it is probable that NO also hasroles in immune defense in renal tissue. NO also acts in host de-fense and epithelial function of other types of secretory epitheliain vertebrates, e.g., airway epithelial cells in the respiratory tract

increased survival of E. coli-infected flies. Batches of 50 flies (c42/UAS-dN1-8; andension (B). Survivors were counted 3 h after inoculation (day 0) and at 24 h intervalsatment were expressed as percentage of starting number. Data are expressed as %

y circles) and UAS-dN1-8 (grey triangles). � (p < 0.05) indicates data significantlylar to that obtained for Oregon R (wild-type flies); E. coli-challenged parental lines.

120 S.-A. Davies, J.A.T. Dow / General and Comparative Endocrinology 162 (2009) 113–121

(Bove and van der Vliet, 2006), suggesting an evolutionary-con-served role of NO in fluid transport and immune regulation in epi-thelia. Importantly, it is clear that in secretory epithelia, fluidtransport and immune sensing are linked; thus suggesting thatthe osmoregulatory capacity of fluid-transporting epithelia caninfluence the immune status of the animal.

It is possible that in principal cells of Drosophila tubules, theautocrine regulation of NO is specifically intended to ‘shape’ thesignal in order to modulate AMP production and immune function.The autocrine regulation of NO confines the second messenger sig-nal to that cell type in the tubule which is capable of producingAMPs in response to an immune challenge, thus conferring spatialregulation of NO/cGMP signaling.

Endocrine modulators of NO in immune function in bothvertebrates and invertebrates are currently unknown. However,establishing potential neuroendocrine input into the NO/im-mune pathways is of importance, given the central role of in-nate immunity in vertebrates and invertebrates. Recent workhas provided the first demonstration of regulation of the hu-moral innate immune response in Drosophila by hormones. Thiswork shows that Juvenile hormone and 20-hydroxy-ecdysone,which coordinate insect growth and development, also modu-late AMP expression in response to immune challenge in Dro-sophila cell lines and in vivo (Flatt et al., 2008). Thus, it willnot be long before other endocrine modulators of innate immu-nity are discovered, thus expanding the repertoire of regulatorsof innate immunity.

Acknowledgments

Research in the authors’ laboratory is funded by the UnitedKingdom Biotechnological and Biological Sciences Research Coun-cil, BBSRC. We thank the referees for helpful comments andsuggestions.

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