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Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

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In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. Here, we characterize the Drosophila tuSz 1 mutant strain, which mounts an aberrant immune response against its own fat body. We demonstrate that this autoimmunity is the result of two mutations: 1) a mutation in the GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Our data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals.
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Extracellular matrix protein N-glycosylation mediates
immune self-tolerance in Drosophila melanogaster
Nathan T. Mortimer
a,1
, Mary L. Fischer
a
, Ashley L. Waring
a
, Pooja KR
a
, Balint Z. Kacsoh
b
, Susanna E. Brantley
c
,
Erin S. Keebaugh
d
, Joshua Hill
a
, Chris Lark
a
, Julia Martin
a
, Pravleen Bains
a
, Jonathan Lee
a
,
Alysia D. Vrailas-Mortimer
a
, and Todd A. Schlenke
e
a
School of Biological Sciences, Illinois State University, Normal, IL 61790;
b
Epigenetics Institute, Department of Cell and Developmental Biology, University
of Pennsylvania, Philadelphia, PA 19104;
c
Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305;
d
Department
of Biology, Emory University, Atlanta, GA 30322; and
e
Department of Entomology, University of Arizona, Tucson, AZ 85719
Edited by Ruslan Medzhitov, Yale University, New Haven, CT, and approved July 26, 2021 (received for review August 17, 2020)
In order to respond to infection, hosts must distinguish pathogens
from their own tissues. This allows for the precise targeting of
immune responses against pathogens and also ensures self-tolerance,
the ability of the host to protect self tissues from immune damage.
One way to maintain self-tolerance is to evolve a self signal and
suppress any immune response directed at tissues that carry this sig-
nal. Here, we characterize the Drosophila tuSz
1
mutant strain, which
mounts an aberrant immune response against its own fat body. We
demonstrate that this autoimmunity is the result of two mutations: 1)
amutationintheGCS1 gene that disrupts N-glycosylation of extra-
cellular matrix proteins covering the fat body, and 2) a mutation in
the Drosophila Janus Kinase ortholog that causes precocious activa-
tion of hemocytes. Our data indicate that N-glycans attached to ex-
tracellular matrix proteins serve as a self signal and that activated
hemocytes attack tissues lacking this signal. The simplicity of this in-
vertebrate self-recognition system and the ubiquity of its constituent
parts suggests it may have functional homologs across animals.
innate immunity
|
self-recognition
|
self-tolerance
|
autoimmunity
|
protein N-glycosylation
Immune systems have evolved to protect organisms from infec-
tion by a range of pathogens (1). One of the defining charac-
teristics of host immunity is the ability to discriminate healthy self
tissue from nonself pathogens. There are at least two mechanisms
by which a host can distinguish its own tissue from that of a
pathogen (2, 3): In nonself recognition, the host deploys receptors
that recognize pathogen-specific molecules; when these receptors
bind their ligands, the pathogen is identified and host immune
mechanisms are triggered. In self recognition, the host deploys
receptors that recognize host-specific molecules; when these re-
ceptors bind the corresponding self ligand, a self tissue is identified
and host immune mechanisms are repressed, a process known as
self-tolerance. Self-recognition is important in protecting self tis-
sue during an immune response and for disposing of diseased or
damaged self tissue.
Blood cells, the unique mobile immune cells of animals, often
act as sentinels of infection and control whether immune re-
sponses are mounted against different tissues (2, 4). Our under-
standing of immune cell function is dominated by examples of
nonself recognition (57). For example, invertebrate blood cells
and most cells from the vertebrate myeloid lineage (i.e., cells re-
sponsible for innate immunity) identify nonself pathogens using
pattern recognition receptors (PRRs), which recognize conserved
pathogen-associated molecular patterns (PAMPs) such as viral
double-stranded RNAs (dsRNAs), bacterial lipopolysaccharides
and peptidoglycans, and fungal β-glucans (812). However, these
cells also contribute to self-recognition (3), allowing hosts to rec-
ognize self tissues and suppress autoimmunity. Although much of
the research uncovering mechanisms of recognition and self-
tolerance has focused on adaptive immune mechanisms (5, 7), a
growing body of work has demonstrated the importance of innate
immune mechanisms in the suppression of autoimmunity (1318).
Successful innate-mediated self-tolerance requires that nonim-
mune tissues produce and display specific self-associated molec-
ular patterns (SAMPs; ref. 19) and that blood cells recognize
SAMPs in these tissues and suppress proimmune signaling. A loss
of self-tolerance occurs when innate immune cells can no longer
recognize SAMPs, when target tissues are unable to display
SAMPs, or when the recognition of self is insufficient to restrain
immunity. The self-tolerance mechanism can also act as an initi-
ator of immunity itself if failure to recognize a self signal on the
pathogen surface triggers an immune response, in what is known
as missing-self recognition(20, 21). It has been proposed that
glycans (complex sugar groups) are likely candidates for SAMP
signals given that they dominate cell surfaces and extracellular
matrices and can be very diverse (19), although the general
mechanisms underlying innate immune-mediated self-tolerance
and missing-self recognition remain largely unexplored.
The fruit fly Drosophila melanogaster has proven an excellent
model for the study of conserved innate immune mechanisms
(2227). Flies are infected by a wide range of pathogens and, being
invertebrates, lack a classical adaptive immune response, allowing
for the study of innate immune mechanisms in isolation. In Dro-
sophila, circulating hemocytes (blood cells) known as plasmato-
cytes actively surveil the hemocoel (body cavity) for damaged
tissues and invading pathogens (4), showing they are capable of
self/nonself discrimination. These macrophage-like immune cells
Significance
The ability of immune cells to distinguish self tissue from nonself
pathogens is a key characteristic of immunity, allowing re-
sponses to be targeted against invading pathogens while pro-
tecting against self-directed immune damage. The recognition of
nonself by innate immune cells has been extensively character-
ized, but the mechanisms that allow for self recognition and self-
tolerance remain largely unexplored. Here, we uncover a self-
tolerance system in Drosophila that relies on the N-glycosylation
of extracellular matrix proteins: immune activity is restrained by
recognition of a self signal and proceeds when encountering self
tissues missing the self signal. This allows the host to recognize
and protect self tissues, destroy aberrant tissue, and, perhaps,
respond to pathogens that evade nonself recognition systems.
Author contributions: N.T.M., M.L.F., and T.A.S. designed research; N.T.M., M.L.F., A.L.W.,
P.K., B.Z.K., S.E.B., E.S.K., J.H., C.L., J.M., P.B., J.L., and A.D.V.-M. performed research;
N.T.M. and T.A.S. analyzed data; and N.T.M. and T.A.S. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
This open access article is distributed under Creative Commons Attribution-NonCommercial-
NoDeriv atives License 4.0 (CC BY-NC- ND).
1
To whom correspondence may be addressed. Email: ntmorti@ilstu.edu.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/
doi:10.1073/pnas.2017460118/-/DCSupplemental.
Published September 20, 2021.
PNAS 2021 Vol. 118 No. 39 e2017460118 https://doi.org/10.1073/pnas.2017460118
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are responsible for phagocytosis of micropathogens (e.g., bacteria)
and encapsulation of macroparasites, during which thousands of
blood cells attack large foreign objects (e.g., parasitoid wasp eggs).
Drosophila phagocytic responses are genetically conserved with
those of vertebrates (28, 29), and the insect encapsulation process
is functionally related to the production of granulomas in verte-
brates, during which natural killer (NK) cells and other blood cells
attack, and occasionally melanize, large foreign tissues (3034).
Thus, Drosophila might also serve as a model for immune self-
tolerance if potential self-recognition systems (including SAMP
production or SAMP recognition) could be targeted and studied.
Here, we focus on Drosophila mutants that mount misdirected
immune responses against their own tissues. Surprisingly, nu-
merous classical Drosophila mutant strains known as melanotic
tumor mutants, in which blood cells attack and encapsulate ab-
errant self tissues due to a deficiency in self-tolerance, were
never fully genetically characterized (3540). Of those still
available in public stock collections (SI Appendix, Table S1), we
decided to characterize the D. melanogaster tumor(1)Suzuki
(tuSz) melanotic tumor strain, which was isolated in a large-scale
temperature-sensitive mutagenesis screen (41, 42). This mutant
displays a temperature-sensitive self-encapsulation phenotype
directed at its own posterior fat body tissue (37). We demon-
strate that the tuSz
1
mutant phenotype is the result of two dis-
tinct mutations: 1) a loss of function mutation in the GCS1 gene,
which disrupts the protein N-glycosylation pathway in the pos-
terior fat body, and 2) a gain-of-function mutation in the hop-
scotch gene (hop), the Drosophila Janus Kinase (JAK) ortholog,
which causes precocious activation of immune cells. Our results
demonstrate that N-glycosylated extracellular matrix (ECM)
proteins serve as SAMPs and that activated innate immune cells
attack tissues that lack these SAMPs.
Results
The tuSz
1
Phenotype Results from a Self-Directed Immune Response.
In accordance with previous work (37), we find that tuSz
1
mutants
display a temperature-sensitive phenotype characterized by the
melanization of posterior fat body tissue. This phenotype first ap-
pears during late larval development (third instar), and the mela-
nized tissue persists throughout the life of the fly (Fig. 1 AC). This
is morphologically reminiscent of the melanotic encapsulation of a
parasitoid wasp egg (Fig. 1D). Drosophila larvae and pupae are
regularly infected by parasitoid wasps in nature (4345), and their
blood cells attack the wasp eggs (46, 47). In this encapsulation
response, plasmatocytes become activated and adhere to the sur-
face of the parasitoid egg (48, 49). Immune stimulation also trig-
gers the production of specialized flattened immune cells known as
lamellocytes (50, 51), which adhere to the plasmatocyte cell layer to
form a multicellular, multilayered capsule around the parasitoid
egg. The capsule is consolidated and melanized, and free radicals
are released into the capsule, leading to parasitoid death (49, 52,
53). The morphological similarity between melanization of self fat
body tissue and wasp eggs suggested that the tuSz
1
mutant phe-
notype may represent a self-directed immune response.
To test whether the tuSz
1
melanization phenotype results from a
melanotic encapsulation of self tissue, we used the eater-eGFP and
msn-mCherry reporter lines to mark plasmatocytes and lamello-
cytes, respectively, in control and tuSz
1
mutant backgrounds. We
find that both plasmatocytes (Fig. 1F) and lamellocytes (Fig. 1G)
adhere to, and form a capsule around, fat body tissue that is un-
dergoing melanization in tuSz
1
mutant larvae, mirroring their role
in the encapsulation of parasitoid wasp eggs (52, 53). By contrast,
there are minimal interactions between plasmatocytes and fat
body tissue in the control background (SI Appendix,Fig.S1), and
lamellocytes are rarely observed in naïve wild-type larvae (54).
This suggests that the tuSz
1
phenotype arises from a loss of self-
tolerance leading to the autoimmune targeting of self tissue by the
encapsulation response.
A similar self-encapsulation response in Drosophila was pre-
viously linked to the loss of ECM (55), leading to the hypothesis
that self-tolerance is mediated by a SAMP associated with the
ECM. To test the presence of the ECM in tuSz
1
mutants, we
expressed GFP fused to either Laminin A (LanA) or Viking
(Vkg) in the tuSz
1
background. LanA and Vkg are Drosophila
orthologs of subunits of the major structural ECM proteins
laminin and collagen, respectively (56), so imaging GFP fusions
with either protein allows us to assay the state of the ECM in
tuSz
1
mutants. In the mutants, the posterior fat body, including
sites of self-encapsulation, is covered by laminin (Fig. 1 HJ) and
collagen (SI Appendix, Fig. S2), demonstrating that the ECM is
intact in the mutant background. This is also observed in the
melanotic tumor mutant tu(2)bw (57), suggesting that, while
necessary, ECM integrity may not be sufficient for self-tolerance.
We therefore hypothesize that the self-tolerance signal may be a
specific posttranslational modification of one or more ECM
proteins and that this modification is disrupted in tuSz
1
mutants.
To understand the mechanism mediating self-tolerance in
Drosophila, we began by further characterizing the tuSz
1
mutant
phenotype. The tuSz
1
mutation was previously mapped to the X
chromosome (37, 58), so we first tested whether the phenotype
was influenced by the sex of the fly. We find no difference in the
penetrance of the self-encapsulation (melanized posterior fat
Fig. 1. The tuSz self-encapsulation phenotype. (AC)IntuSz
1
mutants at 28
°C, posterior fat body tissues are melanized during the third-instar larval
stage (A), and the melanization persists through the pupal (B) and adult
stages (C). The self-encapsulation phenotype is morphologically similar to
the encapsulation of a Leptopilina clavipes (parasitoid wasp) egg by a w
1118
control larva (D). Brightfield (E), eGFP (F), and mCherry (G) images of fat
bodies dissected from tuSz
1
, eater-eGFP, msn-mCherry larvae demonstrate
that plasmatocytes (marked by eGFP expression) and lamellocytes (marked
by mCherry expression) interact with fat body tissue that is undergoing
melanization. Brightfield (H), GFP (I), and merged (J) images of fat bodies
dissected from tuSz
1
; LanA-GFP larvae show that self-encapsulated fat body
tissue nevertheless maintains intact ECM (marked by GFP expression).
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https://doi.org/10.1073/pnas.2017460118 Extracellular matrix protein N-glycosylation mediates immune self-tolerance in
Drosophila melanogaster
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body) phenotype between hemizygous male and homozygous
female flies (SI Appendix, Table S2), so we pooled the sexes for
further analyses. As previously described (37), we find that the
tuSz
1
phenotype is temperature sensitive, with homozygous mu-
tant flies displaying a high penetrance of self-encapsulations at
the restrictive temperature of 28 °C but not at the permissive
temperature of 18 °C (Fig. 2Aand SI Appendix, Table S2). We
additionally find that heterozygous female flies do not display
the self-encapsulation phenotype at 28 °C (Fig. 2Aand SI Ap-
pendix, Table S2), demonstrating that this phenotype is recessive.
The Hemocyte Population Is Altered in tuSz
1
Mutants. This self-
encapsulation phenotype is morphologically similar to the Dro-
sophila encapsulation response against wasp eggs. The encapsula-
tion response is characterized by an increase in hemocyte numbers
and the differentiation of lamellocytes (46, 5961), so we tested
whether the self-encapsulating tuSz
1
mutants showed similar al-
terations in the numbers or ratios of different hemocyte pop-
ulations. We counted the circulating hemocyte subtypes in tuSz
1
and control w
1118
(both strains derived from the wild-type Oregon R
strain) larvae at 18 °C and 28 °C. We find that plasmatocyte counts
are elevated in tuSz
1
mutant larvae raised at 18 °C relative to w
1118
controls raised at 18 °C (Fig. 2B), suggesting that tuSz
1
mutants
have a significantly increased plasmatocyte population. However,
circulating plasmatocyte counts are significantly decreased in tuSz
1
mutants relative to w
1118
controls at 28 °C (Fig. 2B). This difference
is likely due to the fact that, in tuSz
1
mutants at 28 °C, the immune
cells are actively participating in the self-encapsulation response
(Fig. 1F) and are sequestered out of circulation as they adhere to
the developing capsule.
Lamellocytes are Drosophila immune cells that are usually only
produced during an encapsulation response, and their numbers are
significantly increased in tuSz
1
mutant larvae at both temperatures
(Fig. 2C), in agreement with previous findings (37). Notably, these
data functionally separate the production of lamellocytes (occur-
ring at both 18 °C and 28 °C) from the self-encapsulation response
(28 °C only) and suggest that immune cell alterations and self-
encapsulation penetrance are genetically separable aspects of the
tuSz
1
phenotype. To further test the genetic basis of the immune
cell phenotype, we assayed lamellocyte number in heterozygous
(tuSz
1
/w
1118
) flies at 28 °C and found that heterozygous flies have
significantly more lamellocytes than control flies (Fig. 2C),
Fig. 2. Phenotypic characterization of tuSz flies. (A) Penetrance of the self-encapsulation phenotype of w
1118
(control), tuSz
1
, and tuSz
1
/w
1118
heterozygous
flies raised at the indicated temperature. *P<0.05 compared to w
1118
at 28 °C. (BE) Plasmatocyte number (B), lamellocyte number (C), podocyte number (D),
and crystal cell number (E) of the indicated genotypes at the indicated temperatures. *P<0.05 compared to w
1118
at each temperature. Pvalues for Aand C
were determined by Dunnetts test. Pvalues for B,D, and Ewere determined by Welchs two-sample ttest.
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demonstrating that this phenotype is dominant. Lamellocytes can be
produced directly from hemocyte precursors in the lymph gland
(the Drosophila hematopoietic organ) or via the transdifferentiation
of circulating or sessile plasmatocyte populations (50, 59, 60). The
production of lamellocytes within the lymph gland leads to the
dispersal of the lymph gland lobes (50, 61), and we find that
lymph glands are dispersed in tuSz
1
mutant larvae at both tem-
peratures (SI Appendix, Fig. S3). Additionally, podocytes, also
known as prelamellocytes, are plasmatocyte-like cells with fine
cytoplasmic filaments extending outward from the cell body and
are hypothesized to represent an intermediate step when circu-
lating plasmatocytes transdifferentiate into lamellocytes during a
macroparasite immune challenge (54, 59, 62, 63). There is a
significant increase in the production of podocytes in tuSz
1
mu-
tants, but only at 28 °C (Fig. 2D), perhaps contributing to the
production of additional lamellocytes in the restrictive condition.
These findings suggest that lamellocytes are produced via both
routes in tuSz
1
mutants.
Finally, crystal cells are a Drosophila immune cell type in-
volved in the melanization of wounds, and their numbers are
significantly decreased in tuSz
1
mutant larvae at both tempera-
tures (Fig. 2E). The decrease in crystal cells in the absence of
self-encapsulation at 18 °C suggests that this decrease is not due
to the participation of crystal cells in melanotic encapsulation.
We speculate that the loss of crystal cells may result from
changes in signal transduction during the specification of the
shared hemocyte precursor cells in the lymph gland, away from
crystal cell production and to lamellocyte production, a process
known to be regulated by the Notch and JAK-STAT signal
transduction pathways (6467).
The tuSz
1
Phenotype Is Caused by Two Distinct Mutations. Overall,
these phenotypic characterization data lead to the hypothesis
that the tuSz
1
mutant phenotype is dependent on two distinct
mutations: One is a nonconditional, dominant mutation that
results in an alteration in the population of circulating hemo-
cytes, including the production of lamellocytes, which are a
hallmark of the Drosophila encapsulation response. The other is
a temperature-sensitive, recessive mutation that results in an
alteration of the posterior fat body tissue, making it a target of
this activated immune cell response. This second mutation is
likely to affect a gene required for production of a Drosophila
SAMP that serves as a self-tolerance signal.
The tuSz
1
mutation was isolated in a temperature-sensitive
mutagenesis screen (41) and mapped to the X chromosome
(42). Subsequent mapping efforts using meiotic recombination
mapping and complementation testing with large genomic de-
letions mapped the tuSz
1
mutation to a 572-kb region of the X
chromosome containing 80 predicted genes, but despite strong
efforts, the genetic basis of the phenotype was not identified (37,
58). Since that time, the Drosophila genome has been sequenced
and annotated, the protein products of many genes in the
mapped region have been characterized, and mutant strains have
been developed for many of these genes. Furthermore, numer-
ous molecularly defined chromosomal aberration collections
have become available in Drosophila (6872), which enables fine
scale mapping of many classic mutations that had not been
previously localized to the genome (73). We took a candidate
gene approach as well as a mapping approach that makes use of
complementation testing using defined genomic deletions and
duplications. Along with targeted genomic DNA sequencing, we
were able to more precisely map and identify the mutations
leading to the loss of self-tolerance in tuSz
1
mutants.
The Dominant Mutation in tuSz
1
Is a Gain-of-Function Mutation in
hop.The tuSz
1
mutant locus includes hop,theDrosophila homo-
log of JAK, a central member of the JAK-STAT pathway.
Gain-of-function mutations in hop have been demonstrated to
increase hemocyte numbers and cause differentiation to the
lamellocyte type (74, 75). Additionally, JAK-STAT pathway activity
is linked to the melanotic encapsulation immune response (76, 77).
We therefore hypothesized that a mutation in hop may be the
dominant mutation causing constitutive hemocyte production as
part of the self-encapsulation phenotype in tuSz
1
mutant flies.
Sequencing the hop gene from the tuSz
1
strain reveals that the
hop
Sz
allele contains a missense mutation in the JH2 pseudokinase
domain, converting the aspartic acid at position 682 to an aspar-
agine (D682N; Fig. 3A). The JH2 domain is conserved across
species and is required for the autoinhibition of JAK when the
pathway is inactive (78). Mutations in the JH2 domain lead to
gain-of-function JAK activity and are linked to human diseases
including multiple forms of leukemia and other cancers (7982).
We predict that the D682N missense mutation may disrupt JH2
domain function and result in ectopic pathway signaling, as this
residue only shows conservative substitutions between species,
with glutamic acid found at the corresponding position in the
human JAK2 protein (Fig. 3A). To test this prediction, we assayed
JAK-STAT pathway activity using the Stat92E-GFP reporter (83).
This reporter expresses GFP under the control of the Stat92E
transcription factor, and GFP fluorescence intensity gives a rela-
tive readout of pathway activity. The reporter is activated in he-
mocytes from tuSz
1
; Stat92E-GFP mutants but not control
Stat92E-GFP larvae (Fig. 3 BE), suggesting that the hop
Sz
allele
encodes a gain-of-function Hop protein. To test the hypothesis
that the hop
Sz
allele is dominant, we imaged the Stat92E-GFP
reporter in heterozygous tuSz
1
/+; Stat92E-GFP larvae. The re-
porter is activated in this heterozygous background (SI Appendix,
Fig. S4), supporting the dominant nature of the hop
Sz
allele.
To test whether this ectopic activation of the JAK-STAT path-
way is an important contributor to the tuSz
1
phenotype, we ge-
netically decreased the dosage of Stat92E in tuSz
1
mutants and
assayed the self-encapsulation phenotype. Loss of one copy of
Stat92E in tuSz
1
mutant larvae is sufficient to suppress the mutant
phenotype in tuSz
1
;Stat92E
06346
/+ larvae (Fig. 3Fand SI Appendix,
Table S2). To validate this finding and test the tissue specificity of
the role of Stat92E in the tuSz
1
phenotype, we crossed UAS-
Stat92E
RNAi
with tuSz
1
; msn-GAL4 flies to knock down Stat92E
specifically in lamellocytes in the tuSz
1
mutant background. Once
again, we observe a significant decrease in penetrance of the self-
encapsulation phenotype relative to the UAS-GAL4
RNAi
control
cross (Fig. 3Gand SI Appendix,TableS2), suggesting that ectopic
JAK-STAT activation in immune cells plays a role in the produc-
tion of the self-encapsulation phenotype.
Importantly, gain-of-function JAK-STAT signaling alone is in-
sufficient for the self-encapsulation phenotype. The encapsulation
of self tissue is not observed in larvae carrying the widely studied
hop
Tum
gain-of-function mutation (Fig. 3 Hand I), even though
the hop
Tum
and hop
Sz
mutations both lead to the production of
excess plasmatocytes and lamellocytes (Fig. 2, refs. 74 and 75).
Whereas in tuSz
1
mutants the lamellocytes are mainly observed on
the surface of posterior fat body tissue (arrow in Fig. 3H), the
excess lamellocytes in hop
Tum
are dispersed in circulation, occa-
sionally forming small melanized nodules by adhering to each
other (arrow in Fig. 3I). This suggests that the hop
Sz
allele may
serve as a potentiating factor for the loss of self-tolerance, but
there is likely a second mutation responsible for targeting of hop
Sz
lamellocytes to the posterior fat body.
A Conditional, Recessive Loss-of-Function Allele of GCS1 Disrupts
Protein N-glycosylation in tuSz
1
Mutants. To uncover the genetic
basis of the loss of self-tolerance phenotype observed in tuSz
1
mutants, we used molecularly defined chromosomal aberrations to
map the conditional recessive mutation within the previously de-
fined X chromosome region. Five X chromosome deletion strains
that, in combination, uncover the entirety of the previously
mapped 572 kb tuSz
1
locus were tested for their ability to
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https://doi.org/10.1073/pnas.2017460118 Extracellular matrix protein N-glycosylation mediates immune self-tolerance in
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complement the tuSz
1
self-encapsulation phenotype (Fig. 4A).
Three of these deletions fail to complement tuSz
1
,whiletwode-
letions do complement tuSz
1
, with the overlap defining a 24.4-kb
chromosomal region responsible for the self-encapsulation phe-
notype (Fig. 4A). To further narrow down the location of the self-
tolerance locus, we tested whether defined X chromosome ge-
nomic duplications (with small regions of the X chromosome
translocated to the third chromosome) (71) within the mapped
region could rescue the self-encapsulation phenotype in homozy-
gous mutant tuSz
1
flies. Of the seven X chromosome duplication
strains tested, two rescue the self-encapsulation phenotype, while
five fail to rescue, and together, these narrowed the self-tolerance
locus to an even smaller 10.2-kb region containing five candidate
genes (Fig. 4A).
Note that the hop gene is located within 15 kb of the tuSz
1
locus and that in our mapping experiments, we observed that the
DC237 duplication, which includes the newly defined tuSz locus
but excludes hop, was only able to partially rescue the self-
encapsulation phenotype. However, the DC238 duplication,
which includes both the tuSz and hop loci, rescued the phenotype
nearly to control levels (Fig. 4B). This provided additional ex-
perimental evidence that hop
Sz
, along with one other distinct
mutation, are both required for the full tuSz
1
self-encapsulation
phenotype.
We find that two EMS-generated recessive lethal mutations in
the GCS1 gene (84), one of the five genes found within the newly
defined tuSz
1
locus, fail to complement the tuSz
1
self-
encapsulation phenotype (Fig. 4C). Sequencing of the GCS1 lo-
cus from tuSz
1
, including 2 kb up- and downstream (Dataset S1),
reveals 30 sequence variants compared to the D. melanogaster
reference genome (SI Appendix,TableS3). Of these sequence
changes, 27 are observed in the Drosophila Genetics Reference
Panel (85), suggesting they may reflect standing variation in nat-
ural Drosophila populations. To further assess the potential
functional significance of these sequence variants, we sequenced
the GCS1 locus from the w
1118
control strain (Dataset S1), which
complements the tuSz
1
self-encapsulation phenotype (Fig. 2A).
Except for one mutation that is unique to tuSz
1
,thetuSz
1
and
w
1118
GCS1 sequences are identical, suggesting that this lone
mutation may be the causative mutation in tuSz
1
.
The identified mutation in tuSz
1
(referred to as GCS1
Sz
)isa
C-to-T transition 35 bp upstream of the transcription start site of
GCS1. This sequence change may alter transcription factor
binding sites identified in high-throughput transcription factor
mapping projects (8688) (Dataset S2) and falls within an
identified transcription factor hotspot (89), suggesting that gene
expression may be affected in tuSz
1
mutants. To test this, we
stained fat body tissues from early third-instar control w
1118
and
tuSz
1
mutant larvae raised at 28 °C with an α-GCS1 antibody. We
find α-GCS1 strongly stains control posterior fat body tissues
(Fig. 5 Aand B); however, α-GCS1 staining is largely undetect-
able in posterior fat body tissue from early third-instar tuSz
1
mutants (Fig. 5 Cand D). Importantly, α-GCS1 staining is
maintained in anterior fat body tissue in tuSz
1
mutants at 28 °C
(SI Appendix, Fig. S5 Aand B), mirroring the restriction of the
self-encapsulation phenotype to posterior fat body tissue. We
hypothesize that the GCS1
Sz
mutation alters an upstream en-
hancer that regulates GCS1 expression in the posterior fat body
and that the mutation results in the temperature-sensitive dis-
ruption of gene expression. Interestingly, while plasmatocytes
are responsible for coating other fly larval tissues with ECM, the
fat body secretes its own ECM (9092), which is consistent with
Fig. 3. The role of JAK-STAT signaling in tuSz
1
mutants. (A) An amino acid sequence alignment within the JH2 domain of Hop and the Hop orthologs
GA14036 from Drosophila pseudoobscura and JAK2 from humans shows the missense D. melanogaster Hop
Sz
allele. (BE) Brightfield (B) and GFP (C) images
of immune cells from Stat92E-GFP larvae raised at 28 °C show no JAK-STAT pathway activity. Brightfield (D) and GFP (E) images of immune cells from tuSz
1
;
Stat92E-GFP larvae raised at 28 °C show JAK-STAT pathway activity. (F) Penetrance of the self-encapsulation phenotype in tuSz
1
and tuSz
1
;Stat92E
06346
/+ flies
raised at 28 °C. *P<0.05 compared to tuSz
1
.(G) Penetrance of the self-encapsulation phenotype in tuSz
1
; msn-GAL4; UAS-GAL4
RNAi
and tuSz
1
;msn-GAL4;
UAS-Stat92E
RNAi
flies raised at 28 °C. *P<0.05 compared to tuSz
1
; msn-GAL4; UAS-GAL4
RNAi
.Pvalues were determined by generalized linear models. (Hand I)
Lamellocytes (marked by mCherry expression) aggregate around large self-encapsulations in tuSz
1
, msn-mCherry larvae (arrow in H) but are dispersed
throughout hop
Tum
, msn-mCherry larvae (I). Tissue self-encapsulations are not seen in hop
Tum
, msn-mCherry larvae, but small melanized nodules are common
(arrow in I).
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the idea that the GCS1 locus might contain a fat-bodyspecific
enhancer that is mutated in GCS1
Sz
.
GCS1 encodes a mannosyl-oligosaccharide glucosidase, an en-
zyme required in the protein N-glycosylation pathway (93, 94). To
test whether decreased GCS1 expression has a functional outcome
in tuSz
1
mutants, we incubated fat body tissues dissected from early
third-instar w
1118
and tuSz
1
larvae with fluorescein isothiocyanate
(FITC)-conjugated wheat germ agglutinin (WGA), a lectin that
specifically recognizes N-glycosylated proteins (95). The magnified
surfaces of fat body tissue from w
1118
control larvae grown at both
18 °C and 28 °C show WGA binding displaying the characteristic
honeycomb pattern of neighboring fat body cells (Fig. 5 Eand F),
indicating that fat body ECM proteins are N-glycosylated. tuSz
1
mutants display a temperature-dependent loss of this WGA bind-
ing, with binding lost from posterior (but not anterior) fat body
tissue in larvae grown at 28 °C (Fig. 5 Gand Hand SI Appendix,
Fig. S5C). The loss of α-GCS1 staining and WGA binding are
observed in tuSz
1
fat body tissue from younger fly larvae prior to
the onset of melanization. This suggests that loss of ECM protein
N-glycosylation renders tissues susceptible to self-encapsulation,
not that loss of staining is a consequence of tissue melanization.
Furthermore, the loss of WGA reactivity is distinct from the effects
of hop gain-of-function mutations: WGA staining is observed in
heterozygous tuSz
1
/w
1118
larvae (Fig. 5I)andhop
Tum
mutant larvae
(Fig. 5J), both of which have activated immune cells with no self-
encapsulation. These findings are consistent with the maintenance
of self-tolerance in these genotypes (Figs. 2Aand 3I) and suggest
that N-glycosylated ECM proteins may be recognized as SAMPs to
promote self-tolerance.
ECM Protein N-glycosylation Is a Self-Tolerance Mark in Drosophila.
To test the hypothesis that ECM protein N-glycosylation plays a
role in self-tolerance in Drosophila, we disrupted protein
N-glycosylation in gain-of-function hop backgrounds and assayed
for self-encapsulation. First, we used a UAS- GCS1
RNAi
construct in
the tuSz
1
background to knock down GCS1 levels specifically in fat
body tissue by crossing to the c833-GAL4 driver. As previously
discussed, the tuSz
1
self-encapsulation phenotype is temperature
sensitive and only observed at the restrictive temperature of 28 °C
(Fig. 2A), whereas the JAK-STAT gain-of-function phenotype was
seen at all temperatures. Taking into account the temperature-
dependent nature of the GAL4/UAS expression system (96), in
which knockdown is more efficient at higher temperatures, we
raised larvae at 25 °C, at which we predict that tuSz
1
larvae should
have activated JAK-STAT signaling but no self-encapsulation re-
sponse. We find that knockdown of GCS1 in c833-GAL4; UAS-
GCS1
RNAi
larvae at 25 °C results in decreased fat body WGA
binding compared to the c833-GAL4; UAS-GAL4
RNAi
knockdown
control (Fig. 6 Aand B), although to a lesser degree than tuSz
1
mutants at 28 °C (compare Figs. 5Eand 6B). We do not, however,
observe the self-encapsulation phenotype when GCS1 is knocked
down in fat body tissue in this otherwise wild-type background
(n>100), which is consistent with our data suggesting that the hop
Sz
gain-of-function allele is required for the self-encapsulation phe-
notype. We also do not observe the self-encapsulation phenotype in
tuSz
1
;c833-GAL4; UAS-GAL4
RNAi
control knockdown flies raised
at 25 °C, confirming that 25 °C is a permissive temperature for the
temperature-sensitive self-encapsulation phenotype. However, we
do see a strong self-encapsulation phenotype in tuSz
1
;c833-GAL4;
Fig. 4. Mapping the hypothesized tuSz
1
self-tolerance mutation. (A) Sche-
matic demonstrating the mapping of the tuSz
1
recessive mutation on the X
chromosome. Lines are listed by strain name. The hop gene locus is indicated
on the chromosome. (Top) Interrupted segments illustrate the location of
genome deletions in the indicated Drosophila strains. Black lines indicate
deletions that fail to complement the tuSz
1
self-encapsulation phenotype.
Gray lines indicate complementing deletions. (Middle) Blocks represent du-
plicated regions of the X chromosome. Black blocks indicate duplications
that rescue the tuSz
1
self-encapsulation phenotype, and gray blocks indicate
failure to rescue. (Bottom) The deletion and duplication lines indicate a small
locus for the tuSz
1
SAMP mutation. Candidate genes are shown. (B)
Penetrance of the self-encapsulation phenotype of tuSz
1
flies in combination
with the indicated duplication raised at 28 °C. *P<0.05 for the indicated
comparisons. (C) Penetrance of the self-encapsulation phenotype of tuSz
1
heterozygous flies in combination with w
1118
controls or the indicated GCS1
allele raised at 28 °C. *P<0.05 for the indicated comparisons. Pvalues for B
and Cwere determined by Dunnetts test.
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UAS-GCS1
RNAi
flies raised at 25 °C (Fig. 6 Cand D), providing
further support for our hypothesis that GCS1 is required for self-
tolerance. Given that c833-GAL4 is a panfat body driver, we
expected and observed self-encapsulation of both anterior and
posterior fat body tissues in this genotype.
Second, we tested whether enzymes in the protein N-
glycosylation pathway that function downstream of GCS1 also
play a role in the maintenance of self-tolerance. Specifically, we
assayed if mutations in either of the N-glycanprocessing enzymes
Mgat1 or α-Man-IIb are sufficient to cause a loss of self-tolerance
in a second hop gain-of-function mutant background, hop
Tum
.
hop
Tum
is a temperature-sensitive mutant, and at 28 °C, the mu-
tants display an activated immune response (75, 97). However,
unlike tuSz
1
, this immune activation does not lead to the encap-
sulation of self tissue (Fig. 3I), suggesting that self-tolerance is
maintained. As expected, we find that adult hop
Tum
/+ flies raised
at 28 °C display the characteristic melanotic nodule phenotype
(44.6 ±4.5% of flies have melanized nodules) but very rarely
encapsulate self tissues (Fig. 6E). However, the removal of a single
copy of either Mgat1 (Fig. 6 Eand F)orα-Man-IIb (Fig. 6 Eand
G)inthehop
Tum
/+ background in flies raised at 28 °C leads to a
significant increase in the proportion of adults with the self-
encapsulation phenotype. These findings suggest that the disrup-
tion of the N-glycosylation pathway leads to a loss of self-
tolerance, supporting our hypothesis that ECM protein
N-glycosylation serves as a self-tolerance signal. Interestingly,
though the hop
Tum
and Mgat1/α-Man-IIb mutant combinations
might have led to melanotic encapsulation of other host tissues,
the self-encapsulations we observed in these genotypes are pre-
dominantly localized to fat body tissue, which might be more
sensitive to N-glycosylation processing enzyme dosage than other
tissues given that it secretes its own ECM (9092).
Discussion
A Two-Step Model for Maintaining Self-Tolerance in Drosophila.In
this work, we have investigated the Drosophila tuSz
1
mutant strain.
tuSz
1
is a temperature-sensitive mutant, and at the restrictive
temperature, posterior fat body tissue is melanotically encapsu-
lated by hemocytes in a reaction similar to the antiparasitoid im-
mune response. We found that the tuSz
1
phenotype is caused by
two tightly linked mutations: a nonconditional, dominant
gain-of-function mutation in hop that leads to ectopic immune
activation and a temperature-sensitive, recessive mutation in
GCS1 that leads to loss of protein N-glycosylation of the ECM
overlaying the posterior fat body. These data lead us to propose a
two-step model in which immune activation and the loss of SAMP
presentation/recognition are both necessary for the breakdown of
self-tolerance (Fig. 7). In a naïve wild-type larva, neither condition
is met and self-tolerance is maintained (Fig. 7A). In the case of the
tuSz
1
mutant, the posterior fat body lacks appropriate ECM pro-
tein N-glycosylation and is targeted by constitutively activated
hemocytes for encapsulation (Fig. 7B). This two-step model is also
reflected in the hop
Tum
mutant background, in which the simul-
taneous disruption of N-glycosylation in this immune-activated
background results in tissue self-encapsulation similar to the
tuSz
1
mutant (Fig. 6 EG).
Interestingly, previous work (55) also documented the neces-
sity of at least two signals for self-encapsulation in Drosophila.In
that case, both the loss of the ECM (with its glycosylated pro-
teins) and the disrupted positional integrity of the underlying fat
body cells (potentially mimicking a wound) were required for
immune cells to become activated and encapsulate the self tissue
(Fig. 7C). A similar loss of ECM and underlying cell integrity was
also found in the classical melanotic tumor mutant tu(2)W (98,
99). This model, in which at least two factors are required for
self-encapsulation, may explain why the several classically de-
scribed self-encapsulation mutants (SI Appendix, Table S1), un-
like virtually all other types of visible Drosophila mutants, were
never successfully mapped (38).
The disruption of either factor in the two-step model in iso-
lation is not sufficient to cause self-encapsulation. This can be
seen in parasitoid wasp infected larvae; the wounding associated
with parasitoid infection leads to immune activation (59, 60), but
in the absence of SAMP disruption, the fly is able to specifically
encapsulate the parasitoid egg while protecting against self-
encapsulation (Figs. 1Dand 7D). Conversely, while internal tis-
sue damage in a naïve larva does attract hemocyte interactions,
in the absence of an immune stimulus, this does not lead to self-
encapsulation, but rather the hemocytes attempt to repair the
damaged tissue (100). That blood cells err on the side of fixing
disrupted self tissue rather than treating it as pathogenic and
encapsulating it unless another stress signal is also present sug-
gests that flies may have evolved a multi-input system to safe-
guard against spurious encapsulation (100, 101).
Fig. 5. Fat body GCS1 expression and protein N-glycosylation. (AD) Posterior fat body tissue dissected from early third-instar w
1118
(Aand B) and tuSz
1
(C
and D) larvae raised at 28 °C prior to tissue melanization. (Aand C) Brightfield images of dissected fat body tissue demonstrating the absence of tissue
melanization. (Band D) Corresponding fluorescent images of α-GCS1 antibody staining. (EJ) Magnified posterior fat body tissue dissected from early third-
instar larvae and stained by FITC-WGA to assay protein N-glycosylation. w
1118
fat body from larvae raised at 28 °C (E)or1C(F) is positive for FITC-WGA
staining. tuSz
1
fat body tissue is negative for FITC-WGA at 28 °C (G) but positive at 18 °C (H). tuSz
1
/w
1118
heterozygous (I) and hop
Tum
(J) larvae raised at 28 °C
are positive for FITC-WGA staining, indicating that hop mutations do not affect fat body ECM protein N-glycosylation.
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A Conserved Mechanism of Innate Self-Tolerance? D. melanogaster
immune responses have proventobeanexcellentmodelfor
understanding the mechanisms underlying conserved innate
immune responses, including those of humans (23, 102). Our
findings on Drosophila self-tolerance may also be relevant to
human innate self-tolerance. Indeed, data from a range of
studies are consistent with the idea that protein glycosylation is
a mediator of vertebrate immune responses, and cell-surface
glycans have been proposed as candidate SAMPs for the in-
nate immune response to distinguish healthy self tissues from
aberrant or foreign tissues even if the mechanisms are not
entirely understood (3, 19, 103105). Protein-linked sugar
groups should presumably fit this role well, as they can take on
diverse combinations of sugar residues and branching patterns
(19, 106).
Protein N-glycosylation is a complex multistep process (106,
107) that begins with the addition of a presynthesized glycosyl
precursor to the protein at an asparagine residue. This nascent
glycan is then trimmed back to a core glycan structure, a process
that is initiated by the activity of GCS1 (108). The core glycan is
then elaborated with the addition of multiple carbohydrate
groups to give rise to a variety of final structures, with hybrid and
complex type N-glycans among the most prevalent (109111).
Glycan elaboration begins with the activity of Mgat1, which leads
to the production of hybrid type N-glycans (112). These hybrid
N-glycans can be further processed by the α-mannosidase-I and -II
Fig. 6. Loss of GCS1 leads to loss of self-tolerance. (Aand B) Posterior fat body tissue dissected from larvae raised at 25 °C and stained with FITC-WGA to assay
protein N-glycosylation. Control c833-GAL4; UAS-GAL4
RNAi
tissue is positive for FITC-WGA staining (A). c833-GAL4; UAS-GCS1
RNAi
shows a mosaic loss of FITC-
WGA staining, where * marks fat body cells with decreased staining (B). (C)tuSz
1
; c833-GAL4; UAS-GCS1
RNAi
flies raised at 25 °C show melanized self-
encapsulations (indicated by the arrow). (D) Penetrance of the self-encapsulation phenotype in control tuSz
1
; c833-GAL4; UAS-GAL4
RNAi
and tuSz
1
;c833-GAL4;
UAS-GCS1
RNAi
flies raised at 25 °C. *P<0.05 compared to tuSz
1
; c833-GAL4; UAS-GAL4
RNAi
.(E) Penetrance of the self-encapsulation phenotype in hop
Tum
/+,
hop
Tum
/+;Mgat1
KG02444
/+, and hop
Tum
/+;α-Man-IIb
MI09613
/+ flies raised at 28 °C. *P<0.05 compared to hop
Tum
/+.(F)hop
Tum
/+;Mgat1
KG02444
/+ flies raised at
28 °C show melanized self-encapsulations (indicated by the arrow). (G)hop
Tum
/+;α-Man-IIb
MI09613
/+ flies raised at 28 °C show melanized self-encapsulations
(indicated by the arrow). Pvalues (Dand E) were determined by generalized linear models.
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family of enzymes to produce paucimannose N-glycans, which can
serve as complex N-glycan precursors and are further elaborated
by downstream enzymes to give rise to the final complex N-glycan
structure (113). Our data suggest that disruption of any of the
genes encoding key N-glycanprocessing enzymes will be associ-
ated with the loss of self-tolerance in Drosophila. Similarly, the loss
of the α-mannosidase-II (αM-II)geneinmiceislinkedwiththe
development of an autoimmune phenotype that is likened to
systemic lupus erythematosus (114116). Like the tuSz
1
mutant,
the αM-II mouse phenotype arises due to alterations in protein
N-glycosylation in nonimmune tissues and is mediated by innate
immune cells (115). Altered patterns of protein N-glycosylation
are also observed in additional mouse models of autoimmune
disease (117, 118) and have been linked to autoimmune disease in
human patients (119123).
The ECM is a conserved structure made up of numerous
proteins, many of which are N-glycosylated, including laminin
and collagen (124127). A role for the ECM in mediating self-
tolerance has been previously proposed: The encapsulation of
self tissues in D. melanogaster is also observed following RNA
interference (RNAi) knockdown of genes encoding the ECM
proteins laminin and collagen, supporting the idea that SAMPs
reside in the ECM (55). The role of the ECM in self-tolerance is
further emphasized by tissue transplantation studies in Dro-
sophila.Drosophila larvae are largely tolerant of conspecific tis-
sue transplants, but this tolerance is abolished when tissues are
first treated with collagenase to disrupt the ECM, leading to the
specific encapsulation of treated tissues (128). Reactivity to
ECM proteins is also associated with various forms of human
autoimmune disease (129131). Based on these data, we propose
that the N-glycosylation of ECM proteins may serve as a con-
served self-tolerance signal for innate immune mechanisms and
that loss of ECM protein N-glycosylation may lead to loss of self-
tolerance and, consequently, autoimmune disease in a diverse
range of species.
Glycosylation and Missing-Self Recognition. An alternative means by
which hosts can recognize pathogens is missing-self recognition.
Instead of tracking pathogen diversity with numerous recognition
receptors, as in nonself recognition, missing-self recognition does
not rely on tracking pathogens at all, but only on specifically
recognizing self and attacking tissues that lack the self signal.
Further, unlike germ lineencoded forms of nonself recognition,
missing-self recognition systems allow host species to respond to
novel pathogen types that they have never encountered in their
evolutionary history. Protein glycosylation plays an important role
in the handful of identified missing-self recognition systems of
vertebrates (3, 19, 132135). The most well-known case of missing-
self recognition involves the interaction between vertebrate NK
cells and host MHC class I (MHCI) proteins (136). All vertebrate
cells produce MHCI to display any possible antigens present in
their cytoplasm to T cells, but intracellular pathogens often sup-
press host cell MHCI expression to prevent their molecules from
being displayed (137). NK cells are lymphoid-type cells that induce
cytolysis in infected host cells (138). In an uninfected state, rec-
ognition of properly glycosylated MHCI inhibits NK cell cytolysis
of host cells, but in an infected state in which host cells are missing
the MHCI self signal, the NK cell inhibitory receptors fail to
recognize self,and the infected host cells are lysed, an effective
means of killing intracellular pathogens that have suppressed host
MHC signaling (21, 132, 139, 140).
As of yet, there are no examples of missing-self immune rec-
ognition systems in invertebrates (141, 142), and it has been
hypothesized that invertebrate immune systems rely largely on
PRRs for nonself recognition of pathogens (143). Still, inverte-
brates do mount immune responses against a variety of inani-
mate objects like oil droplets, sterile nylon, and charged
chromatography beads as well as tissue transplants from other
insect species (128, 144147). All of these foreign bodies pre-
sumably lack distinct PAMPs, suggesting that invertebrates have
some sort of missing-self recognition system. Additionally, while
multiple antimicrobial PRRs have been identified in the Dro-
sophila genome (11), PRRs targeting macroparasites like para-
sitoid wasps have not yet been discovered (52). Our model of
self-recognition suggests that following parasitoid infection, ac-
tivated immune cells assess all exposed tissue surfaces for the
self-tolerance glycan signal and that the absence of this Dro-
sophila SAMP on parasitoid wasp eggs might be the cue that
targets them for melanotic encapsulation (Fig. 7D).
Fig. 7. Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naïve wild-type larvae.
(B)IntuSz
1
mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hop
Sz
gain-of-function mutation. The loss of
protein N-glycosylation in posterior fat body tissue due to the GCS1
Sz
mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-
tolerance described by Kim and Choe (55), the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D)
Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting
host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP.
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Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila
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Materials and Methods
Insect Strains. D. melanogaster used in this study were maintained on
standard Drosophila medium on a 12-h lightdark cycle. The D. mela-
nogaster strains and sequence analysis are described in more detail in SI
Appendix,Supplementary Materials and Methods.
Imaging. To determine GCS1 expression in fat body tissue, fat bodies were
dissected from early third-instar larvae in phosphate-buffered saline (PBS),
fixed in 4% paraformaldehyde for 10 min, and permeabilized in PBS + 0.1%
Triton-X100 (PBSTx). Tissues were then blocked in 5% normal goat serum in
PBSTx and stained with the α-GCS1/MOGS antibody (Sigma-Aldrich, Prestige
Antibodies HPA011969) diluted to 1:100 in blocking solution.
To assay tissue protein N-glycosylation, we dissected fat body tissues from
early third-instar larvae in PBS and stained them with 100 mg/mL FITC-
conjugated wheat germ agglutinin (FITC-WGA; Vector Laboratories, FL-
1021) for 3 min at room temperature (53). Stained tissues were washed three
times with Drosophila Ringers solution prior to imaging.
To assay hemocyte localization, whole mount larvae carrying the fluo-
rescent hemocyte marker strain msn-mCherry (lamellocytes) were imaged in
PBS. To assay hemocytefat body interactions, fat body tissue from larvae
carrying the eater-eGFP (plasmatocytes) and msn-mCherry markers were
dissected and imaged under paraffin oil.
Hemocyte Counts. To determine the numbers of different hemocyte subtypes,
five late third-instar larvae were rinsed in Drosophila Ringers solution and
bled into 20 μL PBS + 0.01% phenylthiourea. Hemocytes were transferred to
a disposable hemocytometer (Incyto C-Chip DHC-N01) and allowed to ad-
here for 30 min. Hemocytes from each sample were counted from sixteen
0.25 ×0.25 ×0.1 mm squares. Experiments were performed in triplicate, as
described in ref. 53. The different hemocyte types were identified by their
stereotypical morphologies (148).
Statistical Analysis. To analyze phenotypic penetrance data, we used gen-
eralized linear models with quasibinomial errors for parametric data, and
ANOVA of aligned rank transformed data for comparisons involving geno-
types with a penetrance proportion of 0 or 1. For experiments with multiple
comparisons, Dunnetts test was used for pairwise comparisons to the w
1118
control genotype.
Welchs two-sample ttests were used to compare immune cell count data
between two genotypes. For multiple comparisons, immune cell count data
were analyzed by ANOVA followed by Dunnetts test for pairwise compar-
isons. All statistics were done in the R statistical computing environment
(149) using the multcomp (150) and ARTool (151) packages. Graphs were
produced using the ggplot2 package (152).
Data Availability. All study data are included in the article and/or supporting
information.
ACKNOWLEDGMENTS. We thank two anonymous reviewers, as well as
members of the N.T.M. and T.A.S. laboratories, for detailed and constructive
feedback that greatly improved this manuscript. Research reported in this
publication was supported by NIH Awards R35GM133760 to N.T.M. and
R01AI081879 to T.A.S. Stocks obtained from the Bloomington Drosophila
Stock Center (NIH P40OD018537) were used in this study. The Illinois State
University Confocal Microscopy Facility was funded by NSF Grant DBI-
1828136.
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Multiple sclerosis (MS) is an inflammatory autoimmune disorder affecting the central nervous system (CNS), with unresolved aetiology. Previous studies have implicated N-glycosylation, a highly regulated enzymatic attachment of complex sugars to targeted proteins, in MS pathogenesis. We investigated individual variation in N-glycosylation of the total plasma proteome and of IgG in MS. Both plasma protein and IgG N-glycans were chromatographically profiled and quantified in 83 MS cases and 88 age-and sex-matched controls. Comparing levels of glycosylation features between MS cases and controls revealed that core fucosylation (p = 6.96 × 10 −3) and abundance of high-mannose structures (p = 1.48 × 10 −2) were the most prominently altered IgG glycosylation traits. Significant changes in plasma protein N-glycome composition were observed for antennary fucosylated, tri-and tetrasialylated, tri-and tetragalactosylated, high-branched N-glycans (p-value range 1.66 × 10 −2-4.28 × 10 −2). Classification performance of N-glycans was examined by ROC curve analysis, resulting in an AUC of 0.852 for the total plasma N-glycome and 0.798 for IgG N-glycome prediction models. Our results indicate that multiple aspects of protein glycosylation are altered in MS, showing increased proinflammatory potential. N-glycan alterations showed substantial value in classification of the disease status, nonetheless, additional studies are warranted to explore their exact role in MS development and utility as biomarkers.
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