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Loss of self-tolerance leads to altered gene expression and IMD pathway activation in Drosophila melanogaster

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Loss of self-tolerance leads to altered gene expression and IMD pathway activation in Drosophila melanogaster

Abstract

Immune self-tolerance is the ability of a host's immune system to recognize and avoid triggering immune responses against self-tissue. This allows the host to avoid self-directed immune damage while still responding appropriately to pathogen infection. A breakdown of self-tolerance can lead to an autoimmune state in which immune cells target healthy self-tissue, leading to inflammation and tissue damage. In order to better understand the basic biology of autoimmunity and the role of the innate immune system in maintaining self-tolerance, we have recently characterized the Drosophila melanogaster tuSz autoimmune mutant. This mutant strain can serve as a model of innate immune mediated self-tolerance, and here we identify transcripts that are deregulated in flies experiencing a loss of self-tolerance. We found that these changes include the ectopic activation of pro-inflammatory signaling through the Relish/NFκB transcription factor, alterations in transcripts encoding proteins predicted to mediate organismal metabolism, and a downregulation of transcripts linked to developmental processes. This study can provide insight into the transcriptional and physiological changes underlying self-tolerance and autoimmunity.
Loss of self-tolerance leads to altered gene expression and IMD pathway activation in
Drosophila melanogaster
Authors: Pooja Kr and Nathan T. Mortimer
Affiliation: School of Biological Sciences, Illinois State University, Normal, IL, 61790, USA
*Correspondence: Nathan T. Mortimer, School of Biological Sciences, Campus Box 4120,
Illinois State University, Normal, IL 61790, USA, ntmorti@ilstu.edu
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ABSTRACT
Immune self-tolerance is the ability of a host’s immune system to recognize and avoid
triggering immune responses against self-tissue. This allows the host to avoid self-directed
immune damage while still responding appropriately to pathogen infection. A breakdown of self-
tolerance can lead to an autoimmune state in which immune cells target healthy self-tissue,
leading to inflammation and tissue damage. In order to better understand the basic biology of
autoimmunity and the role of the innate immune system in maintaining self-tolerance, we have
recently characterized the Drosophila melanogaster tuSz autoimmune mutant. This mutant
strain can serve as a model of innate immune mediated self-tolerance, and here we identify
transcripts that are deregulated in flies experiencing a loss of self-tolerance. We found that
these changes include the ectopic activation of pro-inflammatory signaling through the
Relish/NF B transcription factor, alterations in transcripts encoding proteins predicted to
mediate organismal metabolism, and a downregulation of transcripts linked to developmental
processes. This study can provide insight into the transcriptional and physiological changes
underlying self-tolerance and autoimmunity.
INTRODUCTION
The maintenance of self-tolerance, the process in which self-tissues are identified and
self-directed immune mechanisms are repressed, is an important characteristic of the immune
system that protects an organism from pathogen infection (Medzhitov and Janeway 2002;
Romagnani 2006). The breakdown of the immune system’s ability to discriminate healthy self-
tissue from non-self pathogens can lead to damage of a host’s own tissues by the immune
system. Multiple studies have revealed the importance of self-tolerance and failure of this
system has been implicated in a variety of autoimmune disorders and other immune mediated
diseases. Although much of the research uncovering mechanisms of immune recognition and
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self-tolerance has focused on adaptive immune mechanisms, a growing body of work has
demonstrated the importance of innate immune mechanisms in the suppression of
autoimmunity (Waldner 2009; Toubi and Vadasz 2019).
Innate immune responses are evolutionarily conserved and serve as a first line of
defense against infection (Hoffmann et al. 1999). The innate immune system relies upon germ-
line encoded receptors, signaling components and other molecules for the recognition and
destruction of pathogens. When activated, these receptors induce intracellular signaling that
results in the activation of genes involved in host defense. The inappropriate activation and
targeting of these innate immune effectors against self-tissue can contribute to autoimmune
pathology (Toubi and Vadasz 2019). However, mechanisms underlying innate immune
mediated self-tolerance and the genes associated with the maintenance of this system remain
largely unexplored.
The genetic model organism Drosophila melanogaster is a valuable model for
understanding conserved innate immune mechanisms (Lemaitre and Hoffmann 2007). Flies are
host to a wide range of pathogens including bacteria, fungi, viruses, and parasites and use both
humoral and cell mediated immune responses to combat these pathogens (Brennan and
Anderson 2004). In Drosophila, hemocytes (immune cells) known as plasmatocytes are found in
circulation, and following stimulation, surveil the hemolymph (body cavity) for the presence of
invading pathogens and tissue damage. These macrophage-like plasmatocytes are responsible
for the phagocytosis of microbial pathogens and are involved in the encapsulation response
against macroparasites including parasitoid wasp eggs (Mortimer et al. 2012; Honti et al. 2014;
Parsons and Foley 2016). Another immune cell type, the lamellocytes, are generally not present
in a healthy fly larva, but are induced following parasitoid wasp infection and are required for
melanotic encapsulation of parasitoid eggs and other foreign objects (Mortimer et al. 2012;
Honti et al. 2014; Anderl et al. 2016).
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Drosophila hemocytes also play a role in self-recognition and maintaining self-tolerance.
We have recently characterized the Drosophila tuSz1 temperature sensitive mutant strain which
mounts a self-directed encapsulation response at the restrictive temperature (Mortimer et al.
2021). We found that the loss of self-tolerance in tuSz1 is due to two genetic changes. The first
is a gain of function mutation in hopscotch (hop), the Drosophila orthologue of Janus Kinase
(JAK). The JAK-STAT signaling pathway plays a key role in immune priming, and the hopSz
allele leads to hemocyte activation and the ectopic production of lamellocytes in naïve larvae
(Mortimer et al. 2021). These hemocyte phenotypes are also seen in the hopTum gain of function
allele (Hanratty and Dearolf 1993; Luo et al. 1995), but interestingly, even though hopTum and
tuSz1 mutants both have hemocyte activation and excess lamellocyte production, hopTum
mutants do not display an autoimmune phenotype (Mortimer et al. 2021). This would suggest
that JAK-STAT mediated immune activation alone is insufficient for the loss of self-tolerance,
and that loss of self-tolerance and immune priming are genetically separable. The second
mutation in the tuSz1 strain is a conditional loss of function mutation in the GCS1 gene. At the
restrictive temperature, the GCS1Sz allele leads to disruption of protein N-glycosylation of
extracellular matrix proteins (Mortimer et al. 2021). Together these findings define a system in
which Drosophila self-tolerance is maintained by the ability of primed hemocytes to sense a self-
signal that is encoded in the N-glycosylation of extracellular matrix proteins, and the loss of this
self-recognition leads to the melanotic encapsulation of self-tissues. Thus, hemocytes can
mediate both the activation of an immune response and the maintenance of self-tolerance
thereby regulating immune homeostasis.
Here, we utilize the Drosophila tuSz1 and hopTum mutant strains to identify key
transcriptional changes underlying immune priming, self-tolerance, and autoimmunity.
Differentially expressed transcripts often reveal disease-induced changes (Porcu et al. 2021), so
our analysis of transcript abundance changes in these mutants may allow us to identify
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processes that occur in autoimmune reactions following the loss of self-tolerance. Our results
further suggest that the pro-inflammatory Immune deficiency (Imd) signaling pathway may be
dysregulated in the tuSz1 loss of self-tolerance phenotype.
MATERIALS AND METHODS
Insect Stocks
The D. melanogaster strains w1118 (BDSC: 5905), tuSz1 (BDSC: 5834),
PBac{GFP.FPTB-Rel}VK00037 (Rel-GFP) (BDSC: 81268), hopTum (BDSC: 8492) were obtained
from the Bloomington Drosophila Stock Center (BDSC). All fly stocks were maintained on
standard Drosophila medium on a 12-hour light-dark cycle. The study also uses the parasitoid
wasp Leptopilina boulardi (strain Lb17) which is maintained on the Canton S D. melanogaster
strain.
RNA Isolation
Total RNA was extracted from late third instar w1118, tuSz1 and hopTum larvae raised at
28º. In total, three biological replicates were prepared with each biological replicate consisting of
20 pooled larvae. RNA was extracted from whole larvae using Trizol (Thermo Fisher Scientific)
followed by QIAGEN RNeasy Micro clean-up (QIAGEN; both according to manufacturer’s
instructions). RNA concentration was measured using a nanodrop and the integrity was verified
using agarose gel electrophoresis. The RNA samples were sent to Novogene (Sacramento, CA)
for RNA sequencing (150bp paired end reads, Illumina).
Differential Gene Expression Analysis
Sequence data were obtained in the form of FASTQ reads and analyzed using the
Galaxy web platform (https://usegalaxy.org/) (Afgan et al. 2018). Initial quality control was
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performed using FastQC (version 0.11.8) (Andrews 2010) to trim reads below a quality score
threshold of 30. Reads were then mapped to the D. melanogaster reference genome dm6 using
HISAT2 (version 2.1.0) (Kim et al. 2015). The resulting BAM format files were analyzed
using featureCounts (version 1.6.3) (Liao et al. 2014) to generate read counts for each gene in
the reference genome annotation. The counts generated by featureCounts were subsequently
used to determine the differentially expressed genes using DESeq2 (version 2.11.40.6) (Love et
al. 2014). The results were filtered and significant differentially expressed genes (DEGs) were
identified after applying significance cut-offs of a false discovery rate (FDR) < 0.05 and log2 fold
change 1 (for upregulated transcripts) or -1 (for downregulated transcripts).
Functional Annotation
The Gene Ontology (GO) Resource (http://geneontology.org/) and PANTHER
classification system (Ashburner et al. 2000; Mi et al. 2013; Gene Ontology Consortium 2021)
were used for GO term analysis of each list of DEGs. The FDR threshold to determine term
enrichment was 0.05.
Statistical Analysis
All statistical analyses described in the following sections were done in the R statistical
computing environment (R Core Team 2021) as detailed. Graphs were produced using the
ggplot2 and ggvenn R packages (Wickham 2009; Yan 2021).
Quantitative PCR (qPCR)
qPCR was used to confirm the differential expression of selected genes of interest.
Primers for individual genes were designed according to the FlyPrimerBank (Hu et al. 2013) and
obtained from Integrated DNA Technologies (Table S1). Ten larvae from each genotype raised
at 28º were collected and total RNA was extracted using Trizol (Thermo Fisher Scientific). RNA
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concentration was measured using a Nanodrop and samples were normalized to 2.5 µg for
reverse transcription into cDNA using SuperScript IV (Thermo Fisher Scientific). cDNA samples
were diluted 1:100 in nuclease-free water and 1 µl was used as a template for qPCR
experiments. qPCR was performed using the PowerUp SYBR Green Supermix (Thermo Fisher
Scientific). Samples were run as technical triplicates and a total of three biological replicates
were performed for each primer in each genotype.
For comparison across genotypes, transcript levels for the genes of interest were
normalized to the Arp1 housekeeping control from the same sample. Within each biological
replicate, the Ct value was calculated by subtracting the Ct value of Arp1 from the Ct value of
each experimental primer set in each genotype. Differences in transcript Ct values were tested
across biological replicates using mixed linear models, with genotype as a fixed effect and
biological replicate as a random effect using the lmerTest package (Kuznetsova et al. 2017) in
R. Relative quantification was calculated as 2-Ct (where Ct is the difference between the
tuSz1 Ct values and w1118 control Ct values for each gene of interest).
Rel Localization
To determine Rel subcellular localization in hemocytes in control and mutant larvae,
w1118 and tuSz1 unmated females were crossed to Rel-GFP males and the crosses were raised
at 28ºC. From these crosses we selected w1118 /Y; Rel-GFP/+ control larvae and tuSz1/Y; Rel-
GFP/+ mutant larvae. Five male third instar larvae from each genotype were bled in PBS and
hemocytes were stained using DAPI (1:500; Invitrogen) to mark nuclei and mounted in
Vectashield mounting medium. To determine Rel localization in hemocytes of parasitoid infected
larvae, late second instar w1118 /Y; Rel-GFP/+ larvae were placed on 35mm Petri dishes filled
with Drosophila medium only (uninfected control) or together (infected) with L. boulardi wasps at
25°C. Larvae were dissected at 72-hours post infection (hpi) in PBS and hemocytes were
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stained using DAPI mounted in Vectashield for imaging. To determine the Rel-GFP expression
in fat body tissue, anterior and posterior regions of fat bodies from w1118/Y; Rel-GFP/+ and
tuSz1/Y; Rel-GFP/+ larvae raised at 28° were dissected in PBS and mounted in Vectashield
mounting medium. Cells and tissues were imaged using a Leica SP8 confocal microscope in the
ISU Confocal Microscopy Facility to assay Rel localization.
The relative proportion of Rel-GFP signal coming from the nucleus vs cytoplasm (N/C
ratio) was calculated using Fiji (Schindelin et al. 2012). Image analysis was performed as
described in (Noursadeghi et al. 2008). Specifically, the DAPI stain was used to mask the
nucleus allowing for mean cytoplasmic and nuclear fluorescence intensity values to be
independently determined. The N/C ratio was then calculated based on the ratio of mean
nuclear intensity to mean cytoplasmic intensity. Cells with an N/C ratio greater than 1.5 were
considered to have nuclear Rel-GFP localization and cells with an N/C ratio less than 1.5 were
considered to have diffuse Rel-GFP (Rel-GFP exclusion from the nucleus was not observed).
The proportion of cells with nuclear Rel-GFP localization in the tuSz1 genetic background was
compared to w1118 using Fisher's exact test in R.
RESULTS AND DISCUSSION
Identification of differentially expressed genes in tuSz1 and hopTum
To gain a better understanding of the changes involved in JAK-STAT mediated immune
priming and the consequences of the loss of self-tolerance, we performed RNA sequencing on
w1118, tuSz1 and hopTum larvae raised at 28º to assess differential transcript abundance. The
resulting sequence reads were mapped to the D. melanogaster genome (Table S2) to quantify
transcript levels. We separately compared transcript levels in each mutant genotype to the w1118
control genotype (Tables S3-S6). Both tuSz1 and hopTum have gain of function mutations in hop,
so any genes that are differentially expressed in both genotypes are likely to be regulated by the
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JAK-STAT pathway. We found 832 shared genes that were differentially expressed in both
tuSz1 and hopTum mutants compared to wildtype, with 384 genes that were upregulated, and 438
genes that were downregulated (Figure 1). Because the JAK-STAT pathway plays a key role in
immune activation (Agaisse and Perrimon 2004), these identified genes might be relevant to the
priming of the immune response.
To begin characterizing these putative immune activation genes, we conducted Gene
Ontology (GO) analysis to identify the biological processes and molecular functions that are
enriched among the common set of differentially expressed genes (Table 1). Analysis of genes
that were upregulated in both tuSz1 and hopTum identified GO terms that have been linked to
host immunity including defense response to other organism (GO:0098542) and response to
external stimulus (GO:0009605). General immune response genes identified in these categories
include five members of the antimicrobial peptide (AMP)-like Bomanin gene family (BomBc1,
BomS1, BomS2, BomS4, BomT1) (Clemmons et al. 2015) and the Cecropin family AMP gene
CecC (Tryselius et al. 1992), along with several immune receptors (NimB2, NimC1, NimC4)
(Kurucz et al. 2007; Somogyi et al. 2010), the prophenoloxidase family members PPO1 and
PPO3 (Nam et al. 2008; Dudzic et al. 2015), and the induced by infection (IBIN) gene (Valanne
et al. 2019). We also found differential expression of important immune related signaling
pathways such as the JAK-STAT signaling pathway components dome, et and upd3 (Myllymäki
and Rämet 2014), the Toll pathway genes nec and SPE (Kambris et al. 2006), and Rac2 and
Rap1 which are linked to hemocyte function (Williams et al. 2005; Huelsmann et al. 2006). The
tuSz1 and hopTum mutations both lead to the ectopic production of lamellocytes, and accordingly,
we see lamellocyte related genes such as ItgaPS4, ItgaPS5, and drip upregulated in these
genotypes (Stofanko et al. 2010; Tattikota et al. 2020). The proteolysis (GO:0006508) GO term
is also enriched among genes upregulated in both tuSz1 and hopTum. Notably, 29 members of
the S1A serine protease family are significantly upregulated in both tuSz1 and hopTum, including
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CG10764 and CG4793 which have recently been shown to be involved in the cellular immune
response to parasitoid infection (Kr et al. 2021).
The induction of host immunity is associated with life history tradeoffs, primarily in the
redistribution of energy resources from development or reproduction to the production of an
immune response (Zuk and Stoehr 2002). We found that genes involved with cellular lipid
metabolism (GO:0044255) are enriched among the transcripts upregulated in tuSz1 and hopTum,
and conversely that genes linked to development, including regulation of multicellular
organismal development (GO:2000026), tissue morphogenesis (GO:0048729), and cell fate
commitment (GO:0045165) are among those transcripts downregulated in both tuSz1 and
hopTum (Table 1). The upregulation of transcripts involved in metabolism and downregulation of
transcripts linked to developmental processes possibly reflects the immune response-
development tradeoff, and suggests that this tradeoff may be regulated in part by JAK-STAT
signaling or downstream processes. Further study of these genes may help to provide an
understanding of the energy trade-offs and other physiological changes that occur during
immune responses.
Loss of self-tolerance in tuSz1 mutants results in differential gene expression
Despite the similarities between the tuSz1 and hopTum mutants, tuSz1 specifically
displays a loss of self-tolerance phenotype (Mortimer et al. 2021), so we wanted to investigate
transcripts that were specifically differentially expressed in tuSz1 to identify genes that might be
involved in innate immune self-tolerance and autoimmune mechanisms. Out of 1154 transcripts
that were differentially expressed in tuSz1 but not hopTum (referred to as tuSz1 specific DEGs),
we found 670 transcripts that were significantly upregulated and 484 transcripts that were
significantly downregulated (Figure 1).
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In contrast to the list of upregulated transcripts in common to tuSz1 and hopTum, GO Term
analysis of these tuSz1 specific DEGs (Table 2) failed to find enrichment of any GO categories
associated with positive regulation of host immunity. Indeed, one of the most highly enriched
terms was the negative regulation of antimicrobial peptide biosynthetic process (GO:0002806).
This term was linked to peptidoglycan recognition protein (PGRP) encoding genes, and we
found that six PGRP gene transcripts are upregulated in tuSz1 mutants. There are 13 members
of this family in the Drosophila genome (Table 3), which primarily play roles in the regulation of
the Toll and Imd immune signaling pathways (Kurata 2014). There are five PGRP genes that
encode negative regulators of Imd signaling, and we found that all five of these are specifically
upregulated in tuSz1 mutants, with the sixth altered PGRP playing an unknown role (Table 3).
The finding that whereas positive regulators of immunity were identified as upregulated genes in
both tuSz1 and hopTum, the enrichment of genes involved in negative regulation of the Imd
pathway specifically in tuSz1 mutants suggests a differential role of immune mechanisms in
immune priming and the autoimmune response to a loss of self-tolerance. We additionally found
26 genes that map to the chitin-based cuticle development (GO: 0040003) term among the
tuSz1 specific DEGs, including members of the Lcp, Acp, Twdl, Cpr, and Ccp gene families.
Expression of cuticle genes has been linked to infection in D. melanogaster and other insects,
although a role in host immunity has yet to be identified (Yadav et al. 2017; Hou et al. 2021;
Pantha et al. 2021).
Many of the additional enriched GO categories among the tuSz1 specific DEGs are
associated with stress responses and metabolism, perhaps providing insight into physiological
changes in tuSz1 mutant larvae due to the occurrence of an autoimmune response (Table 2).
We found an enrichment of oxidoreductase activity (GO: 0016491) genes, including the
glutathione transferase (GST) encoding genes GstD2, GstD6, GstD7, GstD8, GstE1, GstE3,
GstE5, and GstE7 (Low et al. 2007). We also found that 18 cytochrome P450 (CYP450)
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encoding genes are upregulated in tuSz1 mutants. CYP450s are involved in a wide range of
cellular activities including roles in hormone signaling and detoxification of xenobiotics (Bergé et
al. 1998; Chung et al. 2009). The specific upregulation of these stress response genes suggests
that loss of self-tolerance may lead to the production of an internal oxidizing environment.
As mentioned previously, metabolic changes often accompany immune responses, and
we found that two general regulators of host metabolism, the Adipokinetic hormone receptor
(AkhR) (Bharucha et al. 2008) and Linking immunity and metabolism (Lime) (Mihajlovic et al.
2019), are upregulated in tuSz1 mutants. Lime encodes a transcription factor that plays a key
role in metabolic changes following infection. Accordingly, we found that genes associated with
a wide range of metabolic activities including generation of precursor metabolites and energy
(GO:0006091), carbohydrate derivative metabolic process (GO:1901135), alpha-amino acid
metabolic process (GO:1901605) and lipid metabolic process (GO:0006629) were enriched
among those upregulated in tuSz1 larvae (Table 2). Further analysis of these genes and the
mechanisms they mediate will help to identify the metabolic pathways involved in the response
to a loss of self-tolerance.
Along with upregulated transcripts, transcripts that are downregulated in these
genotypes may also provide insight into the cellular activities that are altered. We found that
genes associated with tissue morphogenesis (GO:0048729) and the establishment of tissue
polarity (GO:0007164) are enriched among the downregulated tuSz1 specific DEGs (Table 2).
The loss of cellular polarity and disrupted tissue organization has also been observed in both
autoimmune mouse models and patients with autoimmune conditions such as inflammatory
bowel disease (Ahmad et al. 2017; Klunder et al. 2017; Guo and Shen 2021). This may suggest
that tissue structural maintenance is a key self-tolerance checkpoint, in agreement with previous
findings in Drosophila (Rizki and Rizki 1974; Kim and Choe 2014; Mortimer et al. 2021).
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Relish/NF B is activated in immune tissues in tuSz1 mutants
Based on the specific upregulation of PGRP genes that act as negative regulators of the
Imd pathway (Table 3) (Bischoff et al. 2006; Zaidman-Rémy et al. 2006; Maillet et al. 2008), we
hypothesized that Imd signaling may be dysregulated in the tuSz1 loss of self-tolerance
phenotype. While the role of the Imd pathway in mediating the Drosophila humoral immune
response to Gram-negative bacteria has been well established (Lemaitre et al. 1995), the
mechanism by which it may participate in the response to loss of self-tolerance is unknown. The
tuSz1 autoimmune mutant may therefore provide an effective model to begin exploring the role
of the Imd pathway in self-tolerance and autoimmune reactions.
To verify the elevated expression of PGRP genes in tuSz1 larvae, we assayed the
expression of PGRP-SC1a and PGRP-SC2 in tuSz1 and w1118 by qPCR. As expected, both
PGRP-SC1a and PGRP-SC2 were significantly upregulated in tuSz1 compared to w1118 (PGRP-
SC1a: 11.3-fold upregulated, p < 2.2e-16; PGRP-SC2: 1.3-fold upregulated, p = 0.0094) (Figure
2A-B). To further evaluate the ability of qPCR to detect altered expression in tuSz1 mutants, we
also assayed transcript levels of scrib (a cell polarity gene) and observed the expected
downregulation in tuSz1 larvae compared to w1118 (1.7-fold downregulated, p = 1.4 x 10-8)
(Figure 2C).
The Imd pathway mainly responds to infection via differential expression of target genes
(Kleino and Silverman 2014). A previous study has identified genes whose expression is
modified by constitutive Imd pathway activation (Davoodi et al. 2019). We found that 58 of the
324 genes identified in this study also showed differential expression in tuSz1 larvae (Table S7),
including three of the identified PGRP genes (PGRP-LB, PGRP-LF, PGRP-SC2), and other
immune genes such as AttA, Rala, Gbp1 and GILT2. These findings provide support for the
hypothesis that Imd signaling is dysregulated in tuSz1 mutants.
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Gene expression downstream of Imd is largely mediated by the activation of Relish
(Rel), a D. melanogaster member of the NF- B family of transcriptional activators (Hetru and
Hoffmann 2009). Rel activation is regulated by signal-dependent translocation into the nucleus.
Whereas full-length Rel protein is autoinhibited and remains in the cytoplasm, Imd activation
results in cleavage of Rel and the production of an active N-terminal fragment, which
translocates to the nucleus to drive gene expression (Stöven et al. 2000). To determine whether
Rel is activated in tuSz1 mutants, we used a Rel-GFP fusion protein to track the subcellular
localization within the main immune tissues, the hemocytes and fat body. This reporter has GFP
fused to the N-terminal domain of Relish, and GFP localization in the nucleus versus cytoplasm
gives a relative readout of pathway activity.
Rel-GFP expression was observed in plasmatocytes in tuSz1/Y; Rel-GFP/+ and w1118 /Y;
Rel-GFP/+ larvae (Figure 3A-B). We found that Rel-GFP was significantly more likely to be
concentrated in the nucleus in plasmatocytes from tuSz1/Y; Rel-GFP/+ larvae compared to w1118
/Y; Rel-GFP/+ control larvae (Fisher’s exact test: p = 1.23x10-5; odds ratio = 9.02) (Figure 3D),
suggesting that Imd/Rel activation is induced in tuSz1 mutants. In addition, Rel-GFP expression
and nuclear localization were observed in lamellocytes from tuSz1/Y; Rel-GFP/+ larvae (Figure
3C), suggesting that the Imd pathway is also active in this hemocyte type in the tuSz1 mutant
background.
To determine if this Rel activation is common to cellular immune responses in
Drosophila or if it may be specific to loss of self-tolerance reactions, we assayed Rel-GFP
expression and localization in the immune cells of w1118 /Y; Rel-GFP/+ larvae during the
response to parasitoid wasp infection. w1118 /Y; Rel-GFP/+ larvae were infected by Leptopilina
boulardi and hemocytes were isolated and imaged 72hpi. We observed minimal Rel-GFP
expression in both plasmatocytes (Figure 4A-B) and lamellocytes (Figure 4C) in these infected
larvae. Circulating non-hemocyte cells (marked by an asterisk* in Figure 4B-C) showed strong
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Rel-GFP expression and acted as an imaging control. These results suggest that Rel and the
Imd pathway are not likely to be involved in the cellular immune response to parasitoid infection.
In agreement with this finding, a previous study has shown that Rel mutants have a normal
encapsulation response against parasitoid wasp infection (Hedengren et al. 1999).
To test whether Rel activation is specific to hemocytes or seen in other immune tissues
in tuSz1 mutants, we imaged fat body tissue dissected from tuSz1/Y; Rel-GFP/+ and w1118 /Y;
Rel-GFP/+ larvae raised at 28°. We observed minimal Rel-GFP expression throughout the fat
body of control larvae (Figure 5A). The tuSz1 self-encapsulation phenotype is specific to the
posterior fat body tissue in mutant larvae, so we imaged both anterior and posterior fat body
regions. We found that similar to control, Rel-GFP showed minimal expression in anterior fat
body tissue from tuSz1/Y; Rel-GFP/+ larvae (Figure 5B). However, Rel-GFP expression was
elevated in posterior fat body tissue, including at the sites of self-encapsulation, in tuSz1/Y; Rel-
GFP/+ larvae (Figure 5C).
Rel/NF B transcriptional activity is associated with pro-inflammatory signaling in
Drosophila and other species (Libert et al. 2006; Lawrence 2009; Ganesan et al. 2011;
Myllymäki et al. 2014). The observed Rel activation suggests that inflammation is a feature of
loss of self-tolerance responses in Drosophila, a mechanism that is conserved in human
autoimmune disease (Zhang et al. 2017; Furman et al. 2019). Interestingly, tuSz1 mutants
appear to attempt to block pro-inflammatory Rel activity by inducing transcription of negative
regulators of the Imd pathway. A similar mechanism is seen in the establishment of immune
tolerance; immune tolerance can be defined as an attempt by the host to mitigate the effects of
infection rather than eliminating the infecting pathogen (Ayres and Schneider 2012), and is often
accompanied by a downregulation of pro-inflammatory signaling (Sears et al. 2011; Vale et al.
2014). Indeed, a recent study has revealed that the downregulation of Imd signaling plays an
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important role in establishing immune tolerance in Drosophila (Prakash et al. 2021), highlighting
this possible relationship.
The constitutive activation of NF- B is associated with various chronic inflammatory
conditions, autoimmune diseases, and cancer (Baker et al. 2011; Zhang et al. 2017; Sun 2017).
Studies using in vivo mouse models and human patients with autoimmune disorders have
demonstrated that NF- B signaling is implicated in the pathogenesis of a number of
autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple
sclerosis, and systemic lupus erythematosus (Atreya et al. 2008; Zubair and Frieri 2013;
Miraghazadeh and Cook 2018; Zhou et al. 2020; Ilchovska and Barrow 2021). The involvement
of aberrant NF- B activity in disease has attracted focus in the therapeutic targeting of this
signaling pathway (Viatour et al. 2005; Lin et al. 2010). Thus, our model may serve as a suitable
system to further understand the role of NF- B in autoimmunity and allow for investigation into
its full potential as a therapeutic target.
noncoding RNAs are differentially expressed in hopTum and tuSz1 mutants
Similar to a recent study looking at the transcriptional response to aging and viral
infection in Drosophila (Sheffield et al. 2021), we found widespread changes in the expression
of predicted noncoding RNAs (ncRNAs) in tuSz1 and hopTum larvae (Figure 6A-B). ncRNAs are a
class of RNA molecules that are transcribed from the genome but are not translated into
proteins. Major classes of ncRNAs include ribosomal RNAs (rRNAs), transfer RNAs (tRNAs),
small ncRNAs, and long ncRNAs (Cech and Steitz 2014). While the roles of rRNAs and tRNAs
are well characterized, multiple studies have found that small and long ncRNAs participate in a
range of biological processes such as genome imprinting, organismal development, and
disease pathogenesis (Wang and Chang 2011; Anastasiadou et al. 2018; Fasolo et al. 2019),
as well as immune regulation (Atianand et al. 2017; Yuan et al. 2018).
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ncRNAs are categorized according to their transcript size, with small ncRNAs being
<200 nucleotides (nt). These small ncRNA species include microRNAs (miRNAs), small nuclear
RNAs (snRNAs), and small nucleolar RNAs (snoRNAs), and only a limited number of small
RNAs are differentially expressed in tuSz1 and hopTum larvae (Table 4). On the other hand, a
large number of long ncRNAs (lncRNAs; >200 nt) show differential expression in these mutants
(Figure 6C-D). Amongst the lncRNAs upregulated in both tuSz1 and hopTum, Induced by infection
(IBIN) is the only lncRNA that has been studied in the context of Drosophila immunity, although
it has been proposed that IBIN encodes a short peptide that confers its function (Valanne et al.
2019; Ebrahim et al. 2021). The significant upregulation of lncRNAs in both genotypes highlights
the potential importance of lncRNAs in mediating immune priming and autoimmune responses.
CONCLUSION
Immune self-tolerance is essential for maintaining immune homeostasis, and breakdown
of self-tolerance mechanisms results in autoimmunity and other immune related diseases.
Although great progress has been made in understanding self-tolerance in the context of
adaptive immunity, innate immune mechanisms regulating self-tolerance and its role in
autoimmunity remain less well characterized. In this study we have identified genes that are
differentially expressed following loss of self-tolerance in the Drosophila melanogaster tuSz1
mutant. These results provide insight into the biological processes that take place in tuSz1
mutant larvae and may contribute toward a better understanding of the events that take place in
loss of self-tolerance autoimmune reactions.
DATA AVAILABILITY
RNAseq data will be made available through the NCBI Gene Expression Omnibus (submission
pending). The authors are happy to make these data available during the review process, and
we will update with the NCBI GEO accession number when available.
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ACKNOWLEDGEMENTS
We thank Joshua Hill and Elise N. Le for their assistance on this project. The ISU Confocal
Microscopy Facility was funded by NSF grant DBI-1828136. Stocks obtained from the
Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study.
FUNDING
Research reported in this publication was supported by the National Institute of General Medical
Sciences of the National Institutes of Health under Award Number R35GM133760 to NTM, a
Grant-in-Aid of Research from the National Academy of Sciences administered by Sigma Xi,
The Scientific Research Society to PK, and the Illinois State University College of Arts and
Sciences University Research Grants program.
FIGURE LEGENDS
Figure 1. Venn diagrams illustrating the numbers of transcripts that are (A) upregulated and (B)
downregulated in the indicated genotypes in comparison with w1118 control larvae.
Figure 2. Mean relative quantification ± standard error of PGRP-SC1a (A), PGRP-SC2 (B) and
scrib (C) transcript levels in tuSz1 mutant larvae compared to w1118 controls. Transcripts were
quantified by qPCR, * indicates p < 0.05 relative to w1118 control.
Figure 3. Localization of the Rel-GFP fusion protein in hemocytes. Hemocytes were dissected
from w1118/Y; Rel-GFP/+ and tuSz1/Y; Rel-GFP/+ larvae raised at 28°C, and stained with
propidium iodide (PI). Shown are representative images of hemocytes: brightfield (A-C), GFP to
visualize Rel-GFP localization (A’-C’) and PI to mark nuclei (A’’-C’’). Rel-GFP shows enriched
nuclear localization in plasmatocytes (B-B’’) and lamellocytes (C-C’’) in tuSz1/Y; Rel-GFP/+, but
not in plasmatocytes (A-A’’) in w1118/Y; Rel-GFP/+. Note that w1118/Y; Rel-GFP/+ larvae do not
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produce lamellocytes. (D) Bar graph showing the proportion of hemocytes with a predominantly
nuclear Rel-GFP localization (see Materials and Methods for analysis details). Complete
genotypes tuSz1/Y; Rel-GFP/+ and w1118/Y; Rel-GFP/+ are simplified to tuSz1 and w1118 for
clarity. * indicates p < 0.05 relative to w1118/Y; Rel-GFP/+ control.
Figure 4. Expression of the Rel-GFP fusion protein in hemocytes from parasitoid infected
w1118/Y; Rel-GFP/+ larvae. Brightfield (A-C) and GFP (A’-C’) images. The Rel-GFP fusion
protein is barely detectable in plasmatocytes (A, B) and lamellocytes (C). Strong Rel-GFP
expression is seen in an unknown non-hemocyte cell type (indicated by * in B and C).
Figure 5. Expression of the Rel-GFP fusion protein in the fat body dissected from w1118/Y; Rel-
GFP/+ and tuSz1/Y; Rel-GFP/+ larvae raised at 28°C. Minimal Rel-GFP expression is observed
in fat body tissue dissected from w1118/Y; Rel-GFP/+ (A-A’) or anterior fat body tissue dissected
from tuSz1/Y; Rel-GFP/+ (B-B’) larvae. Increased Rel-GFP expression is observed in posterior
fat body tissue dissected from tuSz1/Y; Rel-GFP/+ (C-C’) larvae. The posterior fat body is the
site of the loss of self-tolerance in tuSz1 mutant larvae, and the characteristic self-encapsulation
phenotype is evident in (C).
Figure 6. Venn diagrams illustrating the numbers of differentially expressed ncRNA transcripts
that are (A) upregulated and (B) downregulated, and the numbers of differentially expressed
lncRNA transcripts that are (C) upregulated and (D) downregulated in the indicated genotypes
in comparison with w1118 control larvae.
REFERENCES
Afgan, E., D. Baker, B. Batut, M. van den Beek, D. Bouvier et al., 2018 The Galaxy platform for
accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic
Acids Research 46: W537–W544.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Agaisse, H., and N. Perrimon, 2004 The roles of JAK/STAT signaling in Drosophila immune
responses. Immunol. Rev. 198: 72–82.
Ahmad, R., M. F. Sorrell, S. K. Batra, P. Dhawan, and A. B. Singh, 2017 Gut permeability and
mucosal inflammation: bad, good or context dependent. Mucosal Immunol 10: 307–317.
Anastasiadou, E., L. S. Jacob, and F. J. Slack, 2018 Non-coding RNA networks in cancer. Nat
Rev Cancer 18: 5–18.
Anderl, I., L. Vesala, T. O. Ihalainen, L.-M. Vanha-aho, I. Andó et al., 2016 Transdifferentiation
and proliferation in two distinct hemocyte lineages in Drosophila melanogaster larvae
after wasp infection. PLOS Pathogens 12: e1005746.
Andrews, S., 2010 FastQC: A quality control tool for high throughput sequence data.
Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler et al., 2000 Gene ontology: tool for
the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29.
Atianand, M. K., D. R. Caffrey, and K. A. Fitzgerald, 2017 Immunobiology of Long Noncoding
RNAs. Annu Rev Immunol 35: 177–198.
Atreya, I., R. Atreya, and M. F. Neurath, 2008 NF-kappaB in inflammatory bowel disease. J
Intern Med 263: 591–596.
Ayres, J. S., and D. S. Schneider, 2012 Tolerance of infections. Annual Review of Immunology
30: 271–294.
Baker, R. G., M. S. Hayden, and S. Ghosh, 2011 NF- B, inflammation, and metabolic disease.
Cell Metab 13: 11–22.
Bergé, J. B., R. Feyereisen, and M. Amichot, 1998 Cytochrome P450 monooxygenases and
insecticide resistance in insects. Philos Trans R Soc Lond B Biol Sci 353: 1701–1705.
Bharucha, K. N., P. Tarr, and S. L. Zipursky, 2008 A glucagon-like endocrine pathway in
Drosophila modulates both lipid and carbohydrate homeostasis. J Exp Biol 211: 3103–
3110.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Bischoff, V., C. Vignal, B. Duvic, I. G. Boneca, J. A. Hoffmann et al., 2006 Downregulation of the
Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2. PLoS
Pathog 2: e14.
Brennan, C. A., and K. V. Anderson, 2004 Drosophila: The genetics of innate immune
recognition and response. Annual Review of Immunology 22: 457–483.
Cech, T. R., and J. A. Steitz, 2014 The noncoding RNA revolution-trashing old rules to forge
new ones. Cell 157: 77–94.
Chung, H., T. Sztal, S. Pasricha, M. Sridhar, P. Batterham et al., 2009 Characterization of
Drosophila melanogaster cytochrome P450 genes. Proc Natl Acad Sci U S A 106: 5731–
5736.
Clemmons, A. W., S. A. Lindsay, and S. A. Wasserman, 2015 An effector peptide family
required for Drosophila Toll-mediated immunity. PLOS Pathogens 11: e1004876.
Davoodi, S., A. Galenza, A. Panteluk, R. Deshpande, M. Ferguson et al., 2019 The immune
deficiency pathway regulates metabolic homeostasis in Drosophila. J Immunol 202:
2747–2759.
Dudzic, J. P., S. Kondo, R. Ueda, C. M. Bergman, and B. Lemaitre, 2015 Drosophila innate
immunity: regional and functional specialization of prophenoloxidases. BMC Biol 13: 81.
Ebrahim, S. A. M., G. J. S. Talross, and J. R. Carlson, 2021 Sight of parasitoid wasps
accelerates sexual behavior and upregulates a micropeptide gene in Drosophila. Nat
Commun 12: 2453.
Fasolo, F., K. Di Gregoli, L. Maegdefessel, and J. L. Johnson, 2019 Non-coding RNAs in
cardiovascular cell biology and atherosclerosis. Cardiovasc Res 115: 1732–1756.
Furman, D., J. Campisi, E. Verdin, P. Carrera-Bastos, S. Targ et al., 2019 Chronic inflammation
in the etiology of disease across the life span. Nat Med 25: 1822–1832.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Ganesan, S., K. Aggarwal, N. Paquette, and N. Silverman, 2011 NF- B/Rel proteins and the
humoral immune responses of Drosophila melanogaster. Curr Top Microbiol Immunol
349: 25–60.
Gendron, C. M., and S. D. Pletcher, 2017 MicroRNAs mir-184 and let-7 alter Drosophila
metabolism and longevity. Aging Cell 16: 1434–1438.
Gene Ontology Consortium, 2021 The Gene Ontology resource: enriching a GOld mine. Nucleic
Acids Res 49: D325–D334.
Guo, C., and J. Shen, 2021 Cytoskeletal organization and cell polarity in the pathogenesis of
Crohn’s disease. Clinic Rev Allerg Immunol 60: 164–174.
Hanratty, W. P., and C. R. Dearolf, 1993 The Drosophila Tumorous-lethal hematopoietic
oncogene is a dominant mutation in the hopscotch locus. Mol. Gen. Genet. 238: 33–37.
Hedengren, M., BengtÅsling, M. S. Dushay, I. Ando, S. Ekengren et al., 1999 Relish, a central
factor in the control of humoral but not cellular immunity in Drosophila. Molecular Cell 4:
827–837.
Hetru, C., and J. A. Hoffmann, 2009 NF- B in the immune response of Drosophila. Cold Spring
Harb Perspect Biol 1: a000232.
Hoffmann, J. A., F. C. Kafatos, C. A. Janeway, and R. A. Ezekowitz, 1999 Phylogenetic
perspectives in innate immunity. Science 284: 1313–1318.
Honti, V., G. Csordás, É. Kurucz, R. Márkus, and I. Andó, 2014 The cell-mediated immunity of
Drosophila melanogaster: Hemocyte lineages, immune compartments, microanatomy
and regulation. Developmental & Comparative Immunology 42: 47–56.
Hou, Z., F. Shi, S. Ge, J. Tao, L. Ren et al., 2021 Comparative transcriptome analysis of the
newly discovered insect vector of the pine wood nematode in China, revealing putative
genes related to host plant adaptation. BMC Genomics 22: 189.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Hu, Y., R. Sopko, M. Foos, C. Kelley, I. Flockhart et al., 2013 FlyPrimerBank: an online
database for Drosophila melanogaster gene expression analysis and knockdown
evaluation of RNAi reagents. G3 (Bethesda) 3: 1607–1616.
Huelsmann, S., C. Hepper, D. Marchese, C. Knöll, and R. Reuter, 2006 The PDZ-GEF Dizzy
regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila
embryo. Development 133: 2915–2924.
Ilchovska, D. D., and D. M. Barrow, 2021 An Overview of the NF-kB mechanism of
pathophysiology in rheumatoid arthritis, investigation of the NF-kB ligand RANKL and
related nutritional interventions. Autoimmun Rev 20: 102741.
Kambris, Z., S. Brun, I.-H. Jang, H.-J. Nam, Y. Romeo et al., 2006 Drosophila immunity: A
large-scale in vivo RNAi screen identifies five serine proteases required for Toll
activation. Current Biology 16: 808–813.
Kim, M. J., and K.-M. Choe, 2014 Basement membrane and cell integrity of self-tissues in
maintaining Drosophila immunological tolerance. PLoS Genet 10:.
Kim, D., B. Langmead, and S. L. Salzberg, 2015 HISAT: a fast spliced aligner with low memory
requirements. Nat Methods 12: 357–360.
Kleino, A., and N. Silverman, 2014 The Drosophila IMD pathway in the activation of the humoral
immune response. Dev Comp Immunol 42: 25–35.
Klunder, L. J., K. N. Faber, G. Dijkstra, and S. C. D. van IJzendoorn, 2017 Mechanisms of cell
polarity-controlled epithelial homeostasis and immunity in the intestine. Cold Spring Harb
Perspect Biol 9: a027888.
Kr, P., J. Lee, and N. T. Mortimer, 2021 The S1A protease family members CG10764 and
CG4793 regulate cellular immunity in Drosophila. microPublication Biology 2021:.
Kurata, S., 2014 Peptidoglycan recognition proteins in Drosophila immunity. Dev. Comp.
Immunol. 42: 36–41.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Kurucz, É., R. Márkus, J. Zsámboki, K. Folkl-Medzihradszky, Z. Darula et al., 2007 Nimrod, a
putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Current
Biology 17: 649–654.
Kuznetsova, A., P. B. Brockhoff, and R. H. B. Christensen, 2017 lmerTest package: Tests in
linear mixed effects models. Journal of Statistical Software 82: 1–26.
Lawrence, T., 2009 The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb
Perspect Biol 1: a001651.
Lemaitre, B., and J. Hoffmann, 2007 The host defense of Drosophila melanogaster. Annual
Review of Immunology 25: 697–743.
Lemaitre, B., E. Kromer-Metzger, L. Michaut, E. Nicolas, M. Meister et al., 1995 A recessive
mutation, immune deficiency (imd), defines two distinct control pathways in the
Drosophila host defense. Proc. Natl. Acad. Sci. U.S.A. 92: 9465–9469.
Liao, Y., G. K. Smyth, and W. Shi, 2014 featureCounts: an efficient general purpose program for
assigning sequence reads to genomic features. Bioinformatics 30: 923–930.
Libert, S., Y. Chao, X. Chu, and S. D. Pletcher, 2006 Trade-offs between longevity and
pathogen resistance in Drosophila melanogaster are mediated by NFkappaB signaling.
Aging Cell 5: 533–543.
Lin, Y., L. Bai, W. Chen, and S. Xu, 2010 The NF-kappaB activation pathways, emerging
molecular targets for cancer prevention and therapy. Expert Opin Ther Targets 14: 45–
55.
Love, M. I., W. Huber, and S. Anders, 2014 Moderated estimation of fold change and dispersion
for RNA-seq data with DESeq2. Genome Biology 15: 550.
Low, W. Y., H. L. Ng, C. J. Morton, M. W. Parker, P. Batterham et al., 2007 Molecular evolution
of glutathione S-transferases in the genus Drosophila. Genetics 177: 1363–1375.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Luo, H., W. P. Hanratty, and C. R. Dearolf, 1995 An amino acid substitution in the Drosophila
hopTum-l Jak kinase causes leukemia-like hematopoietic defects. EMBO J 14: 1412–
1420.
Maillet, F., V. Bischoff, C. Vignal, J. Hoffmann, and J. Royet, 2008 The Drosophila
peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway
activation. Cell Host Microbe 3: 293–303.
Medzhitov, R., and C. A. Janeway, 2002 Decoding the patterns of self and nonself by the innate
immune system. Science 296: 298–300.
Mi, H., A. Muruganujan, J. T. Casagrande, and P. D. Thomas, 2013 Large-scale gene function
analysis with the PANTHER classification system. Nat Protoc 8: 1551–1566.
Mihajlovic, Z., D. Tanasic, A. Bajgar, R. Perez-Gomez, P. Steffal et al., 2019 Lime is a new
protein linking immunity and metabolism in Drosophila. Developmental Biology 452: 83–
94.
Miraghazadeh, B., and M. C. Cook, 2018 Nuclear Factor-kappaB in autoimmunity: man and
mouse. Frontiers in Immunology 9: 613.
Mortimer, N. T., M. L. Fischer, A. L. Waring, P. Kr, B. Z. Kacsoh et al., 2021 Extracellular matrix
protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster.
PNAS 118:.
Mortimer, N. T., B. Z. Kacsoh, E. S. Keebaugh, and T. A. Schlenke, 2012 Mgat1-dependent N-
glycosylation of membrane components primes Drosophila melanogaster blood cells for
the cellular encapsulation response. PLoS Pathog 8: e1002819.
Myllymäki, H., and M. Rämet, 2014 JAK/STAT pathway in Drosophila immunity. Scand J
Immunol 79: 377–385.
Myllymäki, H., S. Valanne, and M. Rämet, 2014 The Drosophila Imd signaling pathway. The
Journal of Immunology 192: 3455–3462.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Nam, H.-J., I.-H. Jang, T. Asano, and W.-J. Lee, 2008 Involvement of pro-phenoloxidase 3 in
lamellocyte-mediated spontaneous melanization in Drosophila. Mol Cells 26: 606–610.
Noursadeghi, M., J. Tsang, T. Haustein, R. F. Miller, B. M. Chain et al., 2008 Quantitative
imaging assay for NF- B nuclear translocation in primary human macrophages. J
Immunol Methods 329: 194–200.
Pantha, P., S. Chalivendra, D.-H. Oh, B. D. Elderd, and M. Dassanayake, 2021 A tale of two
transcriptomic responses in agricultural pests via host defenses and viral replication. Int
J Mol Sci 22: 3568.
Paredes, J. C., D. P. Welchman, M. Poidevin, and B. Lemaitre, 2011 Negative regulation by
amidase PGRPs shapes the Drosophila antibacterial response and protects the fly from
innocuous infection. Immunity 35: 770–779.
Parsons, B., and E. Foley, 2016 Cellular immune defenses of Drosophila melanogaster. Dev.
Comp. Immunol. 58: 95–101.
Porcu, E., M. C. Sadler, K. Lepik, C. Auwerx, A. R. Wood et al., 2021 Differentially expressed
genes reflect disease-induced rather than disease-causing changes in the
transcriptome. Nat Commun 12: 5647.
Prakash, A., K. M. Monteith, and P. F. Vale, 2021 Negative regulation of IMD contributes to
disease tolerance during systemic bacterial infection in Drosophila:, 2021.09.23.461574
p.
R Core Team, 2021 R: A language and environment for statistical computing.
Rizki, T. M., and R. M. Rizki, 1974 Topology of the caudal fat body of the tumor-w mutant of
Drosophila melanogaster. J. Invertebr. Pathol. 24: 37–40.
Romagnani, S., 2006 Immunological tolerance and autoimmunity. Intern Emerg Med 1: 187–
196.
Schindelin, J., I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair et al., 2012 Fiji: an open-
source platform for biological-image analysis. Nature Methods 9: 676–682.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Sears, B. F., J. R. Rohr, J. E. Allen, and L. B. Martin, 2011 The economy of inflammation: when
is less more? Trends in Parasitology 27: 382–387.
Sheffield, L., N. Sciambra, A. Evans, E. Hagedorn, C. Goltz et al., 2021 Age-dependent
impairment of disease tolerance is associated with a robust transcriptional response
following RNA virus infection in Drosophila. G3 Genes|Genomes|Genetics 11:.
Sokol, N. S., P. Xu, Y.-N. Jan, and V. Ambros, 2008 Drosophila let-7 microRNA is required for
remodeling of the neuromusculature during metamorphosis. Genes Dev 22: 1591–1596.
Somogyi, K., B. Sipos, Z. Pénzes, and I. Andó, 2010 A conserved gene cluster as a putative
functional unit in insect innate immunity. FEBS Lett 584: 4375–4378.
Stofanko, M., S. Y. Kwon, and P. Badenhorst, 2010 Lineage tracing of lamellocytes
demonstrates Drosophila macrophage plasticity. PLoS ONE 5: e14051.
Stöven, S., I. Ando, L. Kadalayil, Y. Engström, and D. Hultmark, 2000 Activation of the
Drosophila NF- B factor Relish by rapid endoproteolytic cleavage. EMBO Rep 1: 347–
352.
Sun, S.-C., 2017 The non-canonical NF- B pathway in immunity and inflammation. Nat Rev
Immunol 17: 545–558.
Tattikota, S. G., B. Cho, Y. Liu, Y. Hu, V. Barrera et al., 2020 A single-cell survey of Drosophila
blood (B. Lemaître, A. Akhmanova, & B. Lemaître, Eds.). eLife 9: e54818.
Toubi, E., and Z. Vadasz, 2019 Innate immune-responses and their role in driving autoimmunity.
Autoimmun Rev 18: 306–311.
Tryselius, Y., C. Samakovlis, D. A. Kimbrell, and D. Hultmark, 1992 CecC, a cecropin gene
expressed during metamorphosis in Drosophila pupae. Eur J Biochem 204: 395–399.
Valanne, S., T. S. Salminen, M. Järvelä-Stölting, L. Vesala, and M. Rämet, 2019 Immune-
inducible IBIN connects immunity and metabolism in Drosophila melanogaster. PLOS
Pathogens 15: e1007504.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Vale, P. F., A. Fenton, and S. P. Brown, 2014 Limiting Damage during infection: lessons from
infection tolerance for novel therapeutics. PLOS Biology 12: e1001769.
Viatour, P., M.-P. Merville, V. Bours, and A. Chariot, 2005 Phosphorylation of NF-kappaB and
IkappaB proteins: implications in cancer and inflammation. Trends Biochem Sci 30: 43–
52.
Waldner, H., 2009 The role of innate immune responses in autoimmune disease development.
Autoimmun Rev 8: 400–404.
Wang, K. C., and H. Y. Chang, 2011 Molecular mechanisms of long noncoding RNAs. Mol Cell
43: 904–914.
Wickham, H., 2009 ggplot2: Elegant graphics for data analysis. Springer-Verlag, New York.
Williams, M. J., I. Ando, and D. Hultmark, 2005 Drosophila melanogaster Rac2 is necessary for
a proper cellular immune response. Genes to Cells 10: 813–823.
Yadav, S., S. Daugherty, A. C. Shetty, and I. Eleftherianos, 2017 RNAseq analysis of the
Drosophila response to the entomopathogenic nematode Steinernema. G3 (Bethesda)
7: 1955–1967.
Yan, L., 2021 ggvenn: Draw Venn Diagram by “ggplot2.”
Yuan, X., N. Berg, J. W. Lee, T.-T. Le, V. Neudecker et al., 2018 MicroRNA miR-223 as
regulator of innate immunity. J Leukoc Biol 104: 515–524.
Zaidman-Rémy, A., M. Hervé, M. Poidevin, S. Pili-Floury, M.-S. Kim et al., 2006 The Drosophila
amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24:
463–473.
Zhang, Q., M. J. Lenardo, and D. Baltimore, 2017 30 years of NF- B: a blossoming of relevance
to human pathobiology. Cell 168: 37–57.
Zhou, Y., C. Cui, X. Ma, W. Luo, S. G. Zheng et al., 2020 Nuclear Factor B (NF- B)-Mediated
Inflammation in Multiple Sclerosis. Front Immunol 11: 391.
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted November 20, 2021. ; https://doi.org/10.1101/2021.11.19.469298doi: bioRxiv preprint
Zubair, A., and M. Frieri, 2013 NF- B and systemic lupus erythematosus: examining the link. J.
Nephrol. 26: 953–959.
Zuk, M., and A. M. Stoehr, 2002 Immune defense and host life history. Am. Nat. 160 Suppl 4:
S9–S22.
.CC-BY-NC-ND 4.0 International licenseavailable under a
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Table 1. Enriched Gene Ontology (GO) Terms among DEG in common to both tuSz1
and hopTum
GO Term Fold enrichment FDR
Upregulated genes
Defense response to other organism (GO:0098542) 2.58 0.014
Response to external stimulus (GO:0009605) 2.11 2.6 x10-4
Proteolysis (GO:0006508) 2.87 1.2x10-9
Cellular lipid metabolic process (GO:0044255) 2.43 0.022
Downregulated genes
Developmental process (GO:0032502) 1.59 4.7x10-4
Regulation of multicellular organismal development
(GO:2000026) 2.24 0.015
Tissue morphogenesis (GO:0048729) 2.15 0.029
Cell fate commitment (GO:0045165) 2.99 0.005
Table 2. Enriched GO Terms among tuSz1 specific DEGs
GO Term Fold enrichment FDR
Upregulated genes
Negative regulation of antimicrobial peptide
biosynthetic process (GO:0002806) 12.42 0.031
Chitin-based cuticle development (GO:0040003) 2.89 4.9x10-6
Oxidoreductase activity (GO:0016491) 2.80 5.4x10-13
Generation of precursor metabolites and energy
(GO:0006091) 2.32 0.023
Carbohydrate derivative metabolic process
(GO:1901135) 2.14 5.0x10-5
Alpha-amino acid metabolic process (GO:1901605) 3.29 0.006
Lipid metabolic process (GO:0006629) 1.98 0.003
Downregulated genes
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Tissue morphogenesis (GO:0048729) 2.43 2.3x10-4
Establishment of tissue polarity (GO:0007164) 4.17 0.049
Appendage development (GO:0048736) 2.63 0.004
Central nervous system development (GO:0007417) 2.53 0.042
Cell differentiation (GO:0030154) 1.53 0.043
Pattern specification process (GO:0007389) 2.09 0.035
Regulation of cell communication (GO:0010646) 1.83 0.025
Positive regulation of stem cell proliferation
(GO:2000648) 9.6 0.003
Metamorphosis (GO:0007552) 2.42 0.002
Table 3. Expression of PGRP family genes in tuSz1 and hopTum compared to w1118
control larvae.
Gene Pathway Function tuSz1 log2FC hopTum log2FC
PGRP-LA IMD Positive Regulator -0.38 -0.30
PGRP-LB IMD Negative Regulator 1.83* -0.18
PGRP-LC IMD Positive Regulator -1.12 -0.63
PGRP-LD Unknown Unknown -2.77* -0.81
PGRP-LE IMD Positive Regulator -.018 -0.75
PGRP-LF IMD Negative Regulator 1.37* 0.32
PGRP-SA Toll Positive Regulator 1.67 0.99
PGRP-SB1 IMD Immune Effector -0.92 -1.95
PGRP-SB2 IMD Immune Effector Not detected Not detected
PGRP-
SC1a IMD Negative Regulator 3.46* 1.70
PGRP-
SC1b IMD Negative Regulator 3.38* 1.51
PGRP-SC2 IMD Negative Regulator 1.23* -0.19
PGRP-SD Toll Positive Regulator 0.66 -0.83
* indicates FDR < 0.05 compared to w1118 control; log2FC = log2 Fold Change
Table 4. Number of differentially expressed ncRNAs of each class that are upregulated
and downregulated in the indicated genotype in comparison with w1118 control larvae.
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tuSz1 hopTum Both mutants
ncRNA Class Up Down Up Down Up Down
lncRNAs 55 136 53 130 14 68
miRNAs 0 5 4 1 0 1
snRNAs 1 2 3 1 1 1
snoRNAs 2 2 4 1 2 1
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