Muscle function and homeostasis require macrophage-derived cytokine inhibition of AKT activity in Drosophila

Unpaired ligands are secreted signals that act via a GP130-like receptor, domeless, to activate JAK-STAT signaling in Drosophila. Like many mammalian cytokines, unpaireds can be activated by infection and other stresses and can promote insulin resistance in target tissues. However, the importance of this effect in non-inflammatory physiology is unknown. Here, we identify a requirement for unpaired-JAK signaling as a metabolic regulator in healthy adult Drosophila muscle. Adult muscles show basal JAK-STAT signaling activity in the absence of any immune challenge. Macrophages are the source of much of this tonic signal. Loss of the dome receptor on adult muscles significantly reduces lifespan and causes local and systemic metabolic pathology. These pathologies result from hyperactivation of AKT and consequent deregulation of metabolism. Thus, we identify a cytokine signal from macrophages to muscle that controls AKT activity and metabolic homeostasis.

Here, we identify a physiological requirement for Dome signaling in adult muscle. We observe that 56 adult muscles show significant JAK/STAT signaling activity in the absence of obvious immune 57 challenge and macrophages seem to be a source of this signal. Inactivation of dome on adult muscles 58 significantly reduces lifespan and causes muscular pathology and physiological dysfunction; these 59 result from remarkably strong AKT hyperactivation and consequent dysregulation of metabolism. We 60 thus describe a new role for JAK/STAT signaling in adult Drosophila muscle with critical importance in 61 healthy metabolic regulation. Therefore, we stained thorax muscles with the neutral lipid dye LipidTox. In 14 day old flies, we 90 detected numerous small neutral lipid inclusions in several muscles, including the large jump muscle 91 (TTM), of 24B-Gal80 ts >dome ∆ flies ( Fig 1D). 92

Muscle dome activity is required for normal systemic homeostasis 93
Having observed lipid inclusions in adult muscles, we analysed the systemic metabolic state of 24B-94 Gal80 ts >dome ∆ flies. We observed significant reductions in total triglyceride, glycogen and free sugar 95 (glucose + trehalose) in these animals (Fig 1E, F). The reduction in free sugar was not detectable in 96 any dissected solid tissue, suggesting that it was due to a reduction in hemolymph sugar ( Fig 1G). 97 Reduced hemolymph sugar could result from increased tissue glucose uptake. In this case, it should 98 be reflected in an increased metabolic stores or metabolic rate. Since metabolic stores were 99 decreased in our flies, we tested metabolic rate by measuring respiration. CO2 production and O2 100 consumption were both significantly increased in 24B-Gal80 ts >dome ∆ flies, indicating an overall 101 increase in metabolic rate ( Fig 1H) We examined the activity of these signaling mechanisms in legs (a tissue source strongly enriched in 109 muscle) from 24B-Gal80 ts >dome ∆ flies. We found an extremely strong increase in abundance of the 110 60-kDa form of total and activated (S505-phosphorylated) AKT (Fig 1I, J). This change was also seen in 111 legs from Mef2-Gal80 ts >dome ∆ flies, confirming that dome functions in muscles (Fig S1H, I). We also 112 saw this effect in flies carrying a different insertion of the dome ∆ transgene, under the control of a 113 third muscle-specific driver, MHC-Gal4, though the effect was weaker (Fig S1J). These MHC-114 Gal4>dome ∆ (II) animals were also short-lived relative to controls (Fig S1K). 115 Elevated total AKT could result from increased transcript abundance or changes in protein 116 production or stability. We distinguished between these possibilities by assaying Akt1 mRNA; Akt1 117 transcript levels were elevated in 24B-Gal80 ts >dome ∆ muscle, but only by about 75%, suggesting that 118 the large effect on AKT protein abundance must be, at least in part, post-transcriptional ( Fig S1L). 119 Similarly, AKT hyperactivation could be driven by insulin-like peptide overexpression; however, we 120 assayed the expression of Ilp2-7 in whole flies and observed that none of these peptides were 121 significantly overexpressed (Fig S1M-R). 122 Unlike AKT, the amino-acid-responsive TORC1/S6K and the starvation-responsive AMPK pathway 123 showed no significant difference in activity in 24B-Gal80 ts >dome ∆ flies (Fig 1K, L). However, flies with 124 AMPK knocked down in muscle did exhibit mild AKT hyperactivation ( Fig S2A). 125 To identify signaling mediators acting between Dome and AKT, we first tested activity of the MAPK-126 ERK pathway, which can act downstream of the JAK kinase Hop (Luo et al., 2002). We found an 127 insignificant reduction in ERK activity in 24B-Gal80 ts >dome ∆ flies ( Fig 1M). We then assayed survival 128 and AKT activity in flies with hop (JAK), Dsor1 (MEK) and rl (ERK) knocked down in adult muscle. rl 129 and Dsor1 knockdown gave mild or no effect on survival and pAKT (Fig S2B, C). In contrast, hop 130 knockdown phenocopied the milder dome ∆ transgene with regard to survival and pAKT (Fig S2D, E). 131 We further analysed the requirement for hop in muscle dome signaling by placing 24B-Gal80 ts >dome ∆ 132 on a genetic background carrying the viable gain-of-function allele hop Tum-l . Flies carrying hop Tum-l 133 alone exhibited no change in lifespan, AKT phosphorylation, or muscle lipid deposition (Fig 2A-C). 134 However, hop Tum-l completely rescued lifespan and pAKT levels in 24B-Gal80 ts >dome ∆ flies ( Fig 2D, E), 135 indicating that the physiological activity of muscle Dome is mediated via Hop and that this signal is 136 required, but not sufficient, to control muscle AKT activity. 137

Increased AKT activity causes the effects of dome inhibition 138
The phenotype of 24B-Gal80 ts >dome ∆ flies is similar to that previously described in flies with loss of 139 function in Pten or foxo (Demontis and Perrimon, 2010; Mensah et al., 2015), suggesting that AKT 140 hyperactivation might cause the dome loss of function phenotype; however, to our knowledge, direct 141 activation of muscle AKT had not previously been analysed. We generated flies with inducible 142 expression of activated AKT (myr-AKT) in adult muscles (w;tubulin-Gal80 ts /+;24B-Gal4/UAS-myr-AKT 143 [24B-Gal80 ts >myr-AKT]) (Stocker et al., 2002). These animals phenocopied 24B-Gal80 ts >dome ∆ flies 144 with regard to lifespan, climbing activity, metabolite levels, metabolic rate, and muscle lipid 145 deposition (Fig 3A-F). 146 We concluded that AKT hyperactivation could cause the pathologies seen in 24B-Gal80 ts >dome ∆ flies. 147 We next tested whether reducing AKT activity could rescue 24B-Gal80 ts >dome ∆ flies. We generated 148 flies carrying muscle-specific inducible dominant negative dome (UAS-dome ∆ ) with dsRNA against 149 Akt1 (UAS-AKT-IR). These flies showed significantly longer lifespan than 24B-Gal80 ts >dome ∆ and 24B-150 Gal80 ts >AKT-IR flies, similar to all control genotypes analyzed ( Fig 3G). Dome and AKT antagonism 151 synergised to control the mRNA level of dome itself, further suggesting strong mutual antagonism 152 between these pathways ( Fig S3A). 153 AKT hyperactivation should reduce FOXO transcriptional activity. To test whether this loss of FOXO 154 activity caused some of the pathologies observed in 24B-Gal80 ts >dome ∆ flies, we increased foxo gene 155 dosage by combining 24B-Gal80 ts >dome ∆ with a transgene carrying a FOXO-GFP fusion protein under 156 the control of the endogenous foxo regulatory regions. These animals exhibited rescue of 157 physiological defects and lifespan compared to 24B-Gal80 ts >dome ∆ flies (Fig 3H-J). They also 158 exhibited increased dome expression (Fig S3B). The effects of these manipulations on published foxo 159 target genes were mixed ( Fig S3B); the strongest effect we observed was that Dome blockade 160 increased upd2 expression, consistent with the observation that FOXO activity inhibits upd2 161 expression in muscle (none of the other genes tested have been shown to be FOXO targets in 162 muscle) (Zhao and Karpac, 2017). This may explain some of the systemic effects of Dome blockade. 163 The effect of the foxo transgene was stronger than expected from a 1.5-fold increase in foxo 164 expression, so we further explored the relationship between FOXO protein expression and AKT 165 phosphorylation. We found that 24B-Gal80 ts >dome ∆ markedly increased FOXO-GFP abundance, so 166 that the increase in total FOXO was much greater than 1.5-fold ( Fig S3C). This drove an apparent 167 feedback effect, restoring AKT in leg samples of foxo GFP ;24B-Gal80 ts >dome ∆ flies to near-normal levels 168 ( Fig S3D). Dome. 174 We found plasmatocytes close to STAT-GFP-positive leg muscle (Fig 4A, B). This, and the prior 175 published data, suggested that plasmatocytes might produce relevant levels of dome-activating 176 cytokines in steady state. We then overexpressed upd3 in plasmatocytes and observed a potent 177 increase in muscle STAT-GFP activity (Fig 4C), confirming that plasmatocyte-derived upd signals were 178 able to activate muscle Dome. 179 To determine the physiological relevance of plasmatocyte-derived signals, we assayed STAT-GFP 180 activity in flies in which plasmatocytes had been depleted by expression of the pro-apoptotic gene 181 reaper (rpr) using a temperature-inducible plasmatocyte-specific driver line (w;tub-Gal80 ts ;crq-Gal4).

182
STAT-GFP fluorescence and GFP abundance were reduced in legs of plasmatocyte-depleted flies (crq-183 Gal80 ts >rpr) compared to controls (crq-Gal80 ts /+) (Fig 4D, E). Activity was not eliminated, indicating 184 that plasmatocytes are not the only source of muscle STAT-activating signals. 185 We then examined the lifespan of flies in which we had depleted plasmatocytes in combination with 186 various upd mutations and knockdowns. Plasmatocyte depletion gave animals that were short-lived 187 ( Fig 4F). (This effect was different from that we previously reported, possibly due to changes in fly 188 culture associated with an intervening laboratory move (Woodcock et al., 2015).) The lifespan of 189 these animals was further reduced by combining plasmatocyte depletion with null mutations in upd2 190 and upd3; plasmatocyte-replete upd2 upd3 mutants exhibited near-normal lifespan ( Fig 4F). 191 Similarly, plasmatocyte depletion drove muscle lipid accumulation, and upd2 upd3 mutation 192 synergised with plasmatocyte depletion to further increase muscle lipid ( Fig 4G). However, depleting 193 plasmatocytes in upd2 upd3 mutants failed to recapitulate the effects of muscle Dome inhibition on 194 whole-animal triglyceride, free sugar, and glycogen levels ( Fig S4A, B). This could be due to 195 antagonistic effects of other plasmatocyte-derived signals. 196 We attempted to pinpoint a specific Upd as the relevant physiological ligand by examining  activity, first testing mutants in upd2 and upd3 because upd1 mutation is lethal. However, these 198 mutants, including the upd2 upd3 double-mutant, were apparently normal ( Fig S4C). We then tested 199 plasmatocyte-specific knockdown of upd1 and upd3; these animals were also essentially normal (Fig  200  S4D), and plasmatocyte upd1 knockdown did not reduce lifespan ( Fig 4H). However, plasmatocyte-201 specific upd1 knockdown gave significant compensating increases in expression of upd2 and upd3 202 ( Fig 4I). In keeping with this, combining plasmatocyte-specific upd1 knockdown with mutations in 203 upd2 and upd3 reduced lifespan ( Fig 4J) and also reduced STAT-GFP activity in these flies ( Fig S4F). 204 Our results indicate that plasmatocytes are an important physiological source of the Upd signal 205 driving muscle Dome activity in healthy flies, and suggest that upd1 may be the primary relevant 206 signal in healthy animals. However, plasmatocytes are not the only relevant source of signal, and Upd 207 mutual regulation prevents us from pinpointing a single responsible signal. 208

Discussion 209
Here we show that upd-dome signaling in muscle acts via AKT to regulate physiological homeostasis 210 in Drosophila. were used for imaging, using either the 10x/NA0.4 objective, or the 20x/NA0.5 objective. Images 295 were acquired with a resolution of either 1024x1024 or 512x512, at a scan speed of 400Hz. Averages 296 from 3-4 line scans were used, sequential scanning was employed where necessary and tile scanning 297 was used in order to image whole flies. For imaging of whole live flies, the flies were anaesthetized 298 with CO2 and glued to a coverslip. Flies were kept on ice until imaging. For measuring mean 299 fluorescence intensity, a z-stack of the muscle was performed and the stack was projected in an 300 average intensity projection. Next the area of the muscle tissue analyzed was defined and the mean 301 fluorescent intensity within this area was measured. Images were processed and analysed using 302 Image J. 303

RNA isolation and Reverse Transcription 304
For RNA extraction three whole flies or three thoraces were used per sample. After anaesthetisation, 305 the flies were smashed in 100µl TRIzol (Invitrogen), followed by a chloroform extraction and 306 isopropanol precipitation. The RNA pellet was cleaned with 70% ethanol and finally solubilized in 307 water. After DNase treatment, cDNA synthesis was carried out using the First Strand cDNA Synthesis 308 Kit (Thermo Scientific) and priming with random hexamers (Thermo Scientific normalized to the value of the loading control gene, Rpl1, prior to further analysis. 317 The following primer sequences have been used in this study: 318 5'-tccaccttgaagaagggcta-3' 5'-ttgcggatctcctcagactt-3' 319

Smurf Assay 320
Smurf assays with blue-coloured fly food were performed to analyse gut integrity in different 321 genotypes. Normal fly food, as described above, was supplemented with 0.1% Brilliant Blue FCF 322 (Sigma Aldrich). Experimental flies were placed on the blue-coloured fly food at 9AM and kept on the 323 food for 2 h at 29°. After 2 h the distribution of the dye within the fly was analysed for each 324 individual. Flies without any blue dye were excluded, flies with a blue gut or crop were identified as 325 "non-smurf" and flies which turned completely blue or showed distribution of blue dye outside the 326 gut were classified as "smurf". 327 Hybridoma Bank, used as an unpurified supernatant at 1:3,000; used as a loading control for all 338 blots). Primary antibodies were diluted in TBST containing 5% BSA and incubated over night at 4°. 339

Thin Layer Chromatography (TLC) for Triglycerides 345
Groups of 10 flies were used per sample. After CO2 anaesthesia the flies were placed in 100µl of ice-346 cold chloroform:methanol (3:1). Samples were centrifuged for 3 min at 13,000 rpm at 4°, and then 347 flies were smashed with pestles followed by another centrifugation step. A set of standards were 348 prepared using lard (Sainsbury's) in chloroform:methanol (3:1) for quantification. Samples and 349 standards were loaded onto a silica gel glass plate (Millipore), and a solvent mix of hexane:ethyl 350 ether (4:1) was prepared as mobile phase. Once the solvent front reached the top of the plate, the 351 plate was dried and stained with an oxidising staining reagent containing ceric ammonium 352 heptamolybdate (CAM) (Sigma Aldrich). For visualization of the oxidised bands, plates were baked at 353 80° for 20 min. Baked plates were imaged with a scanner and triglyceride bands were quantified by 354 densitometry according to the measured standards using Image J. 355 Respiration in flies was measured using a stop-flow gas-exchange system (Q-Box RP1LP Low Range 374

Measurement of Glucose, Trehalose and Glycogen
Respirometer, Qubit Systems, Ontario, Canada, K7M 3L5). Ten flies from each genotype were put 375 into an airtight glass tube and supplied with our standard fly food via a modified pipette tip. Each 376 tube was provided with CO2-free air while the 'spent' air was concurrently flushed through the 377 system and analysed for its CO2 and O2 content. All vials with flies were normalized to a control vial 378 with food but no flies inside. In this way, evolved CO2 per chamber and consumed O2 per chamber 379 were measured for each tube every ~ 44 min (the time required to go through each of the vials in 380 sequence) 381  unpaired T-test. 535  Gal80 ts >dome ∆ flies at 29°, pooled from three independent experiments. Log-Rank test (24B-573