A novel mode of induction of the humoral innate immune response in drosophila larvae

A novel mode of induction of the humoral innate immune response in Drosophila larvae RESEARCH ARTICLE A novel mode of induction of the humoral innate immune response in Drosophila lar[.]

RESEARCH ARTICLE

A novel mode of induction of the humoral innate immune response

in Drosophila larvae

Hiroyuki Kenmoku1,*, Aki Hori1,2,*, Takayuki Kuraishi1,3,4,5,*,‡,§and Shoichiro Kurata1,‡

ABSTRACT

Drosophila adults have been utilized as a genetically tractable model

organism to decipher the molecular mechanisms of humoral innate

immune responses In an effort to promote the utility of Drosophila

larvae as an additional model system, in this study, we describe a novel

aspect of an induction mechanism for innate immunity in these larvae.

By using a fine tungsten needle created for manipulating

semi-conductor devices, larvae were subjected to septic injury However,

although Toll pathway mutants were susceptible to infection with

Gram-positive bacteria as had been shown for Drosophila adults, microbe

clearance was not affected in the mutants In addition, Drosophila

larvae were found to be sensitive to mechanical stimuli with respect to

the activation of a sterile humoral response In particular, pinching with

forceps to a degree that might cause minor damage to larval tissues

could induce the expression of the antifungal peptide gene Drosomycin;

notably, this induction was partially independent of the Toll and immune

deficiency pathways We therefore propose that Drosophila larvae

might serve as a useful model to analyze the infectious and

non-infectious inflammation that underlies various inflammatory diseases

such as ischemia, atherosclerosis and cancer.

KEY WORDS: Innate immunity,Drosophila, Larvae

INTRODUCTION

Drosophila adults have been used as a leading model organism to

investigate molecular mechanisms of innate immunity (Lemaitre and

Hoffmann, 2007; Buchon et al., 2014) since it was first demonstrated

in 1996 that the Toll pathway, which was initially characterized as an

essential pathway for dorsoventral patterning in Drosophila embryos

(Anderson et al., 1985a,b), was required for the induction of the

antifungal peptide gene Drosomycin (Drs) upon fungal infection

(Lemaitre et al., 1996) In particular, Drosophila adult models have

contributed to identifying genes required for the humoral innate

immune responses and for the production of antimicrobial peptides

(AMPs) and melanization factors (Lemaitre et al., 1995; Rämet and Hultmark, 2014) In Drosophila adults, AMP induction upon challenge with microbes is controlled by two distinct signaling pathways, the Toll and immune deficiency (IMD) pathways (Lemaitre and Hoffmann, 2007; Valanne et al., 2011; Myllymäki et al., 2014) The Toll pathway is required for the induction of Drs and for survival following systemic infection with Gram-positive bacteria or fungi (Ferrandon et al., 2007) Specifically, the recognition of lysine-type peptidoglycans or β-glucans from microbes by the PGRP-SA/GNBP1 complex or by GNBP3 in the hemolymph activates modular serine protease (ModSP), followed by activation of Spätzle (Spz)-processing enzyme and cleavage of Spz, a protein ligand of the Toll receptor (Gottar et al., 2002, 2003, 2006; Jang et al., 2006; Buchon et al., 2009b) In addition, so-called ‘danger signals’ also activate the Toll pathway through the protease Persephone (Psh) For example, exogenous danger signals such as PR1 secreted from pathogenic fungi, as well as endogenous danger signals generated in apoptosis-deficient mutants, lead to the activation of Psh and subsequent processing of Spz (Chamy et al., 2008; Ming et al., 2014; Obata et al., 2014) The active form of Spz induces conformational changes in the Toll receptor, activates Toll intracellular signaling (Kanoh et al., 2015b) and ultimately leads to the nuclear translocation of nuclear factor-kappa B (NF- κB) proteins Dif and Dorsal, inducing the expression of antimicrobial peptide genes including Drs (Lindsay and Wasserman, 2014) Conversely, the IMD pathway recognizes diaminopimelic acid-type peptidoglycans derived from Gram-negative bacteria via peptidoglycan recognition protein (PGRP)-LC and PGRP-LE (Kleino and Silverman, 2014) These receptors facilitate downstream signaling via the adaptor protein IMD, activate the NF- κB protein Relish, and induce the expression of antimicrobial peptides such as Diptericin (Dpt) (Paquette et al., 2010) Notably, these pathways are essentially characterized in Drosophila adults.

In contrast, Drosophila larvae have been largely utilized for dissecting cellular immune responses, particularly for nematode and wasp infections (Paddibhatla et al., 2010; Arefin et al., 2015; Kucerova et al., 2015; Hillyer, 2016) Insect hemocytes, representing blood cells, are composed of three cell types: plasmatocytes, crystal cells, and lamellocytes These play central roles in cellular immunity by phagocytosing bacteria (plasmatocytes), involvement in the melanization process (crystal cells) and forming capsules around wasp eggs, a process referred to as encapsulation (lamellocytes) (Honti

et al., 2014; Gold and Brückner, 2015; Parsons and Foley, 2016) For example, recent studies have begun to unravel the complex encapsulation processes by using Drosophila larvae upon infection with parasitoid wasps such as Leptopilina boulardi (Kari et al., 2016).

In addition, the fat body, an immune-responsive organ in flies functionally resembling the mammalian liver, expresses edin and utilizes Toll signaling to control the numbers of plasmatocytes (Schmid

et al., 2014; Vanha-aho et al., 2015) Finally, JAK-STAT signaling in somatic muscles is important for inducing the encapsulation reaction and controls the number of circulating lamellocytes (Yang et al., 2015).

Received 12 July 2016; Accepted 20 January 2017

1Department of Molecular Biopharmacy and Genetics, Graduate School of

Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.2

Graduate School of Medical Sciences, Kanazawa University, Ishikawa 920-1192, Japan

3

Department of Microbiology and Immunology, Keio University School of Medicine,

Tokyo 160-8582, Japan.4

Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Ishikawa 920-1192, Japan.5

PRESTO, Japan Science and Technology Agency, Tokyo 102-0076, Japan

*These authors contributed equally to this work

§Senior author

‡

Authors for correspondence (tkuraishi@staff.kanazawa-u.ac.jp;

kurata@m.tohoku.ac.jp)

A.H., 0000-0001-5375-6678; T.K., 9493-6082; S.K.,

0000-0002-0301-872X

This is an Open Access article distributed under the terms of the Creative Commons Attribution

License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution and reproduction in any medium provided that the original work is properly attributed

By contrast, only a handful of studies have been published related

to use of the Drosophila larval model of bacterial infection to

analyze humoral immune responses (Ferrandon et al., 1998;

Manfruelli et al., 1999; Ligoxygakis et al., 2002; Shia et al.,

2009; Yamamoto-Hino et al., 2015; Yamamoto-Hino and Goto,

2016) Because these studies implicate intriguing differences in

terms of the induction mechanisms of AMPs between larvae and

adults, a larval model might thus have the potential to identify novel

molecular mechanisms However, it is possible that the limited

numbers of publications on larval bacterial infection might partly be

due to technical difficulties in the manufacture of uniform tungsten

wires sharpened by electrolysis and their use in introducing

infections (Romeo and Lemaitre, 2008) without causing severe

damage that leads to the death of the larvae Consistent with this

likelihood, the survival and colony-forming assays upon systemic

infection in larvae have been seldom reported Here, we present a

method to perform larval infection using a tungsten needle provided

by a manufacturer that produces pins for testing semi-conductor

devices By using this uniform and solid needle, we were able to

successfully perform and investigate bacterial infection in

Drosophila larvae In addition, we found that mechanical stimuli

generated by pinching larvae with forceps resulted in the sterile

induction of a antimicrobial peptide, providing a novel model for

non-infectious activation of the humoral innate immune response.

RESULTS

The Toll pathway is required for survival against

Gram-positive bacterial infection in larvae but not for bacterial

removal

To easily and consistently perform infection using third instar larvae,

we employed a fine tungsten needle used for the examination of

semiconductor devices With this needle, over 80% of larvae were able

to survive following a clean injury in the wild type and in Toll pathway

and IMD pathway mutants (Fig 1A) By pricking larvae with a needle

dipped into a pellet of Gram-positive bacteria Staphylococcus

saprophyticus, we found that Toll pathway mutants were susceptible

to the infection (Fig 1B), although the number of bacteria in the

infected whole mutant larvae after any time point was similar to that in

the wild type (Fig 1C) These results suggest that the Toll pathway is

dispensable for bacterial clearance in larvae, showing a sharp contrast

to the results from Drosophila adults in which the Toll pathway is

required for the removal of bacteria upon Gram-positive bacterial

challenge Notably, although the induction of the antifungal peptide

gene Drs was slightly lower in Toll pathway mutants than in wild-type

larvae, substantial induction of Drs still remained in the mutants

(Fig 1D), consistent with the results of Manfruelli et al (1999).

We next challenged larvae with Gram-negative bacteria using the

needle Fig 1E and F show that IMD mutant larvae were not

sensitive to infection with Ecc15, although the induction of the

antibacterial peptide gene Dpt was almost abrogated in the mutant.

From these results, we conclude that survival, AMP expression and

bacterial number upon bacterial infection by septic injury with a

tungsten needle could be consistently measured in Drosophila

larvae, and that the role of the Toll pathway was somewhat different

during this process compared with the adult infection model.

Pinching by forceps induces the expression of AMP in larvae

We found that clean injury with the needle induced the expression of

Drs and Dpt (Fig 2A,B) Furthermore, even pinching larvae using

forceps, a normal means of handling larvae, caused strong Drs

induction (Fig 2A) Time-course experiments showed that Drs

expression was induced from 2 h, maximized at 4 h and continued

to 12 h (Fig 2C) The level of Drs after eclosion was not increased compared with the level in untreated flies (Fig S4A) After pinching, 10% of larvae showed small melanized spots (Fig 2D), although extremely weakly pinched larvae did not show melanization and the level of Drs induction was marginal (Fig S4B), implying that pinching might cause minor injury in larval tissues Next, we examined which tissues exhibited Drs expression Fig 2E shows that Drs reporter larvae exhibited GFP signals in the whole fat body and that the position of pinching was not connected with Drs induction Consistent with this result, quantitative real-time-polymerase chain reaction (real-time qPCR) analysis showed that the induction of Drs was detected in the fat body dissected out from other tissues (Fig 2F) These results indicate that Drs is induced in the fat body upon pinching with forceps.

As Drosophila possess commensal bacteria (Kuraishi et al., 2013), Drs induction by pinching might be caused by such infections To assess this possibility, germ-free larvae (Fig 2G) were used for pinching experiments Fig 2H shows that the level of induction of Drs in germ-free larvae was not reduced compared with that in conventionally reared larvae, indicating that Drs expression

is sterilely induced by pinching with forceps Next, we performed microarray analysis using pinched larvae in order to examine whether Drs was uniquely induced by pinching or whether other defense response genes that respond to infection in adults (De Gregorio et al., 2001, 2002) were also induced We found that in addition to Drs, several immune-related genes such as IM1, IM3, IM10 and Attacin were induced over 10-fold upon pinching with forceps (Table 1) In addition, stress responsive genes such as TotA, TotB and TotC were induced in the larvae This result suggests that pinching larvae with forceps induces a humoral innate immune response that is similar to that observed in systemic infection in adults We also noticed that a number of chitin metabolic genes were also downregulated upon pinching (Table 2).

pinching with forceps

Next, we asked which signaling pathway is involved in the induction of Drs upon larval pinching Real-time qPCR analysis showed that the level of induction in the spz mutant or dMyd88 mutant was approximately half that of the wild-type larvae (Fig 3A) In contrast, the induction of Drs was comparable to that in the wild-type in the Dif mutant, or psh and modSP double mutants (Fig 3A) These results suggest that certain Toll pathway components are partly required for the induction of Drs upon pinching We next investigated IMD pathway mutants and found that larvae of the pgrp-le and pgrp-lc double mutant, imd mutant or relish mutant exhibited normal Drs induction after pinching (Fig 3B) Furthermore, the level of induction of Drs in the double mutant larvae for imd and spz, or for relish and spz was almost the same as that in the spz single mutant (Fig 3D), suggesting that the Toll and IMD pathways did not have a redundant role in pinching-induced Drs expression We further investigated the involvement of the JAK-STAT, JNK, p38, dFOXO and pro-PO pathways, all of which suggested a role for AMP induction or host defense under certain conditions (Kim et al., 2002; Buchon et al., 2009a; Becker et al., 2010; Chen et al., 2010; Binggeli et al., 2014; Parisi et al., 2014) Inhibition of the JAK-STAT pathway by using

an upd2 and upd3 double mutant did not reduce the induction of Drs

in pinched larvae (Fig 3C), implying that the JAK-STAT pathway may be dispensable for Drs induction Similarly, the normal Drs induction observed upon pinching in larvae with an eiger mutation

or c564-GAL4-driven expression of a dominant negative form of Disease

Bsk implied that there was no requirement of the JNK pathway in

Drs induction (Fig 3E) The level of Drs induction was also same in

wild-type larvae as in the larvae of p38a, p38b and p38c (Fig 3F),

dfoxo (Fig 3G) and PPO (Fig 3H) mutants, indicating that the p38,

dFOXO and pro-PO pathways played no role in the induction of Drs

following larval pinching with forceps.

Sensory neurons and hemocytes are dispensable for the

When pinching larvae with forceps, we touched the larval cuticle under which the web of sensory neurons exists, prompting us to examine the role of sensory neurons in pinching-induced Drs expression We first ablated sensory neurons by expressing the

Fig 1 Systemic infection inDrosophila larvae by septic injury with a fine tungsten needle (A) Survival analysis of larvae upon clean injury Oregon R wild-type larvae, wild-type control y w, Toll pathway mutant Difnmc, psh and modSPKOdouble mutant, and the IMD pathway mutant relishE20were used (B) Survival analysis

of larvae upon septic injury with S saprophyticus Larvae of Oregon R, Difnmc, modSPKOand imd1mutant were used (C) Colony forming unit (CFU) assay

before (0 h) and after septic injury with S saprophyticus at the indicated time points Larvae of y w, Difnmc, and psh1and modSPKOdouble mutant were used (D) Real-time qPCR analysis of antimicrobial peptide Drs expression upon septic injury with S saprophyticus at the indicated time points with larvae of Oregon

R, Difnmc, and the psh1and modSPKOdouble mutant (E) Survival analysis of larvae upon septic injury with Ecc15 Larvae of Oregon R, Difnmc, modSPKOand imd1

mutants were used Each survival curve is representative of at least two independent experiments of 60 larvae each (A,B,E) P-values were calculated using the log-rank test (F) Real-time qPCR analysis of antimicrobial peptide Dpt expression upon septic injury with Ecc15 at the indicated time points with larvae of Oregon

R, Difnmc, and imd1mutants Data are representative of more than two independent experiments performed in 20 larvae (C,D,F) (*P<0.05)

apoptosis-inducing genes reaper and hid with a pan-sensory neuron

GAL4109(2)80driver (Fig 4A, Fig S5) or class IV sensory neuron

ppk-GAL4 driver Fig 4A shows that the level of induction of Drs in

larvae with sensory neurons ablated by either driver was the same as

that in the wild type We next inhibited the function of sensory

neurons with the same drivers by expressing temperature-sensitive

Shibire (Kitamoto, 2001) However, no effect was again observed

on Drs induction when neurotransmission was suppressed

(Fig 4B) Conversely, we then monitored Drs expression using

larvae in which the sensory neurons were artificially activated by

expressing the dTrpA1 ion channel (Hamada et al., 2008) Fig 4C

shows that Drs was not induced in the activated larvae without

pinching These results collectively suggest that sensory neurons are not involved in the induction of Drs upon larval pinching with forceps.

As Spz has been suggested to be secreted from hemocytes (Shia

et al., 2009), we tested the role of hemocytes in the induction of Drs upon pinching We observed that hemocyte-specific expression of reaper and hid mediated by using a hml Δ-GAL4 driver effectively ablated hemocytes in larvae (Fig 4D) Using these larvae, we next examined the induction of Drs upon pinching and found that no difference could be detected between wild-type and hemocyte-ablated larvae with respect to the level of Drs induction (Fig 4E) Consistent with this, inhibition of the phagocytic function of

Fig 2 Characterization of antimicrobial peptide induction following pinching larvae with forceps (A-C) Real-time qPCR analysis of Drs expression (A,C)

or Dpt expression (B) upon clean injury or pinching larvae with forceps at the indicated time points with larvae of Oregon R, w1118and y w Pinching was performed with larvae of w1118in C (D) Melanization spots after pinching larvae with forceps after 4 h The indicated magnification of the objective lens was used;

arrowheads indicate melanization spots The right bar graph shows the percentage of larvae that exhibited melanization spots Data were analyzed by Student’s t-test and values represent the means±s.e of three independent experiments with 30 larvae each (E) Drs-GFP reporter analysis using a fluorescence

stereomicroscope upon pinching larvae with forceps after 4 h Larvae of Oregon R, Drs-GFP Dpt-lacZ (DD1); c564-GAL4, DD1; Dif1and DD1; Difnmcwere used Brightfield (top row) and fluorescence images (bottom 2 rows) of single larva or multiple larvae (bottom row); the green signal indicates GFP fluorescence (F) Real-time qPCR analysis of Drs expression following pinching of Oregon R, w1118, and y w larvae with forceps at the indicated time points The larval fat body was dissected out from other tissues (G,H) Real-time qPCR analysis of bacterial genomic DNA coding for 16S rRNA, which was normalized by the

Drosophila genomic region for 12S RNA from conventionally reared or germ-free Oregon R larvae before pinching (G), or of Drs expression following pinching larvae with forceps at 4 h with conventionally reared or germ-free Oregon R larvae (H) Data were analyzed by the Student’s t-test and values represent the means

±s.e of three independent experiments with 10 larvae each (F,G) *P<0.05

hemocytes by expressing Shibire (Awasaki and Ito, 2004) had no

effect on the induction of Drs upon pinching (Fig 4E) These results

suggest that hemocytes are dispensable for the induction of Drs after

pinching with forceps.

DISCUSSION

In this study, we present a method by which systemic bacterial

infection can be performed easily and consistently in Drosophila

larvae, thus providing another genetically tractable model to

decipher infectious diseases In this model, we show that the role

of the Toll pathway in resistance against systemic infection in

Drosophila larvae differs from that in adults to a certain extent.

Specifically, the Toll pathway is likely to be required for tolerance

(Ayres and Schneider, 2012) but not for resistance against

Gram-positive bacteria, and exhibits partial involvement in the induction

of AMPs; the latter being consistent with suggestions from a

previous study (Manfruelli et al., 1999) Drs, an antimicrobial

peptide whose expression is under the control of the Toll pathway, is

strongly induced upon Gram-positive bacterial infection, although

Drs is only active against fungi but not bacteria (Fehlbaum et al.,

1994) One possibility to explain the former discrepancy might be

that certain genes induced upon infection, including Drs, might

function in conjunction to confer resistance and tolerance to adults

and larvae, albeit with as-yet unknown mechanisms This point

should be elucidated in future research.

While performing these infection studies, we serendipitously

found that the humoral innate immune response is activated in

Drosophila larvae by modest mechanical stimuli; i.e by pinching

larvae with forceps, as they are commonly handled AMP

expression induced by pinching in larvae is sterile and partially

independent from known innate immune signaling; these conclusions are supported by the following evidence: (1) the induction of Drs was observed in germ-free larvae upon pinching; (2) substantial induction of Drs remained in double mutants for the Toll and IMD pathways; (3) normal induction of Drs occurred after pinching in p38, JNK, JAK-STAT, dFOXO and pro-PO pathway mutants; and (4) hemocytes were dispensable for the induction of Drs upon pinching These observations support the assertion that Drosophila larvae possess a novel mode of induction of the humoral innate immune response that might represent a good model for studying the mechanisms underlying sterile inflammation Although we demonstrated that Dif, a Drosophila NF- κB essential for the induction of Drs upon systemic infection in adults, was not involved in pinching-induced Drs expression

in larvae, we could not rule out the possible involvement of NF- κB

in transactivating Drs expression, as we were unable to examine the redundant role of the other NF- κB proteins, Dorsal and Relish, because of the unavailability of viable lines The dependency on NF- κB remains a question to be solved in future studies Furthermore, we were unable to identify the essential genes required for the induction of Drs upon pinching Unbiased genetic screening might therefore be necessary to unravel the molecular mechanism underlying this phenomenon.

Sterile inflammation is believed to contribute to many pathological conditions such as chronic inflammatory diseases including cancer (Rock et al., 2010) In Drosophila, several studies

Table 1 Top 20 genes upregulated after pinching larvae with forceps

Probe Set ID Gene symbol

Gene ontology biological process

Fold change (4 h vs 0 h)

Pinching

Clean injury

to Gram-negative bacterium

humoral response

At 4 h after pinching or clean injury to y w larvae, expression profiles were

analyzed by DNA microarray using whole larvae The table shows the top 20

genes that were upregulated upon pinching Probe set IDs, gene symbols,

Gene ontology biological processes, and the fold change in gene expression

compared with no treatment (0 h) are indicated

Table 2 Top 20 genes downregulated after pinching larvae with forceps

Fold change (4 h vs 0 h)

Probe set ID

Gene symbol

Gene ontology biological

Clean injury

development

development

−6.8 1640975_at Lcp65Ag2 Chitin-based cuticle

development

−6.2

−4.6

−2.7

process

−5.8

process

−5.6

process

−5.7

−7.2

development

−5.0 1639362_s_at CG10476/

CG18606

At 4 h after pinching or clean injury to y w larvae, their expression profiles were analyzed by DNA microarray using whole larvae The table shows the top 20 genes that were downregulated upon pinching Probe set IDs, gene symbols, Gene ontology biological processes, and the fold change in gene expression compared with no treatment (0 h) are indicated

Fig 3 See next page for legend Disease

have established a sterile inflammation model in larvae (Shaukat

et al., 2015) Ming et al (2014) established a larval model for sterile

AMP induction using a caspase mutant They showed that the

induction of Drs is solely dependent on Spz and Persephone,

suggesting that the molecular mechanism of Drs induction in this

mutant is different from our ‘pinching’ model In addition, Hauling

et al (2014) and Parisi et al (2014) reported that tumors can induce

the expression of AMPs Parisi et al (2014) demonstrated that the

induction of Drs is dependent on eiger and spz, both of which are

not essential for our pinching-induced expression of Drs.

Furthermore, Kanoh et al (2015a) showed that Drosophila larvae possess another intrinsic ligand for the Toll receptor in addition to Spz, although its molecular nature has not yet been identified Together, these reports suggest that Drosophila larvae possess multiple modes of induction of AMPs in response to various sterile stimuli that activate innate immunity.

In the current study, we showed that pinching stimuli can induce AMP expression; however, the physiological relevance of this phenomenon has not yet been elucidated The larvae of Drosophila melanogaster in the wild are expected to be exposed to serious likelihood of attack by parasitoid wasps Thus, mechanical stimuli might be considered as a potential infectious danger, suggesting that even small injuries resulting from oviposition might be able to activate AMP expression Consistent with this, Schmid et al (2014) recently showed that overactivation of Toll signaling could provoke

a cellular immune defense that has potential importance in the response to wasp infection.

In conclusion, we demonstrate in this study that Drosophila larvae represent a suitable model in which to perform microbial infection by using a fine and uniform tungsten needle and to assess sterile induction of the humoral immune response by pinching larvae with forceps In particular, because pinching-induced AMP expression is likely to be dependent on an as-yet uncharacterized

Fig 4 Sensory neurons and hemocytes are dispensable for the induction ofDrs upon pinching (A) Fluorescence microscopy (right) of larval sensory neurons around the mouth hook Larvae of +/+; GAL4109(2)80 UAS-mCD8::GFP/+ and UAS-rpr UAS-hid/+; GAL4109(2)80 UAS-mCD8::GFP/+ were used Green indicates the GFP signal and white dotted lines indicate the outlines of each larva Scale bar: 100 µm (A-C) Real-time qPCR analysis of Drs expression after pinching larvae with forceps for larvae of y w, UAS-rpr UAS-hid ; GAL4109(2)80UAS-mCD8::GFP/+, UAS-rpr UAS-hid; ppk-GAL4/+ (second instar stage), UAS-rpr UAS-hid ;; ppk-GAL4/+ (third instar stage) at 0 or 4 h (A, left); larvae of UAS-lacZ/GAL4109(2)80UAS-mCD8::GFP, UAS-Shits/GAL4109(2)80UAS-mCD8:: GFP, with or without pinching and with or without heat shock at 30°C for 4 h (hs) (B); larvae 4 h after treatment in UAS-lacZ/GAL4109(2)80UAS-mCD8::GFP, UAS-Shits/GAL4109(2)80UAS-mCD8::GFP, with or without pinching or heat shock treatment at 37°C for 2 min (hs) (C) Data are representative of at least two independent experiments and were analyzed by the Student’s t-test; values represent the means±s.e of triplicate samples with 10 larvae each (D) Fluorescence microscopy observation of larval hemocytes Larvae of hmlΔ-GAL4 UAS-2×EGFP/UAS-lacZ, and UAS-rpr UAS-hid/+; hmlΔ-GAL4 UAS-2×EGFP/+ were used Green indicates the GFP signal and white dotted lines indicate the outlines of each larva Scale bar: 200 µm (E) Real-time qPCR analysis of Drs expression at 4 h after pinching larvae with forceps of hmlΔ-GAL4 2×EGFP/lacZ, rpr hid/+; hmlΔ-GAL4 2×EGFP/+, hmlΔ-GAL4 2×EGFP/+; UAS-Shits/+, with or without pinching or treatment at 29°C for 2 days (hs) Data are representative of at least two independent experiments and were analyzed by the Student’s t-test; values represent the means±s.e of triplicate samples with 10 larvae each

Fig 3.Drs induction in several mutant larvae upon pinching with forceps

(A-H) Real-time qPCR analysis of Drs expression after pinching larvae with

forceps at 4 h with larvae of Oregon R, w1118, spzrm7, spzΔ8-1, psh1;;modSPKO,

Difnmc, Dif1and dMyd88kra1(A); larvae of y w, pgrp-le and pgrp-lc double

mutant (LE112;;LCΔ), imd1, and relishE20(B); larvae of y w and upd2Δupd3Δ(C);

larvae of Oregon R, y w, imd1, spzrm7, imd1;spzrm7, reslihE20, and spzrm7

relishE20(D); larvae of Oregon R, y w, bsk1/CyO, eiger1, GPF-IR, or bskDN

driven by c564-GAL4 tubP-GAL80ts(E); larvae of y w, p38a13, p38b156A,

p38c7B1, p38a13p38b156A, and p38b156A/CyO ; p38c7B1(F); larvae of y w,

dFoxo21, and dFoxo21/dFoxow24(G); and larvae of y w, PPO1Δ, PPO2Δ, and

PPO1ΔPPO2ΔPPO3Δ(H) Data are representative of at least two independent

experiments and were analyzed by Student’s t-test; values represent the

means±s.e of triplicate samples with 10 larvae each *P<0.05; n.s., not

significant

molecular mechanism, our model might be useful to decipher the

complex mechanisms that regulate sterile inflammation, which has

considerable importance for the treatment of inflammatory diseases

in humans such as ischemia, atherosclerosis and cancer.

MATERIALS AND METHODS

Fly stocks and maintenance

Drosophila stocks were maintained in standard corn meal-yeast agar

Ferrandon) (Rutschmann et al., 2000) and dMyd88[kra1] (a gift from Dr

Jean-Luc Imler) (Charatsi et al., 2003) were used As IMD pathway mutants,

Lemaitre) (Davis et al., 2008; Chen et al., 2010; Chakrabarti, et al., 2014).

The following transgenic flies were used: Drs-GFP Dpt-lacZ (a gift from Dr

UAS-mCD8::GFP (a gift from Dr Tadashi Uemura) (Gao et al., 1999), ppk-GAL4

on 2nd instar stage (BDSC, 32078), ppk-GAL4 on 3rd instar stage (BDSC,

Awasaki) (Kitamoto, 2001), UAS-rpr UAS-hid (a gift from Dr Shigeo

Hayashi) (Zhou et al., 1997), UAS-dTrpA1 (a gift from Dr Paul Garrity)

Difnmcallele

Shireen-Anne Davies) (Osborne et al., 1997) we happened to find that the

S saprophyticus However, trans-heterozygotes of the deficiency line that

(Fig S1A) Consistent with this, Drs induction upon S saprophyticus in

ED243 trans-heterozygotes were perfectly normal (Fig S1B) These results

immunity-mediating component) and performed mapping with several

deficiency lines on the second chromosome We found that deficiencies that

covered the Dif and Dorsal locus, such as Df(2L)ED1161 or Df(J4), could

reported not to be required for Drs induction in adults upon infection, we

foreign sequence of approximately 5 kb, possibly representing an accidental

transposable element insertion at the third exon of Dif-RC (Fig S1D and E),

using several primers as shown in Fig S1D or as follows:

These results indicate that nmc represents a serendipitous mutation of the

Dif gene As the induction levels of Drs upon systemic infection with

spzΔ8-1andDrsΔ7-17mutants

spz or Drs mutants were generated using the CRISPR/Cas9 system as described in Kondo and Ueda (2013) A double-gRNA vector was constructed using pBFv-U6.2, pBFv-U6.2B, and the following primers: spz_in1_1_F: 5′-CTT CGT GCT TGT CTT AAG AAG ACA-3′; spz_ in1_1_R: 5′-AAA CTG TCT TCT TAA GAC AAG CAC-3′; spz_ex1_1_F: 5′-CTT CGC AGG TGA TTG GCG GAT CGG-3′; spz_ex1_1_R: 5′-AAA CCC GAT CCG CCA ATC ACC TGC-3′; Drs-int1-1-F; 5′-CTT CGA AAA GGT TCT CAC GGA GCT-3′; Drs-int1-1-R: 5′-AAA CAG CTC CGT GAG AAC CTT TTC-3′; Drs-ex1-1-F: 5′-CTT CGC AGC CCC AGT CTG AAG TGC-3′; Drs-ex1-1-R: 5′-AAA CGC ACT TCA GAC TGG GGC TGC-3′ The constructed vector was used to generate the U6-spz-gRNA line,

BestGene) The U6-spz-gRNA line was crossed to nos-Cas9 (National Institute of Genetics, CAS-0001) as described in Kondo (2014) to generate candidate deletion lines Genomic DNAs of each candidate mutant were screened by PCR to check for the deletion (Fig S2) using the following primes: spz-Fw: 5′-GGA ACT GCT AGA ACA ACT ATG GA-3′; spz-Rv: 5′-CAG TAA CAC CAG CTA CCA GT-3′; Drs-Fw: 5′-GTG ACT GCA CAT GTA TCA TCA TAA TTT G-3′; and Drs-Rv: 5′-GTA GGT CGG GAA

found to have a 210 bp deletion that includes the start codon (Fig S2); thus,

we used these lines as spz or Drs null mutants, respectively.

was crossed to UAS-dTrpA1 and maintained at 18°C until they had developed into third instar larvae In ppk-GAL4, third instar larvae were

were incubated in a water bath at 37°C twice for 2 min (10 min intervals at 25°C), then maintained at 25°C for 4 h and used for assays.

water bath at 32°C for 5 min and experiments were performed at 30°C with warmed equipment Pinched larvae were moved to agar plates and maintained at 25°C.

mCD8::GFP, or hmlΔ-GAL4 2×EGFP were crossed to rpr UAS-hid and maintained at 18°C Third instar larvae were incubated at 29°C for two days to induce apoptosis and observed under a stereo fluorescence microscope (M205FA, Leica, Wetzlar, Germany) to check the decrease of GFP signal, or used for the assays.

To inhibit exocytosis and phagocytic activity of hemocytes, hmlΔ-GAL4

instar larvae were incubated at 29°C for two days, the experiments were performed at 25°C, and the pinched larvae were soon moved to agar plates and maintained at 29°C.

Microbial infection and pinching

The following pathogens were used for infection: Ecc15 (IFO3830) and

S saprophyticus (GTC0205) For larval infection, overnight bacterial cultures were concentrated by centrifugation, the pellet was washed with phosphate-buffered saline (PBS), and the larvae were then placed on a cold agar plate and pricked with a fine tungsten needle until complete penetration was achieved (Seimi, Sendai, Japan; total length: 43.5 mm; diameter: 0.2 mm; taper length: 2.5 mm) (Fig S3A) that had been dipped in a pellet of concentrated bacteria (Fig S3B) and moved to sealed Petri dishes containing apple juice agar The needle was frequently changed before it became dull.

To monitor survival, 60 larvae of each genotype were incubated at 29°C after infection and the surviving larvae were every 2 h during transfer to fresh apple juice plates.

To assess the bacterial load in larvae, a colony-forming unit (CFU) assay was performed Larvae were collected and their surfaces were sterilized with

500 μl nutrient broth (NB) bacterial medium, serially diluted, and plated

onto NB medium plates.

For pinching larvae, MilliQ water was poured into Drosophila vials and

the water and larvae were moved to Petri dishes The middle part of third

instar larvae were gently (0.2-0.25 MPa, Prescale, Fujifilm, Tokyo, Japan)

pinched by forceps (Dumont, 0108-5-PO) for about 1 s (Fig S3C), and then

the larvae were moved to sealed Petri dishes containing apple juice agar.

Melanization spots and GFP signals in larvae after pinching were observed

using a fluorescent stereo microscope.

Rearing the axenic fly line

To obtain germ-free larvae, embryos were washed with bleach as described

in Broderick et al (2014) Briefly, embryos were rinsed in 70% ethanol for

1 min, placed in a 2.5% solution of sodium hypochlorite for 2 min, and then

washed with 70% ethanol for 2 min Embryos were then rinsed in sterile

MilliQ water Embryos were transferred to sterile foods and developed to

larvae.

To check the axenic state, bacterial DNA was extracted from whole larvae

and assessed by real-time qPCR using 16S rRNA primers (Suau et al.,

1999).

Total RNA isolation, real-time qPCR, and microarray analysis

Larvae infected with bacteria or pinched by forceps were collected Total

RNA (1 µg), isolated from around 10 larvae using TRIzol reagent (Thermo

Fisher Scientific, Waltham, MA, USA), was used for cDNA synthesis with

ReverTra Ace reverse transcriptase (Toyobo Ltd., Osaka, Japan) and oligo

(dT) 15 primers (Promega, Madison, WI, USA) Using first-strand cDNA

Diagnostics, Roswell, GA, USA) rpL32 was used as the internal control.

The following primers were used for real-time qPCR (F=forward,

For microarray analysis, total RNA from Drosophila larvae homogenized

in TRIzol was isolated using an RNeasy kit (Qiagen, Venlo, The

Netherlands) The RNA quality was checked using an Agilent

Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) Total

using an IVT Labeling Kit (Affymetrix, Santa Clara, CA, USA) Affymetrix

washed, stained and scanned (Goto et al., 2010) Data were analyzed by R

software (https://www.r-project.org/).

Statistical analysis

Statistical analyses were performed using the Student’s t-test or log-rank

test, and P<0.05 was considered significant.

Acknowledgements

We are grateful to Drs Bruno Lemaitre, Jean-Marc Reichhart, Dominique Ferrandon,

Masayuki Miura, Marc Tatar, Tadashi Uemura, Manabu Ote, Takeshi Awasaki,

Shigeo Hayashi and Shireen-Anne Davies, as well as the Bloomington Stock

Center, the Genetic Strain Research Center of National Institute of Genetics, and the

Vienna Drosophila RNAi Center for fly stocks We thank Drs Kenya Honda, Koji

Atarashi, Hirotaka Kanoh, Nichole Broderick, Takeshi Awasaki, Hirofumi Furuhashi

and Masayuki Miura for discussions and suggestions We thank Fumi Shishido and

Ryo Watanabe for technical support We would also like to thank Editage (www

editage.jp) for English language editing

Competing interests

The authors declare no competing or financial interests

Author contributions

H.K., A.H., and T.K conceived this study; H.K, A.H., and T.K designed the

experiments; A.H and T.K found that pinching larvae with forceps induced

Drosomycin expression; A.H and T.K performed the experiments in Figs 1 and 2; H.K performed the experiments in Figs 2-4; H.K., A.H., and T.K analyzed the data; T.K wrote the draft; H.K and A.H prepared the figures; all authors finalized the manuscript; T.K led the entire project; and S.K oversaw the study

Funding This work was supported by the Japan Science and Technology Agency (JST) of PRESTO‘Chronic Inflammation’, and by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT)

Data availability Microarray data have been deposited in GEO under accession number GSE94668 (available at: www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94668)

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