Role Of Updl And FMRFamide In Regulation Of Sleep And Immune Response In Drosophila

ObjectiveIt is common belief that sleep is a vital part of our life and is indispensable forhuman health. Meanwhile, sleep is a complex process that the function of sleep remainselusive and there is scant information about the signaling transduction pathways that areinvolved in it. Many researchers believe that immune genes promote sleep, while sleep isalso associated with the immune physical and pathological status. In the past two decades,studies on the reciprocal relationship between sleep and immune system have revealedthat these two systems are intricately intertwined and in close communications viamediators such as cytokines. Besides, there is a causal relationship betweeninflammatory factors like IL-1β，TNF-αand the increased sleep phenomenon. However,the underlying mechanism as well as the function of this increased sleep during recoveryprocess is not clear.We have often observed in clinics that at the early phase of injury, inflammation orinfection, the patients are tend to have fever, sleepiness, and lack of appetite or thewillingness to communicate.Mounts of studies have confirmed that fever is a protectiveresponse which could inhibit the virus replication and enhance the immune function. Thequestion then arises iswhether the acute sleep response associated with acute infection ismerely coincidental or whether it is recuperative for immune function, enhancing theability of the host to defend against immunological challenge.In the first chapter of the thesis, I will explain the role of cytokine Upd1inregulation of Drosophila sleep and the immune function. I will introduce the advantagesof using Drosophila melanogasterasa virtually ideal organism for sleep andimmunebehavioral experiments. By understanding the previous studies on the cytokinesand JAK-STAT signaling pathway in both mammals and flies, I will introduce how mycurrent project is come up.In the present study, we take advantage of Drosophila’sUAS/GAL4specific expression system to manipulate the expression of Upd1and thenegative regulators of JAK-STAT signaling pathway and assess the impact ofJAK-STAT signaling pathway onthe baseline sleep, acute sleep response induced byinjury or infection and theimmune function. And the results are as follows:1、 The peripheral cytokine Upd1inhibit the baseline daily sleep of the DrosophilaBy employing the classical UAS/Gal4expression system, we investigated the effectof cytokine Upd1overexpression on the baseline sleep of Drosophila. To begin with, wecrossed the virgin female S1106-Gal4and Yolk-Gal4flies with the male UAS-Upd1fliesto get the offspring flies with Upd1specifically expressed in the fat body cells. Aftermonitoring the baseline sleep for7-10days, we analyzed the daily and night sleep of eachfly.The result revealed that the daily sleep in flies with Upd1overexpressed in the fatbody was greatly reduced compared to the sibling control. There was no statisticaldifference in the night time sleep, which implied that the Upd1might inhibit the dailybaseline sleep.2、The peripheral cytokine Upd1regulates sleep via activating the centralJAK-STAT signaling pathwayTo explore whether JAK-STAT pathway mediates the sleep changes by Upd1, weexamined the expression of STAT92E protein in the Drosophila brain afteroverexpressing Upd1in the fat body cells. By employing the balancer, we crossedYolk-Gal4>UAS-Upd1female flies with male flies carrying10XSTAT92E-dGFPreporter and after screening for several times, we got two different genotypes:10×STAT-dGFP/+, Yolk/UAS-Upd1and10×STAT-dGFP/+, UAS-Upd1/TM6B.The firstgenotype of flies overexpressed Upd1in the fat body and carried the10XSTAT92E-dGFP reporter. The latter genotype flies was used as the siblingcontrol.The expression of GFP in the fly brain could reflect the activity of STAT92E.Theimmunostaining result revealed that, comared to the sibling control, Stat92E protein washighly activated in sleep regulating regions like mushroom body and the parsintercerebaralis in the10×STAT-dGFP/+, Yolk/UAS-Upd1flies. The immunostainingresult revealed from the molecular level that the JAK-STAT signaling pathway wasactivated after overexpressing Upd1in the fat body.Next, we explored the functional effect of the activation of JAK-STAT pathway. Weused the Gene-Switch driver MB-Switch to overexpress the truncated form of theDomless receptor in the mushroom body to antagonist the signaling pathway, thusinhibiting the effect of Upd1. Surprisingly, we found that the result was just reversed andthe daily sleep was increased when compared to the vehicle control.The overall results encouraged us to hypothesize boldly that the cytokine Upd1secreted from the fat body could arrive the central sleep regulating regions through the lymphatic circulation and activate the JAK-STAT pathway which further mediating thesleep changes.3、The peripheral cytokine Upd1regulates the acute sleep changes duringimmune response via JAK-STAT pathwayIn the following study, we want to see whether the JAK-STAT pathway is involvedin regulating the acute sleep changes during immune response. Previous studies haveshowed that aseptic injury could stimulate the host immune response. So in the presentpaper, we injected flies with aseptic PBS, and then analysed the induced acute sleepresponses. When compared with the sibling control, we found that the acute sleepresponse in flies with Upd1overexpressed in the fat body was greatly reduced. However,when we interfered with the expression of Upd1in the fat body, the effect was reversed,and the acute sleep responses were increased. Next, when we overexpressed the negativeregulator of the JAK-STAT pathway-SOCS36E and the truncated form of the receptorDomeΔCyt to block the pathway in pars intercerebralis with Dilp2-Gal4driver, the acutesleep response induced by injury was also increased, compared to the parental control.4、 The peripheral cytokine Upd1enhances the immune function duringbacterial infection via JAK-STAT pathwayTo investigate how the Upd1influence the immune system, we manipulated theexpression of Upd1in the fat body and assessed the survival outcome after injecting withGram-negative bacteria of Serratia Marcescens. Comared with the sibling control group,Upd1overexpressed flies had better survival, and the survival outcome was reversedwhen interfering with the Upd1in the fat body. Consistent with this result, we found thatblocking the JAK-STAT pathway with either UAS-DomΔcyt or UAS-SOCS36E, thesurvival rate was decreased when compared with the parental line.In summary, we here provide evidence to show that the evolutionarily conservedpathway of JAK-STAT signaling pathway is involved in the regulation of interactionbetween sleep and innate immune function in Drosophila. The novelty of this study is thatwe firstly provide direct evidence showing that the peripheral cytokine Upd1couldactivate the central JAK-STAT pathway to regulate sleep and the immune function.In the second chapter, we investigated the role of the neuropeptidePhe-Met-Arg-Phe-NH2(FMRFamide) in the Drosophila’s sleep and immune responseinduced by stressful stimuli. It is one of the most abundant neuropeptide in invertebrateand has been implicated in the mediation of a variety of behavioral and physiological functions. Recent study in C.elegant just demonstrated that FMRFamide-relatedneuropeptide were involved in the hypoxia-enhanced sensory perception, revealing a rolein a stress-induced adaptive behavior. To finish this, we employed FMRFamidereceptormutant flies FRMBO4659and the wild type W1118flies to examine the behavior changesagainst different stimuli like heat shock and septic injury. The methods and results are asfollows:1、The acute sleep response is reducedin FRMBO4659flies after heat shockWe put both the FRMBO4659and W1118flies into37℃incubator at ZT18for1h.Thenwe take them out and put back into25℃incubator and record the sleep behavior foranother7-10days. We find that flies in both groups sleeps more after heat shock, but theacute sleep response after heat shock reduced dramatically in the FR mutant flies whencompared with the W1118. However, there is no statistical difference in the survivaloutcome between the two groups.2、 The acute sleep responseflies and the survival rate aredecreased inFRMBO4659after bacterial infectionWe inject both the FRMBO4659and W1118flies with the same load of Serratiamarcescens at ZT18and record the sleep behavior for another7-10days. We find that theacute sleep response in the FR mutant flies is reduced dramatically after heat shock whencompared with the W1118. Meanwhile, the survival rate is also decreased in FFRMBO4659flies.3、The bacteria clearance ability in FRMBO4659flies is reduced after bacterialinfectionInorder to measure the bacterial clearance ability of the flies, we collect8-10fliesimmediately after infection and homogenize in LB solution and pour over the solid LBplate to count the colony forming unit. With the same way, we collect the survived flies24hours after infection. Statistical analysis shows that there is no difference between thecounts at0h. However, the colony counts with the FRMBO4659flies are much greater thanthe wild type, which reveals that the bacterial clearance ability of the FRMBO4659flies aredecreased.Principal findings and summaryIn this study, the effects of JAK-STAT pathway as well as the FMRFamideneuropeptide on sleep and immune system are determined by using the sleep model of Drosophila melanogaster. We provide evidence to show that the peripheral cytokine Upd1could inhibitthe physical sleep as well as the acute sleep response induced in immuneresponse via JAK-STAT signaling pathway in the central sleep regulating area.Besides,Upd1could enhance the immune function of the flies. Moreover, we show thatFMRFamide neuropeptide is also involved in the regulation of Drosophila acute sleepresponse induced by stressful stimuli and the acute sleep responses are associated withthe survival outcome during bacterial infection. In conclusion, these findings indicate acomplex interaction between sleep and the immune response in Drosophila and suggestthat sleep during an immune response might be beneficial for the recovery process. Thisstudy opens new avenues to study the role of specific cytokines and neuropeptide in sleepregulation and shed light on the further study to investigate the mechanism of theinteraction between sleep and the immune system.