10 1016@j Cmet 2018 05 026

Article
The Drosophila Immune Deficiency Pathway

Modulates Enteroendocrine Function and Host
Metabolism
Graphical Abstract Authors
Layla Kamareddine, William P. Robins,
Cristin D. Berkey, John J. Mekalanos,
Paula I. Watnick
Correspondence
paula.watnick@childrens.harvard.edu
In Brief
Here Kamareddine et al. show that an
innate immune pathway in
enteroendocrine cells of the Drosophila
melanogaster intestine is activated by the
intestinal microbiota and the microbial
metabolite acetate. Activation of this
pathway increases expression of the
endocrine peptide tachykinin to promote
host metabolic homeostasis.
Highlights
d The innate immune deficiency (IMD) pathway regulates
tachykinin transcription
d Microbial activation of enteroendocrine IMD signaling

mediates metabolic homeostasis
d Acetate restores IMD pathway activation and metabolic

homeostasis in axenic flies
d Acetate signaling through IMD depends on the membrane-

associated receptor PGRP-LC
Kamareddine et al., 2018, Cell Metabolism 28, 1–14

September 4, 2018 ª 2018 Elsevier Inc.
https://doi.org/10.1016/j.cmet.2018.05.026
Please cite this article in press as: Kamareddine et al., The Drosophila Immune Deficiency Pathway Modulates Enteroendocrine Function and Host
Metabolism, Cell Metabolism (2018), https://doi.org/10.1016/j.cmet.2018.05.026
Cell Metabolism
Article
The Drosophila Immune Deficiency

Pathway Modulates Enteroendocrine
Function and Host Metabolism
Layla Kamareddine,1 William P. Robins,2 Cristin D. Berkey,1 John J. Mekalanos,2 and Paula I. Watnick1,2,3,*
1Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
2Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA
3Lead Contact
*Correspondence: paula.watnick@childrens.harvard.edu
https://doi.org/10.1016/j.cmet.2018.05.026
SUMMARY Communication between the innate immune and enteroendo-

crine (EE) systems of the mammalian intestine has been
Enteroendocrine cells (EEs) are interspersed be- proposed based on the following collection of observations
tween enterocytes and stem cells in the Drosophila (Worthington, 2015). Toll-like receptors are expressed on EEs
intestinal epithelium. Like enterocytes, EEs express and respond to activation by increasing transcription of genes
components of the immune deficiency (IMD) innate encoding cytokines and EE peptides (Bogunovic et al., 2007;
immune pathway, which activates transcription of Larraufie et al., 2017; Palazzo et al., 2007; Selleri et al., 2008).
Furthermore, EEs regulate host metabolism in response to
genes encoding antimicrobial peptides. The discov-
inflammation (Gagnon et al., 2015; Zietek and Rath, 2016). How-
ery of large lipid droplets in intestines of IMD
ever, these lines of communication are poorly understood.
pathway mutants prompted us to investigate the Because of its facile genetics and conserved cell types, the
role of the IMD pathway in the host metabolic model Drosophila melanogaster is an ideal host for mechanistic
response to its intestinal microbiota. Here we provide studies of intestinal physiology. The Drosophila intestine is
evidence that the short-chain fatty acid acetate composed of enterocytes, EEs, and stem cells (Ohlstein and
is a microbial metabolic signal that activates Spradling, 2006). Many of the peptides produced by EEs have
signaling through the enteroendocrine IMD pathway been identified (Veenstra et al., 2008; Veenstra and Ida, 2014),
in a PGRP-LC-dependent manner. This, in turn, in- and, recently, cells that produce the EE peptide Tachykinin (Tk)
creases transcription of the gene encoding the endo- have been shown to regulate glucose and lipid metabolism (Am-
crine peptide Tachykinin (Tk), which is essential for cheslavsky et al., 2014; Song et al., 2014).
Key studies describe the impact of intestinal microbes on
timely larval development and optimal lipid meta-
dipteran metabolism. The intestinal microbiota of Drosophila
bolism and insulin signaling. Our findings suggest
raised under standard conditions in the laboratory is composed
innate immune pathways not only provide the first predominantly of Lactobacillus and Acetobacter species, which
line of defense against infection but also afford the promote growth and development (Shin et al., 2011; Storelli
intestinal microbiota control over host development et al., 2011). Due to the absence of this microbiota, axenic flies
and metabolism. have altered insulin signaling and lipid metabolism, and this
can be reversed by provision of the microbial metabolite acetate
(Hang et al., 2014; Shin et al., 2011). However, the mechanism
INTRODUCTION underlying these observations has not been elucidated.
The Drosophila immune deficiency (IMD) signaling pathway is
As tiny intermediaries that guide hosts in their utilization of die- an innate immune pathway similar to the TNF innate immune
tary nutrients, intestinal microbiota cross economic boundaries signaling pathway of mammals (Kleino and Silverman, 2014;
through the profound effects they have on metabolic syndrome Myllymaki et al., 2014). The most proximal components of the
and malnutrition (Fandriks, 2017; Kau et al., 2015; Ridaura IMD pathway are the diaminopimelic acid-type peptidoglycan-
et al., 2013). One means by which intestinal microbiota commu- sensing receptors PGRP-LC and PGRP-LE (Choe et al., 2002,
nicate with their hosts is through metabolic byproducts that arise 2005; Gottar et al., 2002; Kaneko et al., 2006; Ramet et al.,
from bacterial catabolism of the host diet. Short-chain fatty 2002). PGRP-LC is a membrane-associated receptor that
acids, in particular, are almost exclusively derived from intestinal senses peptidoglycan polymers in the extracellular space, while
bacteria (Koh et al., 2016). These metabolites are recognized by PGRP-LE, a cytoplasmic protein, senses transported mono-
specific G protein-coupled receptors on enteroendocrine cells meric peptidoglycan (Kaneko et al., 2006). These receptors inter-
(EEs). These cells secrete small enteroendocrine peptides that face with adaptors that activate cleavage and phosphorylation of
modulate local and systemic lipid and carbohydrate metabolism the transcription factor Relish (Rel) by the caspase 8 homolog
to maintain host homeostasis (Bolognini et al., 2016; Miyamoto Dredd and the complex formed by the I-kappaB kinase g homo-
et al., 2016). log kenny (key) and I-kappaB kinase b, respectively. Rel then
Cell Metabolism 28, 1–14, September 4, 2018 ª 2018 Elsevier Inc. 1

Figure 1. Enteroendocrine IMD Pathway Signaling Regulates Intestinal Lipid Droplet Accumulation and Tk Expression
(A) Representative fluorescence images showing DAPI and BODIPY staining (DAPI/Lipid) or DAPI staining and Tk immunofluorescence (DAPI/Tk) in the anterior
midgut of control (yw) and IMD pathway mutant (RelE20, PGRP-LC D5, and PGRP-LE112) flies. Ten intestines were examined for each genotype. Scale bars, 50 mm.
(B and C) Quantification of normalized total fluorescence in the anterior midgut (AMG) of the indicated flies after incubation with BODIPY. Measurements
represent the mean of five intestines. Error bars represent the standard deviation.
(D and E) Quantification of fluorescent cells in the anterior midgut (AMG) of the indicated flies after incubation with an anti-Tk antibody and a fluorescent sec-
ondary antibody (Tk+ cells). The horizontal bar marks the mean.
(legend continued on next page)
2 Cell Metabolism 28, 1–14, September 4, 2018
translocates to the nucleus where it activates transcription of mutant and, to a lesser extent, a PGRP-LE mutant also demon-
many genes, including several encoding antimicrobial peptides. strated defects in intestinal lipid trafficking in the anterior but
The IMD pathway is expressed in EEs (Dutta et al., 2015). We not posterior midgut (Figures 1A, 1C, S1E, and S1F). This is
hypothesized that microbe-mediated signaling through this consistent with the previous observation that PGRP-LC plays
pathway might link metabolisms of host and microbe. Here we a more important role in IMD pathway signaling in the anterior
show that IMD pathway signaling specifically in EEs that express midgut, while PGRP-LE dominates signaling in the posterior
the enteroendocrine peptide Tk activates Tk and insulin-like pep- midgut (Bosco-Drayon et al., 2012). We also noted greatly
tide 3(ilp3) transcription, mobilization of intestinal lipids, insulin depleted lipids in the fat bodies of all IMD pathway mutants
signaling, and development. The metabolic phenotype of IMD that could be due either to decreased fat body lipid uptake
pathway mutants is similar to that of germ-free flies. While ace- and synthesis or increased mobilization of fat body lipids to
tate was previously known to restore metabolic homeostasis to non-adipose tissue such as the intestine (Figures S1G and
axenic flies, here we show that acetate does so by activating S1H). These observations suggested that microbe-associated
IMD pathway signaling at the level of PGRP-LC. Furthermore, molecular patterns might control host metabolism via IMD
we show that IMD pathway activation in EEs is essential for pathway signaling.
normal development. These observations provide evidence The phenotype of IMD pathway mutants was reminiscent of in-
that, while the innate immune system is essential for defense testinal lipid droplet accumulation caused by knockdown of Tk in
against intestinal pathogens, it also provides a landline for meta- EEs (Song et al., 2014). To determine whether insufficient Tk
bolic communications between microbe and host. might be responsible for the abnormal lipid distribution observed
in IMD pathway mutant flies, we used immunofluorescence to
RESULTS visualize Tk-expressing cells. Considerably fewer Tk-expressing
cells were identified in anterior midgut of IMD pathway mutant
An Innate Immune Signaling Pathway Regulates flies compared with control flies (Figures 1A, 1D, 1E, S1C, and
Transcription of the Gene Encoding the S1I). No change in Tk-expressing cells was observed in the pos-
Enteroendocrine Peptide Tk in the Drosophila Intestine terior midgut (Figures S1E and S1J).
A meta-analysis of studies comparing the transcriptomes of The observed reduction in Tk-containing cells upon IMD
uninfected, infected, and diet-restricted control and/or IMD pathway inhibition could indicate increased Tk release from
pathway mutant flies revealed substantial overlap and differen- EEs or decreased production. To assess IMD pathway control
tial regulation of lipid metabolism genes (Figure S1A and Tables of Tk transcription, we used qRT-PCR to measure Tk transcript
S1, S2, S3, and S4) (Berkey et al., 2009; Buchon et al., 2009; abundance. We found that Tk transcription was significantly
Chatterjee et al., 2014). Furthermore, electron microscopy decreased in the intestines of all IMD pathway mutants except
demonstrated structures suggestive of lipid droplets in the en- PGRP-LE112 (Figures 1F–1H). To determine whether the IMD
terocytes of IMD pathway mutants (Figure S1B). We hypothe- pathway regulates transcription of all enteroendocrine peptides
sized that canonical IMD pathway signaling might control lipid or just a subset, we measured transcription of Diuretic hormone
storage in enterocytes. To test this, we visualized lipid droplets 31 (Dh31), which is co-expressed with Tk, as well as AstA, AstC,
in the midguts of control and IMD pathway mutant flies using and Burs, which are expressed in distinct EEs (Figures S1K–S1N)
boron-dipyrromethene (BODIPY), a hydrophobic fluorescent (Veenstra et al., 2008). Transcription of Dh31 was decreased,
dye, and quantified total fluorescence. IMD pathway mutants whereas transcription of the other peptide-encoding genes
consistently demonstrated lipid droplets in the anterior but was unchanged or increased. These results suggest that IMD
not posterior midgut, whereas no lipid droplets were visible in pathway control of enteroendocrine peptide expression is
the intestines of control flies (Figures 1A, 1B, and S1C–S1F). restricted to a subset of EEs.
We questioned whether PGRP-LC and PGRP-LE, the cell mem- To eliminate the possibility that this decrease in Tk tran-
brane-associated and cytoplasmic IMD pathway peptido- scription was simply due to an effect of the IMD pathway on
glycan receptors, respectively, might be involved in this pheno- EE numbers, we enumerated EEs in the intestines of IMD
type (Kaneko et al., 2006). In fact, we found that a PGRP-LC pathway mutants (Figures S2A and S2B). Overall numbers of
(F–H) Quantification of Tk transcription by qRT-PCR in the intestines of control (yw) or mutant flies (DreddB118, key1, RelE20, PGRP-LCD5, and PGRP-LE112) flies.
Measurements were normalized to the transcript levels of control flies. Ten intestines were used for each replicate. Measurements represent the mean of three
independent biological replicates. Error bars represent the standard deviation.
(I) Representative fluorescence images showing DAPI and BODIPY staining (DAPI/Lipid) or DAPI staining and Tk immunofluorescence (DAPI/Tk) in the anterior
midgut of driver-only control (Tk>) or Rel (Tk>RelRNAi), PGRP-LC (Tk>LCRNAi) and PGRP-LE (Tk>LERNAi) knockdown flies. Scale bars, 50 mm. Ten intestines were
examined for each genotype.
(J and K) Quantification of normalized total fluorescence in the anterior midgut (AMG) of the indicated control or knockdown flies after incubation with BODIPY.
Measurements represent the mean of five intestines. Error bars represent the standard deviation.
(L and M) Quantification of fluorescent cells in the anterior midgut (AMG) of the indicated control or knockdown flies after incubation with an anti-Tk antibody and a
fluorescent secondary antibody (Tk+ cells). The horizontal bar marks the mean.
(N and O) Quantification of Tk transcription by qRT-PCR in the intestines of control or RNAi knockdown flies as indicated. Ten intestines were used for each
replicate. Measurements represent the mean of three independent biological replicates. Error bars represent the standard deviation.
An unpaired t test (B, D, and J), one-way ANOVA with Dunnett’s multiple comparison test (C, E, F, K, N, and O), Welch’s t test (G, H, and L), and Kruskal-Wallis with
Dunn’s multiple comparisons test (M) were used to evaluate statistical significance. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant. See also
Figures S1 and S2 and Tables S1, S2, S3, S4, and S5.
Cell Metabolism 28, 1–14, September 4, 2018 3

Figure 2. IMD Pathway Signaling in EEs

Controls Tk Transcription, Ilp3 Transcrip-
tion, and Insulin Signaling
(A–C) Quantification of Ilp3 transcription by qRT-
PCR in the intestines of control or IMD pathway
mutant flies with the indicated genotypes. Mea-
surements were normalized to the transcript levels
of control yw flies. Ten intestines were used for
each replicate. Measurements represent the mean
of three independent biological replicates. Error
bars represent the standard deviation.
(D and E) Quantification of Ilp3 transcription by
qRT-PCR in the intestines of control or RNAi
knockdown flies as indicated. Measurements were
normalized to the transcript levels of control yw
flies. Ten intestines were used for each replicate.
Measurements represent the mean of three inde-
pendent biological replicates. Error bars represent
the standard deviation.
(F–I) Western blot analysis of total and phosphor-
ylated Akt1 (pAkt1) levels in flies with the indicated
genotypes. Samples were prepared from ten
whole flies. Tubulin was used as a loading control.
See also Figures S3 and S4.
Welch’s t test (B and C) or one-way ANOVA with
Dunnett’s multiple comparison test (A, D, and E)
was used to evaluate statistical significance. **p <
0.01, ****p < 0.0001. ns, not significant.
mately 2-fold (Figure S2P). We then inves-

tigated co-expression of Diptericin A
(DptA) and Tk in V. cholerae-infected flies
expressing DptA-GFP. Although expres-
sion of GFP in EEs was quite low, we
could detect co-localization with Tk
under these conditions (Figure S2Q). We
EE in the intestines of these flies were similar to those of con- conclude that the IMD pathway regulates both DptA and Tk tran-
trol flies. scription in EEs in response to intestinal bacteria.
We questioned whether regulation of EE peptide expression
and lipid metabolism by the IMD pathway was the result of IMD Pathway Inactivation in Tk-Expressing EEs Disrupts
IMD pathway activation in EEs. To assess this, we used RNAi Systemic Insulin Signaling and Metabolic Homeostasis
technology to knock down components of the IMD pathway in Tk has been shown to increase Ilp3 transcription (Amcheslav-
EEs using the EE-specific Tk driver (Tk>) (Figures S2C–S2G) sky et al., 2014). We, therefore, hypothesized that IMD
(Song et al., 2014). We then assessed intestinal lipid accumula- pathway-mediated activation of Tk expression might similarly
tion and quantified Tk-expressing cells. Knockdown of IMD modulate signaling through the insulin/insulin-like signaling
pathway components specifically in Tk-expressing EEs resulted (IIS) pathway. To explore this possibility, we measured
in lipid accumulation in enterocytes and decreased Tk transcrip- transcription of the genes encoding the Drosophila insulin-like
tion and expression in the anterior midgut (Figures 1I, 1L–1O, peptides (Ilp) 2, 3, 5,and 6 in the intestines of IMD pathway
and S2H–S2J). No change in either lipid accumulation or Tk mutant and knockdown flies. Transcription of Ilp3 but not the
expression was observed in the posterior midgut (Figures other Ilps was drastically reduced in the intestines of IMD
S2K–S2M). There was also no change in lipid accumulation in pathway mutants (Figures 2A–2C and S3A–S3C). Knockdown
the fat body when IMD pathway components were knocked of IMD pathway components in Tk-producing EEs also greatly
down in EEs (Figures S2N and S2O). This suggests that lipid decreased Ilp3 transcription (Figures 2D and 2E). We ques-
accumulation in the fat body is modulated by IMD signaling in tioned whether a reduction in Ilp3 transcription might be asso-
a manner that is independent of Tk-producing EEs in the anterior ciated with decreased signaling through the IIS pathway. IIS
midgut. leads to phosphorylation of AKT. Therefore, we assessed levels
We reasoned that, if the IMD pathway activated Tk transcrip- of phosphorylated Akt1 (pAkt1) by western blot using a phos-
tion, oral infection with a low-virulence pathogen such as a phorylation-specific antibody. We found that mutation of IMD
quorum sensing-competent Vibrio cholerae strain should in- pathway components (Figures 2F and 2G) and IMD pathway
crease Tk transcription (Kamareddine et al., 2017). We found knockdown in Tk-expressing EEs (Figures 2H and 2I) resulted
that this infection increased intestinal Tk transcription approxi- in lower levels of pAkt1 than those observed for control flies.

In contrast, knockdown of the IMD pathway in the fat body had for normal Drosophila development and metabolic homeostasis
no effect on pAkt1 levels (Figure S3D). (Hang et al., 2014; Shin et al., 2011). Although the only pathogen-
Decreased signaling through the IIS has been associated with associated molecular pattern known to activate the IMD
hyperglycemia and hyperlipidemia. To determine whether IMD pathway is peptidoglycan, a component of the bacterial cell
control of Tk and Ilp3 transcription might affect these parame- wall, we were struck by the similarities between the metabolic
ters, we measured total glucose and triacylglycerol (TAG) levels phenotypes of IMD pathway mutants, axenic flies, and axenic
in control and IMD pathway mutant flies. We found that levels of flies re-colonized with an Acetobacter mutant unable to produce
glucose and TAG were elevated in IMD pathway mutants (Fig- acetate (Shin et al., 2011). This led us to hypothesize that the
ures S3E and S3F). Knockdown of IMD pathway components short-chain fatty acid acetate activates the IMD pathway in Tk-
in Tk-expressing EEs also increased TAG levels, but resulted in expressing EEs. To test this, we generated axenic flies (Fig-
much smaller increases in glucose levels, which only reached ure S5E) and correlated metabolic effects of acetate treatment
statistical significance for Tk>DreddRNAi flies (Figures S3G– with IMD pathway activation. As had been previously shown,
S3J). In contrast, knockdown of Rel in the fat body had no effect axenic flies demonstrated intestinal lipid accumulation (Figures
on total glucose and TAG levels (Figures S3K and S3L). The 3A and 3B) (Hang et al., 2014). This was accompanied by a
metabolic difference between IMD pathway mutant and EE-spe- decrease in Tk-expressing EEs in the anterior midgut (Figures
cific knockdown flies is most likely multifactorial, resulting from 3A and 3C). Supplementation of the axenic fly diet with acetate
complete, as opposed to partial, knockdown of the IMD pathway diminished intestinal lipid accumulation and increased Tk and
in EEs and global, as opposed to tissue-specific, knockdown of Ilp3 transcription (Figures 3A–3D). This was correlated with
the IMD pathway. restoration of Akt1 phosphorylation and normalization of TAG
To eliminate the possibility that the phenotype observed in and glucose levels (Figures 3E–3G). We predicted that, if host
IMD pathway mutant and knockdown flies resulted from a devel- modulation of metabolism by acetate was the result of IMD
opmental defect, we used a temperature-sensitive driver to pathway activation, it should cause nuclear localization of Rel
confirm that conditional knockdown of Rel in EEs in adulthood in intestinal cells. To test this, we diminished the intestinal micro-
resulted in a phenotype similar to that of flies with constitutive biota with an antibiotic cocktail and compared the location of Rel
IMD pathway knockdown in EEs (Figure S4). This supports a in the presence and absence of dietary acetate. As shown in Fig-
direct rather than developmental role for the IMD pathway in ure 3H, antibiotic treatment eliminated nuclear localization of Rel
regulation of host metabolism. Our results suggest that IMD in the intestines of conventionally raised flies, resulting in an
pathway signaling in Tk-expressing EEs is essential for mainte- appearance similar to that of RelE20 flies. Oral administration of
nance of host metabolic homeostasis. V. cholerae increased Rel translocation, as did acetate supple-
mentation. Although not limited to EEs, we documented that
Knockdown of the IMD Pathway in Tk-Expressing EEs Rel translocation was present in EEs (Figure S5F).
Does Not Alter Host Metabolism Indirectly by Increasing To determine whether acetate-induced nuclear localization of
Numbers of Intestinal Bacteria Rel resulted in increased transcription of IMD pathway targets,
Because the IMD pathway activates expression of antimicrobial we measured expression of several IMD pathway-regulated
peptides that limit growth of intestinal bacteria (Ryu et al., 2006), antimicrobial peptides. In fact, we found that acetate increased
we considered the possibility that the IMD pathway might regu- transcription of the genes encoding a subset of IMD-regulated
late metabolism indirectly by decreasing the population of intes- antimicrobial peptides including DptA, Attacin A (AttA), Cecropin
tinal microbiota. To assess this, we first quantified colony-form- A1 (CecA1), and Drososin but not Metchnikowin or Defensin
ing units of Acetobacter and Lactobacillus species, the main (Def) (Figure 3I). Previous investigators have demonstrated that
components of the Drosophila microbiota, in the intestines of transcription of DptA, AttA, and CecA1 can be activated by Rel
control and IMD mutant flies. As expected, IMD pathway mu- homodimers, whereas Def is best activated by Rel/Dorsal or
tants had increased numbers of both Acetobacter and Lactoba- Rel/Dif heterodimers (Han and Ip, 1999). Therefore, our observa-
cillus species in their intestines (Figures S5A and S5B). However, tion is consistent with a specific effect of acetate in activating
in Tk>RelRNAi flies, the bacterial burden was similar to that of the Rel. To further support a role for the IMD pathway in regulation
driver-only control (Figures S5C and S5D). These data suggest of host metabolism by acetate, we tested whether knockdown
that, while the IMD pathway does limit the bacterial population of Rel in Tk-expressing EEs could block acetate-mediated
in the gut, knockdown of the IMD pathway specifically in Tk-ex- rescue of the metabolic derangements of axenic flies. As shown
pressing EEs does not. Therefore, modulation of microbial in Figures 4A–4C, the ability of acetate to mobilize intestinal TAG
numbers in the Drosophila intestine is unlikely to contribute to and activate Tk expression in axenic flies was dependent on a
the metabolic disturbance caused by IMD pathway knockdown functional IMD pathway.
in Tk-expressing EEs. Activation of the intestinal IMD pathway by acetate supple-
mentation could indicate contamination with peptidoglycan. To
IMD Pathway Activation in EEs by Acetate Restores rule this out, we performed two controls. First, we introduced ac-
Metabolic Homeostasis to Axenic Flies etate into the hemolymph by septic injury using V. cholerae and
Two commensal intestinal bacteria of the fly, Acetobacter pomo- PBS as positive and negative controls, respectively, and
rum and Lactobacillus plantarum, have previously been shown to assessed IMD pathway activation through DptA transcription
regulate host metabolism and development (Erkosar et al., 2015; (Figure 4D). Induction of DptA transcription in acetate and
Shin et al., 2011; Storelli et al., 2011), and there is strong evi- PBS-treated flies was similar, while that in V. cholerae-treated
dence that the acetate produced by Acetobacter is essential flies was significantly greater. This shows that our acetate

Figure 3. Dietary Acetate Activates IMD Pathway Signaling and Restores Metabolic Homeostasis to Axenic Flies
(A) Representative fluorescence images showing DAPI staining and BODIPY staining (DAPI/Lipid) and Tk immunofluorescence (DAPI/Tk) in the anterior midgut of
axenic (GF) control flies (yw) fed fly food alone or supplemented with 50 mM acetate (Ac). Ten intestines were examined for each genotype. Scale bar, 50 mm.
(B) Quantification of normalized total fluorescence in the anterior midgut (AMG) of the indicated flies after incubation with BODIPY. Measurements represent the
mean of five intestines. Error bars represent the standard deviation.

Figure 4. IMD Pathway Knockdown in EE

Prevents the Metabolic and Transcriptional
Response of Axenic Flies to Acetate
(A) Representative fluorescence images showing
DAPI staining and BODIPY staining (DAPI/Lipid)
and Tk immunofluorescence (DAPI/Tk) in the
anterior midgut of axenic (GF) driver-only flies
(Tk>) or Tk-expressing EE-specific Rel knock-
down (Tk>RelRNAi) flies fed fly food alone or sup-
plemented with 50 mM acetate (Ac). Ten intestines
were examined for each genotype. Scale bar,
50 mm.
(B) Quantification of normalized total fluorescence
in the anterior midgut (AMG) of the indicated flies
incubated with BODIPY. Measurements represent
the mean of five intestines. Error bars represent
the standard deviation.
(C) Quantification of fluorescent cells in the ante-
rior midgut (AMG) of the indicated flies incubated
with an anti-Tk antibody and a fluorescent sec-
ondary antibody (Tk+ cells). The horizontal bar
represents the mean.
(D) Quantification of Diptericin A (DptA) tran-
scription by qRT-PCR in control, PBS or acetate
supplemented (Ac), and V. cholerae-infected (Vc)
flies. Acetate, PBS, and V. cholerae were intro-
duced into the hemolymph by septic injury. Mea-
surements were normalized to transcript levels of
control flies. Measurements represent the mean of
three independent biological replicates. Error bars
represent the standard deviation. Ten flies were
used for each replicate.
(E) Quantification of Diptericin A (DptA) transcrip-
tion by qRT-PCR in flies administered Antibiotic
(ABX), Acetate (Ac), acid hydrolyzed Acetate (Ac-H), Vibrio cholerae (Vc), acid hydrolyzed Vc (Vc-H), muramyl dipeptide (MDP), or acid hydrolyzed MDP (MDP-H).
Measurements were normalized to transcript levels of ABX-treated flies. Measurements represent the mean of three independent biological replicates. Error bars
represent the standard deviation. Ten flies were used for each replicate.
One-way ANOVA followed by a Dunnett’s test (B, C, D, and E) was used to calculate significance. *p < 0.05, **p < 0.01, ****p < 0.0001. ns, not significant.
solution does not activate the systemic immune system as acetate, V. cholerae, and MDP with or without hydrolytic treat-
contaminating peptidoglycan would be predicted to do. Pepti- ment and measured DptA transcription. As shown in Figure 4E,
doglycan can be hydrolyzed by prolonged heating at low acid hydrolysis completely eliminated activation of DptA tran-
pH. As a second test, we subjected solutions of acetate, scription by V. cholerae and MDP ingestion. However, acid hy-
V. cholerae, and muramyl dipeptide (MDP) to acid hydrolysis. drolysis did not alter induction of DptA transcription by acetate.
We then supplemented the food of antibiotic-treated flies with Taken together, these experiments indicate that IMD pathway
(C) Quantification of fluorescent cells in the anterior midgut of the indicated flies incubated with an anti-Tk antibody and a fluorescent secondary antibody (Tk+
cells). The horizontal bar marks the mean.
(D) Quantification of Tk and Ilp3 transcription by qRT-PCR in the intestines of axenic (GF) flies treated as in (A). Measurements were normalized to transcript levels
of unsupplemented flies and represent the mean of three independent biological replicates. Error bars represent the standard deviation. Ten intestines were used
for each replicate.
(E) Western blot analysis of total and phosphorylated Akt1 (pAkt1) levels in conventionally raised (CV) and axenic (GF) flies treated as indicated in (A). Ten whole
flies were used for the preparation of each sample. Tubulin was used as a loading control.
(F and G) Quantification of systemic triglycerides and glucose levels in conventionally (CV) raised and axenic (GF) flies treated as indicated in (A). Five flies were
used in each of three independent biological replicates. Measurement represents the mean, and error bars represent the standard deviation. yw (CV) was used as
the reference to assess statistical significance.
(H) Representative immunofluorescent images of the intestines of flies obtained using an antibody that recognizes the Rel 68 cleavage product and stained with
DAPI. Control (yw) or RelE20 mutants were examined. Flies were raised conventionally and examined directly (CV) or after antibiotic treatment (ABX). Additional
treatments included dietary acetate supplementation (Ac) and oral infection with a quorum sensing-competent V. cholerae strain (Vc). Ten intestines were
examined for each condition. Scale bar, 50 mm.
(I) Quantification of Diptericin A (DptA), Attacin A (AttA), Cecropin A1 (CecA1), Drosocin (Dro), Defensin (Def), and Metchnikovin (Mtk) transcription by qRT-PCR in
the intestines of flies treated as noted in (A). Measurements were normalized to the transcript levels of flies that were not administered acetate. Ten intestines were
used for each replicate. Measurements represent the mean of three independent biological replicates. Error bars represent the standard deviation.
Welch’s t test (B, C, D, and I) or one-way ANOVA followed by Dunnett’s multiple comparisons test (F and G) was used to assess statistical significance. *p < 0.05,
**p < 0.01 ***p < 0.001, ****p < 0.0001. ns, not significant. See also Figure S5.

Figure 5. Constitutive Rel Transcription in Tk-Expressing EEs Is Sufficient to Restore Metabolic Homeostasis to Axenic Flies
(A) Quantification of transcription of the indicated IMD pathway components by qRT-PCR in the intestines of axenic flies (GF) with or without dietary supple-
mentation with 50 mM acetate (Ac).
(B) Quantification of Diptericin A (DptA) transcription by qRT-PCR in the intestines of conventional (CV) or axenic (GF) control flies (yw), driver-only flies (Act> and
Tk>), or flies expressing full-length Rel either ubiquitously (Act>Rel) or in Tk-expressing EE (Tk>Rel). Measurements were normalized to transcript levels of

activation by acetate is not the result of peptidoglycan contam- in axenic flies, and this was rescued by constitutive Rel transcrip-
ination and that ingested acetate activates the IMD pathway tion in Tk-expressing EEs (Figures 5H–5J).
locally but not systemically. However, this does not rule out a We questioned whether differential regulation of Rel was a
role for peptidoglycan in activation of Tk transcription. unique characteristic of acetate supplementation. Differential
regulation of Rel was also observed in the intestines of conven-
Increased Relish Expression Is Sufficient to Activate the tionally raised IMD pathway mutants (Figure 5K) and in trans-
IMD Pathway and Rescue Metabolic Dysregulation in genic flies with knockdown of IMD pathway components in Tk-
Axenic Flies expressing EEs (Figure 5L). This suggests that increased Rel
Overexpression of full-length Rel in S2 cells has previously been transcription may be a general characteristic of IMD pathway
shown to induce a modest increase in signaling through the activation.
IMD pathway (Dushay et al., 1996). We questioned whether ace- Taken together, these observations suggest that, in the
tate might amplify signaling through the IMD pathway by absence of intestinal bacteria, increased expression of full-
increasing expression of IMD pathway components. In fact, we length Rel in Tk-expressing EEs provides sufficient IMD pathway
found that acetate supplementation of germ-free flies did result activation to restore metabolic homeostasis. However, because
in a modest 2-fold increase in Rel transcription (Figure 5A). The increased Rel expression is a general feature of IMD pathway
basal level of intestinal IMD pathway signaling present in the un- activation, it is unlikely to be the sole mechanism by which ace-
infected host is sufficient to maintain host metabolic homeosta- tate acts.
sis. We reasoned, therefore, that the small increase in Rel expres-
sion upon acetate supplementation might be sufficient to restore Acetate Depends on PGRP-LC to Increase Signaling
metabolic homeostasis to axenic flies. To test this, we generated through the IMD Pathway
axenic control and transgenic flies with ubiquitous (Act>Rel) or Although acetate is structurally quite different from peptido-
cell-type-specific (Tk>Rel) expression of Rel. Overexpression of glycan, we considered the possibility that acetate might act on
full-length Rel in the EEs of axenic flies yielded levels of DptA tran- or through either PGRP-LC or PGRP-LE to activate the IMD
scription approximately 2.5-fold higher than those observed in pathway. To test this, we suppressed intestinal bacterial popula-
the intestines of conventionally raised control flies (Figure 5B). tions in control, PGRP-LC, and PGRP-LE mutant flies by admin-
As previously observed, germ-free control flies accumulated istering an antibiotic cocktail. This resulted in lipid accumulation
lipids in their intestines (Figures 5C and 5D) (Hang et al., 2014). in the gut and a decrease in Tk-expressing cells similar to that
This was coupled with a decrease in the number of Tk-express- observed in axenic flies (Figures 6A–6C). When the diet of these
ing EEs (Figures 5C and 5E). Constitutive Rel transcription tar- flies was supplemented with acetate in addition to antibiotics,
geted to Tk-expressing EEs reversed these phenotypes (Figures lipid accumulation and Tk expression were rescued in control
5C–5E). In addition, Tk and Ilp3 transcription was also decreased and PGRP-LE mutant flies but not in PGRP-LC mutant flies.
in the intestines of germ-free control flies, and this was rescued Furthermore, specific knockdown of PGRP-LC but not PGRP-
by constitutive Rel transcription in Tk-expressing EEs (Figures LE in Tk-expressing EEs blocked Tk expression and mobilization
5F and 5G). of intestinal lipid by acetate (Figures 6A, 6D, and 6E).
Axenic flies have increased TAG stores and glucose levels As additional evidence of a specific interaction with the PGRP-
accompanied by decreased insulin signaling (Shin et al., 2011). LC receptor, we measured transcription of PGRP-LC and PGRP-
We questioned whether this might also be rescued by activation LE in the intestines of axenic flies with and without acetate sup-
of the IMD pathway in EEs. As previously reported, we observed plementation. In fact, acetate treatment increased expression of
decreased IIS, increased glucose levels, and TAG accumulation PGRP-LC but not PGRP-LE (Figure S6A). This difference is
conventionally-raised control flies. Measurements represent the mean of three independent biological replicates. Error bars represent the standard deviation. Ten
intestines were used for each replicate.
(C) Representative fluorescence images of DAPI staining and BODIPY staining (DAPI/Lipid) and Tk immunofluorescence (DAPI/Tk) in the anterior midgut of axenic
driver-only control (Tk>) flies or flies with Rel constitutively transcribed in Tk-expressing EEs (Tk>Rel). Scale bar, 50 mm. Ten intestines were examined for each
genotype.
(D) Quantification of normalized total fluorescence in the anterior midgut (AMG) of axenic (GF) driver-only flies (Tk>) or flies expressing full-length Rel in Tk-
expressing EE (Tk>Rel) incubated with BODIPY. Measurements represent the mean of five intestines. Error bars represent the standard deviation.
(E) Quantification of fluorescing cells in the anterior midgut of axenic (GF) driver-only flies (Tk>) or flies expressing full-length Rel in Tk-expressing EE (Tk>Rel)
incubated with an anti-Tk antibody and a fluorescent secondary antibody (Tk+ cells). The horizontal bar represents the mean.
(F and G) Transcript levels of Tk and Ilp3 measured by qRT-PCR in the intestines of conventionally raised (CV) or axenic flies (GF) with the genotypes described in
(B). Measurements were normalized to those of conventionally raised yw flies and represent the mean of three independent biological replicates. Ten intestines
were used for each replicate. Error bars represent the standard deviation.
(H) Western blot analysis of total and phosphorylated Akt1 (pAkt1) levels in conventionally raised (CV) or axenic (GF) flies with the genotypes as noted in (B).
Samples were prepared from ten flies for each condition. Tubulin was used as a loading control.
(I–L) Quantification of systemic triglycerides and glucose levels in conventionally raised (CV) or axenic (GF) flies with the genotypes described in (B). Five flies were
used for each of three biological replicates. Measurements represent the mean, and error bars represent the standard deviation. Quantification of Rel tran-
scription by qRT-PCR in the intestines of (K) control (yw) and IMD pathway mutants PGRP-LCD5 (LCD5), PGRP-LE112 (LE112) (L) EE-specific driver-only control
(Tk>), or Dredd (Tk>DreddRNAi), key (Tk>keyRNAi), PGRP-LC (Tk>PGRP-LCRNAi), and PGRP-LE (Tk>PGRP-LERNAi) knockdown flies. For pooled data representing
three biological replicates, the mean and standard deviation are shown. Ten intestines were used for each biological replicate.
Welch’s t test (A, B, D, F, and G), unpaired t test (E), or one-way ANOVA followed by Dunnett’s multiple comparisons test (I, J, K, and L) was used to assess
statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Figure 6. Acetate Functions Upstream of PGRP-LC to Activate the IMD Pathway

(A) Representative fluorescence images showing DAPI staining and BODIPY staining (DAPI/Lipid) and Tk immunofluorescence (DAPI/Tk) in the anterior midgut of
conventionally-raised flies. Where noted, flies were treated with antibiotics (ABX) and acetate (Ac). The following genotypes were examined: control (yw), PGRP-
LCD5 (LCD5), and PGRP-LE112 (LE112) mutant flies as well as Tk> driver-only flies, Tk>PGRP-LCRNAi (Tk>LCRNAi), and Tk> PGRP-LERNAi (Tk>LERNAi). Ten intestines
were examined for each genotype. Scale bar, 50 mm.
(B and C) Normalized total fluorescence in the anterior midgut (AMG) of the indicated flies incubated with BODIPY. Measurements represent the mean of five
intestines. Error bars represent the standard deviation.
(D and E) Quantification of fluorescing cells in the anterior midgut (AMG) of the indicated flies incubated with an anti-Tk antibody and a fluorescent secondary
antibody (Tk+ cells). The horizontal bar represents the mean.
One-way ANOVA followed by Dunnett’s multiple comparisons test (B and D) or Kruskal-Wallis with Dunn’s multiple comparisons test (C and E) was used to assess
statistical significance. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001. ns, not significant. See also Figure S6.
specific to acetate treatment since over-expression of Rel had Activation of the IMD Pathway Is Required for Normal
no effect on transcript levels of either PGRP-LC or PGRP-LE Development
(Figures S6B and S6C). Based on these experiments, we Defective insulin signaling, such as we observed in IMD
conclude that acetate depends on PGRP-LC to increase IMD pathway-deficient flies, is known to stunt growth and delay
pathway signaling. development (Bohni et al., 1999; Brogiolo et al., 2001; Rulifson

Figure 7. IMD Pathway Activation in EEs Is Essential for Normal Development

(A) Representative images of 5- and 8-day-old larvae and pupae, respectively. Scale bar, 0.1 cm.
(B) Quantification of pupal lengths of control (yw) and IMD pathway mutant (DreddB118, key1, and RelE20). Horizontal bars represent the mean measurement.
(C) Time to larval pupation in hours (hr) for yw control and noted IMD pathway mutant flies.
(D) Representative images of 5-day old control (yw) and IMD pathway mutant adult female and male flies. Scale bar, 0.1 cm.
(E and F) Quantification of the body weight of 5-day old control (yw) and IMD pathway mutant adult female and male flies. Horizontal bars represent the mean
measurement.
(G) Time to larval pupation in hours (hr) of axenic control flies (yw), driver-only flies (Act> and Tk>), flies expressing full-length Rel either ubiquitously (Act>Rel) or in
Tk-expressing EE (Tk>Rel). Flies were either fed fly food alone or supplemented with acetate (Ac).
(H) Quantification of pupal lengths of flies with genotype and treatment as indicated in (G). Horizontal bars represent the mean measurement.

et al., 2002; Shin et al., 2011). In agreement with this, we noted ucts allows coordination of the metabolisms of the host and its
that IMD pathway mutant larvae were significantly smaller than intestinal microbiota.
control flies (Figures 7A and 7B) and exhibited delayed pupation Dietary supplementation with acetate increases transcription
(Figure 7C). Furthermore, emerging IMD pathway mutant adults of IMD pathway components and activates the intestinal IMD
were also smaller and lighter than control flies (Figures 7D–7F). pathway. This activation depends on the presence PGRP-LC
We questioned whether this developmental delay was a func- but not PGRP-LE. PGRPs bind polymeric peptidoglycan within
tion of a larval metabolic disturbance similar to that observed for a specific binding pocket (Guan et al., 2005a, 2005b). It seems
adults. In fact, we also observed lipid accumulation, decreased unlikely that acetate would bind directly to the receptor at the
Tk expression, and decreased Ilp3 transcription in the intestines peptidoglycan binding site. There is evidence, however, that
of IMD pathway mutant larvae (Figures S7A–S7E). We hypothe- increased expression of the receptor PGRP-LE activates the
size that the impact of the IMD pathway on development may IMD pathway (Takehana et al., 2002). Similarly, increased
reflect its role in regulating metabolic homeostasis in larval expression of PGRP-LC may contribute to activation of IMD
stages. pathway signaling by acetate. Additionally, dietary acetate could
promote a post-transcriptional modification of the PGRP-LC re-
ceptor such as acetylation or could bind to an EE-enriched mem-
Dietary Acetate and IMD Pathway Activation in EEs
brane-associated co-receptor that interacts with PGRP-LC to
Accelerates Development of Axenic Flies
activate the IMD pathway. Our study provides evidence for the
The developmental program of axenic flies is delayed and can be
first mechanism but does not rule out contributions from mech-
accelerated by bacterial acetate generation in the intestine (Shin
anisms such as the latter two.
et al., 2011). Therefore, we questioned whether development of
The importance of the intestinal IMD innate immune signaling
axenic flies might also be rescued by constitutive expression of
pathway in protection against invading bacteria is well estab-
full-length Rel either ubiquitously or within Tk-expressing EEs. As
lished (Ha et al., 2005; Ryu et al., 2006). In the setting of acute
previously observed, pupation of germ-free flies was signifi-
infection, this pathway is activated by receptors that sense and
cantly delayed. This delay was rescued by dietary acetate
respond to bacterial cell wall peptidoglycan (Aggrawal and Sil-
supplementation as well as constitutive ubiquitous or EE-spe-
verman, 2007). Here we describe a very different role for host
cific Relish expression (Figure 7G). In addition, the pupal size
IMD pathway activation in ensuring a suitable host response to
and adult weight of axenic flies were also increased (Figures
metabolites produced by bacterial fermentation of the host
7H–7J). We conclude that dietary acetate supplementation as
diet. It is interesting to consider how a host might respond appro-
well as constitutive Rel expression can rescue the develop-
priately to these two very disparate situations using the same
mental defect caused by the absence of intestinal bacteria.
signaling pathway. We previously showed that V. cholerae con-
Taken together, our results show that the IMD pathway,
sumes intestinal acetate in the setting of a very heavy pathogen
commonly considered to function primarily in defense against in-
burden (Hang et al., 2014). In contrast, commensal bacteria,
testinal pathogens, may have a novel and equally important role
which are present in much lower numbers, secrete acetate into
as a means of communication between the host and its
the intestine (Shin et al., 2011). One possibility is that the IMD
commensal intestinal bacteria that accelerates development
pathway monitors the relative abundance of bacterial metabo-
and maintains metabolic homeostasis.
lites and peptidoglycan in the intestinal lumen and, thus,
distinguishes between commensal colonization and pathogenic
DISCUSSION invasion. Another possibility is that IMD pathway receptors, acti-
vation thresholds, and regulatory outputs are cell-type specific.
IMD pathway components are expressed in EEs and activated in A third possibility is that there is regional IMD pathway speciali-
response to intestinal bacteria (Dutta et al., 2015). We hypothe- zation. For instance, IMD pathway signaling in the anterior
sized that the IMD pathway in EEs might provide a mechanism midgut may regulate host metabolism, while IMD pathway
by which intestinal bacteria could regulate host metabolism. signaling in the posterior midgut may function in host defense.
Here we show that decreased IMD pathway knockdown in Tk- These are rich avenues of future investigation.
expressing EEs decreases transcription of the gene encoding Our findings provide a mechanism by which both commensal
the enteroendocrine peptide Tk, which has previously been and pathogenic intestinal bacteria may ensure or disrupt optimal
shown to modulate intestinal lipid metabolism (Song et al., host nutrient utilization and suggest that simple dietary modifica-
2014). This, in turn, leads to lipid droplet accumulation in enter- tions, such as increased ingestion of fermentable carbohydrates,
ocytes. A similar phenotype is observed in axenic flies. We pro- may be implemented to counteract the metabolic derangements
vide evidence that supplementation of the axenic fly diet with the caused by disruptions or imbalances of the intestinal microbiota.
microbial fermentation product acetate increases signaling This has profound implications for the treatment of metabolic
through the IMD pathway by a PGRP-LC-dependent mechanism disorders, the effect of protracted antibiotic treatment on child-
to reverse this metabolic disturbance. We propose that activa- hood development and adult metabolic health, and the meta-
tion of EE IMD pathway signaling by bacterial metabolic prod- bolic ramifications of intestinal dysbioses.
(I and J) Quantification of the body weight of 5-day-old adult female and male flies with genotype and treatment as indicated in (G). Horizontal bars represent the
mean measurement.
Kruskal-Wallis with Dunn’s multiple comparisons test (B, E, F, H, I, and J) was used to calculate statistical significance. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns,
not significant. See also Figure S7.

Limitations of Study Received: January 8, 2018

Although the fruit fly is a well-studied and highly tractable model Revised: April 16, 2018
Accepted: May 25, 2018
organism of proven utility in understanding mechanisms of
Published: June 21, 2018
innate immunity, the relevance of molecular mechanisms uncov-
ered in this work to human disease remains to be demonstrated REFERENCES
experimentally. Extending our findings to a mammalian model
would better establish their generalizability. Aggrawal, K., and Silverman, N. (2007). Peptidoglycan recognition in
Drosophila. Biochem. Soc. Trans. 35, 1496–1500.
STAR+METHODS Amcheslavsky, A., Song, W., Li, Q., Nie, Y., Bragatto, I., Ferrandon, D.,
Perrimon, N., and Ip, Y.T. (2014). Enteroendocrine cells support intestinal
stem-cell-mediated homeostasis in Drosophila. Cell Rep. 9, 32–39.
Detailed methods are provided in the online version of this paper
Berkey, C.D., Blow, N., and Watnick, P.I. (2009). Genetic analysis of Drosophila
and include the following:
melanogaster susceptibility to intestinal Vibrio cholerae infection. Cell
Microbiol 11, 461–474.
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING Bogunovic, M., Dave, S.H., Tilstra, J.S., Chang, D.T., Harpaz, N., Xiong, H.,
Mayer, L.F., and Plevy, S.E. (2007). Enteroendocrine cells express functional
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
Toll-like receptors. Am. J. Physiol. Gastrointest. Liver Physiol. 292,
B Drosophila Husbandry and Stocks G1770–G1783.
d METHOD DETAILS Bohni, R., Riesgo-Escovar, J., Oldham, S., Brogiolo, W., Stocker, H., Andruss,
B Transcriptomic Meta-Analyses B.F., Beckingham, K., and Hafen, E. (1999). Autonomous control of cell and or-
B Electron Microscopy gan size by CHICO, a Drosophila homolog of vertebrate IRS1-4. Cell 97,
B Sample Preparation and Fluorescence Microscopy 865–875.
B Western Blot Analysis Bolognini, D., Tobin, A.B., Milligan, G., and Moss, C.E. (2016). The pharma-
B Acetobacter and Lactobacillus sp. Intestinal Burden cology and function of receptors for short-chain fatty acids. Mol. Pharmacol.
B Metabolic Assays 89, 388–398.
B RNA Extraction and Quantitative Reverse-Transcrip- Bosco-Drayon, V., Poidevin, M., Boneca, I.G., Narbonne-Reveau, K., Royet,
tion PCR J., and Charroux, B. (2012). Peptidoglycan sensing by the receptor PGRP-
LE in the Drosophila gut induces immune responses to infectious bacteria
B Generation of Axenic Flies
and tolerance to microbiota. Cell Host Microbe 12, 153–165.
B Oral and Systemic Bacterial Infection, Acetate, and
Broderick, N.A., Buchon, N., and Lemaitre, B. (2014). Microbiota-induced
Muramyl Dipeptide Supplementation changes in Drosophila melanogaster host gene expression and gut
B Peptidoglycan Hydrolysis morphology. MBio 5, e01117-14.
B Developmental Assays
Brogiolo, W., Stocker, H., Ikeya, T., Rintelen, F., Fernandez, R., and Hafen, E.
d QUANTIFICATION AND STATISTICAL ANALYSIS (2001). An evolutionarily conserved function of the Drosophila insulin receptor
and insulin-like peptides in growth control. Curr. Biol. 11, 213–221.
SUPPLEMENTAL INFORMATION Buchon, N., Broderick, N.A., Poidevin, M., Pradervand, S., and Lemaitre, B.
(2009). Drosophila intestinal response to bacterial infection: activation of
Supplemental Information includes seven figures and five tables and can be host defense and stem cell proliferation. Cell Host Microbe 5, 200–211.
found with this article online at https://doi.org/10.1016/j.cmet.2018.05.026. Chang, J.T., and Nevins, J.R. (2006). GATHER: a systems approach to inter-
preting genomic signatures. Bioinformatics 22, 2926–2933.
ACKNOWLEDGMENTS Chatterjee, D., Katewa, S.D., Qi, Y., Jackson, S.A., Kapahi, P., and Jasper, H.
(2014). Control of metabolic adaptation to fasting by dILP6-induced insulin
This work was supported by NIH R21 AI109436 and R01 AI112652 (P.I.W.) and signaling in Drosophila oenocytes. Proc. Natl. Acad. Sci. USA 111,
NIH R01AI018045 to J.J.M. Many stocks from the Bloomington Drosophila 17959–17964.
Stock Center (NIH P40OD018537) and Vienna Drosophila Resource Center
Choe, K.M., Lee, H., and Anderson, K.V. (2005). Drosophila peptidoglycan
were used in this study. Ilp3 and Tk antibodies were generously provided by
recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune
Dr. Jan-Adrianus Veenstra. The 12G10 anti-alpha-tubulin antibody developed
receptor. Proc. Natl. Acad. Sci. USA 102, 1122–1126.
by J. Frankel and E.M. Nelson at The University of Iowa and the mouse mono-
clonal anti-Pros (MR1A)-c developed by C.Q. Doe at the University of Oregon Choe, K.M., Werner, T., Stoven, S., Hultmark, D., and Anderson, K.V. (2002).
were obtained from the Developmental Studies Hybridoma Bank, created by Requirement for a peptidoglycan recognition protein (PGRP) in Relish activa-
the NICHD of the NIH and maintained at The University of Iowa, Department tion and antibacterial immune responses in Drosophila. Science 296, 359–362.
of Biology, Iowa City, IA 52242. Microscopy was performed at the Microscopy Dushay, M.S., Asling, B., and Hultmark, D. (1996). Origins of immunity: relish, a
Resources on the North Quad (MicRoN) core facility at Harvard Medical compound Rel-like gene in the antibacterial defense of Drosophila. Proc. Natl.
School. Acad. Sci. USA 93, 10343–10347.
Dutta, D., Dobson, A.J., Houtz, P.L., Glasser, C., Revah, J., Korzelius, J., Patel,
AUTHOR CONTRIBUTIONS P.H., Edgar, B.A., and Buchon, N. (2015). Regional cell-specific transcriptome
mapping reveals regulatory complexity in the adult Drosophila midgut. Cell
L.K., C.D.B., and P.I.W. designed experiments. L.K. and C.D.B. performed ex-
Rep 12, 346–358.
periments. W.P.R. and J.J.M. carried out meta-analysis of existing transcrip-
tomic data. L.K., W.P.R., and P.I.W. wrote the manuscript. All authors Erkosar, B., Storelli, G., Mitchell, M., Bozonnet, L., Bozonnet, N., and Leulier,
approved the submission. F. (2015). Pathogen virulence impedes mutualist-mediated enhancement of
host juvenile growth via inhibition of protein digestion. Cell Host Microbe 18,
445–455.
DECLARATION OF INTERESTS
Fandriks, L. (2017). Roles of the gut in the metabolic syndrome: an overview.
The authors declare no competing interests. J. Intern. Med. 281, 319–336.

Gagnon, J., Sauve, M., Zhao, W., Stacey, H.M., Wiber, S.C., Bolz, S.S., and Palazzo, M., Balsari, A., Rossini, A., Selleri, S., Calcaterra, C., Gariboldi, S.,
Brubaker, P.L. (2015). Chronic exposure to TNFalpha impairs secretion of Zanobbio, L., Arnaboldi, F., Shirai, Y.F., Serrao, G., et al. (2007). Activation
glucagon-like Peptide-1. Endocrinology 156, 3950–3960. of enteroendocrine cells via TLRs induces hormone, chemokine, and defensin
Gendrin, M., Aidman-Remy, A., Broderick, N.A., Paredes, J., Poidevin, M., secretion. J. Immunol. 178, 4296–4303.
Roussel, A., and Lemaitre, B. (2013). Functional analysis of PGRP-LA in Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., and Ezekowitz, R.A.
Drosophila immunity. PLoS One 8, e69742. (2002). Functional genomic analysis of phagocytosis and identification of a
Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J.A., Drosophila receptor for E. coli. Nature 416, 644–648.
Ferrandon, D., and Royet, J. (2002). The Drosophila immune response against Ridaura, V.K., Faith, J.J., Rey, F.E., Cheng, J., Duncan, A.E., Kau, A.L., Griffin,
Gram-negative bacteria is mediated by a peptidoglycan recognition protein. N.W., Lombard, V., Henrissat, B., Bain, J.R., et al. (2013). Gut microbiota from
Nature 416, 640–644. twins discordant for obesity modulate metabolism in mice. Science 341,
Guan, R., Roychowdury, A., Ember, B., Kumar, S., Boons, G.J., and Mariuzza, 1241214.
R.A. (2005a). Crystal structure of a peptidoglycan recognition protein (PGRP)
Rulifson, E.J., Kim, S.K., and Nusse, R. (2002). Ablation of insulin-producing
in complex with a muramyl tripeptide from Gram-positive bacteria.
neurons in flies: growth and diabetic phenotypes. Science 296, 1118–1120.
J. Endotoxin Res. 11, 41–46.
Ryu, J.H., Ha, E.M., Oh, C.T., Seol, J.H., Brey, P.T., Jin, I., Lee, D.G., Kim, J.,
Guan, R., Wang, Q., Sundberg, E.J., and Mariuzza, R.A. (2005b). Crystal struc-
Lee, D., and Lee, W.J. (2006). An essential complementary role of NF-kappaB
ture of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolu-
pathway to microbicidal oxidants in Drosophila gut immunity. EMBO J. 25,
tion. J. Mol. Biol. 347, 683–691.
3693–3701.
Ha, E.M., Oh, C.T., Bae, Y.S., and Lee, W.J. (2005). A direct role for dual oxi-
dase in Drosophila gut immunity. Science 310, 847–850. Selleri, S., Palazzo, M., Deola, S., Wang, E., Balsari, A., Marincola, F.M., and
Rumio, C. (2008). Induction of pro-inflammatory programs in enteroendocrine
Han, Z.S., and Ip, Y.T. (1999). Interaction and specificity of Rel-related proteins
cells by the Toll-like receptor agonists flagellin and bacterial LPS. Int. Immunol.
in regulating Drosophila immunity gene expression. J. Biol. Chem. 274,
20, 961–970.
21355–21361.
Hang, S., Purdy, A.E., Robins, W.P., Wang, Z., Mandal, M., Chang, S., Shin, S.C., Kim, S.H., You, H., Kim, B., Kim, A.C., Lee, K.A., Yoon, J.H., Ryu,
Mekalanos, J.J., and Watnick, P.I. (2014). The acetate switch of an intestinal J.H., and Lee, W.J. (2011). Drosophila microbiome modulates host develop-
pathogen disrupts host insulin signaling and lipid metabolism. Cell Host mental and metabolic homeostasis via insulin signaling. Science 334,
Microbe 16, 592–604. 670–674.
Kamareddine, L., Wong, A.C.N., Vanhove, A.S., Hang, S., Purdy, A.E., Kierek- Simon, R., Lam, A., Li, M.C., Ngan, M., Menenzes, S., and Zhao, Y. (2007).
Pearson, K., Asara, J.M., Ali, A., Morris, J.G., Jr., and Watnick, P.I. (2017). Analysis of gene expression data using BRB-ArrayTools. Cancer Inform.
Activation of Vibrio cholerae quorum sensing promotes survival of an 3, 11–17.
arthropod host. Nat. Microbiol. 3, 243–252. Song, W., Veenstra, J.A., and Perrimon, N. (2014). Control of lipid metabolism
Kaneko, T., Yano, T., Aggarwal, K., Lim, J.H., Ueda, K., Oshima, Y., Peach, C., by tachykinin in Drosophila. Cell Rep. 9, 40–47.
Erturk-Hasdemir, D., Goldman, W.E., Oh, B.H., et al. (2006). PGRP-LC and Storelli, G., Defaye, A., Erkosar, B., Hols, P., Royet, J., and Leulier, F. (2011).
PGRP-LE have essential yet distinct functions in the Drosophila immune Lactobacillus plantarum promotes Drosophila systemic growth by modulating
response to monomeric DAP-type peptidoglycan. Nat. Immunol. 7, 715–723. hormonal signals through TOR-dependent nutrient sensing. Cell Metab 14,
Kau, A.L., Planer, J.D., Liu, J., Rao, S., Yatsunenko, T., Trehan, I., Manary, 403–414.
M.J., Liu, T.C., Stappenbeck, T.S., Maleta, K.M., et al. (2015). Functional char-
Takehana, A., Katsuyama, T., Yano, T., Oshima, Y., Takada, H., Aigaki, T., and
acterization of IgA-targeted bacterial taxa from undernourished Malawian chil-
Kurata, S. (2002). Overexpression of a pattern-recognition receptor, peptido-
dren that produce diet-dependent enteropathy. Sci. Transl Med. 7, 276ra224.
glycan-recognition protein-LE, activates imd/relish-mediated antibacterial de-
Kleino, A., and Silverman, N. (2014). The Drosophila IMD pathway in the acti- fense and the prophenoloxidase cascade in Drosophila larvae. Proc. Natl.
vation of the humoral immune response. Dev. Comp. Immunol. 42, 25–35. Acad. Sci. USA 99, 13705–13710.
Koh, A., De Vadder, F., Kovatcheva-Datchary, P., and Backhed, F. (2016).
Veenstra, J.A., Agricola, H.J., and Sellami, A. (2008). Regulatory peptides in
From dietary fiber to host physiology: short-chain fatty acids as key bacterial
fruit fly midgut. Cell Tissue Res 334, 499–516.
metabolites. Cell 165, 1332–1345.
Veenstra, J.A., and Ida, T. (2014). More Drosophila enteroendocrine peptides:
Larraufie, P., Dore, J., Lapaque, N., and Blottiere, H.M. (2017). TLR ligands and
orcokinin B and the CCHamides 1 and 2. Cell Tissue Res 357, 607–621.
butyrate increase Pyy expression through two distinct but inter-regulated
pathways. Cell Microbiol 19, https://doi.org/10.1111/cmi.12648. Wang, Z., Berkey, C.D., and Watnick, P.I. (2012). The Drosophila protein
Miyamoto, J., Hasegawa, S., Kasubuchi, M., Ichimura, A., Nakajima, A., and mustard tailors the innate immune response activated by the immune defi-
Kimura, I. (2016). Nutritional signaling via free fatty acid receptors. Int. J. ciency pathway. J. Immunol. 188, 3993–4000.
Mol. Sci. 17, 450. Worthington, J.J. (2015). The intestinal immunoendocrine axis: novel cross-
Myllymaki, H., Valanne, S., and Ramet, M. (2014). The Drosophila Imd signaling talk between enteroendocrine cells and the immune system during infection
pathway. J. Immunol. 192, 3455–3462. and inflammatory disease. Biochem. Soc. Trans. 43, 727–733.
Ohlstein, B., and Spradling, A. (2006). The adult Drosophila posterior midgut is Zietek, T., and Rath, E. (2016). Inflammation meets metabolic disease: gut
maintained by pluripotent stem cells. Nature 439, 470–474. feeling mediated by GLP-1. Front Immunol. 7, 154.

STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit polyclonal Drosophila Akt Antibody Cell Signaling Technology Cat# 9272, RRID:AB_329827
Rabbit polyclonal Phospho-Drosophila Akt Cell Signaling Technology Cat# 4054, RRID:AB_331414
(Ser505) Antibody
Mouse monoclonal anti-Pros (MR1A)-c Developmental Studies Hybridoma Bank Cat# Prospero (MR1A), RRID:AB_528440
Goat polyclonal anti-Rabbit IgG (H+L) Molecular Probes Cat# A-11012, RRID: AB_141359
Secondary Antibody, Alexa Fluor 594
conjugate
Goat polyclonal anti-Rabbit IgG (H+L) Molecular Probes Cat# A-11008, RRID: AB_143165
conjugate
Goat polyclonal anti-Mouse IgG (H+L) Thermo Fisher Scientific Cat# R37121, RRID: AB_2556549
conjugate
Rabbit anti-Tk (DTK) Kind Gift from Jan Adrianus Veenstra Veenstra et al., 2008
Anti-alpha-tubulin Developmental Studies Hybridoma Bank Cat# 12G10 anti-alpha-tubulin, RRID:
AB_1157911
Rabbit anti-Rel68 Paula I. Watnick’s lab Wang et al., 2012
Anti-rabbit IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7074, RRID: AB_2099233
Anti-mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat# 7072, RRID: AB_331144
Chemicals, Peptides, and Recombinant Proteins
Bacto-Agar VWR Cat# 214010
Formaldehyde Thermo Fisher Scientific Cat# NC9658705
Phosphate buffered saline (PBS) TEKnova Cat# P0191
Bovine Serum Albumin Sigma-Aldrich Cat# A8022
Triton X-100 Sigma-Aldrich Cat# 9002-93-1
DAPI (40 ,6-diamidino-2- Invitrogen Cat# D1306
phenylindole,Dihydrochloride)
BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8- Invitrogen Cat# D3922
Pentamethyl- 4-Bora-3a,4a-Diaza-s-
Indacene)
Vectashield Antifade Mounting Medium Vector Laboratories Cat# H-1000
4x Laemmli Sample Buffer Biorad Cat# 161-0747
4-20% precast Polyacrylamide gels Biorad Cat# 456-1096
Molecular Biology Grade Ethanol Thermo Fisher Scientific Cat# BP28184
Tween20 American Bioanalytical Cat# 9005-64-5
DeMan, Rogosa and Sharpe (MRS) Sigma-Aldrich Cat# 69966
TE Buffer Affymetrix-usb Cat# 75834
Sulfuric acid Sigma-Aldrich Cat# 320501
TRIzol Reagent Thermo Fisher Scientific Cat# 15596026
TURBO DNAse (2U/L) Thermo Fisher Scientific Cat# AM2238
Sucrose Sigma-Aldrich Cat# S9378
Brewer’s Yeast ICN Biomedicals Cat# 903312
Germicidal Bleach VWR Cat# 89501-620
Grace‘s insect medium Thermo Fisher Scientific Cat#11667-037
Gluteraldehyde EMS Cat# 16210
Picric acid EMS Cat# 19550
(Continued on next page)
Cell Metabolism 28, 1–14.e1–e5, September 4, 2018 e1

Continued
REAGENT or RESOURCE SOURCE IDENTIFIER
Sodium cacodylate buffer EMS Cat# 11650
Calcium chloride EMS Cat# 12340
Paraformaldehyde EMS Cat# 19208
Sodium Acetate Thermo Fisher Scientific Cat# 127-09-3
Muramyl dipeptide InvivoGen Cat# 53678-77-6
Hydrochloric acid Thermo Fisher Scientific Cat #A481212
Critical Commercial Assays
Glucose (GO) Assay Kit Sigma-Aldrich Cat# GAGO20
Triglyceride Reagent Sigma-Aldrich Cat# T2449
Glycerol Reagent Sigma-Aldrich Cat# F6428
Glycerol Standard Sigma-Aldrich Cat# G7793
SuperScript III First-Strand Synthesis Invitrogen Cat#11752-050
SuperMix for qRT-PCR
iTaq Universal SYBR Green Supermix Biorad Cat# 1725121
Trans-Blot Turbo RTA Mini PVDF Biorad Cat# 1704272
Transfer Kit
Experimental Models: Drosophila Strains
yw and w1118 Kind gift from Jonathan Kagan N/A
DreddB118 Kind gift from Norbert Perrimon N/A
key1 Kind gift from Neil Silverman N/A
RelE20 Bloomington Drosophila Stock Center BL 9457
PGRP-LCD5 Bloomington Drosophila Stock Center BL 36323
PGRP-LE112 Bloomington Drosophila Stock Center BL 33055
PGRP-LERNAi line Bloomington Drosophila Stock Center BL 60038
PGRP-LCRNAi line Bloomington Drosophila Stock Center BL 33383
RelRNAi line VDRC VDRC49413
TkRNAi line TRiP-Harvard Medical School JF01818
DreddRNAi line Bloomington Drosophila Stock Center BL 34070
keyRNAi line Bloomington Drosophila Stock Center BL 35572
UAS-Rel Kind gift from Wan Jae Lee N/A
Actin-Gal4 Kind gift from Norbert Perrimon N/A
Tk-Gal4 Kind gift from Norbert Perrimon N/A
Gal80ts prosv1-Gal4 Kind gift from Bruce Edgar N/A
DptA-GFP Kind gift from Jean Marc Reihhart N/A
Software and Algorithms
BRB-Array tools v4.5.1 Harvard Medical School N/A
Graphpad Prism 7.00 Boston Children’s Hospital N/A
Other
Ampicillin, Sodium Salt Thermo Fisher Scientific Cat# 69-52-3
Tetracycline Hydrochloride IBI Scientific Cat# 64-75-5
Streptomycin, Sulfate Sigma-Aldrich Cat# 3810-74-0
Rifampicin Calbiochem Cat# 557303
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Dr. Paula I. Watnick, at
Harvard Medical School-Boston Children’s Hospital (Paula.watnick@childrens.harvard.edu).
e2 Cell Metabolism 28, 1–14.e1–e5, September 4, 2018

EXPERIMENTAL MODEL AND SUBJECT DETAILS
Drosophila Husbandry and Stocks

All Drosophila procedures conformed to ASL2 guidelines approved by Boston Children’s Hospital Institutional Biosafety Committee.
Flies used in experiments were maintained on standard fly food (16.5 g/L yeast, 9.5 g/L soy flour, 71 g/L cornmeal, 5.5 g/L agar,
5.5 g/L malt, 7.5% corn syrup and 0.4% propionic acid) at 25 C, 70% humidity in a 12 hr day-night cycle. Female flies were used
in all experiments. The controls yw and w1118 as well as DreddB118 and key1 fly stocks were kind gifts from Jonathan Kagan, Norbert
Perrimon and Neal Silverman, respectively. RelE20 (BL 9457), PGRP-LCD5 (BL 36323), PGRP-LE112 (BL 33055), PGRP-LCRNAi
(BL 33388), PGRP-LERNAi (BL 60038), DreddRNAi (BL 34070), and keyRNAi (BL 35572) fly lines were obtained from the Bloomington
Drosophila Stock Center (http://flystocks.bio.indiana.edu/). The RelRNAi line (VDRC49413) was obtained from Vienna Drosophila
Resource Center (http://stockcenter.vdrc.at/) and the TkRNAi line (JF01818) from the TRiP at Harvard Medical School (http://www.
flyrnai.org/TRiP-HOME.html). UAS-Rel was a kind gift from Won Jae Lee. Drivers used in this study are: Actin-Gal4 and Tk-Gal4
(Kind gifts from Norbert Perrimon), and Tub-Gal80ts; Prosv1-Gal4 (prosv1ts) (kind gift from Bruce Edgar). For prosv1ts> RelRNAi trans-
gene expression was induced by shifting flies from 18 C to 29 C. DptA-GFP was a kind gift from Jean Marc Reichhart. Where noted,
flies were supplemented with an antibiotic cocktail of 500 mg/mL Ampicillin (Thermo Fisher Scientific 69-52-3), 50 mg/mL Tetracycline
(IBI Scientific 64-75-5), 200 mg/mL Rifampicin (Calbiochem 557303), and 100 mg/mL Streptomycin (Sigma-Aldrich 3810-74-0), or with
50 mM sodium acetate (Thermo Fisher Scientific 127-09-3) for 3 days.
METHOD DETAILS
Transcriptomic Meta-Analyses
Raw microarray data comparing the transcriptomes of uninfected and Erwinia Ecc15-infected larvae and adult flies and Vibrio chol-
erae-infected adult flies were analyzed using BRB-Array tools v4.5.1 (Berkey et al., 2009; Broderick et al., 2014; Gendrin et al., 2013;
Simon et al., 2007). All genes called significantly upregulated in infected WT versus RelE20 and key mutants (more than or equal to
2-fold) were extracted and compared to the list of statistically upregulated genes in the fat bodies of starved flies (more than or equal
to 5-fold) (Chatterjee et al., 2014). The vetted list of genes upregulated and identified as significant in more than one group were eval-
uated for function and ontology (Chang and Nevins, 2006).
Electron Microscopy
Fly midguts were dissected into Grace’s insect medium and transferred immediately to FGP fixative that consists of 1.25% formal-
dehyde (Thermo Fisher Scientific 11667-037), 2.5% glutaraldehyde (EMS, 16210), 0.03% picric acid (EMS, 19550), 0.05-0.16M
sodium cacodylate buffer (EMS 11650), 0.03% calcium chloride (EMS 12340), for 2 hr at room temperature. Midguts were then
washed overnight at 4 C in sodium cacodylate buffer (EMS 11650). Samples were embedded, and ultra-thin sections were prepared
for examination on a Tecnai G2 Spirit BioTWIN electron microscope.
Sample Preparation and Fluorescence Microscopy

The fat body and intestines of 5- to 7-day-old female IMD mutants and transgenic flies were dissected in 1x phosphate buffered saline
(PBS) (Teknova P0191), fixed in 4% formaldehyde (Thermo Fisher Scientific NC9658705)/PBS for 30 min, washed with 0.1% Triton
X-100 (Sigma-Aldrich 9002-93-1)/PBS, blocked for 1 hr at room temperature in blocking buffer (2% BSA/ 0.1% Triton X-100/ PBS).
Gut tissues were incubated with primary antibodies overnight at 4 C and then stained with a fluorescence-conjugated secondary
antibody. Both guts and fat bodies were then stained with DAPI (1:1000, Invitrogen D1306) and BODIPY 493/503 (1mg/mL, Invitrogen
D3922) in blocking buffer for 1 hr at room temperature, washed with 0.1% Triton X-100/PBS, and mounted in Vectashield mounting
medium (Vector Laboratories H-1000). Images were collected using a Zeiss LSM 780 confocal microscope. Primary and secondary
antibodies used in this study are: rabbit anti-Tk (1:500, kind gift from Jan Adrianus Veenstra), mouse anti-Prospero (MR1A)-c (1:100,
Developmental Studies Hybridoma Bank AB_528440), rabbit anti-Rel68 (1:100, generated in the Watnick lab), Alexa 594 conjugated
a-rabbit secondary antibody (1:500, Thermo Fisher Scientific A-11012), Alexa 594 conjugated a-mouse secondary antibody (1:500,
Thermo Fisher Scientific R37121) and Alexa 488 conjugated a-rabbit secondary antibody (1:500, Thermo Fisher Scientific A-11008).
For assessment of lipid accumulation in fat bodies or intestines, total fluorescence was quantified and normalized by dividing by the
total area included in the measurement. Background fluorescence/unit area was subtracted from this measurement. At least 5 guts or
fat bodies were scored for each genotype. Researchers who carried out the microscopy were not blinded.
Western Blot Analysis

Ten whole female flies (5-7 days old) were homogenized in PBS (Teknova P0191). Protein samples were reduced and denatured in
SDS-PAGE sample buffer (BioRad 161-0747) for 20 min 95 C, separated on a 4-20% polyacrylamide gel (BioRad 456-1096) and
transferred to a PVDF membrane (BioRad 1704272). Extracts were immunoblotted with rabbit anti-AKT (1:1000, Cell Signaling
Technologies 9272), rabbit anti-Ser505-phospho-dAKT (1:1000, Cell Signaling Technologies 4054), mouse anti-b-Tubulin (1:2500,
Developmental Studies Hybridoma Bank AB_579794), anti-mouse and anti-rabbit IgG horseradish peroxidase-conjugated second-
ary antibodies (1:5000, Cell Signaling Technologies, 7074 and 7072, respectively).

Acetobacter and Lactobacillus sp. Intestinal Burden

Groups of 7 flies per genotype were collected, surface sterilized with 70% ethanol (Thermo Fisher Scientific. BP28184), washed
3 times with 0.1% Tween 20 (American Bioanalytical 9005-64-5)/PBS (Teknova P0191), and homogenized in PBS. Serial dilutions
of the homogenates were plated on deMan, Rogosa and Sharpe (MRS) plates (Sigma-Aldrich 69966) and the numbers of colony-
forming units (CFUs) of Acetobacter and Lactobacillus were counted.
Metabolic Assays
Five female flies per genotype (5-7 days old) fed on standard fly food were homogenized in 100 mL TE buffer (500 mM Tris pH 7.5,
50mM EDTA + 0.1% Triton X-100, Affymetrix-usb 75834), and samples were heat-treated at 95 C for 20 min to inactivate endoge-
nous enzymatic activity. All metabolic assays were conducted using commercial kits following the manufacturers’ instructions. For
glucose assays: 30 mL of glucose standards (0.01 to 0.16 mg/mL) and diluted test samples (1:5 parts) were loaded into a clear-bottom
96-well plate. 100 mL of GO reagent (Sigma-Aldrich GAGO-20) were added to each well. Plates were incubated at 37 C for 1 hr. The
reaction was stopped by adding 100 mL of 12 N sulfuric acid (Sigma-Aldrich 320501) into each well, and the absorbance was
measured at 540 nm using a microplate spectrophotometer (Bio-Rad, Benchmark Plus). For TAG assays: 20 mL of glycerol standards
(0.125-0.5 mg/mL) (Sigma-Aldrich G7793) as well as test samples were incubated at 37 C for 1 hr with either 20 mL TE buffer or tri-
glyceride reagent (Sigma-Aldrich T2449). Samples were then centrifuged at max speed for 3 min, and 30 mL of each sample was
loaded into a clear-bottom 96-well plate. 100 mL of free glycerol reagent (Sigma-Aldrich F6428) was further added to each sample,
the plate was incubated at 37 C for 5 min, and absorbance was measured at 540 nm using a microplate spectrophotometer (Bio-Rad,
Benchmark Plus).
RNA Extraction and Quantitative Reverse-Transcription PCR

To evaluate transcription of Drosophila genes, total RNA was extracted from 10-15 whole flies or intestines using TRIzol reagent
(Thermo Fisher Scientific 15596026) according to the supplier’s instructions, and contaminant genomic DNA was removed by DNase
I (Thermo Fisher Scientific AM2238) treatment. First-strand cDNA was produced from 500 ng-1 mg of total RNA using the SuperScript
III cDNA synthesis kit (Invitrogen 117520-050) as described by the manufacturer. qRT-PCR was performed using iTaq SYBR Green
(Bio-Rad 1725121) on a StepOnePlus real-time PCR system (Applied Biosystems). The primers used in qRT-PCR are listed in
Table S5. Relative gene expression values were calculated using the comparative CT method and RP49 was used as an internal
control.
Generation of Axenic Flies

Freshly laid eggs were collected on agar plates (VWR 214010), Sucrose (Sigma-Aldrich S9378), Brewer’s yeast extract (ICN Biomed-
icals 903312) supplemented with yeast paste. In a laminar flow hood, eggs were surface sterilized by 2 washes (5 mins each) with
0.6% hypochlorite/1:10 dilution of germicidal bleach (VWR 89501-620), followed by 3 washes with sterile water. Eggs were then
aseptically transferred to sterile vials containing autoclaved standard Drosophila food. PCR using universal 16S rRNA primers
was used to confirm the germ-free condition of the flies (Hang et al., 2014).
Oral and Systemic Bacterial Infection, Acetate, and Muramyl Dipeptide Supplementation
For oral bacterial infection, acetate and muramyl dipeptide supplementation, flies were placed for 3 days in fly vials containing cel-
lulose acetate plugs infused with 2.3 mL of LB broth harboring 50 mM sodium acetate (Thermo Fisher Scientific 127-09-3), 10 mg/mL
muramyl dipeptide (InvivoGen 53678-77-6), or a 1:10 dilution of an overnight culture of V. cholerae. 30 female flies were used per
condition and divided evenly between three vials.
Septic injury was used to induce systemic infection for experiments as noted. In those experiments, after being anesthetized with
CO2, the dorsal thorax of 30 flies was pierced with a fine needle dipped in a solution containing 1X PBS (TEknova P0191), 50mM
sodium acetate (Thermo Fisher Scientific 127-09-3), or LB broth harboring a 1:10 dilution of an overnight culture of V. cholerae. Flies
were then kept in food vials for 3 hr after which experiments were performed.
Peptidoglycan Hydrolysis
To eliminate the possibility of peptidoglycan contamination, the acetate solution along with other positive controls were subject to
total acid hydrolysis. Briefly, solutions containing 50 mM sodium acetate (Thermo Fisher Scientific 127-09-3), 10 mg/mL muramyl
dipeptide (InvivoGen 53678-77-6), and a 1:10 dilution of an overnight culture of V. cholerae were placed in 4N HCl (Thermo Fisher
Scientific A481212) at 100 C for 16 hr, resulting in peptidoglycan hydrolysis to amino acids and hydrolytically resistant peptides.
The hydrolyzed solutions were adjusted to an approximate pH of 7 prior to oral administration to flies as previously described.
Developmental Assays
The time to pupation was quantified by scoring the number of emerging pupae in each genotype, twice each day. The length of 5-day-
old larvae and 8-day-old pupae was measured under the microscope using a size metric ruler. 5 days post eclosion, individual male
and female flies were weighed to the nearest mg, using a Toledo (MX5) microbalance (Mettler). The density of flies in our cultures was
e4 Cell Metabolism 28, 1–14.e1–e5, September 4, 2018

carefully controlled as follows. Ten female and 10 male flies were placed in each vial at the beginning of the experiment. To prevent
the development of differences in population as a result of differences in eclosion rates between groups, newly emerged offspring
were collected daily and transferred to new vials.
QUANTIFICATION AND STATISTICAL ANALYSIS
Measurements represent the mean of at least three biological replicates in all graphs, and error bars represent the standard deviation.
As appropriate, a two-tailed Student’s t test, Welch’s t test (unequal variance), Mann-Whitney test (non-parametric), one-way ANOVA
with post-hoc Dunnett’s test, or Kruskal Wallis with a post-hoc Dunn’s multiple comparison test was used to calculate significance.
All statistical analysis was carried out using Graphpad Prism 7.00 software with the exception of the transcriptomic meta-analyses,
which were carried out using BRB-Array tools v4.5.1. The statistical test applied is noted in the figure legends. For all tests, a p < 0.05
was considered significant. Asterisks reflecting the calculated p values are shown above each measurement, and ns indicates that
differences between measurements were not statistically significant. No data collected were excluded from any experimental or sta-
tistical analysis. Tests for normality or outlier tests were not performed.

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Article

The Drosophila Immune Deficiency Pathway

d Microbial activation of enteroendocrine IMD signaling

d Acetate restores IMD pathway activation and metabolic

d Acetate signaling through IMD depends on the membrane-

Kamareddine et al., 2018, Cell Metabolism 28, 1–14

The Drosophila Immune Deficiency

SUMMARY Communication between the innate immune and enteroendo-

Cell Metabolism 28, 1–14, September 4, 2018 ª 2018 Elsevier Inc. 1

Cell Metabolism 28, 1–14, September 4, 2018 3

Figure 2. IMD Pathway Signaling in EEs

mately 2-fold (Figure S2P). We then inves-

4 Cell Metabolism 28, 1–14, September 4, 2018

Cell Metabolism 28, 1–14, September 4, 2018 5

(legend continued on next page)

Figure 4. IMD Pathway Knockdown in EE

Cell Metabolism 28, 1–14, September 4, 2018 7

(legend continued on next page)

Cell Metabolism 28, 1–14, September 4, 2018 9

Figure 6. Acetate Functions Upstream of PGRP-LC to Activate the IMD Pathway

10 Cell Metabolism 28, 1–14, September 4, 2018

Figure 7. IMD Pathway Activation in EEs Is Essential for Normal Development

(legend continued on next page)

12 Cell Metabolism 28, 1–14, September 4, 2018

Limitations of Study Received: January 8, 2018

Cell Metabolism 28, 1–14, September 4, 2018 13

14 Cell Metabolism 28, 1–14, September 4, 2018

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Cell Metabolism 28, 1–14.e1–e5, September 4, 2018 e1

CONTACT FOR REAGENT AND RESOURCE SHARING

e2 Cell Metabolism 28, 1–14.e1–e5, September 4, 2018

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Drosophila Husbandry and Stocks

Sample Preparation and Fluorescence Microscopy

Western Blot Analysis

Cell Metabolism 28, 1–14.e1–e5, September 4, 2018 e3

Acetobacter and Lactobacillus sp. Intestinal Burden

RNA Extraction and Quantitative Reverse-Transcription PCR

Generation of Axenic Flies

e4 Cell Metabolism 28, 1–14.e1–e5, September 4, 2018

QUANTIFICATION AND STATISTICAL ANALYSIS

Cell Metabolism 28, 1–14.e1–e5, September 4, 2018 e5

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