SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

MUCOSAL IMMUNOLOGY Copyright © 2020

The Authors, some
Specialization of mucosal immunoglobulins rights reserved;
exclusive licensee
in pathogen control and microbiota homeostasis American Association
for the Advancement
occurred early in vertebrate evolution of Science. No claim
to original U.S.
Government Works
Zhen Xu1,2*, Fumio Takizawa1,3*, Elisa Casadei4, Yasuhiro Shibasaki1, Yang Ding1,
Thomas J. C. Sauters4, Yongyao Yu1, Irene Salinas4†, J. Oriol Sunyer1†

Although mammalian secretory immunoglobulin A (sIgA) targets mucosal pathogens for elimination, its interac-
tion with the microbiota also enables commensal colonization and homeostasis. This paradoxical requirement in
the control of pathogens versus microbiota raised the question of whether mucosal (secretory) Igs (sIgs) evolved
primarily to protect mucosal surfaces from pathogens or to maintain microbiome homeostasis. To address this
central question, we used a primitive vertebrate species (rainbow trout) in which we temporarily depleted its
mucosal Ig (sIgT). Fish devoid of sIgT became highly susceptible to a mucosal parasite and failed to develop com-

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pensatory IgM responses against it. IgT depletion also induced a profound dysbiosis marked by the loss of
sIgT-coated beneficial taxa, expansion of pathobionts, tissue damage, and inflammation. Restitution of sIgT levels
in IgT-depleted fish led to a reversal of microbial translocation and tissue damage, as well as to restoration of micro-
biome homeostasis. Our findings indicate that specialization of sIgs in pathogen and microbiota control occurred
concurrently early in evolution, thus revealing primordially conserved principles under which primitive and modern
sIgs operate in the control of microbes at mucosal surfaces.

INTRODUCTION in higher vertebrates or represent instead convergently evolved func-

Mucosal surfaces of vertebrates are inhabited by incredibly dense and
complex populations of commensal microbiota and are also continu-
ously exposed to a myriad of pathogens. Mucosal (secretory) immu-
noglobulins (sIgs) constitute the main humoral adaptive immune
molecules contributing to the homeostasis and defense of mucosal
surfaces (1, 2). In mammals, the central sIg is secretory IgA (sIgA)
(1). sIgA plays two seemingly paradoxical functions: On the one hand,
sIgA targets mucosal pathogens for their neutralization and clear-
ance (1, 3–5), whereas, on the other hand, sIgA interaction with the
microbiota enables commensal colonization, persistence, and homeo-
stasis (6–8). Moreover, sIgA contributes to, but is not essential for,
protection of mucosal surfaces against pathogens, as evidenced by the
very modest increased susceptibility to respiratory and intestinal in-
fections observed in patients with selective sIgA deficiency (sIgAdef)
(9). In contrast, recent studies have shown that sIgA is indispens-
able for the preservation of microbiota homeostasis, because profound
microbiome changes have been reported in IgA-deficient mice and
humans (10–15). Such changes are likely due to the involvement of
IgA in microbiota colonization (6) and to the coating of both bene-
ficial bacteria and pathobionts by sIgA (8, 11, 16). These paradoxical
requirements for sIgA in the control of pathogens versus microbiota
raise the question of whether sIgs initially evolved to protect muco-
sal surfaces from pathogens or arose instead to establish and main-
tain homeostatic relationships with microbiota. Moreover, whether
those roles were independently acquired during the evolution of sIgA
1
Department of Pathobiology, School of Veterinary Medicine, University of Penn-
sylvania, Philadelphia, PA 19104, USA. 2Department of Aquatic Animal Medicine,
College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei 430070, China.
3
Faculty of Marine Science and Technology, Fukui Prefectural University, Obama,
Fukui 917-0003, Japan. 4Center for Evolutionary and Theoretical Immunology (CETI),
Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: sunyer@vet.upenn.edu (J.O.S.); isalinas@unm.edu (I.S.)
tions common to all vertebrate sIgs remains to be determined.
Whereas the role of innate immunity in mucosal host-microbial
pathogenic or mutualistic interactions has often benefited from
studies in invertebrates and lower vertebrates (17, 18), the study of
sIg function in such interactions has almost exclusively been limited
to mammalian sIgA (1, 8). Because teleost fish represent the oldest
living bony vertebrates with an adaptive immune system similar to
that of mammals, fish represent a unique model to understand crit-
ical aspects of Ig evolution and function (19–21). Teleosts have three
bona fide Ig isotypes (i.e., IgM, IgD, and IgT), have B and T lym-
phocytes, and respond to pathogens by inducing antigen-specific
antibody (Ab) (i.e., IgM and IgT) responses (21, 22). However, in
contrast to warm-blooded vertebrates, fish lack organized lymphoid
structures (i.e., lymph nodes and Peyer’s patches), and B cells in fish
do not undergo isotype class switching (22). Up until recently, it was
thought that bony fish were devoid of specialized sIgs. Breaking this
paradigm, we identified teleost IgT as the most ancient Ab class spe-
cialized in mucosal immunity (23). We found that IgT is expressed
by a unique subset of IgT+ B cells that do not express IgD or IgM,
and, analogously to IgA, IgT exists in the serum as a monomer and
in a polymeric form in mucosal secretions (23). We determined that
in contrast to sIgM or sIgD, sIgT was highly induced at mucosal
surfaces by pathogens (23–25). Critically, we also showed that sIgT
is the predominant Ig isotype coating a large portion of the fish micro-
biota (23–25).
To interrogate whether early sIgs emerged primarily to control
pathogens or to establish homeostatic relationships with the micro-
biota, here, we developed a novel teleost fish model in which we
were able to selectively deplete IgT in adult animals. In addition to
representing the only nonmammalian model system devoid of its
main sIg, our IgT-depleted fish also embody the only vertebrate
model in which an sIg can be temporarily depleted during adulthood
rather than permanently from birth (i.e., all existing mammalian

Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020 1 of 17

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
models lacking sIgA). The effects of IgT depletion were analyzed on
the gill mucosa, a bona fide mucosal surface where we have pre-
viously demonstrated the local induction of pathogen-specific IgT
responses (24) that is also known to harbor a rich and diverse micro-
biota (26). Overall, our findings demonstrate that sIgT is required
for the control of mucosal pathogens and for the preservation of
microbiota homeostasis. Thus, our results indicate that pathogen
and microbiota control at mucosal sites are guided by primordially
conserved principles that emerged early in vertebrate evolution and
that are inherent to both primitive and modern sIgs.
RESULTS
Strategy to generate IgT-depleted fish
We previously generated an antitrout IgT monoclonal Ab (mAb)
(23) (a mouse IgG2b mAb) that was exploited here to deplete IgT+
B cells in vivo. The antitrout IgT mAb was used in conjunction with
an anti-mouse IgG2b trout antiserum (TAs) generated by immu-
nizing fish with purified mouse IgG2b (fig. S1A, top). The speci-
ficity of TAs was assessed by enzyme-linked immunosorbent assay
(ELISA) (fig. S1B) and Western blotting (fig. S1C, lane 3). More-
over, flow cytometric analysis using mouse antitrout IgM mAb re-
vealed that the TAs specifically recognized the mouse antitrout IgT
mAb on IgT+ B cells (fig. S1, D and E), but trout control serum
(TCs) did not (fig. S1F). We then tested the capacity of the anti-IgT
mAb to deplete IgT+ B cells in vivo, in combination with the TAs or
TCs (Fig. 1A). At a dose of 10 g per fish, the anti-IgT mAb in com-
bination with the TCs or the TAs depleted 90.6 ± 5.8% and 96.1 ±
4.1% of blood IgT+ B cells, respectively, indicating that addition of
TAs had a minor but significant positive effect when compared
with the addition of TCs in the overall depletion efficiency (Fig. 1B,
left, red dashed box). In contrast, IgT+ B cells were not depleted in
fish treated with 10 g per fish of isotype IgG2b control Ab in combi-
nation with either TCs or TAs (Fig. 1B, left). None of the treatments
affected the percentage of IgM+ B cells (Fig. 1B, right), supporting
the specificity of IgT+ B cell depletion. For the in vivo studies per-
formed thereafter, we used a dose of 25 g per fish of anti-IgT mAb
or its respective isotype control, followed by injection 24 hours later
with 30 l of a pooled TAs. Using these conditions, the percentage
of IgT+ B cell depletion achieved in blood was consistently over
95%, whereas the percentage of IgM+ B cells was not altered (Fig. 1,
C to E). In conclusion, we developed a strategy to selectively and con-
sistently deplete IgT+ B cells that did not alter the percentage of
IgM+ B cells.
IgT+ B cell and IgT depletion persisted for several weeks
in all analyzed organs and fluids, respectively
To evaluate the degree and duration of IgT+ B cell depletion in both
systemic and mucosal lymphoid organs, as well as its effect on se-
rum IgT and gill mucus sIgT levels, we performed a time course
study. When compared with control levels, IgT+ B cells were depleted
to a high degree (87.7 to 97.6%) in all tested tissues from day 1 up
until day 28 after depletion treatment (Fig. 2, A to C, left). Thereafter,
IgT+ B cell numbers started to recover, and by day 56 after depletion
treatment, they were similar to levels in control fish. For all three
tissues tested, the percentage of IgM+ B cells remained unchanged
throughout the treatment, indicating a lack of a compensatory re-
sponse by IgM+ B cells (Fig. 2, A to C, right). The absence of IgT+
B cells in depleted but not control animals was confirmed by immu-
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
nofluorescence microscopy studies (Fig. 2, D to F). Although the
depletion of IgT+ B cells occurred quickly (1 to 3 days), the percent-
age of IgT depletion from serum (Fig. 3A) and gill mucus (Fig. 3D)
did not become significant until day 14 after depletion treatment
(48.8 to 72.6% depletion compared with controls). The percentage
of IgT depletion reached its peak 21 to 28 days after depletion treat-
ment (78.2 to 92.3%), with IgT levels being completely restored by
day 56. In contrast to IgT, serum and gill mucus IgM and IgD levels
remained unchanged at all times after depletion treatment, confirm-
ing a lack of IgM and IgD compensatory responses (Fig. 3, B, C,
E, and F). No mortalities were observed throughout the 56-day pe-
riod in either fish group (fig. S2). In conclusion, the IgT+ B cell de-
pletion treatment induced a near complete depletion of IgT+ B cells
and IgT Ig in all lymphoid tissues and fluids analyzed, respectively,
and did not induce measurable compensatory responses from other
Ig isotypes.
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IgT depletion significantly increased the susceptibility
of fish to Ichthyophthirius multifiliis infection
We have previously shown that trout infected with the mucosal par-
asite I. multifiliis (Ich) generates strong local parasite-specific sIgT
responses in the gill mucosa, whereas parasite-specific IgM re-
sponses are primarily detected in the serum (24). To determine
whether sIgT is critical for protecting the gill epithelium against this
parasite, we first generated immune fish (i.e., fish exposed to three
successive rounds of Ich infection; Fig. 4A). Immune fish were then
divided into two groups: One group underwent IgT depletion treat-
ment (as shown in Fig. 1A), whereas the other (nondepleted group)
was injected instead with isotype control Abs and TAs (Fig. 4A).
After 14 days, both groups were reexposed to the parasite, and
thereafter, Ig responses, pathogen load, and fish mortalities were re-
corded (Fig. 4A). Whereas nondepleted immune fish contained sig-
nificant parasite-­specific sIgT titers in the gill mucus, as expected
(Fig. 4B), the IgT-­depleted immune group did not (Fig. 4C). In contrast,
IgT depletion did not have any effect on the low parasite-­specific
IgM titers in the mucus (Fig. 4, B and C) or the high parasite-specific
IgM titers in the serum (fig. S3, A and B). Critically, the parasite
load increased markedly in the IgT-depleted immune group when
compared with that of nondepleted immune fish (Fig. 4, D and E),
which was consistent with the higher expression of the Ich 18S ri-
bosomal RNA (rRNA) gene in the gills of IgT-depleted immune
fish (Fig. 4F). In addition, upon challenge with Ich, the IgT-depleted
immune group had a significantly higher cumulative mortality when
compared with that of nondepleted immune fish (Fig. 4G). Overall,
these results indicate that sIgT is critical for parasite control at the
gill mucosa.
Decreased sIgT coating of microbiota correlates
with bacterial translocation into the gill tissue
Mammalian sIgA coats and homeostatically regulates microbiota at
mucosal surfaces (8). We have previously shown that sIgT is the
predominant sIg coating the gill microbiota (24). Here, upon IgT
depletion, we observed a remarkable reduction (65.3%) in the per-
centage of sIgT-coated microbiota by day 7 when compared with
control fish (Fig. 5A). This decrease in sIgT coating was maximal
between days 14 and 28 after depletion treatment when coating was
reduced to 81.7 to 86.9% of control levels (Fig. 5A). After day 28, the
percentage of microbiota coated by sIgT started to recover and was
fully restored by day 56 (Fig. 5A), thus mirroring the presence of
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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A Depletion strategy B
30 60
25
Isotype
control Abs 20

(% in lymphocytes)
(% in lymphocytes)
15 40

IgM B cells
IgT B cells
10
OR 5

+
+
4 * 20
Anti-IgT mAbs
2

0 0
1 day 1 day s
As s As
s s s s s s
TC TC
s s TC TA TC TA TC TA
+ +
T
+ +
T TC TA + + + + + +
C C g) g) )+ )+ C C g) g) g) g)
Anti-Ms IgG2b Trout control e- e- g g e- e-
yp yp yp yp ( ( (1 (1
trout antiserum serum ot ot ( (
(1 (1 ot ot gT gT
Is Is gT gT Is Is i-I i-I Ig
T
(TAs) injection (TCs) injection t i-I t i-I Ig
T
IgT t t Ig
T i-
An An ti- ti- An An ti- nt
An An An A
7 days 7 days

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In v iv o d e p le tio n tre a tm e n t In v iv o d e p le tio n treatment
Check % of IgT+ and IgM+ B cells

C D E
Control IgT-depleted
105 34.6 105 42.0 60
Control
150
Control
IgT-depleted

% of B cells (relative to control)

IgT-depleted
***
(% in lymphocytes)

104 104
Anti-IgM

Anti-IgM

40 100
B cells

103 103

16.9 0.06 ***

20 50
102 102

0 0

0 0
0 103 104 105 0 103 104 105 + + + +
IgT IgM IgT IgM
Anti-IgT Anti-IgT
Fig. 1. Development of a trout IgT+ B cell depletion model. (A) Scheme of the strategy used to deplete trout IgT+ B cells. Fish were injected with either isotype control
Abs (mouse IgG2b) or with antitrout IgT mAbs. A day after, half of the fish from both groups were injected with anti-mouse (Ms) IgG2b TAs and the other half with TCs.
Seven days later, the percentage of IgT+ and IgM+ B cells from blood leukocytes of all fish was analyzed by flow cytometry. (B) Percentage of IgT+ (left) or IgM+ B cells (right)
in blood leukocytes from trout injected with isotype control (Isotype-C) Abs (10 g per fish) or with mouse antitrout IgT mAbs (2 or 10 g per fish). Isotype control– and
antitrout IgT–treated groups were thereafter injected with TAs or TCs (n = 10 to 12 fish per group) as depicted in (A). The groups injected with anti-IgT mAb (10 g) are
shown in red dashed box. (C) Flow cytometry of blood leukocytes from control (left) and IgT+ B cell–depleted (right) fish. Numbers adjacent to outlined areas indicate the
percentage of IgM+ cells (top left) or IgT+ cells (bottom right) in the lymphocyte population. (D) The percentage of IgT+ or IgM+ B cells in the lymphocyte population of
control or IgT-depleted fish (n = 10 fish per group). (E) The percentage of IgT+ and IgM+ B cells in the IgT-depleted group relative to that of control fish (means ± SEM;
n = 10 fish). Fish in (C) to (E) were injected with either 25 g of antitrout IgT mAb (IgT-depleted) or 25 g of the isotype control Ab (control), followed by injection with TAs.
Each symbol (B and D) represents an individual fish; small horizontal red lines (B and D) indicate the means (±SEM). Data are pooled from two independent experiments
(B) or representative of two independent experiments (C to E). Statistical analysis was performed by unpaired Student’s t test. *P < 0.05, ***P < 0.001.

sIgT in gill mucus of IgT-depleted fish (Fig. 3D). The percentage of The gills of IgT-depleted fish show signs of tissue damage
sIgM-coated bacteria increased significantly from day 21 after de- and inflammation
pletion when compared with that of control fish, and it remained We next assessed whether the translocation of microbiota into the
significantly elevated throughout the rest of the sampling period gill tissue in IgT-depleted fish promoted tissue damage and/or in-
(Fig. 5B). In contrast, sIgD-coating levels in the control group were flammation. Although moderate changes in gill histology were ob-
not different from those found in IgT-depleted fish (Fig. 5C). In the served at 1 week after depletion, significant signs of tissue damage
absence of sIgT coating, significant levels of bacteria translocated were detected at 3 weeks, as evidenced by wider and shorter second-
from the mucus layer across the gill epithelium, especially by week ary lamellae with edema and signs of inflammation (Fig. 6, A to C).
3 after depletion (Fig. 5, D to F, and fig. S4). Moreover, translocated In addition, the expression of several key proinflammatory cyto-
bacteria entered the systemic circulation, as evidenced by signifi- kines was increased, mostly 3 weeks after depletion (Fig. 6D). Spe-
cant increases in lipopolysaccharide (LPS) levels in the serum of a cifically, we detected a 1.67- to 2.81-fold increase relative to control
substantial number of IgT-depleted fish at week 3 after depletion fish for il1b, tnfa1/2, il17a/f1b, il17a/f2a, il17a/f2b, il17a/f3,
treatment (Fig. 5G, right). These results demonstrate a critical role il17a/f1b, and il21a transcripts. The only proinflammatory gene
of sIgT in containing microbiota within the mucosal areas and in up-regulated at week 1 after depletion was il4/13b1 (i.e., 1.98-fold
avoiding its systemic dissemination. increase), whereas the only mildly up-regulated anti-inflammatory

Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020 3 of 17

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A D Control PBL IgT-depleted PBL

IgT, IgM, Nuclei 50 µm

B E
Control head kidney IgT-depleted head kidney

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IgT, IgM, Nuclei 50 µm
C F
Control gill IgT-depleted gill

IgT, IgM, Nuclei 50 µm

+ + +
Fig. 2. Analysis of IgT and IgM B cells in the blood, head kidney, and gill upon IgT B cell depletion treatment. (A to C) Percentage of IgT+ (left) and IgM+ B cells
(right) within the lymphocyte population of the blood (PBL) (A), head kidney (HKL) (B), and gill (C) leukocytes in control and IgT-depleted fish (n = 10 to 12 fish per group).
Data are representative of at least three independent experiments (means ± SEM). Statistical analysis was performed by unpaired Student’s t test. **P < 0.01, ***P < 0.001.
(D to F) Immunofluorescence microscopy of trout blood leukocytes (D), head kidney tissue (E), and gill tissue (F), stained for IgT (green) and IgM (red), from control (left)
and IgT-depleted (right) fish. Nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Isotype-matched control Ab staining in fig. S2. Scale bars, 50 m. Data
are representative of at least three independent experiments.

gene (1.41-fold increase) was tgf1a at 21 days after depletion. Trans-
location and dysbiosis of microbiota at mammalian mucosal surfaces
have often been associated with increases in antimicrobial peptide
(AMP) gene expression (27). Similarly, at 3 weeks after depletion, we
observed moderate increases (1.29- to 1.91-fold relative to control
fish) in transcript levels of two LPS binding proteins (lbp/bpi1 and
lbp/bpi2), the AMP cathelicidin (cath2), the antimicrobial molecule
lysozyme (lyz), and nucleotide binding oligomerization domain 2
(nod2b), an intracellular bacterial pattern recognition receptor. Other
cytokines and AMP genes remained unchanged throughout the deple-
tion period (fig. S5). To evaluate whether the observed impact on tissue
damage and homeostasis was due to a potential impairment in Toll-like
receptor (TLR) signaling in IgT-depleted fish, we injected control and
IgT-depleted animals with three agonists of TLR signaling, includ-
ing lipoteichoic acid (LTA), flagellin, and resiquimod (R848). We
observed no differences in the mRNA expression levels of il1b, il6,
il8, and tnfa 1/2 in the gills between control and IgT-­depleted fish
upon treatment with any of three TLR agonists (fig. S6), thus suggest-
ing that TLR responses remain intact in IgT-depleted trout. Together,
our results underscore the importance of sIgT in the maintenance of
mucosal barrier integrity and suggest that in the absence of sIgT, the
loss of tissue integrity is not related to a defect in TLR signaling.
sIgT targets taxonomically distinct subsets of bacteria
To identify which bacterial taxa are coated by mammalian sIgA, re-
cent substantial inroads have been made using the powerful IgA
sequencing (IgA-seq) technique that enables the sorting and taxa
identification of sIgA-coated bacteria (16). As a first step to under-
stand the involvement of sIgT in microbiota homeostasis upon sIgT
depletion, here, we adapted the IgA-seq technique to identify the trout
gill bacteria taxa coated by sIgT (IgT-seq). To this end, we sorted
sIgT+ microbiota from healthy fish and subjected it to 16S ribosomal
DNA (rDNA) Illumina sequencing. IgT-seq revealed that sIgT coated
a well-defined subset of bacteria [~300 different operational taxo-
nomic units (OTUs)], which accounted for about 25% of all the
microbial diversity present in the gills (fig. S7A and table S1). The sIgT-­
targeted bacterial community was composed mostly of anaerobes
and facultative anaerobes and was highly enriched in Actinobacteria

Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020 4 of 17

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
A B 10,000
/ml)
/ml)
***
*
*** **
D E
/ml)
/ml)
*** *** *** **
Fig. 3. IgT, IgM, and IgD concentration in serum and gill mucus upon IgT+ B cell depletion treatment. (A to C) Concentration of IgT (A), IgM (B), and IgD (C) in serum
of control and IgT-depleted fish. (D to F) Concentration of sIgT (D), sIgM (E), and sIgD (F) in gill mucus of control and IgT-depleted fish. n = 10 to 12 fish per group. Data are
representative of at least three independent experiments (means ± SEM). Statistical analysis was performed by unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.
and Firmicutes (24 and 13%, respectively) compared with the total
bacterial community (0.62 and 0.23%, respectively) (Fig. 7A and fig.
S7B). Bacteroidetes, Cyanobacteria, and Fusobacteria, in turn, were
more abundant in the total bacterial community (24.1, 6.4, and
7.3%, respectively) compared with the sIgT-coated bacterial com-
munity (7.3, 1.3, and 2.2%, respectively) (Fig. 7A and fig. S7B). Pro-
teobacteria, the most common phylum in the trout gill microbiome
(26), was the predominant phylum in both groups, accounting for
54.3% of the diversity in the total bacterial community and 46.2% in
the sIgT-coated community (Fig. 7A and fig. S7B). At the order level,
sIgT coated potentially pathogenic bacteria (Fig. 7B and table S2).
Examples included members of the orders Enterobacteriales (5.5%
sIgT-coated versus 0.12% total bacteria) and Pseudomonadales
(11.5% sIgT-coated versus 1.3% total bacteria) (Fig. 7, B to D). In
contrast, the order Flavobacteriales, which contains several well-
known fish pathogens, was significantly enriched in the total bacteria
(19.7%) compared with the sIgT-coated community (4.8%) (Fig. 7,
B and E). The sIgT-coated microbial community was also enriched
in potentially beneficial taxa known to produce short-chain fatty
acids (SCFAs), including Propionibacteriales (13.36% sIgT-coated
versus 0.1% total bacteria) and Clostridiales (1.4% sIgT-coated versus
0.14% total bacteria) (Fig. 7, B, F, and G). There was also an enrich-
ment trend in other SCFA-producing bacteria, including members
of the order Bacillales (9.2% sIgT-coated versus 0.03% total bacteria)
(Fig. 7, B and H) and the order Lactobacillales (1.6% sIgT-coated
versus <0.1% total bacteria) (table S2), although these changes did
not reach statistical significance. The top 75 differentially abundant
OTUs further showed that subsets of bacteria that are preferentially
targeted by sIgT in the gills include both beneficial bacteria such as
Propionibacterium sp., Corynebacterium sp. (28), and Paenibacillus sp. (29),
as well as disease-driving taxa such as Candidatus Branchiomonas,
Acinetobacter sp., Serratia sp., Veillonela sp., Streptococcus sp., and
Stenotrophomonas sp. (Fig. 7I) (30, 31). Linear discriminant analysis (LDA)
effect size (LEfSe) method showed that the sIgT-­coated community is
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
C
/ml)
F
/ml)
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enriched in 16 taxonomic features including Firmicutes, Bacilli,
Clostridiales, Lactobacillales, Enterobacteriales, and Pseudomonadales
(Fig. 7J and fig. S7C). These results indicate that sIgT coats a broad
but well-defined range of bacteria with both beneficial and patho-
genic characteristics.
IgT depletion induces a profound dysbiosis in the gill
We next asked whether the observed translocation of bacteria in
IgT-depleted fish (Fig. 5, D and E) reflected a defect in maintenance
of microbiota homeostasis. 16S rDNA sequencing of gill tissue of
control and IgT-depleted trout showed changes in the microbial
community with kinetics that mirrored those of tissue damage and
bacterial translocation. Although no significant changes in Shannon
diversity index (a metric that weights the numbers of species by their
relative evenness data) were detected 1 week after depletion (fig. S8A),
the gill bacterial community composition of IgT-depleted animals
already showed some minor differences compared with that of con-
trol fish by this time point. At the phylum level (fig. S8B), we ob-
served a decrease in the overall abundance of Proteobacteria as a
result of IgT depletion (65% in controls versus 33.7% in IgT-depleted)
and increases in Bacteroidetes abundance (8.4% in controls versus
19.5% in IgT-depleted), suggesting that sIgT may be critical for Pro-
teobacteria colonization at least in the initial stages of dysbiosis. A
total of seven OTUs had significantly different abundances in both
experimental groups (table S3). For instance, we observed signifi-
cant decreases in the abundance of Caulobacteriales (3.6% control
versus 0.2% IgT-depleted) and Sphingomonadales (14.2% control
and 5% IgT-depleted) and moderate but not significant increases in
Flavobacteriales (8.7% control and 16.1% IgT-depleted) (fig. S8C
and table S4). In addition, Flavobacterium sp. and Deefgea sp. abun-
dance increased in IgT-depleted trout, while it decreased in the
family Moraxellaceae (fig. S8D). Abundance changes at the genus
level resulting from week 1 after depletion treatment can be found
in table S5. LEfSe analysis and cladogram representation highlighted
5 of 17
SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

Fig. 4. IgT depletion significantly in- A Depletion treatment

creases pathogen load and fish mor-
Antibody Trout antisera (TAs) injection
talities upon Ich infection. (A) Scheme Ich infection
injection (anti-Ms IgG2b TAs)
Ich infection Sampling
of the experimental strategy. Immune
Nondepleted immune fish
fish (fish that survived monthly infec- 4 weeks 1 day 21 days
14 days Ig responses (b, c)
tions by bath with ~1000 theronts per
fish, during a 3-month period) were in- Isotype 10 days
control Abs Pathogen load (d, e, f)
jected with either isotype control Abs IgT-depleted immune fish
(nondepleted immune fish) or anti-IgT 3 times exposure 30 days
(immune fish) Mortality (g)
mAb (IgT-depleted immune fish). A day Anti-IgT
after, fish were injected with TAs. At mAbs

14 days after TAs injection, the nonde-

pleted and IgT-depleted groups were B C
infected by bath with Ich (~1000 the­

(relative to uninfected con tr ol)

(relative to uninfected con tr ol)

P arasite-specific Ig binding
P arasite-specific Ig binding

20
ronts per fish), and at 21, 10, and 30 days ** IgT
after infection, different groups of fish IgM
15 IgD
were analyzed for Ig responses, patho-
gen load, and mortalities, respectively.

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10
(B and C) Specific Ig responses against **
the parasite were measured by pull- *
* 5
down assays in which we assessed the * *
IgT-, IgM-, and IgD-specific binding to 0
Ich in dilutions of gill mucus from non- 1:2 1:10 1:50 1:100
depleted (B) and IgT-depleted fish (C). Gill mucus dilutions Gill mucus dilutions
(nondepleted) (IgT-depleted)
Results are presented as relative values
to those of uninfected naive fish (n =
10 to 12 fish per group). (D) Represent­ D E
ative stereomicroscope images of gill
***
arches infected with Ich from nonde-
pleted (left) and IgT-depleted fish (right).
White arrowheads point to Ich trophonts.
(E) Parasites numbers on gills from non-
depleted and IgT-depleted fish upon
infection with Ich (n = 17 fish per group).
Each symbol represents an individual
fish; small horizontal red lines indicate
the mean. (F) Real-time PCR analysis of
Ich 18S rRNA gene in gills from non-
Nondepleted IgT-depleted
depleted and IgT-depleted fish. DNA
abundance was normalized to that of
nondepleted fish, which is set as 1 (n =
10 fish per group). (G) Percentage sur- F G
vival of nondepleted and IgT-depleted ***
(relative to nondepleted fish)

fish infected with Ich. Data (B to F) are

representative of three independent *
experiments (means ± SEM) or two in-
dependent experiments (G). Statistical
analysis was performed by unpaired
Student’s t test (B, C, E, and F), or log-
Ich 18S

rank (Mantel-Cox) test (G). *P < 0.05,

**P < 0.01, ***P < 0.001. −
Nondepleted IgT-depleted

that 12 taxa were more abundant in the control group than in the IgT depletion has moderate effects on the microbial community
IgT-depleted group including Comamonadaceae, Caulobacteriales, composition of the gill early on during the depletion treatment likely
Actinomycales, and Corynebacteriales (fig. S8, E and F). Conversely, due to the incomplete depletion of mucus sIgT at that time point.
these analyses also identified three taxa that were more abundant in the At 3 weeks after depletion, the Shannon diversity index of the
IgT-depleted group than in control fish, including Pseudomonadaceae, IgT-depleted fish gill microbiome was significantly lower (P = 0.026)
Cyanobacteria, and Betaproteobacteria (fig. S8, E and F). One week compared with that of the control group (Fig. 8A), indicating that
after “treatment” (i.e., IgT depletion) was, however, not a significant fewer OTUs contributed to a higher proportion of the overall bacterial
predictor of gill microbial community composition by analysis of abundance in the community in the IgT-depleted group compared
similarities (ANOSIM) with P = 0.763. These results indicated that with controls. At the phylum level, the gill microbial community

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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A Control B C
IgT-depleted Control
* ***
40 40 40 IgT-depleted
*** ** *

Bacteria coated by IgD (%)

Bacteria coated by IgM (%)
Bacteria coated by IgT (%) ** **
30 *** ***
30 30

20 20 20

Control
10 10 10
IgT-depleted

0 0 0
7 14 21 28 42 56 7 14 21 28 42 56 7 14 21 28 42 56
Days after depletion treatment Days after depletion treatment Days after depletion treatment

1 week 3 weeks
Control IgT-depleted F
D 150 *
**

Bacteria count
(10 fields/fish)
100

1 week **

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50 *

0
20 m Control IgT- Control IgT-
depleted depleted
E
G 1 week 3 weeks
15 50 ***
40

(relative to control)

(relative to control)
Endotoxin levels
3 weeks
Endotoxin levels
10
30

20
5
10

Bacteria, DAPI 0
Control IgT-depleted
0
Control IgT-depleted

Fig. 5. IgT depletion leads to a major decrease in sIgT coating of the gill microbiota and to their translocation across the gill epithelium. (A to C) Percentage of
gill bacteria coated by sIgT (A), sIgM (B), or sIgD (C) at days 7, 14, 21, 28, 42, and 56 after depletion treatment (n = 10 to 12 fish per group). Data are representative of at
least two independent experiments (means ± SEM). (D and E) Detection of bacteria by fluorescence in situ hybridization in gill cryosections from control (left) and IgT-depleted
fish (right) at 1 week (D) and 3 weeks (E) after depletion treatment. Eubacteria were detected with Cy5-EUB338 oligoprobe (yellow). Nuclei were stained with DAPI (blue).
Scale bars, 20 m. (F) Translocated bacteria were quantified in gill cryosections of control and IgT-depleted fish (10 microscopy fields per fish, n = 7 per group). (G) Endotoxin
levels were measured by limulus amebocyte lysate (LAL) chromogenic end point assay in serum from control and IgT-depleted fish (n = 25 per group) at 1 week (left) and
3 weeks (right) after depletion treatment. Endotoxin levels in IgT-depleted fish were normalized to those in control fish, which were set as 1. Each symbol (F and G) rep-
resents an individual fish; small horizontal red lines (F and G) indicate the mean. Data are representative of two independent experiments (D to E) or pooled from two
independent experiments (F and G). Statistical analysis was performed by unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

composition of control samples was dominated by Fusobacteria As examples of abundance changes at the genus level, significant
(29.6%), followed by Proteobacteria (21%) and Bacteroidetes (16%) decreases in beneficial taxa such as Propionibacterium sp. (7.6% control
(Fig. 8B). In response to IgT depletion, the community shifted and versus 1% IgT-depleted) and Pseudomonas sp. (1.3% control versus 0.3%
became dominated by Bacteroidetes (34%), followed by Proteo- IgT-depleted) were observed (Fig. 8, E and F, and table S8). LEfSe
bacteria (26%) and Fusobacteria (18.5%) (Fig. 8B). The abundance analysis determined the features more likely to define control and IgT-­
of Firmicutes (shown to be enriched in the sIgT-coated fraction) depleted gill microbial communities and highlighted that in the ab-
dropped from 3.5% in controls to 0.75% in IgT-depleted animals sence of sIgT, Flavobacteriales and Neisseriales expand at the expense
(Fig. 8B). As a result of this shift, the Bacteroidetes-to-Firmicutes of losses in Firmicutes, Pseudomonales, Bacillales, Pasterurellales, and
ratio increased ~10-fold in IgT-depleted animals. At the order level, Propionibacteriaceae (Fig. 8, G and H). Combined, these data indicate
IgT depletion resulted in significant reductions in the abundance of that sIgT depletion has profound and complex effects on the micro-
Actinomycetales (12.3% control versus 2.5% IgT-depleted) and ben- bial community composition in the gill of rainbow trout. Critically,
eficial SCFA producers such as Bacillales (2.8% control versus 0.16% sIgT deficiency triggered a state of dysbiosis characterized by loss of
IgT-depleted) (Fig. 8C and table S6). On the other hand, other orders potential beneficial SCFA-producing bacteria and expansion of patho-
were found at higher abundances. As examples, significant expansions bionts, underscoring the importance of sIgT in maintaining coloni-
of the orders Flavobacteriales (7.9% control versus 35.3% IgT-depleted) zation of beneficial microbes and controlling disease-driving bacteria.
and Neisseriales (1.1% control versus 4.3% IgT-depleted) were observed
in IgT-depleted gills (Fig. 8, C and D, and table S6). Overall, a total IgT recovery is sufficient to restore tissue
of 39 OTUs showed significantly different abundances between both and microbiome homeostasis
experimental groups indicating a global state of dysbiosis in the gills In our model, gill IgT+ B cells recover almost completely by 8 weeks
3 weeks after IgT depletion (Fig. 8E and table S7) (ANOSIM, P = 0.028). after depletion treatment (Fig. 2). This enabled us to ask whether

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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A D il1b il17a/f1b
Control IgT-depleted
1 week

3 weeks
1 week
il17a/f2a il17a/f2b il17a/f3
1 week

B 3 weeks

il4/13b1 il21a
3 weeks
1 week

3 weeks

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C
1 week 3 weeks 1 week

3 weeks
Pathology score

Pathology score

1 week

3 weeks

Fig. 6. IgT depletion leads to damage and inflammation of the gill tissue. (A and B) Hematoxylin and eosin staining of gill paraffin sections in control (left) and IgT-depleted
(right) fish at 1 week (A) and 3 weeks (B) after depletion treatment. (C) Pathology score of gill tissue in control and IgT-depleted fish at 1 week (left) and 3 weeks (right)
after depletion treatment (means ± SEM; n = 6 fish per group). (D and E) Real-time PCR analysis of cytokine genes (D) and AMP genes (E) in gill tissue from control and
IgT-depleted fish. Expression levels in IgT-depleted fish were normalized to those in control fish, which were set as 1 (means ± SEM; n = 8 fish per group). Data are repre-
sentative of two independent experiments. Statistical analysis was performed by unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.

sIgT recovery is sufficient to restore tissue and microbiome homeo-
stasis in trout gills. To this end, we sampled fish at 13 weeks after
depletion treatment to ensure that IgT recovery was complete. At
that time point, the percentage of IgT+ B cells had recovered to the
levels of control fish both in the gills and head kidney (fig. S9, A and
B), and this recovery paralleled that of IgT protein levels both in the
serum and gill mucus (fig. S9, C and F). Meanwhile, the percentage
of IgM+ B cells remained unchanged in these tissues (fig. S9, A and
B). The levels of microbiota coated by sIgT were also fully restored
(fig. S9I). The increased levels of sIgM-coated bacteria observed in
IgT-depleted fish from weeks 3 to 8 after depletion (Fig. 5B) had
faded away and were comparable with levels observed in control fish
(fig. S9J). As previously observed for week 8 after depletion (Fig. 5C),
levels of microbiota coated by sIgD remained unchanged (fig. S9K).
Restoration of IgT levels after 13 weeks after depletion treatment
was sufficient to revert bacterial translocation levels to those of con-
trol fish (fig. S9L), as reflected also by the absence of systemic LPS in
the IgT-recovered fish (fig. S8M). Absence of translocated bacteria
correlated also with a complete reversal of tissue damage in the gills
(fig. S9N). These data indicate that the deleterious effects in tissue
homeostasis induced by the lack of sIgT are reversed upon the re-
covery of physiological sIgT levels in the IgT-depleted group after
13 weeks of the IgT depletion treatment.
With regard to the microbiome composition, by week 13 after
depletion, there were no significant differences in the Shannon diver-
sity index of the gill microbial community of control and IgT-depleted
fish (P = 0.235) (fig. S10A). The lack of dysbiosis was reflected both at
the phylum level where no significant differences were observed be-
tween both groups (fig. S10B and table S9), as well as at the order level
where only three OTUs were significantly different between both groups,
albeit with marginal significance (P = 0.048), including Staphylococcus
sp., Cetabacterium sp., and an unassigned OTU (fig. S10C and tables
S10 and S11). Thus, by week 13 after depletion, the gill microbial
community composition of IgT-depleted fish was not significantly
different from that of control animals (ANOSIM, P = 0.584). Combined,
the microbiome results indicate that restoring sIgT levels is sufficient
to reestablish the balance in the microbial ecosystem of the gills.
DISCUSSION
sIgs emerged in both primitive and modern vertebrates in response
to common selective pressures and physiological demands imposed by
pathogens and commensals at mucosal surfaces. However, the func-
tion of sIgs in vertebrates in limiting and regulating microbes at mucosal
sites is not well understood (1, 8, 32). In that regard, mammalian
sIgA appears to have antagonistic roles in the control of mucosal
microbes because sIgA is important for mucosal pathogen clearance
(1, 3–5), while at the same time, it is required for microbiota coloni-
zation and homeostasis (6–8). Because the selective forces that have
shaped fish and mammalian sIgs are similar, we reasoned that the
study of a primitive sIg could shed light on whether sIgs arose to fight
mucosal pathogens and/or to enable the establishment of a healthy

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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A B C D
Bacillales
100
6.2 5.8
100
14.2
Burkholderiales
Cellvibrionales
***
*
Proteobacteria 37.5
Fusobacteria Chromatiales
Firmicutes 6.5 Clostridiales
Cyanobacteria Corynebacteriales
75 75
Relative abundance (%)

11.6 Cytophagales
46.2 Chlorobi
54.3 Bacteroidetes 7.3 Enterobacteriales
Flavobacteriales
Actinobacteria 13.4
Fusobacteriales
Other 19.7
50 50 Lactobacillales
Micrococcales
13.10 Neisseriales
7.3 9.1
7.6 Propionibacteriales
6.5 7.3 25 Pseudomonadales −5 −5
25 10.5 Rhodobacterales
12.2 Rickettsiales
24.1 24 Sphingobacteriales
19.6
0
Total IgT coated
0
9.2 Sphingomonadales
Xanthomonadales E F
Other

* *

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G H
*

− −

J IgT coated
Total

Fig. 7. sIgT coats specific subset of gill microbiota with beneficial and pathogenic characteristics. Total bacteria and sIgT-coated bacteria from the gill mucosa were
flow cytometrically sorted and high-throughput 16S rDNA sequencing of the V1 to V3 region was performed (IgT-seq). (A) Mean relative abundance at the phylum level
of the total gill bacterial community (total) and the sIgT-coated bacterial community (IgT coated). (B) Mean relative abundance at the order level of the total gill bacterial
community (total) and the sIgT-coated bacterial community (IgT coated). (C to H) Relative abundance (%) of Enterobacteriales (C), Pseudomonadales (D), Flavobacteriales
(E), Proprionibacteriales (F), Clostridiales (G), and Bacillales (H) in the total and sIgT-coated gill bacterial community. Data were analyzed in QIIME 1.8. Differential abun-
dances were determined by unpaired Student’s t test. *P < 0.05, ***P < 0.001. (I) Heatmap of the top 75 OTUs with differential abundances in the total (n = 4 pools, 4 fish
per pool) and sIgT-coated (n = 3 pools, 4 fish per pool) gill bacterial communities. (P < 0.05). The heatmap was generated using the online free tool Heatmapper (70) using
average linkage as a clustering method, followed by the Spearman rank correlation for distance measurement. (J) Cladogram representation of LEfSe analysis showing
bacterial taxa that were significantly associated with the total bacterial community (green) or the sIgT-coated bacterial community (red) (P < 0.05). n = 3 to 4 pools, 4 fish
per pool. Results are representative of two independent experiments.

microbiota. This fascinating question was addressed here by devel-
oping a new teleost fish model in which its mucosal sIg was selec-
tively depleted during adulthood.
To determine the contributions of sIgT in pathogen control and
microbiota homeostasis, we designed a strategy to selectively deplete
IgT+ B cells in rainbow trout, a teleost species. This strategy proved
highly effective in depleting most of the IgT and IgT+ B cells from
all tested fluids and lymphoid organs, respectively, for over a 4-week
period. It is worth pointing out that extended B cell depletions span-
ning several weeks have also been reported in mice and humans with
the use of a single Ab treatment (33, 34). Naive IgA-deficient mice
and patients with sIgAdef show compensatory increased levels of
total IgM and IgG in serum and mucus (9, 35, 36). In contrast, here,
we show that IgM or IgD in serum and mucus did not increase in
IgT depletion, thus indicating a lack of compensatory responses by
other fish Ig isotypes. In conclusion, our IgT depletion model rep-
resents the only vertebrate in vivo model that allows for the tempo-
ral and selective depletion of a specialized sIg during adulthood. In
contrast, all existing IgA-deficient mammalian models lack sIgA from
birth (9, 35). This fundamental difference between our fish and the
current mammalian models allows us to elucidate the function of a
sIg isotype in an animal where both a wild-type mucosal immune
system and a homeostatic microbiota have coexisted and matured
together before the sIg depletion.
Our previous findings in fish have shown a marked compartmen-
talization of pathogen-specific IgT and IgM responses in mucosal

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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

A diversity index B C

D E F

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−
−

−
−

G H

Fig. 8. IgT depletion results in significant gill dysbiosis 3 weeks after depletion. The microbial community composition of control and IgT-depleted gills (n = 5 to
6 per group) was determined by high-throughput 16S rDNA sequencing 3 weeks after IgT depletion treatment. (A) Shannon diversity index (means ± SEM) of the control
and IgT-depleted gill microbial communities. (B) Mean relative abundance at the phylum level of the control and IgT-depleted gill bacterial community. (C) Mean relative
abundance at the order level of the control and IgT-depleted gill bacterial community. (D and F) Relative abundance (%) of Flavobacteriales (D, top) and Neisseriales (D,
bottom), Propionibacterium sp. (F, top), and Pseudomonas sp. (F, bottom) in the control and IgT-depleted gill bacterial community at 3 weeks after depletion. Data were
analyzed in QIIME 1.8. Differential abundances were determined by unpaired Student’s t test. *P < 0.05, **P < 0.01. (E) Heatmap of the top 25 OTUs with significantly dif-
ferent abundance in control and IgT-depleted gills 3 weeks after depletion. The full list of the 39 differentially abundant OTUs can be found in table S7. Each column
represents one individual fish. (G) Bar chart of the log-transformed LDA score of bacterial taxa found to be significantly associated with control and IgT-depleted 3 week
after depletion showing the presence of 15 taxonomical features with higher abundance in controls (red) and 5 taxonomical features with greater abundance in IgT-depleted
gills (green) by LEfSe (P < 0.05). (H) Cladogram representation of LDA analysis in (G) showing the phylogenetic relationships among the bacterial taxa found to be signifi-
cantly associated with control (red) or IgT-depleted gills (green) by LEfSe. Results are representative of two independent experiments.

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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
and systemic areas, respectively, suggesting an important role for
sIgT in the control of mucosal pathogens (23–25). Supporting this
hypothesis, here, we show that depletion of IgT markedly increases
the susceptibility of fish to a mucosal parasite. We did not observe a
compensatory IgM response in IgT-depleted fish. In contrast, IgA-­
deficient mice have been reported to develop stronger IgM titers than
do their littermate controls in several infection models (35, 37–39).
Similarly, respiratory infections in patients with sIgAdef are more
recurrent when these individuals lack sIgM in their lungs (36). More-
over, patients with sIgAdef with high levels of IgM in saliva have
shown a lower infection incidence (40). Because mammalian IgA
arises from class switch recombination (CSR), it is tempting to pro-
pose that the potential compensatory response of IgM in mammals
is derived from IgM+ B cells with the same Ab specificity but which
have not yet class switched. In that regard, we have previously re-
ported that the germline repertoire between fish IgT and IgM is very
different (41), and class-switch recombination does not exist in these
species (22), thus providing a potential explanation for the inability
of IgM to compensate for pathogen-specific IgT responses in fish.
The symbiosis between microbiota and the mucosal surfaces of
all metazoans is one of the most ancient and successful relation-
ships found in nature. Our previous work has shown that similar to
sIgA in mammals, sIgT is the prevalent Ab isotype coating the mi-
crobiota of fish (23–25). As expected, IgT depletion led to a marked
decrease in the sIgT coating of gill microbiota. We observed an in-
crease in the percentage of IgM-coated microbiota from weeks 3 to
6 after depletion treatment, which coincides with the time points at
which sIgT concentration was at its lowest in the gill mucus of IgT-­
depleted fish. This increase in sIgM-coated microbiota could be ex-
plained by several processes. First, the possibility exists for an sIgM
compensatory mechanism similar to what has recently been reported
in human patients with sIgAdef, in which sIgM partially compen-
sates for the lack of sIgA by coating microbiota in a much less tar-
geted and less specific manner (11, 15). Accordingly, in the absence
of sIgT, compensatory sIgM would then coat microbiota subsets that
would otherwise be coated by sIgT. Alternatively, or concurrently,
it is possible that some of the bacterial taxa that expand during dys-
biosis in IgT-depleted fish are those found initially coated by sIgM
in control individuals, which would increase in abundance upon IgT
depletion. Further work is warranted to analyze all the aforemen-
tioned possibilities. In addition to most gill-associated bacteria los-
ing their sIgT coating by 3 weeks after depletion, large numbers of
bacteria translocated within the gill lamellae, and significant amounts
of LPS were found in blood, thus indicating that the potential sIgM-­
mediated compensatory mechanism postulated above is not suffi-
cient to overcome the deleterious effects of dysbiosis. Microbiota
translocation in IgT-depleted fish suggests that sIgT is required for
microbiota colonization, anchoring, and containment of sIgT-coated
microbiota within the mucosal layer of the gills, as suggested also
for sIgA at mammalian mucosal surfaces (6, 8). Alternatively, be-
cause sIgA has also been shown to regulate microbiota composition
by promoting microbiota symbiosis (7), it is likely that dysbiosis
induced in the absence of sIgT may allow the outgrowth of micro-
biota that can then translocate across the mucosal epithelium and
reach systemic sites, as has been observed also in microbiota of mu-
rine models devoid of sIgA (14, 42–44). Patients with sIgAdef have
no increase in LPS serum levels, suggesting the involvement of IgA-­
independent mechanisms implicated in the containment of uncoated
or dysbiotic bacteria in humans (11). It is conceivable, however,
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
that translocation of non–Gram-negative bacteria occurs in these
patients, a possibility that warrants further investigation. In our
teleost model, the significant bacterial translocation observed at week
3 after depletion correlated with the presence of significant tissue
damage and up-regulation of critical proinflammatory cytokines
and AMPs. The latter strongly suggests that the invasion of micro-
biota into the gill tissue results in inflammatory and antimicrobial
responses in the affected area, similar to what has also been described
in murine models lacking sIgA (11, 43). Our studies with TLR ago-
nists imply that the observed tissue damage in sIgT-depleted fish
was not due to a defect in TLR signaling, and thus, further studies
are warranted to understand the mechanisms involved in the loss of
tissue integrity in the absence of sIgT.
Although a large percentage of microbiota are coated by sIgA in
mammals, the functional significance of such coating is poorly un-
derstood (8, 16). Recently, the newly developed IgA-seq technique
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has enabled the identification of IgA-coated taxa (16). Although these
studies are in their infancy and have only been performed in mam-
mals, they have revealed that despite the polyreactive nature of sIgA,
the sIgA-coated microbiota fraction contains a large but well-defined
range of bacteria with distinct taxonomies, whereas entire taxa are
devoid of IgA coating (11, 16, 45). Here, we developed IgT-seq for
our fish model and found that similar to what has been described
for sIgA, sIgT targets a broad but distinct repertoire of bacterial taxa
belonging to seven different phyla that represent ~25% of all OTUs
found in the gills. Comparable with mammalian sIgA (8, 13), sIgT
preferentially targeted Proteobacteria [the most abundant phylum
in fish external microbiomes (26)], followed by Actinobacteria and
specific groups of Firmicutes. In addition, similar to sIgA (8, 16, 45, 46),
sIgT preferentially coated a number of beneficial bacteria, including
several SCFA-producing taxa such as the orders Lactobacillales and
Clostridiales, which have anti-inflammatory properties (47, 48) and
support Ab production in mice (49, 50). Our findings suggest that
SCFA-producing taxa may play a conserved role across vertebrates
in supporting tissue barrier integrity and sIg production. Further
studies are warranted to explore this hypothesis. We also found that
the sIgT-coated microbiota fraction was significantly enriched in
several opportunistic or pathogenic bacteria, akin to the colitogenic
bacteria and pathobionts with inflammatory properties that are highly
coated by mammalian sIgA (8, 16). Overall, our IgT-seq data sup-
port the notion that sIgs evolved to target well-defined taxa of both
beneficial and pathogenic bacteria that form part of a healthy verte-
brate microbiome under homeostatic conditions.
The IgT-seq data demonstrating that sIgT coated a distinct set of
microbial taxa led us to hypothesize a critical role for sIgT in the
preservation of microbiota homeostasis. In support, we found that
the gill microbiome shifted upon sIgT depletion in a time-dependent
fashion that mirrored sIgT levels. The most profound shifts in the
microbial community composition were detected by week 3 after de-
pletion, when mucus sIgT levels were negligible. The observed dys-
biosis was also associated with significant tissue damage, inflammation,
and microbial translocation. The microbiome changes were charac-
terized by significant abundance shifts in a large number of OTUs,
decreased Shannon diversity, and a ~10-fold decrease in the Firmicutes-­
to-Bacteriodetes ratio in IgT-depleted animals. Large decreases in
the Firmicutes-to-Bacteroidetes ratios in mammals are also charac-
teristic of dysbiotic states (51, 52). Similar to patients with sIgAdef
and murine IgA knockout models (11, 15), sIgT deficiency trig-
gered losses of typically beneficial resident taxa, including Bacillales
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[known to secrete large amounts of antimicrobial compounds (53)]
and Propionibacteriaceae [known to produce SCFAs (54)], which,
in our IgT-Seq dataset, were identified as preferentially coated by
sIgT. In agreement with a very recent study in mice demonstrating
the previously unrecognized ability of certain commensals to use
sIgA for mucosal colonization (6), our current findings suggest that
sIgT coating of bacteria likely mediates bacterial colonization under
homeostatic conditions. Further studies are warranted to test this
hypothesis. IgT depletion also resulted in significant expansions of
pathogenic taxa including the members of the orders Flavobacteriales
and Neisseriales. These subsets were not preferentially coated by sIgT
according to IgT-seq, indicating that expansions of pathobionts upon
IgT depletion occur via indirect mechanisms. For instance, the ab-
sence of sIgT may prevent certain microbiota taxa from colonizing
or being contained in the mucosa, thus allowing space and resources
for the outgrowth of non–sIgT-coated microbiota (i.e., pathobionts).
Similarly, expansions of pathobionts, including segmented fila-
mentous bacteria and Clostridia, occur in sIgA-deficient mammals
(10, 12), although, in general, these bacterial taxa appear to be coated
by sIgA (16, 55–57). Overall, our findings demonstrate the previously
unrecognized requirement of a primitive sIg in the maintenance of
microbiota homeostasis and parallel those reported in mammals
lacking sIgA that show also significant alterations in the composi-
tion of their microbiomes.
Although we see important commonalities in the way bacterial
communities shift between our IgT-depleted fish and the current
mammalian models lacking sIgA, it is worth pointing out two sig-
nificant differences. First, Shannon diversity does not change signifi-
cantly in patients with sIgAdef (11, 15), although we find a significant
decrease in that index in sIgT-depleted fish. Second, in mammalian
models lacking sIgA, the expanding pathobionts are generally those
found coated by sIgA in control individuals (11), whereas in IgT-­
depleted fish, the expanding pathobionts are generally found not
coated by sIgT in control fish. These discrepancies likely arise from
the fact that sIgA-deficient mammalian models lack sIgA from birth,
which prevents the initial colonization of many taxa, whereas our
fish model results in the selective and temporary depletion of sIgT
during adulthood. Thus, in our fish model, microbiota diversity is
optimal under homeostatic conditions (i.e., in the presence of sIgT),
but when a perturbation is introduced (i.e., temporal depletion of
sIgT), the existing communities significantly shift in their abun-
dance as reflected by the decreased Shannon diversity. We propose
that our model provides a more physiologically relevant scenario in
which to test the involvement of sIg in host-microbial interactions,
and thus, it may portray a more accurate picture of what may follow
under conditions in which a sIg is temporarily reduced or depleted
in a mucosal surface (e.g., reductions of IgA levels upon stress or
antibiotic treatment) (58, 59). In this respect, our recovery experi-
ments significantly contribute to our understanding of immune-­
mediated regulation of microbial community stability. Microbiota
recovery after a perturbation depends on the type of perturbation
(i.e., agent or condition, duration, and intensity) and the stability
and resilience of the dysbiotic microbial community (60, 61). Our
study shows that restoring sIgT levels is sufficient to restitute a normal
homeostatic gill microbial community in trout. This finding supports
the notion that adaptive immunity promotes stability of microbiomes
at mucosal barriers (62–64) and that this is a well-conserved mech-
anism across all vertebrate species that contain specialized sIgs. In
addition, previous studies have shown that the immune system re-
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
establishes a healthy microbiome by suppressing harmful microbial
species (62–64). Thus, the restitution of a healthy microbiota upon
sIgT depletion may be explained by several mechanisms. On the one
hand, it is possible that increasing sIgT levels during the recovery
period results in a density-dependent regulation of harmful bacteria
that leads to a dampening of positive feedback loops in the commu-
nity, as previously suggested (60). On the other hand, restoration of
sIgT levels probably reenables the colonization of sIgT-dependent
microbiota and the control (through immune exclusion) of poten-
tial pathobionts whose growth and containment within luminal areas
are controlled by sIgT coating. The aforementioned potential mech-
anisms responsible for restoration of microbiome composition and
homeostasis by sIgT warrant further studies. It is worth pointing
that recovery of microbiome homeostasis was accompanied by res-
toration of mucosal tissue integrity. Because recovery of sIgT levels
in IgT-depleted fish led to a reversal in bacterial tissue translocation
Downloaded from http://immunology.sciencemag.org/ by guest on February 14, 2020
and systemic LPS, it is plausible to infer that the tissue damage and
inflammation induced at the peak of the IgT depletion period were
due to the significant dysbiosis and translocation of microbiota. Thus,
sIgT is critical to both maintaining microbiome and mucosal tissue
homeostasis.
In conclusion, our data support the notion that specialization of
sIgs in protection of mucosal sites from pathogens and preservation
of microbiota homeostasis occurred concurrently after the emergence
of bony fish. These data also imply that both sIg functions emerged
early in evolution rather than representing functions recently ac-
quired by sIgA and IgX in tetrapods. Our findings support further
the notion that IgT and IgA are phylogenetically distant Igs that
specialized in mucosal immune responses through convergent evo-
lution (23) and reveal the existence of primordially conserved prin-
ciples by which sIgs of primitive and modern vertebrates control both
pathogens and microbiota. On one hand, sIgT and sIgA are re-
quired to control mucosal pathogens, albeit the role of sIgA in that
area appears to have become less essential throughout evolutionary
time, likely due to the advent of CSR (not present in fish), which
enabled IgM and sIgA to share their antigen-binding sites. On the
other hand, sIgT and sIgA have also evolved to be indispensable in
preservation of microbiota homeostasis at mucosal sites, and the
number of functional commonalities between the two Igs in this area
is remarkable. Most relevantly, both Igs have evolved to coat broad
but exquisitely distinct subsets of beneficial and pathogenic micro-
bial taxa. As a consequence, in the absence of sIgT or sIgA, the micro-
biome undergoes profound dysbiosis that is characterized by losses
of beneficial taxa, large increases in the Bacteroidetes-to-Firmicutes
ratio, and an expansion of pathobionts. Moreover, sIgT- or sIgA-­
mediated dysbiosis is associated with increased microbial transloca-
tion, mucosal tissue damage, and inflammation. In addition to
revealing these primordial principles, our studies offer a unique model
and unbiased phylogenetic dimension to the field, thus providing
the potential to uncover previously unfound mechanisms of micro-
bial control and regulation by sIgs.
MATERIALS AND METHODS
Study design
The objective of this study was to evaluate the involvement of a
primitive fish sIg (sIgT) in the control of mucosal pathogens and the
maintenance of microbiota homeostasis. To this end, we first devel-
oped a fish model lacking IgT. This in vivo model of IgT depletion
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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
was performed in rainbow trout with the injection of antirainbow
trout IgT mAbs and rainbow TAs against mice IgG. Using the newly
generated IgT-depleted fish, we performed parasite (Ich) infections
to evaluate the specific contribution of sIgT in protecting fish against
this mucosal pathogen. We also evaluated the effect of IgT depletion
on mucosal tissue homeostasis and microbiome composition.
Fish maintenance
Rainbow trout (Oncorhynchus mykiss) (2 to 3 g) was obtained from
Troutlodge (Summer, WA) and was maintained as previously de-
scribed (23). Fish were acclimatized for 2 weeks at 15°C in an aerated
recirculating aquarium with internal biofilters and fed daily with dry
pellets at 1% biomass/day. The fish used are of the May strain and
are not genetically inbred. As indicated by Troutlodge, the May strain
is an outbred strain, and it is also a closed population in which no
new genetic material has been introduced for nine generations. This
strain was initiated in the year 2000. All animal procedures were
approved by the Institutional Animal Care and Use Committees of
the University of Pennsylvania.
Production of TAs to mouse IgG2b
Fish (~50 to 100 g) were first immunized with 25 g of mouse IgG2b
(BioLegend) emulsified in Freund’s complete adjuvant (Sigma-Aldrich)
by intraperitoneal injection. Thereafter, fish were injected monthly
with the same amount of antigen emulsified in Freund’s incomplete
adjuvant (Sigma-Aldrich) at least two times. Antisera from immu-
nized fish (TAs) were obtained 2 weeks after the last immunization.
TCs were collected from fish immunized with adjuvant alone fol-
lowing the same immunization scheme described above. Harvested
sera were sterilized with Millex-GV syringe filters (Millipore) and
kept in −80°C until further use. The specificity of TAs was evaluated
by Western blot analysis. Briefly, mouse IgG2b (0.1 g) was resolved
on 4 to 15% Mini-PROTEAN TGX Gels (Bio-Rad) under nonreducing
conditions. The gel was transferred onto Sequi-Blot PVDF mem-
branes (Bio-Rad). The membrane was blocked with 8% skim milk
and incubated with TAs or TCs at a dilution of 1:10,000, followed by
incubation with biotinylated mouse antitrout IgM mAb and then in-
cubation with horseradish peroxidase (HRP)–conjugated streptavidin
(Thermo Fisher Scientific). Immunoreactive bands were visualized
using the HyGLO Chemiluminescent HRP Ab Detection Reagent
(Denville Scientific Products). TAs IgM titers against mouse IgG2b
were determined by ELISA as described previously (23). TAs was
only used for depletion purposes when titers were above 1:204,800.
Depletion of IgT+ B cells and IgT from tissues and body
fluids, respectively
To initially evaluate the IgT+ B cell depletion effect of antitrout IgT
mAbs and TAs in vivo, fish (~2 to 3 g) were intraperitoneally injected
with two different doses (2 or 10 g) of mouse antitrout IgT mAbs
(clone 41.8; IgG2b) (23) or 10 g of mouse IgG2b as isotype control
Ab (BioLegend). After 24 hours, half of the fish from each group
were further injected with 30 l of TAs (titer, ≥1:204,800), whereas
the other half were injected with TCs. Seven days after Ab injection,
blood leukocytes were obtained, and the IgT+ and IgM+ B cell pop-
ulations were stained and evaluated by flow cytometry as described
in Supplementary Methods. After confirmation that effective IgT+
B cell depletion was achieved with the treatment of 10 g of antitrout
IgT mAbs and TAs, we used 2.5 times the amount of the antitrout
IgT mAb (25 g per fish) for the rest of experimental procedure to
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
ensure a consistent and high level of IgT+ B cell depletion in all fish.
A time course of IgT+ B cell depletion from the blood, head kidney,
and gill was thereafter performed using the dose (25 g per fish) of
antitrout IgT mAb or its corresponding isotype control, in combi-
nation with the subsequent injection of TAs in all groups. Concen-
tration of IgT, IgM, and IgD in the serum and gill mucus of these
fish were measured as described in Supplementary Methods.
In vivo treatment of IgT-depleted and control fish with 
TLR agonists
To investigate whether TLR signaling remains intact in IgT-depleted
trout, individual control and IgT-depleted fish (at 3 weeks after de-
pletion treatment) were injected intraperitoneally with phosphate-­
buffered saline (PBS) or LTA from Bacillus subtilis (Sigma-Aldrich;
10 g/g fish), resiquimod (R848, Sigma-Aldrich; 10 g/g fish), or
flagellin from Salmonella typhimurium (InvivoGen; 1 g/g fish)
Downloaded from http://immunology.sciencemag.org/ by guest on February 14, 2020
(n = 7 fish per group). Six hours after injection, fish were euthanized,
and the gills were collected for total RNA extraction. As readouts of
TLR activation, we measured transcript levels of selected fish cytokines
including il1b, il6, il8, and tnfa1/2 using real-time polymerase
chain reaction (PCR) (as described in Supplementary Methods).
IgT-seq and microbiome sequencing from control
and IgT-depleted fish
IgT-seq allows for the identification of taxa-specific microbiota coated
by sIgT. This technique was adapted here for fish from the recently
reported IgA-seq procedure (16). Gill microbiota were collected as
described in Supplementary Methods from healthy fish (2 to 3 g).
To sort sIgT-coated bacteria, we prepared four different pools of
microbiota, each pool containing microbiota from four different fish.
Bacteria from each pool were stained with biotinylated antitrout IgT
mAb for 30 min at 4°C. Stained bacteria were detected with Brilliant
Violet 421–conjugated streptavidin (BioLegend). A portion of the
whole bacterial suspension from each pool was stored as the presort
sample, whereas 500,000 sIgT+ bacteria were sorted per pool, using
a BD FACSAria II flow cytometer (BD Biosciences). Bacteria were
preserved in a nontoxic sucrose lysis buffer (SLB) (65) at −80°C un-
til further use. For total (presort sample) gill bacteria microbiome
analysis, we used four bacterial pools, and for the sIgT-coated bac-
teria microbiome analysis, we used three bacterial pools. Results are
representative of two independent experiments.
To compare the gill microbiome of control versus IgT-depleted fish,
gill tissue from five to six fish per group was collected 1, 3, and 13 weeks
after IgT depletion and control treatment (described above). Tis-
sues were individually preserved in SLB as previously described (65).
To extract DNA from bacteria and gill tissue, we followed the
cetyltrimethylammonium bromide buffer method as previously de-
scribed (66). When using tissue, this was placed in TissueLyser II
(QIAGEN) and homogenized using sterile 3-mm tungsten carbide
beads (QIAGEN). Purified DNA obtained from bacteria or gill tissue
was then resuspended in 30 l of deoxyribonuclease- and ribonuclease-­
free molecular biology grade water. Sample DNA concentration and
purity were measured in a NanoDrop ND 1000 (Thermo Fisher Sci-
entific). Negative controls consisting of SLB only and positive con-
trols consisting of a known mock bacterial community of seven
bacterial species were included in each sequencing run as we have
previously reported (67). Bacterial community composition was de-
termined as by next-generation sequencing of the prokaryotic 16S
rRNA (66). Briefly, total genomic DNA for each sample was diluted
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SCIENCE IMMUNOLOGY | RESEARCH ARTICLE

1 in 10 or 1 in 100 in ribonuclease-free water and amplified in tripli- (serum and gill mucus) were collected, as described in Supplemen-
cate using Illumina adapter fused primers that target V1 to V3 vari- tary Methods, to measure pathogen loads and pathogen-specific Ab
able regions of the prokaryotic 16S rRNA sequences. Gene-specific titers, respectively. Experiments were repeated two or three times.
primer sequences used were as follows: 28F, 5′-GAGTTTGATCNT-
GGCTCAG-3′; 519R, 5′-GTNTTACNGCGGCKGCTG-3′ (where Pathogen load, binding of IgT, IgM, and IgD to Ich,
N = any nucleotide and K = T or G). The amplification was carried and fish mortalities
out with initial activation of the enzyme (5PRIME HotMaster Taq Pathogen load was obtained by counting the number of the Ich par-
DNA Polymerase, Quantabio) at 94°C for 90 s, followed by 33 cycles asites (trophonts) on one side of gill tissue in a double-blind fashion
of the following: 94°C for 30 s, annealing at 52°C for 30 s, and 72°C by two independent researchers using a stereomicroscope. Images
for 90 s, and a 7-min extension cycle at 72°C with a final holding of the gill tissue were acquired using an Olympus SZX12 stereo-
temperature of 4°C. PCR amplicons were purified using the Axygen microscope and DP21 camera (Olympus). The other side of gill tis-
AxyPrep Mag PCR Clean-up Kit (Thermo Fisher Scientific) as per sue of the same fish was used for DNA extraction with the DNeasy
the manufacturer’s instructions. Samples were then indexed by li- Blood & Tissue Kit (QIAGEN) to measure Ich burden by real-time
gating index barcode to Illumina adapters onto the PCR amplicon PCR as described in Supplementary Methods. Primer sequences are
using the Nextera XT Index Kit v2 Set A (Illumina). DNA concen- shown in table S12.
trations in each sample were quantified, pooled, and adjusted to a To assess whether nondepleted and IgT-depleted fish infected

Downloaded from http://immunology.sciencemag.org/ by guest on February 14, 2020

DNA concentration of 200 ng/l. Pooled samples were purified again with Ich parasites contained pathogen-specific Igs, we measured the
using the Axygen AxyPrep Mag PCR Clean-up Kit and sequenced capacity of IgT, IgM, and IgD from their serum and gill mucus to
in an Illumina MiSeq platform at the Translational Science Center bind to Ich using pulldown assays as previously described by us (24).
at University of New Mexico Health Sciences Center. Briefly, parasites (~100 tomonts) were preincubated with a solution
of 0.5% bovine serum albumin (BSA) in PBS (pH 7.2) for 2 hours at
Microbiome sequence analysis and statistics 4°C. Thereafter, parasites were incubated with diluted gill mucus or
Sequence data were analyzed using Quantitative Insights Into Micro- serum obtained from the different fish groups for 2 hours at 4°C in
bial Ecology (QIIME 1.9) pipeline (68) within the web-based platform a 300-l volume. Dilutions were made with PBS containing 0.5% BSA
Galaxy at the University of New Mexico (69). OTUs were selected (pH 7.2). After incubation, the tomonts were washed with PBS, and
by open reference picking using SUMACLUST method. OTUs were bound proteins were eluted with Laemmli Sample Buffer (Bio-Rad)
aligned in SILVA 16S/18S database with a 97% identity level. To gen- and boiled for 5 min at 95°C. The eluted material was resolved on 4
erate rarefaction curves and assess sampling depth, rarefaction anal- to 15% Mini-PROTEAN TGX Gels (Bio-Rad) under nonreducing
ysis was performed in QIIME using several alpha diversity metrics conditions, and the presence of IgT, IgM, or IgD was detected by
(PD_whole_tree, chao1, and observed_otus). Core diversity analy- Western blotting using the antitrout IgT, IgM, or IgD Abs as de-
sis was run on the remaining samples with a normalized sampling scribed in Supplementary Methods. IgT-, IgM-, and IgD-specific
depth of 5800 sequences for whole-tissue sequencing and 11,000 se- binding to Ich in dilutions of gill mucus and serum were evaluated
quences for IgT-seq samples. Alpha diversity metrics including PD_ by densitometric analysis of immunoblots and presented as relative
whole_tree, chao1, observed_otus, Shannon diversity, and goods_ values to those of uninfected (nonimmune) control fish. Fish
coverage were obtained in QIIME. Nonphylogenetic and phylogenetic (25 fish per group) mortalities were recorded daily for 30 days after
beta diversity analyses were performed in QIIME using the Bray-­ Ich infection.
Curtis metric or the unweighted and weighted UniFrac, respectively.
Principal coordinate analysis and taxonomic summaries were Statistical analysis
produced in QIIME to compare the bacterial community in all experi- The sample sizes were determined on the basis of preliminary studies.
mental groups. Statistical analyses (ANOSIM) were performed in Thus, the sample sizes for each in vivo experiment were determined
QIIME comparing IgT depletion treatment with its corresponding by power analyses to ensure a statistical power of 80 to 100% depend-
age-matched isotype control. Differential abundance analysis was ing on the readout. The sample size and number of independent
performed by unpaired Student’s t test in GraphPad Prism version 6. experiments are indicated in the figure captions. Fish were sampled
Differences were considered statistically significant when P < 0.05. from experimental tanks in a randomized manner, and experiments
were repeated at least twice as described throughout the paper. Fish
Ich parasite isolation and infection were not repeatedly sampled but euthanized at each time point.
Isolation of Ich parasite was performed as previously reported by us Histological examination of hematoxylin and eosin stains to determine
(24, 25). Fish (n = 80) were exposed monthly to ~1000 theronts per pathology scores in gills and parasite counting for pathogen load in
fish over a 3-month period to obtain fish survivors from Ich infec- gills were performed blindly by two independent researchers. No data
tion (immune fish). Mock-infected (uninfected) fish were exposed were excluded. Unpaired Student’s t test and Mantel-Cox test were
to the same tank water but without the parasite. One month after the performed in Prism (GraphPad) for analysis of differences between groups.
last exposure, survivor and uninfected fish were divided each into P values of 0.05 or less were considered statistically significant.
two groups: One group was treated for IgT depletion (IgT-depleted
fish) with anti-IgT mAbs, as described above, whereas the other group SUPPLEMENTARY MATERIALS
was treated with isotype control Abs (nondepleted fish). Fourteen days immunology.sciencemag.org/cgi/content/full/5/44/eaay3254/DC1
after the depletion treatment, nondepleted and IgT-depleted groups Methods
Fig. S1. Generation of TAs to mouse IgG2b.
were infected with the same dose of parasites. At 10 and 21 days after Fig. S2. IgT depletion does not affect the survival rate of IgT-depleted fish.
infection, fish were euthanized with an overdose of tricaine meth- Fig. S3. Ich-specific Ig responses in the serum from nondepleted and IgT-depleted fish after infection.
anesulfonate (MS-222, Syndel), and gill tissue samples and fluids Fig. S4. Control probe staining of trout gill cryosections.

Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020 14 of 17

SCIENCE IMMUNOLOGY | RESEARCH ARTICLE
Fig. S5. Cytokines and AMPs with unchanged expression of gene transcripts in gill tissue from
control and IgT-depleted fish.
Fig. S6. Cytokine expression in gill tissue from control and IgT-depleted fish upon in vivo
treatment with TLR agonists.
Fig. S7. sIgT coats specific subset of bacteria with beneficial and pathogenic characteristics.
Fig. S8. IgT depletion results in mild gill dysbiosis 1 week after depletion.
Fig. S9. Levels of IgT+ B cells, IgT protein, sIgT-microbiota coating, gill microbial translocation,
and tissue damage of IgT-depleted fish revert to those of control fish at 13 weeks after
depletion treatment.
Fig. S10. IgT recovery at 13 weeks after depletion treatment is sufficient to restore microbiome
composition.
Table S1. Alpha diversity metrics in gill sIgT-coated bacteria.
Table S2. Bacterial community composition of trout gill sIgT-coated bacteria at the order level
(in percentage).
Table S3. Significant OTUs 1 week after IgT depletion in trout gill.
Table S4. Bacterial community composition in control and IgT-depleted trout gill at the order
level (in percentage) 1 week after depletion.
Table S5. Bacterial community composition in control and IgT-depleted trout gill at the genus
level (in percentage) 1 week after depletion.
Table S6. Bacterial community composition in control and IgT-depleted trout gill at the order
level (in percentage) 3 weeks after depletion.
Table S7. Significant OTUs 3 weeks after IgT depletion in trout gill.
Table S8. Bacterial community composition in control and IgT-depleted trout gill at the genus
level (in percentage) 3 weeks after depletion.
Table S9. Bacterial community composition in control and IgT-depleted trout gill at the
phylum level (in percentage) 13 weeks after depletion.
Table S10. Significant OTUs 13 weeks after IgT depletion in trout gill.
Table S11. Bacterial community composition in control and IgT-depleted trout gill at the
genus level (in percentage) 13 weeks after depletion.
Table S12. Primer sequences used for real-time PCR.
Data file S1. Raw data.
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Acknowledgments: We thank D. Dinwiddie for sharing the Illumina sequencer; L. Bu for
help with microbiome bioinformatics analysis; J. S. Faust and the staff of the Flow
Cytometry Facility (Wistar Institute) for the cell-sorting procedures; the personnel of the
University Laboratory Animal Resources (ULAR) of the University of Pennsylvania for their
invaluable help in the daily husbandry of our Rainbow Trout; and A. Carty for the excellent
veterinary care of the fish. The antirainbow trout IgD mAb was provided by the
U.S. Veterinary Immune Reagent Network, which was funded by the USDA NIFA #2010-
65121-20649 award. Funding: This work was supported by the NSF grant NSF-
IOS-1457282 to J.O.S., the U.S. Department of Agriculture grant USDA-NIFA-2016-09400 to
J.O.S., the NIH grant NIH 2R01GM085207-09 to J.O.S. and I.S., the NIH grant NIH
P20GM103452 to I.S., the National Natural Science Foundation of China 31879045 to Z.X.,
Japan Society for the Promotion of Science (JSPS) KAKENHI JP19K21158 to F.T., JSPS
Overseas Research Fellowships to F.T. and Y.S., and a grant from the University of New
Mexico’s Initiative for Maximizing Student Development (IMSD) Program to T.J.C.S. Author
contributions: J.O.S. designed the overall study, provided overall direction for the study,
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and organized the writing of the manuscript. I.S. contributed in the design of the study,
analyzed the microbiome data, and contributed to the writing of the manuscript. F.T. and
Z.X. developed the IgT depletion model and analyzed the effects of IgT depletion on
pathogen challenge. E.C. and T.J.C.S. performed the microbiome studies. Y.S. performed
microbiota translocation studies in IgT-depleted fish. Y.D. contributed to the analysis of Ig
coating and performed the microbiota sorting studies. Y.D. and Y.Y. contributed to the
analysis of Ig coating and to the microbiota sorting studies of IgT-recovered fish. F.T., Z.X.,
E.C., Y.S., Y.D., I.S., and J.O.S. analyzed the data and designed the manuscript figures. All
authors contributed in the writing of the manuscript. Competing interests: The authors
declare that they have no competing interests. Data and materials availability: All data
needed to evaluate the conclusions in the paper are present in the paper or the
Xu et al., Sci. Immunol. 5, eaay3254 (2020) 7 February 2020
Supplementary Materials. The 16S rRNA sequencing data were deposited at the NCBI
Sequence Read Archive (SRA) with an accession number of Bioproject PRJNA601439.
Submitted 17 July 2019
Accepted 16 January 2020
Published 7 February 2020
10.1126/sciimmunol.aay3254
Citation: Z. Xu, F. Takizawa, E. Casadei, Y. Shibasaki, Y. Ding, T. J. C. Sauters, Y. Yu, I. Salinas,
J. O. Sunyer, Specialization of mucosal immunoglobulins in pathogen control and microbiota
homeostasis occurred early in vertebrate evolution. Sci. Immunol. 5, eaay3254 (2020).
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Specialization of mucosal immunoglobulins in pathogen control and microbiota homeostasis
occurred early in vertebrate evolution
Zhen Xu, Fumio Takizawa, Elisa Casadei, Yasuhiro Shibasaki, Yang Ding, Thomas J. C. Sauters, Yongyao Yu, Irene Salinas
and J. Oriol Sunyer

Sci. Immunol. 5, eaay3254.

DOI: 10.1126/sciimmunol.aay3254

Downloaded from http://immunology.sciencemag.org/ by guest on February 14, 2020

Managing microbiota to a T
Mucosal surfaces in teleost fish harbor B cells that produce IgT, a secretory mucosal immunoglobulin. To
investigate how IgT influences gill microbiota composition and resistance to infection, Xu et al. developed an efficient
technique for temporarily depleting IgT-producing B cells in rainbow trout. Acute induction of IgT deficiency in trout led to
dysbiosis of commensal gill microbiota, inflammation, tissue damage, and impaired resistance to infection by a mucosal
parasite. The gill pathology triggered by loss of IgT was reversible after IgT levels recovered to the normal range. These
findings demonstrate that the mucosal immunoglobulin species in both teleost fish (IgT) and mammals (mostly IgA) play
evolutionarily conserved roles in the maintenance of healthy commensal microbiota communities and control of
pathogens.

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