www.elsevier.com/locate/yexcr

Experimental Cell Researc

Microarray analysis of immune challenged Drosophila hemocytes

Karin C. Johansson, Christoph Metzendorf, Kenneth Sfderh7ll*

Department of Comparative Physiology, Evolutionary Biology Center, Uppsala University, Norbyvagen 18A, SE-752 36 Uppsala, Sweden

Received 27 August 2004, revised version received 17 December 2004

Available online 2 February 2005

Abstract

Insect hemocytes play multiple roles in immunity and carry out cellular responses like phagocytosis, encapsulation and melanization as

well as producing humoral effector proteins in the first line of defense after injury and invasion of microorganisms. In this work, we used the

Drosophila melanogaster hemocyte-like cell line mbn-2 and Affymetrix Drosophila GeneChips to investigate the transcriptome of a single

type of immune competent tissue exposed to Gram-negative cell wall components (crude LPS) or high dose infection with live Escherichia

coli. We found that gene expression profiles of both treatments overlap but show important differences in expression levels of several genes

involved in immunity. In addition, cell morphology during infection was monitored and revealed distinct alterations in cell shape and

adhesion. Presence of large numbers of bacteria also increased the number of cells taking on crystal cell fate. Taken together, our results

indicate that hemocytes sense and respond differently to purified bacterial surface molecules and infection with live and actively growing

bacteria both at the level of gene expression and in cell behavior.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Drosophila; Microarray analysis; Hemocytes; Crude LPS; E. coli

Introduction

Microorganisms that breach protective epithelial barriers

and invade circulated tissues trigger in all animals an array

of host immune reactions that work in synergy to fight

infection. Insects lack the antibody-based adaptive immun-

ity of mammals and rely solely on the capacity of their

innate immunity for survival in environments that are often

thriving with microbes. In the fruit fly Drosophila mela-

nogaster, the innate immune system is thought to recognize

non-self through a set of germ line-encoded pattern

recognition receptors (PRRs), binding to and raising the

alarm against evolutionary conserved microbial epitopes

like peptidoglycans (PGN), mannans and h-1,3 glucans of

bacterial cell walls and fungi, respectively [1]. The defenses

are broadly divided into humoral and cellular responses. The

humoral branch involves the rapid synthesis of antimicrobial

peptides (AMPs), secreted into the hemolymph mainly from

the fat body and to a lesser extent from circulating blood

0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.yexcr.2004.12.018

* Corresponding author. Fax: +46 18 4716425.

E-mail address: kenneth.soderhall@ebc.uu.se (K. Sfderh7ll).

cells (hemocytes) and surface epithelia [2,3]. In addition,

proteolytic enzymes are released and activated in the

hemolymph inducing localized melanization around wounds

and foreign objects [4]. Cellular defense mechanisms,

executed by circulating hemocytes of different types,

include phagocytosis by plasmatocytes, capsule formation

around larger foreign objects such as parasitoid wasp eggs

by large adherent lamellocytes and immobilization of large

numbers of bacteria by the binding of multiple hemocytes in

a process referred to as nodulation. Capsules and nodules

are subsequently melanized by the prophenoloxidase-acti-

vating system (proPO) [5–9]. When the animal is wounded,

a reaction is induced that involves aggregation of hemocytes

into clots sealing the wound to limit bleeding and the risk of

microbial entry into the hemocoel [10]. Recognition of

different form of PGN activates either of two NF-nB-likeintracellular signaling pathways, Toll and immune defi-

ciency (IMD). The IMD pathway responds mainly to Gram-

negative infection by recognition of diaminopimelic acid

(DAP)-type PGN while Toll responds to Gram-positive

lysine(Lys)-type PGN but also to fungal infection. Activa-

tion of IMD leads to expression of AMPs and PRRs like

h 305 (2005) 145–155

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155146

peptidoglycan recognition proteins (PGRPs). The Toll

pathway controls transcription of the antifungal peptide

drosomycin, PPRs and several serine proteases involved in

melanization. In addition, Toll signaling has been proposed

a role in the cellular defense [11–16]. Together, Toll and

IMD regulate the majority of Drosophila immune genes but

a recent genomic-scale expression analysis of LPS chal-

lenged Drosophila SL2 cells has also demonstrated con-

tributing roles for JNK- and JAK/STAT-signaling in

immunity [17]. To investigate the response to live bacterial

infection in a single type of immune responsive tissue, we

took advantage of a Drosophila hemocyte-like cell line,

malignant blood neoplasm-2 (mbn-2). Mbn-2 was originally

established from circulating larval hemocytes [18] and has

with retained ability to express AMPs and phagocytose

microorganisms proven a valuable in vitro model system for

studies of the immune response in Drosophila [19]. In an

earlier study using mbn-2 cells, we showed that co-infection

with crude LPS and high doses of live enteric bacteria

muted induced transcription of the antibacterial peptide

Diptericin [20]. This effect was attributed to a potentially

novel feature of bacterial pathogenesis, distinct from the

well described type III secretion-dependent injection of

effector proteins that can interrupt or hijack normal host cell

processes like signal transduction, actin polymerization, and

vesicular trafficking to benefit the pathogen [21]. In this

aspect, we decided to take a whole genome approach to find

out whether other genes were affected in the same way at

the level of host gene transcription.

Materials and methods

Cells and bacterial strains

TheDrosophila mbn-2 cell line was maintained at 258C in

Schneider’s Drosophila medium (PAN Biotech GmbH,

Aidenbach, Germany) supplemented with 10% FBS and 2

mM l-glutamine. Cells were routinely passaged every third

day to a density of ~1 � 106 cells/ml. Enteropathogenic

Escherichia coli 2348/69 was obtained from J. Kaper

(University of Maryland School of Medicine, Baltimore,

MD). Bacteria for infection was diluted 1:50 in fresh LB from

an over night culture and grown to mid log phase (OD600nm =

0.5) at 378C with shaking. After centrifugation the bacterial

pellet was resuspended in Schneider’sDrosophila medium to

1 � 1011 CFU/ml. Heat-inactivated bacteria was prepared in

the sameway after 15min incubation in a 658Cwater bath. 48

h prior to infection, mbn-2 cells were treated with 1 AMEcdysone (Sigma-Aldrich, St. Louis, MO) to promote

immune competence as described elsewhere [22].

Microarray analysis

Approximately 5 � 106 mbn-2 cells were challenged

with crude LPS (O127:B8) 10 Ag/ml (Sigma) or a

combination of crude LPS and 1 � 109 CFU of live E.

coli, corresponding to approximately 200 bacteria per

Drosophila cell. Cells were harvested after 6 h (258C) andtotal RNA was isolated using TRIzol (Life Technologies,

Bethesda, MD) followed by RNeasy clean-up (Qiagen,

Valencia, CA). When treated with crude LPS only, cultures

were spiked with 5.5 � 109 CFU heat-killed bacteria just

before harvest to compensate for the higher ratio of

Drosophila versus E. coli-specific RNA in these samples.

Synthesis of cDNA, biotin-labeled cRNA and hybridization

to Affymetrix Drosophila genome GeneChipsk (Affyme-

trix, Santa Clara, CA) was performed by the Affymetrix

core facility (NOVUM, Karolinska Institutet, Huddinge,

Sweden) according to standard protocols. Each infection

experiment was made in two replicates, processed in parallel

and hybridized to two individual GeneChips. One single

GeneChip was hybridized with RNA from naive cells as a

control of basal level gene expression. GeneChips were

scanned with an Affymetrix GeneArrayR scanner. Data

were analyzed using Affymetrix Microarray Suite 5.0

software. To select for robust changes in transcript

abundance, transcripts with detection P values of P b

0.0025 for increased, and P N 0.9975 (Wilcoxon’s Signed

Rank test) for decreased, in both naive control and

challenged cells were included in data analyses. Cluster

analysis (SOM) was used to group genes with similar

expression over the experiment as a whole, focusing on the

bshapeQ of the expression pattern rather than absolute levels.

Replicate chips with RNA from cells treated with crude LPS

(control) and cells infected with live bacteria were pair-wise

compared (n = 4) and hierarchically clustered (GeneWeaver)

to identify unique and common expression changes.

Viability and gross cell morphology of cells was assessed

by phase contrast microscopy and Tryphan blue exclusion.

Northern blot

To confirm microarray predictions, mbn-2 cells were

challenged as described above. RNA gel electrophoresis and

transfer to Hybond-N membranes (Amersham Pharmacia

Biotech, Little Chalfont, UK) was performed according to

standard procedures. Hybridizations with radiolabeled

probes that were either inserts from cDNA clones [23], or

PCR products verified by DNA sequence were performed

under high stringency conditions (6� SSC, 658C). Filterexposure time to IP-screens varied between 16 and 48 h for

the individual probes depending on the signal strength of the

target gene.

Scanning electron microscopy (SEM)

Mbn-2 cells were prepared for electron microscopy

essentially by following the infection protocol above, except

that sterile round glass cover slips were placed in each cell

culture well. Challenge with live or heat-inactivated bacteria

at a dose of approximately 20 bacteria per Drosophila cell

Fig. 1. Genome wide response of Drosophila mbn-2 cells to live bacterial

infection and treatment with crude LPS. Distribution of up- or down-

regulated transcripts in response to either or both treatments.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155 147

was carried out for 1, 2, 3, 4, 5, and 6 h (258C). Cells werefixed in 2.5% glutaraldehyde (48C) and then washed 3 � 15

min in PBS (pH 7.0), post-fixed in 1% osmium tetroxide for

2 h and washed again 3� with PBS. Dehydration was

carried out in an ascending ethanol series to 95% overnight.

Two further dehydration steps in 99% ethanol for 20 min

were carried out before drying in a critical point dryer and

gold coating. Images were captured with a Phillips XL30

electron microscope.

Immunohistochemistry

Mbn-2 cells were seeded onto SuperfrostR Plus micro-

scope slides and challenged with live E. coli and/or crude

LPS as described in the microarray section above. Cell

monolayers were fixed in 3.7% paraformaldehyde/PBS,

washed three times with PBS, blocked with 5% normal

rabbit serum (Dako Cytomation, Glostrup, Denmark)/PBS

and incubated with IgG mouse mAb HC12F6 against

Hyalophora cecropia hemocytes over night at 48C. mAb

HC12F6 recognizes Drosophila crystal cells with very high

specificity both in hemocyte monolayers and in the

hematopoietic organ and co-stains the same cells as a

polyclonal anti-PO Ab raised against Bombyx mori PO.

MabHC12F6 recognizes a band with the right molecular

weight of PO on western blots of Drosophila hemolymph

and can be competed with a B. mori anti-PO antibody that

also binds to Drosophila PO (T. Trenczek, personal

communication). Slides were washed 3� with PBS and

incubated with a FITC-conjugated rabbit anti-mouse Ab

(Dako) (1:1000) for 1 h at 258C and washed again several

times before mounting in Vectashield (Vector Laboratories,

Burlingame, CA). Confocal images were captured with a

Leica TCS SP.

Results and discussion

Microarray gene expression analysis

Affymetrix Drosophila GeneChips were probed with

RNA from mbn-2 cells challenged with live E. coli and

crude LPS or crude LPS only (control). We then compared

the transcriptional responses evoked by bacterial cell wall

products (crude LPS preparations are always contaminated

by PGN) to those matching a live actively proliferating

Gram-negative bacterial infection. Of 13197 unique genes

and ESTs represented on the chip, 4970 (35.6%) were

detected as present in naive mbn-2 cells. Control crude LPS

treatment increased this number to 5188 (37.2%), whereas

infection with bacteria decreased the number of present calls

to 4308 (30.8%). Of the 926 transcripts that were diffe-

rentially expressed in response to either type of challenge,

387 were increased and 539 decreased (Fig. 1). To bring

primary structure to the data and uncover fundamental gene

expression patterns, we used a SOM method for sorting

genes with common responses across the experiment in

clusters. This enabled us to identify several clusters of clear

relevance for immunity, which we grouped in three main

classes: (1) genes that were induced by crude LPS and

further up-regulated by live bacterial challenge; (2) genes

that were only up-regulated by live bacterial challenge; and

(3) genes that were up-regulated by crude LPS but down-

regulated by challenge with live E. coli (Fig. 2).

Class 1—Inducible genes with enhanced expression by live

E. coli challenge

In class 1 (Fig. 2A), we found genes with defined roles

both in humoral and cellular immunity like Attacin-A and

actin binding/cytoskeletal proteins (spire, inscuteable). Up-

regulation of Pvf2, one of three putative ligands to the

Drosophila homologue of the mammalian PDGF/VEGF

receptor suggested active cell proliferation. Overexpression

of Pvf2 in larvae has been shown to induce dramatic

proliferation of circulating hemocytes [24], proposing that

encounter with a massive bacterial infection may trigger

maturation or differentiation of hemocytes from precursor

cells in the mbn-2 system as a way of boosting cell mediated

defenses. Up-regulation of an extracellular sulphatase

(Sulphated) required for developmental patterning of the

embryo as a regulator of Wingless signaling [25] was

unexpected but suggests that it may be playing a yet

undefined role in immunity.

Class 2—Genes induced only by challenge with E. coli

For induction of some genes actual presence of whole

bacteria seemed critical (Fig. 2B). Among them were

several proteins involved in tissue remodeling and stress

signaling including the MAP kinase phosphatase Puckered,

a transcriptional target and negative regulator of JNK

signaling [26]. Up-regulation of several genes in this class

is consistent with results from a microarray study of the

Fig. 2. Gene expression patterns in immune challenged mbn-2 cells. SOM analysis clustered genes with similar expression profiles for different types of

challenge; naRve (N), crude LPS (cL) and bacterial challenge with E. coli (E.c). Clusters of clear relevance for immunity were grouped together: (A) Class 1—

Genes induced by crude LPS and further up-regulated by live bacterial infection. (B) Class 2—Genes induced only by bacterial infection. (C) Class 3—Genes

induced by crude LPS and down-regulated by bacterial infection. Genes within each cluster are listed in the tables. Graphs show the average expression level

for genes in the cluster (–o–), in-cluster variation is visualized by the surrounding lines.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155148

LPS response in SL2 cells by Boutros et al. [17], such as

Ets21C, a member of the ETS (erythroblast transformation

specific)—family of transcription factors and the matrix

metalloproteinase 1 (Mmp 1). However, in contrast to Ref.

[17], Mmp 1 was only induced in the presence of bacteria

and not by treatment with crude LPS alone. Matrix

metalloproteinases have demonstrated roles in tissue

remodeling in Drosophila [27] and have in mammals

been implicated in wound healing responses [28]. Detailed

functional characterization of Ets21C in Drosophila is

currently not available but its human counterpart ERG has

been implicated in development of the central nervous

system [29]. Many genes associated with cytoskeletal

function showed unique up-regulation in the presence

of whole bacteria. One, (CG13503) contains a WH2-

domain—an actin interacting domain found in WASP/

SCAR proteins. D-SCAR and D-WASP are required for

efficient phagocytosis in Drosophila hemocytes [30].

Several proteins with demonstrated or suggested functions

in the development of the nervous system also show this

profile, like the plasma membrane axon guidance receptor

Leak, the RAB GTPase Chrowded and E(spl)m, a member

of the Brd-family proteins that when overexpressed

resemble a Notch loss-of-function phenotype in the eye

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155 149

[31]. Indication of active Toll signaling was given by up-

regulation of the NF-nB-like transcription factor Relish.

Relish is involved in transcription of most of the AMPs

and several of the PGRPs, targets of the Imd pathway,

while the transcriptional control of relish is partly depend-

ent on the Toll pathway during infection [15]. Since active

Toll signaling also controls hemocyte division and differ-

entiation in larvae [16], this is in support of the hypothesis

that large doses of bacteria can elicit cell differentiation in

mbn-2 cells.

Class 3—Genes down-regulated by challenge with E. coli

Somewhat contradictory in a situation with a galloping

bacterial infection and up-regulation of Relish, but in

accordance with our previous results [20] many of the

AMPs; Attacin-A and -C, Diptericin, Drosomycin, and

Drosocin showed induction by crude LPS to varying

degrees but were down-regulated by bacterial challenge

(Fig. 2C). Other immune genes, like the short soluble

PGRPs, -SA, -SB1, and -SD [32], displayed the same

profile. The two serine-type endopeptidases (CG30086,

CG4259) have to our knowledge not previously been

identified as immune responsive. Decrease of cytochrome

P450 at the level of gene expression seems to be a

conserved signature of immune stimulation, since it is

observed in insects [15] as well as in association with

cytokine action in vertebrates [33]. A transcription factor,

Bigmax, involved in regulating autophagic cell death during

normal metazoan development was also found in this class

[34].

Active E. coli infection versus crude LPS treatment evoke

different responses in mbn-2 cells

To account for transcripts missing or of very low

abundance in naive cells, we let the transcriptional profile

mbn-2 cells challenged with crude LPS represent a baseline

to which we compared the profile of bacteria infected cells.

A total of 517 differentially regulated gene transcripts were

detected, as defined by the criteria in the Affymetrix

software (difference call), in four pair-wise comparisons

between replica chips of crude LPS and E. coli challenge.

Of these, 210 were up-regulated and 274 down-regulated.

The relative mRNA expression in E. coli challenged cells

versus crude LPS control varied from 1.3- to 11-fold for up-

regulated genes with the majority of genes (71%) showing

relatively little change with ratios in the range between 1.3-

and 2-fold. For down-regulated genes, 84% had ratios

between �1.2- and �2-fold, 45 genes (18%) had decreased

expression level between 2- and 14-fold. Genes that

changed 2-fold or more are listed in Table 1. A complete

list of genes induced by control crude LPS treatment and

differentially regulated genes by infection with live E. coli

can be found in Tables 2 and 3 in the Supplementary

material.

Up-regulated genes

Strongest up-regulation by live bacterial infection (z4-

fold) was recorded for two transcripts encoding actin

binding proteins (CG32030, CG13505), a matrix metal-

loproteinase 1 (Mmp 1), an extensin-like protein, a lipase

(CG4267), the JNK-signaling protein Puckered and two

transcription factors (Ets21C, CG11798). Many up-regu-

lated genes could be assigned to functional groups

reflecting different aspects of cellular immunity. Adhe-

sion between cells, cells and microbes and cells and the

extracellular matrix, is an essential part of invertebrate

immunity which in the array data is recognized by

several transcripts for extracellular matrix proteins (Glu-

tactin, Trol) and integrins (Scab, aPS4) in addition to

many actin and Ca2+-binding proteins. Arthropods have

open circulatory systems, which makes blood coagulation

one of the most important defense reactions that prevent

the animal from bleeding out when injured and aid in-

limiting the spread of microbes in the hemocoel.

Utilization of transglutaminases in cross-linking of clot-

table proteins and hemocytes seem to be phylogenetically

conserved in all clotting systems studied to date [10].

Expression of the only transglutaminase gene found in

the Drosophila genome (CG7356) was unchanged by

either type of immune challenge (data not shown).

However, an extracellular lipid transport protein

(CG15828) with similarity to Vitellogenin was found to

be up-regulated. Vitellogenin-like proteins have docu-

mented roles in clotting and melanization in other

arthropod species [35,36]. In addition, a number of other

genes with motifs frequently shared between clotting

proteins in different species like the immunoglobulin-

fold, (EGF)-like, sushi-, C-type lectin-, clip-, and

domains similar to von Willebrand Factor (vWF) [37]

were up-regulated (Table 1 and Supplementary material).

We also found up-regulation of Annexin IX, a Ca2+-

regulated protein that interacts with phospholipids of the

cell membrane and the actin cytoskeleton. Reports of

Annexin IX up-regulation in response to immune challenge

has come from two other microarray analyses of bacteria

challenged adult flies [38] and LPS challenged Drosophila

SL2 cells [17]. Furthermore, it came up in a recent

proteomic screening of LPS induced proteins in mbn-2

cells [39]. Together, these results confirm Annexin IX as an

immune responsive gene whose function should be further

studied. Several genes with defined or putative function in

transcriptional regulation like, Psc, Ets21C, Jra, CG11798,

CG6051, were also up-regulated. The increase in expression

of Psc (Posterior sex combs), a negative regulator of the

polycomb group of homeotic genes (PcG), is of special

interest. PcG genes regulate Hox gene expression in

vertebrates and invertebrates during development and the

Psc homologues Bmi-1 and Mel 18 also participate in

hematopoiesis control in the mouse [40]. The first report

that homeobox genes may play a role in macrophage

responses to LPS came from a recent study of a murine cell

Table 1

Genes changed z2 fold in mbn-2 cells after 6 h bacterial infection with

E. coli

Probeset Name Fold change

Up-regulated genes

154230_at CG32030 (actin binding protein) 11.1

147882_at CG4859 (matrix metalloprotinease) 10.9

145744_at CG15423 (extensin-like) 5.8

145628_at CG4267 (lipase) 5.1

143201_at ImpL3 (L-lactate dehydrogenase) 4.7

152980_at CG13503 (actin binding protein) 4.5

154213_at CG11798

(DNA binding Zn-finger protein)

4.3

145249_at CG11291 (hydrolase) 4.2

141732_at Puckered 4.2

143570_at Ets21C 4.0

143215_at Lethal2 3.8

146285_at CG31764 3.6

142455_at CG5835 3.6

143823_at CG1128 (carboxylesterase) 3.5

142989_at CG8008 (permease) 3.4

145941_at Pvf2 3.4

145317_at CG6606 3.2

151822_at Relish 3.0

145291_at Upd3 2.9

142301_s_at CG11711

(Mob1/phocein family protein)

2.8

152747_at Peroxidasin 2.8

150842_at CG15507 2.7

143675_at Inscuteable 2.7

150549_at CG11819 (protein kinase) 2.7

151760_at CG8468 (monocarboxylate porter) 2.6

146869_at CG8046 (transporter) 2.6

147955_at Trio (RhoGEF) 2.5

142517_at CG1225 (RhoGEF3) 2.5

146650_at CG11066 (serine-type endopeptidase) 2.5

152914_at CG3764 2.5

155144_at Cct1

(phosphocholine cytidylyltransferase)

2.5

142383_at CG5411 (cAMP phosphodiesterase) 2.5

141784_at Spire (actin binding protein) 2.4

145286_at Upd2 2.3

147465_at CG33146 2.3

152225_at kraken (serine hydrolase) 2.3

143046_at CG8468 (transporter) 2.3

147969_at CG9184 2.3

148747_at CG13482 2.3

154760_at CG4618 2.2

147786_at chrowded (RAB GTPase) 2.2

154372_at Tamo

(regulator of nuclear protein import)

2.2

152665_at CG3881

(UDP-glucuronosyltransferase)

2.2

143558_at Posterior sex combs

(PcG-transcription factor)

2.2

150090_at Attacin D 2.1

142467_at Trol 2.1

151548_at CG5411 2.1

149206_at CG14567 2.1

152246_at CG7888

(amino acid-polyamine transporter)

2.1

150715_at CG6051 (Zn-finger protein) 2.1

141771_at CG30456 2.1

146503_at TepIV 2.1

144149_at Ady43A (adenosine kinase) 2.1

154124_at CG17723 (cation transporter) 2.0

Table 1 (continued)

Probeset Name Fold change

Up-regulated genes

147628_at CG15673 2.0

141472_at Glutactin 2.0

147124_at CG3884 2.0

143334_at Scab (integrin) 2.0

154097_at Charybde 2.0

Down-regulated genes

145730_at CG16713

(Kunitz-type trypsin inhibitor)

14.1

145676_at CG18557 (chymotrypsin) 4.9

150606_at CG4823

(Alpha-2-macroglobulin family)

4.6

142387_at CG8215 (serine-type endopeptidase) 4.3

143609_at Drosocin 4.0

143443_at Diptericin A 3.8

145731_at CG16712

(Kunitz-type trypsin inhibitor)

3.8

144318_at TepI 3.7

142189_at Cyp6a (cytochrome P450) 3.5

149716_at CG6977 (cadherin-like protein) 3.3

141374_at Attacin-A 3.3

147119_at Cyp9h1 (cytochrome P450) 3.2

151496_s_at CG14527

(metalloendopeptidase-like protein)

3.1

141227_at CG8370 3.0

144882_at PGRP-SA 2.9

149680_at CG17721 2.8

146354_at CG4650 (serine-type endopeptidase) 2.8

153172_at CG9524 2.8

141738_at CG9706 (acetyl-CoA transporter) 2.7

148230_at CG17795 (G protein linked receptor) 2.7

148402_at CG7120 2.7

144016_at NPC1 (lysosomal membrane receptor) 2.5

143411_at Wrinkled (apoptosis activator) 2.5

151598_at CG10077 2.5

145620_at CG4259 (chymotrypsin) 2.4

154586_at CPTI (mitoch. carnitine

palmitoyltransferase I)

2.4

153894_at CG11897 (transporter) 2.4

142654_at Ird5 (cactus kinase) 2.4

143605_at Drosomycin 2.3

142765_at CG5091 (glycosyltransferase) 2.3

154078_at Cyp4e2 (cytochrome P450) 2.3

152886_at CG15658 (LRR-containing protein) 2.3

147105_at CG8594 (chloride channel protein) 2.2

150560_at CG13654 2.2

142988_at CG6770 2.2

143723_at Cyp6a9 (cytochrome P450) 2.2

154443_at CG1667 2.1

141583_at Cyp6a20 (cytochrome P450) 2.1

146011_at CG8451 (Na+-dependent transporter) 2.1

155014_at CG5262 (permease) 2.1

151480_at CG4144 (transporter) 2.1

154187_at Gcl 2.1

148973_at Adgf-A 2.0

153367_at CG3137 (Beta-galactosidase) 2.0

142657_at PGRP-SB1 2.0

Fold change is the mean of transformed signal log2 values of 4 pair-wise

comparisons between replicate GeneChips. Names and molecular func

tions were retrieved from NetAffxk (http://www.affymetrix.com) and

Flybase gene ontology (GO) annotations.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155150

-

Fig. 3. Northern blot confirmation of differentially expressed genes. Total

RNA (15 Ag/lane) from mbn-2 cells, naive (N), challenged with crude LPS

(cLPS) or live E. coli for 6 h was separated by denaturing gel

electrophoresis, immobilized on nylon membranes and hybridized with32P-labeled DNA probes for selected genes. (A) Annexin IX probe revealed

two bands corresponding to CG5730-RB (1264 bp) and CG5730-RA

(1196 bp). (B) Matrix metalloproteinase (Mmp 1) was representative of

genes that increased by co-challenge. (C) Diptericin (Dipt) and Peptido-

glycan recognition protein-SA (PGRP-SA) confirmed the class (3) profile.

(D) Unique down-regulation by bacterial challenge could be confirmed for

the GATA-factor Serpent and (CG4650), a serine-type endopeptidase. (E)

Ethidium bromide staining confirmed equal loading.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155 151

line where several Hox genes were up-regulated by

Salmonella typhimurium infection [41]. Up-regulation of

Psc in Drosophila mbn-2 cells shows that immune

challenge provides a probable input into the developmental

programs regulating proliferation/differentiation of blood

cells that may be evolutionary conserved. Bacterial

challenge also induced the transcription of two Unpaired

(Upd)-related genes, upd3 (CG15062 and CG5963) and

upd2 (CG5988). Upd3 was recently demonstrated to

function as a cytokine produced by hemocytes in response

to septic injury in vivo and necessary for JAK/STAT and

Relish-dependent expression of the stress response Tur-

andot M (TotM) protein in the fat body of adult flies [42].

The function of Upd2 is at present uncertain but a role

similar to Upd3 seems likely. Of the other genes known to

function in the JAK/STAT pathway, only the cytokine-like

receptor Domeless (Dome) was up-regulated, but in

contrast to upd3 and upd2, crude LPS was enough for

up-regulation of (Dome) in mbn-2 cells. Moreover, crude

LPS induced the transcription of another Turandot peptide

TotF in mbn-2 cells (Table 2, Supplementary material).

The only inducible PGRPs in mbn-2 cells appear to be the

extracellular peptidoglycan receptor PGRP-SA which

recognizes (Lys)-type PGN leading to Toll activation

[13], a likely scavenger receptor PGRP-SB1 [43] and

PGRP-SD, which at present still awaits physiological

characterization.

Down-regulated genes

At 6 h post-E. coli challenge, the most negatively

affected genes, (z2-fold down-regulation), included pro-

teases, protease inhibitors, a significant number of AMP

genes in addition to the complement-like thiolester contain-

ing protein (TEP-family) Tep1 and the a-2-macroglobulin-

like CG4823, (Table 1 and Supplementary material). TEP-

family proteins have been proposed to function as opsonins

in the phagocytic defense of Anopheles mosquitoes [44] and

Tep1, -2, and -4 are induced after septic injury of

Drosophila adults [38]. In contrast to Tep1, Tep4 was up-

regulated in mbn-2 cells after bacterial challenge illustrating

differences between in vivo studies using whole animals and

in vitro studies with particular cell lines.

Unaffected immune genes

Neither of the proteins with demonstrated function in

phagocytosis, the scavenger receptor dSR-CI [45] and the

peptidoglycan recognition protein PGRP-LC [46] were

inducible by crude LPS or affected by bacterial infection

with the exception of the Croquemort [47], gene that was

slightly down-regulated by E. coli infection (Table 3 in

Supplementary material). Steady levels of mRNA from the

three phenoloxidase (proPO) genes; Bc, CG8193 and Dox-

A3 was also recorded (data not shown). Like in other

arthropods Drosophila phenoloxidase is produced as a

zymogen that is activated through cleavage by a serine

protease [9]. In contrast to the results of De Gregorio et al.

[38] where all of the putative proPO activating enzymes

(ProPO-AE); CG9733, CG3505, CG3066, CG16705,

CG1102 were up-regulated in flies challenged by septic

injury with a mixture of E. coli and Micrococcus luteus, we

could not detect any differential expression of these

transcripts. This may be because penetration of protective

barriers like cuticle and epithelium induces trauma and is

likely to involve additional tissues in the response.

Northern blot confirmation of the array expression data

To confirm gene expression profiles of the array

hybridization, we selected representative transcripts for

Northern analysis. Overall, there was a good correlation

between the two methods (Fig. 3) Annexin IX expression

followed the class 1 response, that is, induced expression

by crude LPS and further up-regulation by E. coli

infection. We could also confirm that Mmp1 is selec-

tively induced by bacterial infection in mbn-2 cells and

not like in SL2 cells induced by LPS alone [17]. Both

Diptericin and PGRP-SA expression correlated with the

profile of class 3 with strong up-regulation by exposure

to crude LPS and down-regulation by high-dose bacterial

infection.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155152

High-dose infection with live bacteria induces

morphological changes in mbn-2 cells

The initial host response to most bacterial pathogens is

phagocytosis, however non-invasive enteropathogenic E.

coli avoids being engulfed and instead induces a spectacular

reorganization of the host cell actin cytoskeleton forming

characteristic pedestal-like structures, enriched in filamen-

tous actin, on the intestinal epithelial cells that the bacteria

resides on. The basis for these remodeling events is bacterial

adherence to the host cell surface followed by injection of the

receptor Tir (Translocated intimin receptor) and a number of

effector proteins via a type III secretion system. Interaction

Fig. 4. Cell morphology is changed in the presence of live E. coli. SEM of mbn-2

6 h (H) revealed no significant alterations in the overall appearance of the cells co

with live E. coli had a rounded to flattened shape and most remained attached to th

arrows), (D). After 6 h, long filopodia were seen extending from the cells and mi

After 6 h, changes in cell adhesion properties were apparent from the elevated num

extracellular material around the cells (B) and (F). The number of surface associate

that appear to be trapped by hemocyte filopodia (E).

between Tir and the bacterial outer membrane protein intimin

triggers the subsequent steps that lead to actin assembly [48].

We used scanning electron microscopy (SEM) to monitor

bacteria–hemocyte interactions during infection to investi-

gate whetherDrosophila hemocytes are susceptible to E. coli

attachment in the same way and whether any injected effector

proteins could account for some of the gene expression

changes in E. coli challenged mbn-2 cells (Fig. 4). Cells

treated with crude LPS and heat-inactivated bacteria dis-

played the same morphology as naive control cells and had a

rounded to slightly elongated shape associated with attach-

ment to the support. This appearance persisted throughout the

length of the experiment. In contrast, mbn-2 cells exposed to

cells challenged with crude LPS and heat-inactivated E. coli at 1 h (G) and

mpared with naRve control cells (H, inset). After 1 h, most cells challenged

e slide (A) and (C). After 5 h, cells were displaying membrane blebs (white

croparticles (white arrowheads) seen where filopodia had disintegrated (E).

ber of cells that detached from the slide and there was abundant deposits of

d E. coli was higher at 6 h than at any other time point. Note the bacteria (*)

Fig. 5. Immunohistochemistry results. Fixed mbn-2 monolayers were

incubated with mouse mAb HC12F6 against Drosophila PO. Confocal

images after staining with a FITC-conjugated secondary Ab (green) Red

background is autofluorescence. (A) Bacterial infection; about 15–20% of

the cells stained positive 6 h post co-challenge. Note the distribution of PO

in granules (white arrowheads). (B) crude LPS; about 5% of the cells

showed positive staining. (C) Naive control was equal to crude LPS-treated

cells.

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155 153

live bacteria started showing changes in morphology around

4 h with the formation of vesicle-like protrusions on the cell

membrane. At 6 h, long filopodia were extending from the

cells that appeared to be contacting both other cells and

nearby bacteria. In addition, we noticed the formation of

small free vesicles in association with fragmenting filopodia.

Such vesicles have been termed insect microparticles with a

reference to the vertebrate system where microparticles

released from platelets play a role in catalyzing blood

coagulation [49,50]. Release of microparticles from insect

cells have been proposed to promote cell adhesion and take

part in the formation of coagulogen since they are enriched in

hemomucin [49]. Indeed, we observed a marked increase of

deposited extracellular material that resembled fibrous

coagulum between and around the cells in the presence of

live E. coli. Pedestal-like structures as those seen in E. coli-

infectedmammalian cells [48] were not observed on the mbn-

2 cells making it unlikely that filopodia formation is induced

and driven by direct interaction with E. coli. Overall, the

number of cell surface-associated bacteria was lower than

expected in relation to the total number of bacteria present

and we also noted a higher number of cells detaching from the

support under live bacterial infection. Detached cells

aggregated in the suspension as free clusters of varying size.

This observation correlates well with down-regulation of

cadherins, basal lamina, and anticoagulant proteins in the

array data and may reflect a clotting response (Table 1).

The number of crystal cells increase in bacteria infected

mbn-2 cells

Apart from plasmatocytes, which constitute the bulk of

the mbn-2 cell population, other hemocytes like crystal cells

and lamellocytes are not believed to produce AMPs in the

same magnitude. One possible situation that partly could

explain the down-regulation of AMPs in co-challenged cells

would be that a heavy bacterial infection could trigger

transformation or maturation other cell types than plasma-

tocytes in the mbn-2 system. Larval hematopoiesis and

differentiation of prohemocytes into plasmatocytes and

crystal cells in vivo is under control of lineage specific

transcription factors Gcm, Gcm2 (Glial cells missing), and

Lz (Lozenge) and Ush (U-shaped), respectively [51]. Even

levels of Gcm and Gcm2 were consistently found in the

expression data from both bacteria infected and crude LPS

challenged control cells (data not shown). Besides plasma-

tocytes, the presence of crystal cells was indicated by

expression of the proPO genes Bc, Dox-A3, and CG8193

[5]. However, decreased levels of the GATA factor Serpent

(Srp) on one replica chip of bacteria-infected cells (data not

shown) hinted that cell differentiation may be induced in

response to bacterial infection since Srp is expressed by all

prohemocytes and known to influence crystal cell number

negatively [51]. Down-regulation of Srp could be confirmed

by northern blot (Fig. 3) and immunohistochemistry with a

mAb against hemocytes of the moth Hyalophora cecropia,

which recognizes PO in Drosophila crystal cells stained

approximately 15–20% of the cells after bacterial infection

compared with naive and crude LPS treated control cells

where the number of crystal cells was about equal,

approximately 5% (Fig. 5). Taken together, these results

propose that high-dose bacterial challenge can induce a

differentiation process in mbn-2 cells leading to increased

population of cells with a crystal cell-like identity.

Concluding remarks

For efficient orchestration of defense programs in a

multicellular organism, it is essential that cells are able to

respond to multiple cues of an infection and communicate

its status to other immune competent cells and organs. The

above results highlight that hemocytes, as part of the total

immune system in Drosophila, respond with different

behavior suited to the type of threat posed. Down-regulation

of many humoral response genes, including AMPs, PGRPs,

serine proteases and inhibitors and concomitant up-regu-

K.C. Johansson et al. / Experimental Cell Research 305 (2005) 145–155154

lation of genes involved in cytoskeletal rearrangements, cell

proliferation and differentiation, suggests that cellular

response reactions, like increased phagocytosis, clotting,

and nodule formation, are favored when hemocytes are

presented with large numbers of whole bacteria. Combined

with synthesis of endocrine signaling molecules aimed at

the fat body—the main organ for production of humoral

effectors in the systemic defense in vivo, it would arguably

be a more effective way of dealing with a large bacterial

infection. Taken together, our results confirm the potency of

bacterial surface molecules (here in the form of crude LPS)

in activating humoral immunity but also shows that other

factors like bacterial titer should be taken into account as it

contributes to a balanced and adequate defense against

infection and hence, that the fly immune system may not

only discriminate between self and infectious non-self but

also respond to the way non-self is presented.

Acknowledgments

We thank Thomas Werner, Michael Williams, Carl-Johan

Zettervall and Dan Hultmark (UCMP, Ume3 University,

Sweden), Ylva Engstrfm (Department of Molecular Biol-

ogy and Functional Genomics, Stockholm University,

Sweden) and Lage Cerenius for helpful discussions, and

finally Tina Trenczek (Inst. of Zoology, Justus-Liebig

University of Giessen, Germany) for kindly providing us

with the hemocyte antibody.

This work was funded by the Wallenberg Consortium

North and the Swedish Research Council/NT and M.

Appendix A. Supplementary material

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.yexcr.2004.

12.018.

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