Activation of innate immune signalling during development predisposes to inflammatory intestine and shortened lifespan
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Abstract
Early-life inflammatory response is associated with risks of age-related pathologies. How transient immune signalling activity during animal development influences life-long fitness is not well understood. Using Drosophila as a model, we find that activation of innate immune pathway IMD signalling in the developing larvae increases adult starvation resistance, decreases food intake, and shortens organismal lifespan. Interestingly, lifespan is shortened by the IMD activation in the larval gut and fat body, while starvation resistance and food intake are altered by that in neurons. The adult flies developed with IMD activation show sustained IMD activity in the gut, despite complete tissue renewal during metamorphosis. The inflammatory adult gut is associated with a greater amount of Gluconobacter sp., characteristic gut microbiota increased in response to immune activation. Removing gut microbiota by antibiotics attenuates the increase of IMD activity and rescues the shortened lifespan. This study demonstrates a tissue-specific programming effect of early-life immune activation on the adult physiology and organismal lifespan.
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Reviewer #1:
This paper shows that transient genetic induction of the IMD innate immune pathway during Drosophila development, has long term effects on adult health and lifespan. The paper is well-written, the experiments are well designed and executed, and the data are without exception good quality. The data also support the specific conclusions well. The experiments take full advantage of the Drosophila system to pinpoint the effect on lifespan to long term activation of inflammation in the gut, which is interlinked and dependent upon changes in the microbiota. However the analysis is not comprehensive, because neural-specific effects on starvation resistance are not followed …
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Reviewer #1:
This paper shows that transient genetic induction of the IMD innate immune pathway during Drosophila development, has long term effects on adult health and lifespan. The paper is well-written, the experiments are well designed and executed, and the data are without exception good quality. The data also support the specific conclusions well. The experiments take full advantage of the Drosophila system to pinpoint the effect on lifespan to long term activation of inflammation in the gut, which is interlinked and dependent upon changes in the microbiota. However the analysis is not comprehensive, because neural-specific effects on starvation resistance are not followed up, and because the etiology of the changes in microbiota is not mapped out. I should also say that I do not fully agree with the conclusion in the last sentence of the Abstract (the most important general conclusion), that the study "demonstrates a tissue-specific programming effect" of early transient IMD function. Since the lifespan shortening was shown to be dependent upon increased gut Gluconabacter, I would not call this "programming" (though the term is vague enough to mean most anything.) Instead, I would refer to the effect as a host-environment interaction. If it were "programming" of, for instance, the genetic or epigenetic sort, it would not be so easy to reverse.
Response1-1: We thank the reviewer for the fair evaluation of the manuscript. We agreed with the point of "programming" effect: it might be a bit overstatement. We would like to make our conclusion modest and avoid the ambiguous word in the last sentence of the abstract.
A few other minor comments:
- Several experiments, the authors use GFP (Fig S1) or the IMD targets DptA or Dro (Fig S2) to validate the induction of IMD-CA. Why have they not directly measured the expression of IMD-CA. This would seem to be logical and technically easy, by qPCR.
Response1-2: We will perform qPCR of Imd gene.
- In Fig 4 we see and experiment in which animals were "supplemented" with Alkaline Phosphatase, a protein. How was this done and why does it work? Is AP a gut luminal protein?
Response1-3: It is a luminal protein and thus ingestion of the protein works just as endogenous one. This is also proved in the literature (Kühn F et al., JCI insight, 2020). The protein targets, for example, peptidoglycan to attenuate its immuno-stimulative capacity. We will add the explanation in the text.
- The results in Fig 5 are really where the paper begins to determine a mechanism for the lifespan shortening. However, these results are rather weak, and they don't extend very far. The increase in Gluconobacter is mild (Fig 5C), and is not clear in the 16S rRNA sequencing experiment (Fig 5A). Furthermore, it is not clear that Glunconobacter specifically is the source of the lifespan shortening, of just bacteria in general (Fig 5E).
Response1-4: Why we are focusing on this bacterial genus is because we have already shown in our previous paper that increase of Gluconobacter shortens organismal lifespan (Kosakamoto H et al., Cell Reports, 2020). We also reported that Gluconobacter is increased in response to the (necrosis-induced) immune activation, the situation of which is strikingly similar in the larval IMD activation in the present study. As we proved before, we wanted to perform the gnotobiotic/monoassociation experiment here to show sufficiency of the bacterium for the lifespan-shortening phenotype, however preliminary experiments implied that combining Germ-free with the GeneSwitch system is technically difficult as it caused higher lethality. This might be because the drug RU486 shows a different bioavailability/ dynamics in the GF flies.
Significance:
Although this paper addresses in interesting topic using an elegant and effective experimental strategy, the final results (Fig 5) and conclusions are modest. The analysis doesn't extend far enough to demonstrate how long term changes in microbiota arise from short term developmental changes in innate immune activity. Moreover, there is no detailed data concerning how the altered microbiota alter lifespan. Thus, while the results are interesting and the findings open avenues for further studies on the topic, the significance of the paper is modest, in its current state. Further analysis of how the microbiota is permanently changed, and why this affects lifespan, could enhance the paper. However, it is not clear that any simple, quick experiments could dramatically advance the findings from where they are now.
Response1-5: We would like to add the data that IMD activation in the larvae increased the Gluconobacter already in the larval gut. This data mechanistically suggests that microbiome alteration in the larval gut persists into adulthood, demonstrating how larval immune signalling influences adult immune activity. This data should strengthen a concept that even a transient and mild immune activation in juvenile stage can mess up the microbiota and permanently trigger the inflammatory pathology.
Reviewer #2:
In this manuscript, the authors study the impact of ubiquitously activating the IMD pathway only during larval stages on subsequent adult life. They report a shortened lifespan due to IMD pathway activation in the larval gut and a resistance to starvation linked to its activation in the nervous system. While there is apparently no activation of the IMD pathway in very young adult flies, the expression of some IMD-dependent antimicrobial peptide (AMP) genes is reported from 7-10 flies onwards. This expression is lost upon treating the adults with antibiotics, which also rescues the shortened lifespan phenotype. It correlates with a possible increase in the proportion of Gluconobacter in the microbiota.
While the study looks interesting, it is not clear whether the results, especially those of survival studies and RTqPCR experiments, have been replicated in independent experiments. This is essential to warrant their conclusions. In this respect, this reviewer notes some important variability in the lifespan studies (e.g., Fig. 2B vs. Fig. 4E): how do the authors account for a lifespan that is shortened almost by half in Fig. 4E? Also, Fig. S2B is not convincing given the observed variability. More data points are required to reach a conclusion.
Response2-1: We would like to mention that all experiments have been replicated at least twice. We admit that the phenotypes of larval IMD activation such as lifespan shortening effect and inflammatory response in adult gut are indeed quite variable, empirically depending on seasons. This is not surprising to us since many immune-metabolic phenotypes as well as lifespan of the flies are variable between seasons. We assume that this would imply that the effect is through gut microbiota. In Japan, we have a typical seasonal change in the temperature/ humidity that greatly influences gut microbial situation, even though we use an incubator which allows constant temperature/humidity setting. It is therefore we need to carefully compare the phenotype of flies in the same experiment, and this is where the GeneSwitch works effectively.
Regarding Fig. S2B, we could increase the number of samples in Fig. S2B in new experiment.
The authors suggest in their Discussion some kind of epigenetic mechanism transmitting the information of IMD pathway activation having occurred at larval stages. Whether this depends on a change of metabolism remains to be demonstrated, in as much it is likely that there is a major metabolic "reset" occurring during metamorphosis to prepare the individual to the new environmental conditions encountered as an adult. It is also likely that larvae in the wild grow in a microbe-rich slurry and are likely to experience intestinal infections. As noted by the authors themselves on the top paragraph of p7 (line numbers are unreadable), the larval gut is degenerated during metamorphosis and thus the enterocytes that have produced AMPs are no longer present. One possibility would be that there is an early dysbiosis already occurring during larval stages and that the young adults re-infect themselves, for instance through contact with the meconium. The authors' experiments with antibiotics are the key to this study. However, one would like to observe results of the converse experiment, that is, treating larvae with antibiotics (a better control would be to bleach the embryos to generate axenic flies) and then raising the hatched adult flies in a conventional manner. In this way, the authors may determine whether the influence of early IMD pathway activation occurs through "self" mechanisms or whether it entails a contribution from the microbiota. It might also be useful to use reporter transgenes such as Dpt-LacZ to document where in the gut IMD activation takes place in the adult and to monitor whether there is any weak signal that would not be picked up by RTqPCR in newly hatched flies.
Response2-2: We highly appreciate the reviewer for pointing out this important caution. We now checked the dysbiosis in the larval gut (by qPCR of Gluconobacter) and found that it is increased already. For detail, please see our response1-4/1-5. This would strikingly improve the study.
Regarding monitoring IMD activation by the reporter, we plan to do this experiment in our next project. Obviously, a remained question is how epigenetic mechanism in a particular cell/locus mediates the phenotype. This is our next goal and thus lies beyond the scope of this paper.
Specific comments
- The GS system used in this study requires multiple controls, as a study from the Serroude laboratory has reported a driver-dependent leakiness of expression independent of exposure to RU486 (Poirier et al., Aging Cell, 2008). Thus, it would be good to check this with a cross to a UAS-GFP driver and examining the 10 and 40-day time points. The same should be done with antibiotics-treated flies as regards DptA and Drosocin expression (Fig. 5C &D: the age of the adult flies is not specified; it would also be positive to examine the distribution of Acetobacter and Gluconobacter at 10 and 40 days).
Response2-3: We believe the backcrossed UAS-LacZ would be suitable as a control. For key experiments, we checked that RU486's side effect and confirmed it was not the case. What we have not been confident in this respect is the gut microbiota, and therefore we would test whether Gluconobacter is increased just by RU486 or not. Regarding Fig. 5C&D, we used young (day 10-old) flies. We did not examine the Aceto/Gluco at older days, but we assume that they are still in the gut microbiota. How ageing involves microbial change in this and many other contexts is our ongoing project.
- The authors state at the bottom of p6 that JAK-STAT-dependent AMP expression was detected. Fig. 4C shows a significant expression of Drsl2. As far as this reviewer recalls, Buchon et al. had demonstrated a dependence on the JAK-STAT pathway of Drsl3. It would also be worth looking at Turandot genes. As regards an involvement of the Toll pathway, it is not clear whether Drosomycin is significantly expressed as it shows a 32-fold increase in Fig. 4C, yet is not found in Table S2. This issue should be clarified using RTqPCR and it may be worth monitoring also the expression of BomS1.
Response2-4: We would like to add the qRT-PCR of TotA,C, Drs, and BomS1 in the revised manuscript.
Minor points
a) It is surprising to observe an expression driven by the TIGS2 transgene in the larval fat body as it appears to be solely expressed in the intestine in adults. In which epithelial cell type of the intestine is TIGS2 expressed?
Response2-5: We were also surprised (and disappointed indeed) by the fact that TIGS2 shows broader expression pattern in the larvae. As far as we observed, it expresses at least in the enterocytes (strongly in anterior midgut).
b) The authors have carefully defined an optimal dose of RU486 at 1 µM. Why use 20µM Fig. S1, or 50µM (Fig. S6)? Of note, the Flygutseq indicates that Alp9&10 are downregulated in enterocytes upon P. entomophila challenge.
Response2-6: We used 1µM at first, only to have realised that 1µM is too mild to carefully assess the expression pattern of the driver. Thank you for the note, we would cite the paper to generalise our finding.
c) Fig. 1B&C: are the flies used in C) escapers as hardly any flies survive the 5µM RU486 challenge B)?
Response2-7: We prepared more than 1000 embryos for this and many other experiments. One percent of survivors is enough to produce flies in Fig. 1C.
d) Fig. 1D: do the authors know why there is such a difference between DptA and Drosocin?
Response2-8: We greatly appreciate for this comment. There seemed to be a miscalculation here. We have repeated the same experiment again, and now they showed similar magnitude of induction. We would revise this figure.
e) Fig. 2E: the caption does not allow to recognize which curve is LacZ RU and which one is IMD[CA] (dashed line?).
Response2-9: We would amend the caption.
f) Methods: the authors mention that they have dissected crop and Malpighian tubules. As no crop data are reported, does it mean that the crop and MT have been pooled in the same sample; please, clarify.
Response2-10: Sorry for our confusing writing. We have revised the text now to clarify we have "removed" crop and MTs.
Significance:
This study takes place in a context of the influence of infections during early life on subsequent fitness at the adult stage of organisms. With respect to mammals, it is important to note that Drosophila melanogaster undergoes a full metamorphosis that yields a thoroughly novel life form adapted to a new aerial life style. Thus, an influence of the larval stage on the imago is definitely interesting. The senior author has already published interesting work on this topic by showing that oxidative stress experienced during larval stages modifies adult fitness through an indirect action on the larval microbiota. This work is going to be of interest to investigators working on the microbiota and also on intestinal infections, let alone the community of entomologists.
Response2-11: We are really happy to see this comment. We believe that it is important to provide evidence and elucidate mechanisms of how gut microbiota alteration acts as a key factor to exert a life-long effect on the host physiology by a transient event occurred at a juvenile stage.
Drosophila host defense against infections, intestinal infections, host-pathogen interactions
Reviewer #3
Summary
In their manuscript "Activation of innate immune signalling during development predisposes to inflammatory intestine and shortened lifespan" Yamashita et al. have used the Gene Switch system to temporally overexpress imd in Drosophila larval stages and followed the possible effect on adult food intake, starvation resistance and lifespan. Specifically, the authors show that activating the IMD pathway in Drosophila larvae leads to decreased lifespan, lower adult body weight and lower food intake. Furthermore, the authors claim that adult flies develop inflammation in the gut, and, as a consequence, a change in the gut microbiome. The study aims to show the effect of prolonged immune system activation at an early developmental stage on adults.
Major comments
The authors' main conclusion is that IMD activation during development results in adult inflammatory gut, which affects the lifespan of the flies as well as food intake and starvation resistance. Mifepristone (RU486) is used to induce gene expression under GeneSwitch drivers. Using mifepristone is a bit controversial when lifespan effects are being studied. The authors should state that there are various earlier studies showing that mifepristone affects lifespan and also metabolism (e.g. reduces mitochondrial functions and activates AMPK). Although it is fairly reliable that the effects that the authors are seeing are resulting from the IMD pathway activation, it can also be a stress response caused by a combination of mifepristone treatment + IMD activation.
Response3-1: We would like to carefully discuss this possibility by citing the relevant literature.
The authors show that mifeprestone concentration of 5 µM is causing severe lethality and low body weight in DaGS>IMDCA animals. The concentration of 1 µM doesn't give the same effect, but already induces gene expression (as confirmed by imaging in Fig. S1B). Throughout the study, the concentration of 5 µM is still used and the authors claim that the phenotype seen in DaGS>IMDCA animals is suggesting that IMD activation impairs larval growth. However, can this be a case of toxicity/synthetic lethality caused by high concentration of RU486? Why wasn't 1 µM concentration used for the experiments, if it's sufficient to induce gene expression? Is there a possibility of using another temporal induction method causing less stress/toxicity for the flies? Furthermore, authors show that 1 µM mifepristone treatment shortens female lifespan, which is contradictory to the earlier literature. Citations are needed in here. Also, the decrease in female lifespan looks like it is non-significant, what statistics were used in this analysis? The methods section says OASIS2 software was used, but no further details are provided.
Response3-2: We apologise our unclear writing. We used 1 µM throughout the study, not 5 µM to avoid the drug's toxicity. We have not tested other method as GS works well by carefully optimising the RU486 doses. For statistics of lifespan, we would like to add the detailed information in the method section.
Only under 10% of in DaGS>IMDCA flies exposed to 5 µM RU486 eclose, yet in Fig. 1C showing the results of body weight measurements, n=20-50. How were the DaGS>IMDCA flies obtained if under the experimental conditions only a few of them develop successfully? At which developmental stage do the flies die? Why were only male flies used for this experiment?
Response3-3: Please see our Response2-7 We did not carefully check the developmental stage, but it apparently died at early stages of the larva. We usually use male flies for body weight, as female's body weight is understandably affected by the number of eggs inside of the body, making it difficult to discuss the phenotype of developmental growth.
More evidence is needed before concluding that the IMD lifespan effect is coming from the inflammatory intestine. TIGS driver is used to express genes of interest in the gut and fat body. No specific drivers for only the gut or only the fat body are used. Can it be claimed that the effect seen is coming purely from the gut expression? Is it possible that the fat body, which is the main organ responsible for the AMP production is actually responsible for enhanced IMD pathway target AMPs expression (as shown in Fig. S2A; the fold change is higher in the gut that in the fat body)? Was the gut not inflamed or damaged in larvae as there were no upd3 expression?
Response3-4: Thank you for raising this important point. Indeed, we have tried to seek for larval gut- (or fat body)-specific GeneSwitch but no drivers were suitable unfortunately. We admit that our conclusion is not thoroughly backed by the data, so we would carefully discuss this in the revised manuscript. Nevertheless, our new data showing dysbiosis in the larval gut now indicates that this is where the irreversible phenotype resides.
If the authors want to state that the effect is coming from inflammatory gut and that the lifespan effect and feeding/starvation resistance effect is coming from other tissues, why did the authors still decide to use the daughterless driver to study the IMD effect on lifespan, rather than gut or fat body driver, especially if they show that the feeding rate is changed (IMD OE in neurons) as this can also affect the microbiota (which they state is because of inflammatory gut)?
Response3-5: We used DaGS driver simply because it was stronger in terms of the lifespan phenotype. One can assume that the decreased feeding of the DaGS>IMDCA flies might influence the increased Gluconobacter, inflammatory gut, and the shortened lifespan. However, these phenotypes were going to the opposite direction, as decreased feeding theoretically leads to decrease the gut bacteria and extend lifespan. We would like to use a gut-specific (or even cell-type specific) GeneSwitch driver for further mechanistic study, but it may take a huge effort. Our take-home message of the present study is that the juvenile-restricted inflammatory experience causes early dysbiosis, which trigger persistent inflammatory gut in adult, and thereby shortens lifespan. We believe this is adequately supported by the data.
Immune responses are costly and that's one reason why their negative control is so important. The authors could state possible effects between continuously activated immune system (IMD pathway in larvae) and trade-offs in size and life-span in adult flies (+ citations to related studies). The role of constitutively activated IMD in larvae could have been confirmed by using alternative method for activating IMD, e.g. knock out of a negative regulator. Additional controls could have been used, e.g. DaGS background strain without the daughterless driver crossed with the IMDCA , or in the experiment where the gut microbiota was checked (this experiment was lacking the DaGS >LacZ + mifepristone treatment and only had DaGS>IMDCA flies with and without the mifepristone treatment). Usually in Drosophila genetics more control crosses are needed, for e.g. two different constructs of the OE IMD strains e.g. GD and KK backgrounds. The efficiency of the IMD OE could have been directly measured with qPCR and not only shown by measuring the expression of target AMPs.
Response3-6: We would like to make sure the point clearer. The phenotype observed in our study is not related to the trade-off between size and lifespan since we used the 1µM of RU486, which did not affect body size (Fig. 1C) but did shorten the lifespan (by larval but not adult IMD activation). In this sense, we tried to avoid the strong immune activation in the larva as it disturbed the development. Regarding other method for activating IMD, we were not able to use knockouts because we need to make it temporal manipulation in larvae. Alternatively, we had tested PGRP-LC overexpession. When it was expressed strongly in the larvae, it led to the lethality. When it was mild, we observed the shortened lifespan just as in IMDCA overexpression. This new data would support our conclusion well. Please note that we use IMD OE not RNAi (GD and KK lines are RNAi lines).
Regarding gut microbiota, we would like to check whether DaGS>LacZ + RU86 affects Gluconobacter or not. Regarding, efficiency of IMD OE, we would like to perform qPCR of IMD gene.
One of the conclusions drawn is that adults develop gut tissue damage as a result of inflammation. The authors could provide further evidence of this by utilizing microscopy to recognize possible changes in gut epithelia (with appropriate controls).
Response3-7: We appreciate for the suggestion. Somewhat intriguingly, we have not observed any difference in the number of ph3 positive cells, a hallmark of tissue damage-induced ISC proliferation. This is consistent with our preliminary observation that aged flies after larval IMD activation did not show "smurf" phenotype, an indicator of gut barrier dysfunction. In the revised manuscript, we would like to add some qPCR data to test whether upd3/JAK-STAT pathway is activated to detect the tissue damage and carefully discuss the point.
The methods section could be more detailed and clearer to the reader. The statistical analyses used for e.g. survival rates should be described in more detail. The sustained alkaline phosphatase treatment should also be described in more detail, as currently the methods do not clearly state how long the flies were treated with Alp. The description of antibiotic cocktail treatment in the materials and methods should not be under the stocks and husbandry section, as it implies that all flies used were all the time maintained on an antibiotic cocktail
Methods sections could be arranged to resemble more the order of the results sections and more details should be added. It would be challenging to repeat the experiments the way as they have been described.Response3-8: We would like to amend the method section accordingly.
Minor comments
The efficiency of the IMD OE was not directly measured with qPCR, only the expression of target AMPs were measured. The authors should show the activation efficiency of the IMD expression.
Response3-9: Please see our Response1-2
Figure 1B, are these females or males?
Response3-10: It includes both sexes. We add this explanation in the methods.
Fig1 E. in the transcriptome analysis the negative control should have been also treated with mifepristone
Response3-11: Due to financial reason, we could not perform RNAseq analysis for all the samples. We believe showing specific activation of IMD pathway in the IMDCA + RU486 compared the negative control IMDCA -RU486 is sufficient.
For the experiment presented in Fig. S6, females are used, although for the majority of other experiments, only male flies are used?
Response3-12: We have done qPCR in males as well. We add this data in the revised manuscript.
In Fig. S1C, DaGS>GFP expression is induced in 3rd instar larvae by 20 µM RU486. Is concentration this high not toxic for the larvae?
Response3-13: In this experiment, we wanted to see the expression pattern of the driver. Please also see our Response2-6.
The fact that developmental IMD activation increased DptA expression in the adult gut suggested that an irreversible change occurred in this tissue. - what is meant by irreversible change? Can this claim be made?
Response3-14: What we meant by "irreversible" here was that there was a permanent increase of immune activity by the larval IMD activation. It would have been inappropriate to describe the phenotype, so we would avoid this word in the revised manuscript.
Alp results are interesting. Does IAP expression decrease in adults as they age? The authors used "sustained Alp supplementation" to rescue the reduced lifespan phenotype in adults. How long were the flies treated with Alp? This should be mentioned in the materials and methods
Response3-15: According to the literature, IAP expression is decreased during ageing in (Kühn F et al., JCI insight, 2020). In this experiment, we used life-long IAP supplementation (from day 2 onward). This would be mentioned in the revised manuscript.
The description of antibiotic cocktail treatment in the materials and methods should not be under the stocks and husbandry section, as it implies that all flies used were all the time maintained on an antibiotic cocktail.
In the qRT-PCR section, the analysis method could be added (copy number method/ΔΔCt)
Line 49-50 is missing a reference
Line 81, PGAM5 is mentioned without further explaining what it is
Line 229 - what is meant by inflammatory vicious cycles?
Line 314 - what is meant by thrifty phenotype?
In figures showing lifespan, a different color code could be used where yellow and orange/red lines represent different genotypes/treatments; it is hard to visually distinguish the colors that are used at the moment
Figure legend for Fig. 4C - AP could be written out as alkaline phosphatase already here. Also in the legend for Fig. 4 it says E twice (instead of E and then F)
Fig. 5A - a title for x-axis could be added to make it clearer that this represents the proportion of the bacterial taxa in the gut
Fig. S2A - LacZ is mentioned in the description but not shown in the figureResponse3-16: We would amend these in the revised manuscript.
Were there possible cross tissue contaminations in the adult gut samples? where possible contaminations checked e.g. with fatbody specific primers? This should be checked as fatbody is known to produce more AMPs when immune activated, than the gut tissue.
Response3-17: We are well-trained in the dissection of the gut. All the fat body was carefully removed by dissection. Especially when abdominal samples do not show any difference in Fig. 4B, we did not agree that the contamination would explain the data.
CFU analysis: were the flies surface sterilized briefly in ethanol prior dissections?
Response3-18: Yes, flies were surface sterilized by serial washes of 3% bleach and 70% ethanol. We add this procedure in the method section.
Fig2 B-C, the differences between the females and males are not drastic enough to decide to use only males later on. E. typo in starvation. DA>IMD males have decreased starvation resistance without and with the mifepristone treatment?
Response3-19: We decided to use males as females have a slight negative side effect of RU486. DaGS>IMDCA have increased starvation resistance only with the mifepristone treatment. We apologise that our figure caption is not clear. We would amend this in the revised manuscript.
Significance
The topic presented in this manuscript is interesting and relevant for both the fields of aging and immunology and partially explains why early life experiences are important for the wellbeing of the individual later in life. Some of the findings presented in the manuscript are novel, at the same time some of these same issues have been examined in papers related to immune priming/training/memory. The reported findings of the manuscript would be of interest for an audience that is interested about aging and lifespan related issues, as well as immunology and metabolism.
Response3-19: This reviewer's evaluation of the significance of the study is very encouraging. We believe that the phenotypes observed in the manuscript would give wide interest to the biologist working on this hot topic: how early-life event induces later-life health.
Field of expertise: Innate Immunity; Drosophila; Metabolism; Host-Pathogen Interactions; Biomedicine
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Referee #3
Evidence, reproducibility and clarity
Summary
In their manuscript "Activation of innate immune signalling during development predisposes to inflammatory intestine and shortened lifespan" Yamashita et al. have used the Gene Switch system to temporally overexpress imd in Drosophila larval stages and followed the possible effect on adult food intake, starvation resistance and lifespan. Specifically, the authors show that activating the IMD pathway in Drosophila larvae leads to decreased lifespan, lower adult body weight and lower food intake. Furthermore, the authors claim that adult flies develop inflammation in the gut, and, as a consequence, a change in the gut …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary
In their manuscript "Activation of innate immune signalling during development predisposes to inflammatory intestine and shortened lifespan" Yamashita et al. have used the Gene Switch system to temporally overexpress imd in Drosophila larval stages and followed the possible effect on adult food intake, starvation resistance and lifespan. Specifically, the authors show that activating the IMD pathway in Drosophila larvae leads to decreased lifespan, lower adult body weight and lower food intake. Furthermore, the authors claim that adult flies develop inflammation in the gut, and, as a consequence, a change in the gut microbiome. The study aims to show the effect of prolonged immune system activation at an early developmental stage on adults.
Major comments
The authors' main conclusion is that IMD activation during development results in adult inflammatory gut, which affects the lifespan of the flies as well as food intake and starvation resistance. Mifepristone (RU486) is used to induce gene expression under GeneSwitch drivers. Using mifepristone is a bit controversial when lifespan effects are being studied. The authors should state that there are various earlier studies showing that mifepristone affects lifespan and also metabolism (e.g. reduces mitochondrial functions and activates AMPK). Although it is fairly reliable that the effects that the authors are seeing are resulting from the IMD pathway activation, it can also be a stress response caused by a combination of mifepristone treatment + IMD activation. The authors show that mifeprestone concentration of 5 µM is causing severe lethality and low body weight in DaGS>IMDCA animals. The concentration of 1 µM doesn't give the same effect, but already induces gene expression (as confirmed by imaging in Fig. S1B). Throughout the study, the concentration of 5 µM is still used and the authors claim that the phenotype seen in DaGS>IMDCA animals is suggesting that IMD activation impairs larval growth. However, can this be a case of toxicity/synthetic lethality caused by high concentration of RU486? Why wasn't 1 µM concentration used for the experiments, if it's sufficient to induce gene expression? Is there a possibility of using another temporal induction method causing less stress/toxicity for the flies? Furthermore, authors show that 1 µM mifepristone treatment shortens female lifespan, which is contradictory to the earlier literature. Citations are needed in here. Also, the decrease in female lifespan looks like it is non-significant, what statistics were used in this analysis? The methods section says OASIS2 software was used, but no further details are provided.
Only under 10% of in DaGS>IMDCA flies exposed to 5 µM RU486 eclose, yet in Fig. 1C showing the results of body weight measurements, n=20-50. How were the DaGS>IMDCA flies obtained if under the experimental conditions only a few of them develop successfully? At which developmental stage do the flies die? Why were only male flies used for this experiment?
More evidence is needed before concluding that the IMD lifespan effect is coming from the inflammatory intestine. TIGS driver is used to express genes of interest in the gut and fat body. No specific drivers for only the gut or only the fat body are used. Can it be claimed that the effect seen is coming purely from the gut expression? Is it possible that the fat body, which is the main organ responsible for the AMP production is actually responsible for enhanced IMD pathway target AMPs expression (as shown in Fig. S2A; the fold change is higher in the gut that in the fat body)? Was the gut not inflamed or damaged in larvae as there were no upd3 expression?
If the authors want to state that the effect is coming from inflammatory gut and that the lifespan effect and feeding/starvation resistance effect is coming from other tissues, why did the authors still decide to use the daughterless driver to study the IMD effect on lifespan, rather than gut or fat body driver, especially if they show that the feeding rate is changed (IMD OE in neurons) as this can also affect the microbiota (which they state is because of inflammatory gut)?
Immune responses are costly and that's one reason why their negative control is so important. The authors could state possible effects between continuously activated immune system (IMD pathway in larvae) and trade-offs in size and life-span in adult flies (+ citations to related studies). The role of constitutively activated IMD in larvae could have been confirmed by using alternative method for activating IMD, e.g. knock out of a negative regulator. Additional controls could have been used, e.g. DaGS background strain without the daughterless driver crossed with the IMDCA , or in the experiment where the gut microbiota was checked (this experiment was lacking the DaGS >LacZ + mifepristone treatment and only had DaGS>IMDCA flies with and without the mifepristone treatment). Usually in Drosophila genetics more control crosses are needed, for e.g. two different constructs of the OE IMD strains e.g. GD and KK backgrounds. The efficiency of the IMD OE could have been directly measured with qPCR and not only shown by measuring the expression of target AMPs.
One of the conclusions drawn is that adults develop gut tissue damage as a result of inflammation. The authors could provide further evidence of this by utilizing microscopy to recognize possible changes in gut epithelia (with appropriate controls).
The methods section could be more detailed and clearer to the reader. The statistical analyses used for e.g. survival rates should be described in more detail. The sustained alkaline phosphatase treatment should also be described in more detail, as currently the methods do not clearly state how long the flies were treated with Alp. The description of antibiotic cocktail treatment in the materials and methods should not be under the stocks and husbandry section, as it implies that all flies used were all the time maintained on an antibiotic cocktail
Methods sections could be arranged to resemble more the order of the results sections and more details should be added. It would be challenging to repeat the experiments the way as they have been described.
Minor comments
The efficiency of the IMD OE was not directly measured with qPCR, only the expression of target AMPs were measured. The authors should show the activation efficiency of the IMD expression.
Figure 1B, are these females or males?
Fig1 E. in the transcriptome analysis the negative control should have been also treated with mifepristone
For the experiment presented in Fig. S6, females are used, although for the majority of other experiments, only male flies are used?
In Fig. S1C, DaGS>GFP expression is induced in 3rd instar larvae by 20 µM RU486. Is concentration this high not toxic for the larvae?
The fact that developmental IMD activation increased DptA expression in the adult gut suggested that an irreversible change occurred in this tissue. - what is meant by irreversible change? Can this claim be made?
Alp results are interesting. Does IAP expression decrease in adults as they age? The authors used "sustained Alp supplementation" to rescue the reduced lifespan phenotype in adults. How long were the flies treated with Alp? This should be mentioned in the materials and methods
The description of antibiotic cocktail treatment in the materials and methods should not be under the stocks and husbandry section, as it implies that all flies used were all the time maintained on an antibiotic cocktail.
In the qRT-PCR section, the analysis method could be added (copy number method/ΔΔCt)
Line 49-50 is missing a reference Line 81, PGAM5 is mentioned without further explaining what it is Line 229 - what is meant by inflammatory vicious cycles? Line 314 - what is meant by thrifty phenotype?
In figures showing lifespan, a different color code could be used where yellow and orange/red lines represent different genotypes/treatments; it is hard to visually distinguish the colors that are used at the moment
Figure legend for Fig. 4C - AP could be written out as alkaline phosphatase already here. Also in the legend for Fig. 4 it says E twice (instead of E and then F)
Fig. 5A - a title for x-axis could be added to make it clearer that this represents the proportion of the bacterial taxa in the gut
Fig. S2A - LacZ is mentioned in the description but not shown in the figure
Were there possible cross tissue contaminations in the adult gut samples? where possible contaminations checked e.g. with fatbody specific primers? This should be checked as fatbody is known to produce more AMPs when immune activated, than the gut tissue.
CFU analysis: were the flies surface sterilized briefly in ethanol prior dissections?
Fig2 B-C, the differences between the females and males are not drastic enough to decide to use only males later on. E. typo in starvation. DA>IMD males have decreased starvation
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, the authors study the impact of ubiquitously activating the IMD pathway only during larval stages on subsequent adult life. They report a shortened lifespan due to IMD pathway activation in the larval gut and a resistance to starvation linked to its activation in the nervous system. While there is apparently no activation of the IMD pathway in very young adult flies, the expression of some IMD-dependent antimicrobial peptide (AMP) genes is reported from 7-10 flies onwards. This expression is lost upon treating the adults with antibiotics, which also rescues the shortened lifespan phenotype. It correlates with a …
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Referee #2
Evidence, reproducibility and clarity
In this manuscript, the authors study the impact of ubiquitously activating the IMD pathway only during larval stages on subsequent adult life. They report a shortened lifespan due to IMD pathway activation in the larval gut and a resistance to starvation linked to its activation in the nervous system. While there is apparently no activation of the IMD pathway in very young adult flies, the expression of some IMD-dependent antimicrobial peptide (AMP) genes is reported from 7-10 flies onwards. This expression is lost upon treating the adults with antibiotics, which also rescues the shortened lifespan phenotype. It correlates with a possible increase in the proportion of Gluconobacter in the microbiota.
While the study looks interesting, it is not clear whether the results, especially those of survival studies and RTqPCR experiments, have been replicated in independent experiments. This is essential to warrant their conclusions. In this respect, this reviewer notes some important variability in the lifespan studies (e.g., Fig. 2B vs. Fig. 4E): how do the authors account for a lifespan that is shortened almost by half in Fig. 4E? Also, Fig. S2B is not convincing given the observed variability. More data points are required to reach a conclusion.
The authors suggest in their Discussion some kind of epigenetic mechanism transmitting the information of IMD pathway activation having occurred at larval stages. Whether this depends on a change of metabolism remains to be demonstrated, in as much it is likely that there is a major metabolic "reset" occurring during metamorphosis to prepare the individual to the new environmental conditions encountered as an adult. It is also likely that larvae in the wild grow in a microbe-rich slurry and are likely to experience intestinal infections. As noted by the authors themselves on the top paragraph of p7 (line numbers are unreadable), the larval gut is degenerated during metamorphosis and thus the enterocytes that have produced AMPs are no longer present. One possibility would be that there is an early dysbiosis already occurring during larval stages and that the young adults re-infect themselves, for instance through contact with the meconium. The authors' experiments with antibiotics are the key to this study. However, one would like to observe results of the converse experiment, that is, treating larvae with antibiotics (a better control would be to bleach the embryos to generate axenic flies) and then raising the hatched adult flies in a conventional manner. In this way, the authors may determine whether the influence of early IMD pathway activation occurs through "self" mechanisms or whether it entails a contribution from the microbiota. It might also be useful to use reporter transgenes such as Dpt-LacZ to document where in the gut IMD activation takes place in the adult and to monitor whether there is any weak signal that would not be picked up by RTqPCR in newly hatched flies.
Specific comments
- The GS system used in this study requires multiple controls, as a study from the Serroude laboratory has reported a driver-dependent leakiness of expression independent of exposure to RU486 (Poirier et al., Aging Cell, 2008). Thus, it would be good to check this with a cross to a UAS-GFP driver and examining the 10 and 40-day time points. The same should be done with antibiotics-treated flies as regards DptA and Drosocin expression (Fig. 5C &D: the age of the adult flies is not specified; it would also be positive to examine the distribution of Acetobacter and Gluconobacter at 10 and 40 days).
- The authors state at the bottom of p6 that JAK-STAT-dependent AMP expression was detected. Fig. 4C shows a significant expression of Drsl2. As far as this reviewer recalls, Buchon et al. had demonstrated a dependence on the JAK-STAT pathway of Drsl3. It would also be worth looking at Turandot genes. As regards an involvement of the Toll pathway, it is not clear whether Drosomycin is significantly expressed as it shows a 32-fold increase in Fig. 4C, yet is not found in Table S2. This issue should be clarified using RTqPCR and it may be worth monitoring also the expression of BomS1.
Minor points
a) It is surprising to observe an expression driven by the TIGS2 transgene in the larval fat body as it appears to be solely expressed in the intestine in adults. In which epithelial cell type of the intestine is TIGS2 expressed?
b) The authors have carefully defined an optimal dose of RU486 at 1 µM. Why use 20µM Fig. S1, or 50µM (Fig. S6)? Of note, the Flygutseq indicates that Alp9&10 are downregulated in enterocytes upon P. entomophila challenge.
c) Fig. 1B&C: are the flies used in C) escapers as hardly any flies survive the 5µM RU486 challenge B)?
d) Fig. 1D: do the authors know why there is such a difference between DptA and Drosocin?
e) Fig. 2E: the caption does not allow to recognize which curve is LacZ RU and which one is IMD[CA] (dashed line?).
f) Methods: the authors mention that they have dissected crop and Malpighian tubules. As no crop data are reported, does it mean that the crop and MT have been pooled in the same sample; please, clarify.
Significance
This study takes place in a context of the influence of infections during early life on subsequent fitness at the adult stage of organisms. With respect to mammals, it is important to note that Drosophila melanogaster undergoes a full metamorphosis that yields a thoroughly novel life form adapted to a new aerial life style. Thus, an influence of the larval stage on the imago is definitely interesting. The senior author has already published interesting work on this topic by showing that oxidative stress experienced during larval stages modifies adult fitness through an indirect action on the larval microbiota. This work is going to be of interest to investigators working on the microbiota and also on intestinal infections, let alone the community of entomologists.
Drosophila host defense against infections, intestinal infections, host-pathogen interactions
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Referee #1
Evidence, reproducibility and clarity
This paper shows that transient genetic induction of the IMD innate immune pathway during Drosophila development, has long term effects on adult health and lifespan. The paper is well-written, the experiments are well designed and executed, and the data are without exception good quality. The data also support the specific conclusions well. The experiments take full advantage of the Drosophila system to pinpoint the effect on lifespan to long term activation of inflammation in the gut, which is interlinked and dependent upon changes in the microbiota. However the analysis is not comprehensive, because neural-specific effects on …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
This paper shows that transient genetic induction of the IMD innate immune pathway during Drosophila development, has long term effects on adult health and lifespan. The paper is well-written, the experiments are well designed and executed, and the data are without exception good quality. The data also support the specific conclusions well. The experiments take full advantage of the Drosophila system to pinpoint the effect on lifespan to long term activation of inflammation in the gut, which is interlinked and dependent upon changes in the microbiota. However the analysis is not comprehensive, because neural-specific effects on starvation resistance are not followed up, and because the etiology of the changes in microbiota is not mapped out. I should also say that I do not fully agree with the conclusion in the last sentence of the Abstract (the most important general conclusion), that the study "demonstrates a tissue-specific programming effect" of early transient IMD function. Since the lifespan shortening was shown to be dependent upon increased gut Gluconabacter, I would not call this "programming" (though the term is vague enough to mean most anything.) Instead, I would refer to the effect as a host-environment interaction. If it were "programming" of, for instance, the genetic or epigenetic sort, it would not be so easy to reverse.
A few other minor comments:
- Several experiments, the authors use GFP (Fig S1) or the IMD targets DptA or Dro (Fig S2) to validate the induction of IMD-CA. Why have they not directly measured the expression of IMD-CA. This would seem to be logical and technically easy, by qPCR.
- In Fig 4 we see and experiment in which animals were "supplemented" with Alkaline Phosphatase, a protein. How was this done and why does it work? Is AP a gut luminal protein?
- The results in Fig 5 are really where the paper begins to determine a mechanism for the lifespan shortening. However, these results are rather weak, and they don't extend very far. The increase in Gluconobacter is mild (Fig 5C), and is not clear in the 16S rRNA sequencing experiment (Fig 5A). Furthermore, it is not clear that Glunconobacter specifically is the source of the lifespan shortening, of just bacteria in general (Fig 5E).
Significance
Although this paper addresses in interesting topic using an elegant and effective experimental strategy, the final results (Fig 5) and conclusions are modest. The analysis doesn't extend far enough to demonstrate how long term changes in microbiota arise from short term developmental changes in innate immune activity. Moreover, there is no detailed data concerning how the altered microbiota alter lifespan. Thus, while the results are interesting and the findings open avenues for further studies on the topic, the significance of the paper is modest, in its current state. Further analysis of how the microbiota is permanently changed, and why this affects lifespan, could enhance the paper. However, it is not clear that any simple, quick experiments could dramatically advance the findings from where they are now.
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