Specialization of mucosal immunoglobulins in pathogen control and microbiota

homeostasis occurred early in vertebrate evolution

Although mammalian secretory immunoglobulin A (sIgA) targets mucosal pathogens for

elimination, its interaction 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 compensatory 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
microbiome 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.

Mucosal surfaces of vertebrates are inhabited by incredibly dense and complex populations of
commensal microbiota and are also continuously exposed to a myriad of pathogens. Mucosal
(secretory) immunoglobulins (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 clearance (1, 3–5), whereas, on
the other hand, sIgA interaction with the microbiota enables commensal colonization,
persistence, and homeostasis (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 infections observed in patients with selective sIgA
deficiency (sIgAdef) (9). In contrast, recent studies have shown that sIgA is indispensable 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 beneficial 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
mucosal surfaces from pathogens or arose instead to establish and maintain homeostatic
relationships with microbiota. Moreover, whether those roles were independently acquired
during the evolution of sIgA in higher vertebrates or represent instead convergently evolved
functions 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 critical 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 lymphocytes, 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 specialized 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 microbiota (23–25). To interrogate whether early sIgs emerged primarily to control
pathogens or to establish homeostatic relationships with the microbiota, 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 models
lacking sIgA). The effects of IgT depletion were analyzed on the gill mucosa, a bona fide
mucosal surface where we have previously demonstrated the local induction of pathogen-specific
IgT responses (24) that is also known to harbor a rich and diverse microbiota (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.

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 immunizing fish with
purified mouse IgG2b (fig. S1A, top). The specificity of TAs was assessed by enzyme-linked
immunosorbent assay (ELISA) (fig. S1B) and Western blotting (fig. S1C, lane 3). Moreover,
flow cytometric analysis using mouse antitrout IgM mAb revealed 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 combination 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 combination 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 performed 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
consistently deplete IgT+ B cells that did not alter the percentage of IgM+ B cells.

To evaluate the degree and duration of IgT+ B cell depletion in both systemic and mucosal
lymphoid organs, as well as its effect on serum 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 response 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 immunofluorescence microscopy studies
(Fig. 2, D to F). Although the depletion of IgT+ B cells occurred quickly (1 to 3 days), the
percentage 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
treatment (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, confirming a lack of IgM and IgD compensatory responses (Fig. 3, B, C, E, and F).
No mortalities were observed throughout the 56-day period in either fish group (fig. S2). In
conclusion, the IgT+ B cell depletion 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.