Vaccine 36 (2018) 3694–3700

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

IL-22 deficiency increases CD4 T cell responses to mucosal immunization

Scott A. Budda, Lauren A. Zenewicz ⇑
Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

a r t i c l e i n f o a b s t r a c t

Article history: Mucosal vaccines are a promising platform for combatting infectious diseases for which we still lack
Received 26 January 2018 effective preventative measures. Optimizing these vaccines to generate the best protective immune
Received in revised form 27 April 2018 responses with the least complicated immunization regimen is imperative. Mucosal barriers are the first
Accepted 2 May 2018
line of defense against many pathogens and, as such, we looked to their biology for strategies to improve
Available online 5 May 2018
vaccine delivery. Interleukin-22 (IL-22) is a key cytokine in both healthy and inflamed mucosal tissues.
IL-22 promotes epithelial cell proliferation and inhibits apoptosis, upregulates mucin and antimicrobial
Keywords:
peptides, all of which promote mucosal barrier integrity. In this study, we find that IL-22 impairs the
Cytokines
Mucosal vaccination
development of a T cell response during mucosal immunization. Compared to wild-type control mice,
CD4 T cells IL-22 deficient mice had increased antigen-specific CD4 T cell responses to intrarectal immunization
using a protein and cholera toxin adjuvant vaccine. When immunized systemically with the same protein
antigen adsorbed to alum, no differences in the CD4 T cell response between wild-type and IL-22 defi-
cient mice were detected. This suggests that transiently inhibiting IL-22 during mucosal vaccination
could enhance T cell responses. The broad-applicability of this proposed approach would allow for
improvement of many existing mucosal vaccine regimens and have positive implications in the develop-
ment of more efficacious mucosal vaccines.
Ó 2018 Elsevier Ltd. All rights reserved.

1. Introduction IL-22 is an important cytokine in mucosal barrier responses

Mucosal surfaces are the first line of defense against numerous
bacterial, viral and parasitic pathogens. Within the gastrointestinal
(GI) tract, a single-cell layer epithelium provides a vital barrier to
the environment [1,2]. In the lamina propria, scattered immune
cells survey for infection. Within the gut-associated lymphoid tis-
sue (GALT) are organized immune sites at which immune responses
are initiated [3]. These features allow innate and adaptive immune
cells to survey for infection and respond accordingly to protect the
host. Upon antigen re-encounter, memory and effector T cells are
directed to the mucosal membranes through tissue-specific homing
receptors. Therefore, vaccination via systemic methods, such as
subcutaneous injection, are often poor at inducing protection at
mucosal surfaces. Few mucosal vaccines have been approved for
human use; these include oral vaccines against polio, Salmonella,
Vibrio cholerae and a rotavirus, and a nasal vaccine against influenza
[4–6]. We still lack efficacious vaccines for many infectious diseases
that infect via mucosal barriers, including human immune
deficiency virus (HIV) and Mycobacterium tuberculosis.
⇑ Corresponding author at: Department of Microbiology and Immunology,
College of Medicine, University of Oklahoma Health Sciences Center, 940 Stanton
L. Young Blvd, BMSB 1053, Oklahoma City, OK 73104, USA.
E-mail address: lauren-zenewicz@ouhsc.edu (L.A. Zenewicz).
during infection and inflammation [7,8]. The cytokine is produced
by both activated innate and adaptive lymphocytes. In healthy
individuals, the only source of IL-22 are low levels found in the
GI tract, likely due to interactions between the immune cells and
commensal bacteria [9]. IL-22 receptor is expressed mainly by
epithelial cells, and its expression is notably absent from immune
cells of healthy individuals [10]. Therefore, there are no detectable
direct effects of the cytokine on the healthy immune system.
In epithelial cells, recognition of IL-22 induces STAT3 activation,
leading to pro-proliferative and anti-apoptotic pathways, which
aids in epithelial barrier maintenance. IL-22 can be both protective
and immunopathogenic in the GI tract [11–13]. During acute
experimental models of inflammatory bowel disease (IBD), IL-22
is important in protecting the mucosal barrier by induction of
mucins and anti-microbial peptides, as well as maintaining barrier
integrity [11,14]. In contrast, during a model of chronic IBD, IL-22
is responsible for hyperproliferation of epithelial cells and leads to
thickening of the colon [12]. IL-22 is also important in the GI tract
in wound healing, graft versus host disease (GVHD), development
of colorectal cancer and protecting against invasion by several dif-
ferent bacterial pathogens [15–19].
In the course of our studies to determine the function of IL-22
using IL-22 deficient mice, we found that memory-phenotype

https://doi.org/10.1016/j.vaccine.2018.05.011
0264-410X/Ó 2018 Elsevier Ltd. All rights reserved.
 S.A. Budda, L.A. Zenewicz / Vaccine 36 (2018) 3694–3700
CD4 T cells from naïve IL-22 deficient mice had increased cytokine
responses compared to age-matched wild-type mice. As IL-22
receptor is expressed on epithelial cells and is not usually
expressed by immune cells, we proposed that IL-22 has an indirect
effect on in vivo T cell responses. These memory-phenotype cells
most likely developed from encounter of antigen from the GI tract
and this led us to hypothesize that in the absence of IL-22, gener-
ation of CD4 T cell responses from luminal antigens may be
enhanced.
To test this hypothesis, we utilized a mucosal vaccination regi-
men with a model antigen (chicken ovalbumin) with a mucosal
adjuvant (cholera toxin) administered intrarectally. This vaccine
regimen induced an antigen-specific CD4 T cell response. We found
that IL-22 deficient mice had greater antigen-specific T cell
responses to mucosally, but not systemically, administered anti-
gens compared to wild-type control mice. In the absence of IL-
22, the epithelial barrier has increased permeability [19,20], poten-
tially allowing more antigen to encounter the immune system,
resulting in a greater immune response. This suggests that tran-
siently inhibiting IL-22 during mucosal vaccination could enhance
T cell responses, allowing for the better rational design of mucosal
vaccines.
2. Materials and methods
2.1. Mice
Il22 / deficient mice on a C57BL/6 background (20 generations
to NCI C57BL/6Cr) were maintained by heterozygous or homozy-
gous breeding [21]. OT-II mice were obtained from The Jackson Lab-
oratory (Bar Harbor, ME) [22]. C57BL/6 and CD45.1 mice were
purchased from NCI/Charles River (Frederick, MD). Mice were
housed by genotype. Mice within experiments were age- and sex-
matched. Both males and females were used. All experiments were
performed with IACUC approval from Yale University or the Univer-
sity of Oklahoma Health Sciences (OUHSC) IACUC committees. Mice
at OUHSC were housed under Helicobacter-free conditions.
2.2. Ex vivo restimulation of purified CD4+ T cells
After positive selection of CD4+ cells by magnetic beads (Mil-
tenyi Biotec; Auburn, CA), splenocytes were stained with fluores-
cently conjugated Abs (CD4 PECy7, CD25 PE and CD45RB FITC, all
from eBioscience; San Diego, CA). CD4+ CD25 CD45RBlow T cells
were isolated by cell sorting on a FACSVantage (BD Biosciences;
San Jose, CA). Cells were stimulated for 24 h with anti-CD3 (clone
145-2C11) and anti-CD28 (clone X37.51) Ab.
2.3. Immunization
For mucosal immunization, mice were anesthetized with isoflu-
rane and then intrarectally administered 40 ll of sterile PBS con-
taining 10 lg cholera toxin (List Biological Laboratories, Inc.;
Campbell, CA) and 100 lg chicken ovalbumin (Sigma; St. Louis,
MO). 40 ll PBS was given as a control, where indicated. For sys-
temic immunization, mice were injected i.p. with 100 lg ovalbu-
min protein adsorbed to Imject (ThermoFisher Scientific;
Waltham, MA). Where noted, 24 h prior to euthanization, mice
were administered i.p. 200 lg of CD4 Ab (clone GK1.5) depletion
(BioXCell; West Lebanon, NH).
2.4. Ex vivo antigen-specific restimulation of splenocytes
5  106 splenocytes were cultured at 37 °C with 5% CO2 in the
presence or absence of 100 lg/ml ovalbumin for 48 h in either
3695
complete Click’s (Irvine Scientific; Santa Ana, CA) or IMDM (Corn-
ing; Tewksbury, MA) media containing 10% heat-inactivated FBS
(Gemini Bio-Products; West Sacramento, CA), 100 U/ml penicillin
(Gemini Bio-Products), 100 U/ml streptomycin (Gemini Bio-
Products), 50 lM b-mercaptoethanol (ThermoFisher) and 2 mM
glutamine (Corning).
2.5. Flow cytometry
Cells were stained with fluorescently-conjugated Ab in PBS with
1% BSA for 30 min in the dark. Cells were fixed with 2% PFA and
then analyzed on a FACSCalibur (BD Biosciences) or a Stratedigm
S1200Ex flow cytometer (San Jose, CA) and data were analyzed
using FlowJo v.9.6 (Tree Star; Ashland, OR).
2.6. Adoptive transfer of OT-II cells
OT-II cells were labeled with 5 lM CFSE (eBioscience) and then
i.v. injected into CD45.1 recipients. Twenty-four hrs later, mice
were intrarectally administered 40 ll containing 100 lg OVA323-339
peptide (InvivoGen; San Diego, CA) with 10 lg cholera toxin.
2.7. ELISAs
Mouse IFNc, IL-17 and IL-13 ELISAs (eBioscience) and mouse IL-
22 ELISA (Antigenix America; Huntington Station, NY) were per-
formed according to the manufacturers’ protocols.
2.8. Real-time RT-PCR
Cells were harvested in Trizol (Life Technologies; Carlsbad, CA)
and RNA was prepared according to the manufacturer’s protocol.
RNA was DNase treated (Life Technologies) and cDNA was reverse
transcribed using SuperScript II (ThermoFisher) with oligo dT as
the primer. cDNA was used as template in a real-time PCR reaction
using ABI Taqman primer-probes sets on a ABI 7500 Fast real time
PCR machine (Life Technologies). cDNA was semi-quantitated
using the DDCT method with Hprt as an internal control for all
samples.
2.9. Statistics
Values are expressed as mean ± SD. For two-way comparisons, a
standard paired t test was used. For multiple comparisons, one-
way ANOVA with Tukey’s post hoc analysis was used. Significance
was defined as a value of p < 0.05.
3. Results
3.1. Restimulated CD4 T cells from year-old Il22 / mice expressed
higher levels of cytokines compared to cells from wild-type control
mice
IL-22 deficient mice are more susceptible than control mice to
many GI bacterial pathogens [7]. However, as IL-22 is expressed at
low levels in healthy individuals and highly upregulated during
inflammation, it is anticipated that Il22 / mice are generally pheno-
typically normal when housed under specific-pathogen free condi-
tions [21], although some changes, such as altered microbiota,
have been reported [23]. During the course of studying if lack of IL-
22 has long-term effects on mouse health, we examined CD4 T cells,
as these cells are a primary source of the cytokine. We found no dif-
ferences in the overall population of CD4 T cells in the spleens of
year-old Il22+/+ and Il22 / mice, including surface expression of
the effector/memory surface marker CD45RB (Fig. 1A). To measure
3696 S.A. Budda, L.A. Zenewicz / Vaccine 36 (2018) 3694–3700

T cell effector capacity, purified CD45RBlow CD4 T cells were restim-
ulated ex vivo with anti-CD3 and CD28 Abs. CD4 T cells from the
Il22 / mice expressed greater levels of Ifng and Il17a compared to
wild-type control mice (Fig. 1B). Furthermore, secreted levels of IFNc
and IL-17 were also increased in supernatants of Il22 / CD4 T cells
compared to control cells (Fig. 1C). Thus, CD4 T cells from year old
IL-22 deficient mice had greater cytokine capacity than those cells
from wild-type control mice.
As IL-22R is not typically expressed by immune cells [10,21,24],
IL-22 produced by T cells is not expected to have direct effects on T
cells. Our data suggest that something inherent within the mice is
causing changes in the CD4 T cells. Since these mice have exclu-
sively lived in a specific-pathogen free environment and have not
been infected with any known pathogens, we hypothesized that
the effector and memory phenotype CD4 T cells had arisen from
recognition of commensal and food antigens. The well-described
decreased barrier permeability in the absence of IL-22 [19,20],
may lead to increased translocation of antigens and produce a
greater T cell response. This led us to hypothesize that mucosal
vaccination in the absence of IL-22 may be an effective way to
improve vaccine regimens.
3.2. Intrarectal mucosal immunization with a model antigen generated
antigen-specific CD4 T cell responses
In order to study the role of IL-22 in mucosal vaccination, we
designed an immunization regimen in which a model antigen,
chicken ovalbumin, was administered via a method that simulated
food antigen exposure. As we have studied the role of IL-22 in the
colon, in order to bypass the upper GI tract, we decided to intrarec-
tally administer then antigen. Rectal administration of vaccines has
gained traction in recent years, especially in HIV vaccine design
[25]. Young mice (8–12 weeks old) were intrarecetally adminis-
tered ovalbumin with a commonly used mucosal vaccine adjuvant,
cholera toxin, or PBS as a control. Seven days post-immunization,
mice were euthanized and the antigen-specific T cell response
was examined. Splenocytes were cultured ex vivo with or without
ovalbumin for 48 h and secretion of various cytokines into the
supernatants was quantitated by ELISA. Cells from PBS control
mice did not secrete cytokines. Splenocytes from immunized mice
produced cytokines only when cultured in the presence of ovalbu-
min (Fig. 2). We found increased levels of IFNc and IL-13, indicative
of Th1 or Th2 responses, respectively. We also detected IL-17 and
IL-22, suggesting a Th17 response to our immunization regimen.
Thus, intrarectal immunization of mice with ovalbumin with cho-
lera toxin resulted in the generation of a broad T helper antigen-
specific cytokine response.
IFNc, IL-13, IL-17 or IL-22 are not exclusively produced by CD4 T
cells. To more closely examine the CD4 T cell response in our muco-
sal immunization regimen, we examined the role of CD4 T cells in
ex vivo cytokine secretion. Twenty-four hrs prior to euthanization,
we injected immunized mice with anti-CD4 Ab (clone GK1.5) to
selectively deplete CD4 T cells or with PBS as a control (Fig. 3A).
Splenocytes were stimulated ex vivo with or without ovalbumin
for 48 h and then secreted cytokines were quantitated by ELISA.
Splenocytes from mice depleted of CD4 T cells secreted less IFNc,
IL-13, IL-17 and IL-22 (Fig. 3B), suggesting that ovalbumin-
specific CD4 T cells were an important source of these cytokines.

25 CD4+ CD25- CD45RBlow cells/spleen

1.5×106
% CD45RBlow/CD4 T cells

20
1.0×106
15

10
5.0×105
5

0 0.0
Il22+/+ Il22-/- Il22+/+ Il22-/-

0.5 0.20 3500 600

3000
0.4
0.15 2500
IL-17 (pg/ml)
IFNγ (pg/ml)

400
Il17a/Hprt
Ifng/Hprt

0.3
2000
0.10
0.2 1500
200
0.05 1000
0.1
500
0.0 0.00 0 0
/+

/-

/+

/-
/+

/+
/-

/-
2-

2-
2+

2-

2-

2+
2+

2+
Il2

Il2
Il2

Il2
Il2

Il2
Il2

Il2

Fig. 1. CD45RBlow CD4 T cells from aged IL-22 deficient mice had greater cytokine capacity. Year old age- and sex-matched Il22+/+ or Il22 / mice (C57BL/6 background) were
euthanized and splenic CD4 T cells were examined. (A) CD45RB surface expression on CD4+ CD25 T cells (left). The percent of CD4 T cells that were CD45RBlow CD25 out of
all CD4+ cells (middle). The total number of CD4+ CD25 CD45RBlow T cells per spleen (right). (B and C) Sorted CD4+ CD25 CD45RBlow cells were stimulated ex vivo with
anti-CD3 and anti-CD28 Abs. Eighteen hrs later cytokines were evaluated by (B) real time RT-PCR or (C) ELISA. Each point represents one mouse, horizontal line indicates
mean and bars indicate SD. ns = not significant (p > 0.05), *p < 0.05, **p < 0.01 by Student’s t test. Experiment was performed three times with similar results.
 S.A. Budda, L.A. Zenewicz / Vaccine 36 (2018) 3694–3700 3697

50000 8000 α
9.5 11.3
40000
6000

IL-17 (pg/ml)
IFNγ (pg/ml)

30000
13.5
4000 0.37

20000
2000
10000 40000 4000

0 0 30000 3000

IL-17 (pg/ml)
IFNγ (pg/ml)
S

T
+C
PB

+C
PB
20000 2000
VA

VA
O

O
6000 8000 10000 1000

0 0
6000

b
ro

ro
A

A
IL-13 (pg/ml)

IL-22 (pg/ml)

nt
4000

nt
4

4
D
co

D
co
C

C
ti-

ti-
an

an
4000 15000 1000

2000 800
2000

IL-13 (pg/ml)

IL-22 (pg/ml)
10000
600

400
0 0 5000
200
S

S
T

T
+C

+C
PB

PB
VA

VA

0 0
O

b
ro

ro
A

A
nt

nt
4

4
D

D
co

co
C

C
PBS
ti-

ti-
an

an
OVA stimulation PBS
OVA stimulation
Fig. 2. Intrarectal immunization with antigen and cholera toxin induced an
antigen-specific immune response. C57BL/6 mice (8–12 weeks old) were intrarec-
tally administered PBS or 100 lg ovalbumin (OVA) and 10 lg cholera toxin (CT).
Seven days later, mice were euthanized and spleens were harvested. Splenocytes
were cultured ex vivo for 48 h with ovalbumin or, as a control, a volume of PBS equal
to the ovalbumin addition. Cytokines (IFNc, IL-17, IL-13 and IL-22) were quanti-
tated in the supernatant by ELISA. Each point represents one mouse, horizontal line
indicates mean and bars indicate SD. *p < 0.05, **p < 0.01, ****p < 0.0001 by two-way
ANOVA. Experiment was performed three times with similar response.

To further examine the CD4 T cell response, we made use of

OVA-specific T cell receptor (TCR) transgenic OT-II cells [22], which
recognize OVA323-339 presented in the context of I-Ab. CFSE labeled,
CD45.2 OT-II cells were adoptively transferred to congenic CD45.1
mice. The next day, the mice were intrarectally immunized with
OVA323-339 peptide with cholera toxin or as a negative control
PBS. As a positive control, mice were intraperitoneally injected
with OVA323-339 adsorbed to alum. Seven days post-
immunization, we examined the proliferation of the OVA-specific
CD4 T cells by CFSE dilution. Mice that received peptide and alum
immunization had a robust OT-II (CD4+ CD45.2+) T cell response
(Fig. 3C) and greater than 90% of the OT-II cells in the spleen or
mesenteric lymph nodes (MLN) had undergone at least one divi-
sion. In mice intrarectally immunized with peptide and CT a dis-
tinct population of OT-II cells in the both the spleens and MLNs
had undergone at least seven divisions (Fig. 3C), with few cells in
divisions 2 through 6 (Fig. 3C). In contrast, OT-II cells in control
mice intrarectally administered PBS mostly did not undergo divi-
sion. Thus, CD4 T cells recognized antigen administered through
intrarectal immunization.
3.3. IL-22 deficient mice had elevated antigen-specific CD4 T cell
responses to mucosal immunization
With our mucosal vaccine that induces antigen-specific CD4 T
cell responses, we immunized Il22+/+ or Il22 / mice. Seven days
Fig. 3. Intrarectal immunization generated an antigen-specific CD4 T cell response.
(A and B) C57BL/6 mice (8–12 weeks old) were intrarectally administered PBS or
100 lg ovalbumin and 10 lg cholera toxin. Six day later, mice were divided into
two groups and one group received PBS and the other received 200 lg anti-CD4 Ab
(clone GK1.5). Twenty-four hrs later (7 days post-immunization), mice were
euthanized and spleens were harvested. (A) CD4 and CD8 T cells in two
representative spleens by flow cytometry. (B) Splenocytes were cultured for 48 h
with ovalbumin or PBS. Cytokines (IFNc, IL-17, IL-13 and IL-22) were quantitated in
the supernatant by ELISA. Each point represents one mouse, horizontal line
indicates mean and bars indicate SD. ns = not significant (p > 0.05), *p < 0.05, **p <
0.01, ***p < 0.001 by two-way ANOVA. (C) 4  106 CD45.2 OT-II T cells were CFSE
labeled and adoptively transferred to CD45.1 C57BL/6 mice. The next day, mice
were intrarectally immunized with 100 lg OVA323–339 peptide and 10 lg cholera
toxin (CT) or no peptide, or intraperitoneally injected with OVA323–339 peptide
adsorbed to alum. Seven days post-immunization, mice were euthanized and
spleens and mesenteric lymph nodes (MLN) were harvested and analyzed by flow
cytometry. Shown are representative CFSE plots of CD4+ CD45.2+ gated cells.
Similar experiments were performed twice with similar results.
post-immunization we examined the antigen-specific T cell
response. When stimulated ex vivo with ovalbumin, compared to
Il22+/+ controls, splenocytes from Il22 / mice produced greater
3698 S.A. Budda, L.A. Zenewicz / Vaccine 36 (2018) 3694–3700

levels of IFNc, IL-17 and IL-13 (Fig. 4). As a control, we also exam- antigen-specific responses to mucosal vaccination, which included
ined IL-22 and found that cells from Il22 / mice did not secrete a continuum of Th1, Th2 and Th17 responses.
detectable IL-22, as expected (Fig. 4). Thus, Il22 / mice had greater To show that the effect of IL-22 on generation of the antigen-
specific response was specific to mucosal administration of the
antigen, we immunized mice via a different route. Wild-type and
6000
IL-22 deficient mice were intraperitoneally immunized with oval-
60000 bumin protein adsorbed to the adjuvant alum. We chose alum
since like cholera toxin, it was thought to be Th2-skewing, but

IL-17 (pg/ml)
IFNγ (pg/ml)

4000
now is known to also induce Th1 and Th17 responses [26]. Exam-
40000
ination of the resulting ovalbumin-specific T cell response on day
seven post-immunization found no significant difference in the
20000
2000 ovalbumin-specific IFNc or IL-17 response between splenocytes
from IL-22 deficient and wild-type mice (Fig. 5). Thus, alum immu-
nization was not dependent on the cytokine IL-22.
0 0
/+

/-

/+

/-
2-

2-
2+

2+

4. Discussion
Il2

Il2
Il2

Il2

4000 2000 Vaccines have significantly reduced the incidence of many once
common diseases [27]. They are designed to generate rapid, strong,
3000 1500 protective adaptive immune responses that swiftly eliminate
IL-13 (pg/ml)

IL-22 (pg/ml)

pathogens so there are no or minimal disease symptoms. The basis

of most FDA approved vaccines is through humoral immunity.
2000 1000 These vaccines generate strong antibody responses by generating
high titers of antigen-specific B cell responses. Upon challenge,
1000 500 these B cells quickly secrete antibodies that may block the recep-
tors needed to invade cells or aid in the opsinization, neutralization
and destruction of pathogens. On the other hand, for diseases
0 0
against which we still lack efficacious vaccines, it is generally
/+

/+
/-

/-

thought that B cell responses are not sufficient to provide protec-

2-

2-
2+

2+
Il2

Il2
Il2

Il2

tion and that antigen-specific CD4 and/or CD8 T cell responses will
be required to confer protection [27]. CD8 T cells may be involved
PBS in direct killing of infected cells through secretion of granzyme and
OVA stimulation perforin and CD4 T cells may provide functional help to B cells and
CD8 T cells through the secretion of cytokines and other effector
Fig. 4. IL-22 deficient mice had a greater antigen-specific T cell response to molecules. Refining existing vaccine regimens to enhance T cell
intrarectal immunization. Il22+/+ or Il22 / mice (8–12 weeks old) were intrarectally
responses is an efficient way to potentially improve vaccine
administered PBS or 100 lg ovalbumin (OVA) and 10 lg cholera toxin (CT). Seven
days later, mice were euthanized and spleens were harvested. Splenocytes were
efficacy.
cultured for 48 h with ovalbumin or PBS. Cytokines (IFNc, IL-17, IL-13 and IL-22) were Our data show that IL-22 impairs the development of a CD4 T
quantitated in the supernatant by ELISA. Each point represents one mouse, horizontal cell response during mucosal immunization. This suggests that
line indicates mean and bars indicate SD. *p < 0.05, **p < 0.01, ****p < 0.0001 by two- blocking IL-22 during mucosal vaccination could enhance T cell
way ANOVA. Experiment was performed four times with similar results.
and potentially other immune responses. We propose that inclu-

1000 800 2000

750 600 1500

IL-17 (pg/ml)

IL-22 (pg/ml)
IFNγ (pg/ml)

500 400 1000

250 200 500

0 0 0
/+

/-
/+

/+
/-

/-
2-
2-

2-
2+
2+

2+
Il2
Il2

Il2
Il2
Il2

Il2

PBS
OVA stimulation

Fig. 5. IL-22 deficient mice had no detectable alteration in CD4 T cell responses to systemic immunization. Il22+/+ or Il22 / mice (8–12 weeks old) were intraperitoneally
injected with 100 lg ovalbumin adsorbed onto alum. Seven days later, mice were euthanized and spleens were harvested. Splenocytes were cultured for 48 h with ovalbumin
or PBS. Cytokines (IFNc, IL-17 and IL-22) were quantitated in the supernatant by ELISA. Each point represents one mouse, horizontal line indicates mean and bars indicate SD.
ns = not significant (p > 0.05), ****p < 0.0001 by two-way ANOVA. Similar experiments were performed three times with comparable results.
 S.A. Budda, L.A. Zenewicz / Vaccine 36 (2018) 3694–3700
sion of a factor that temporally inhibits IL-22 during the vaccine
regimen to enhance immune responses could improve existing
vaccines or those currently in development. In human subjects,
IL-22 could be transiently inhibited at the time of immunization
by either a neutralizing IL-22 antibody or recombinant IL-22
binding protein (IL-22BP). IL-22BP is a soluble receptor homolog
produced by non-inflammatory dendritic cells (DCs) [18]. It binds
IL-22 with greater affinity than the IL-22 receptor and prevents
IL-22 signaling in epithelial cells in vitro and in vivo [28–30].
Inhibition of IL-22 during vaccination may be especially relevant
for soluble antigen formulations. The access of antigens to the
underlying tissues, and in effect the mucosal immune system, is
impeded by the constantly renewing layer of mucus lining the GI
tract. Inhibition of IL-22 may also improve the efficacy of particu-
late formulations of vaccines by down-regulating expression of
mucin [14]. As IL-22 inhibition would only be transient, no detri-
mental effects would be anticipated for a healthy subject. One
potential negative consequence may be a higher risk for infections,
such as Candida or those of the upper respiratory tract [31,32].
IL-22 is a key immunological factor in mucosal barrier integrity
and the cytokine targets many cell types that comprise the barrier
[33]. IL-22 induces mucin production from goblet cells and antimi-
crobial peptides from Paneth cells, both of which limit potentially
inflammatory microbiota interactions with the barrier [14,19,34].
IL-22 induces increased intestinal stem cell proliferation and
expansion allowing for rapid epithelial cell regeneration [35].
Epithelial cells constitute the greatest number of cells in the muco-
sal barrier and IL-22 limits premature apoptosis, thereby maintain-
ing the cellular barrier [19]. In addition to providing protection
against infection by many diverse GI bacterial pathogens, IL-22
mediates limits in trafficking of commensal bacteria or pathobionts
to the liver and other tissues [36,37]. If bacteria dissemination is
detectable, certainly bacterial structural components and food
antigens may also translocate mediating low grade chronic inflam-
mation. In addition to these functional biological deficiencies,
numerous studies have performed barrier function assays on the
normal and/or inflamed colon in the presence or absence of IL-
22. For example, in vivo permeability assays show that IL-22 defi-
cient mice have greater bloodstream levels of intrarectally or intra-
gastrically administered FITC-dextran [19,20]. IL-22 deficient mice
have alterations in microbiota [23] that may, in part, cause these
changes in barrier integrity, but complementary experiments with
transient neutralization of IL-22 via antibody generally recapitu-
late findings from IL-22 deficient mice. Furthermore, in vitro per-
meability assays using polarized epithelial cell culture show that
addition of recombinant IL-22 increases transcellular epithelial
resistance (TER) [38]. It should be noted that a few studies have
found the converse; in some circumstances IL-22 increases barrier
permeability [39,40]. These IL-22-mediated changes in barrier
integrity may in turn modulate immune responses. In addition to
the findings of this study, a report using a model of Plasmodium
infection, found that DCs from IL-22 deficient mice had a more
activated profile (CD86+ CD80+) than those from wild-type mice
and resulted in slightly greater T cell proliferative responses [41].
Other indirect effects of IL-22 on immune cells could potentially
modulate the outcomes of infection and inflammatory events.
Mucosal vaccines have recently garnered much attention since
they are a prospective vaccination strategy against HIV [5]. HIV is
a mucosal pathogen as transmission occurs through the mucosal
surfaces of the rectal and genital mucosae and furthermore early
infection by any route is accompanied by a transient depletion of
T cells in the GI tract [42]. Mucosal vaccine strategies are also
desirable for combating other prevalent mucosal-associated dis-
eases such as Salmonella and Vibrio cholera. Recent advances in
adjuvants and immunomodulatory cytokines have allowed for
new ways for vaccines to either breach the epithelium or alter
3699
the mucosal immune system [5,6]. Forthcoming studies will inves-
tigate the role of IL-22 during mucosal vaccination at other epithe-
lial barriers, especially the respiratory tract, since new or improved
influenza or M. tuberculosis vaccines are very desirable. Indirect
effects of IL-22 on antibody generation, particularly sIgA, will also
be evaluated. In addition to correlates of protection, protection
itself will also be assessed.
Future successful vaccines will almost certainly be multi-
component vaccines, composed of many parts that synergize to
generate protective immunity [27]. They will generate robust T
and B cell responses that quickly respond to infectious challenge.
We propose that inclusion of a factor that temporally inhibits IL-
22 during vaccination to enhance immune responses can be a part
of this success and future studies will address this vision.
Acknowledgments
We thank Dr. Mark Lang and for discussion and reading of the
manuscript. We acknowledge the Yale University Department of
Immunobiology Flow Cytometry Facility and the OUHSC Flow
Cytometry and Imaging Core for cell sorting and analysis. Research
in the Zenewicz laboratory is supported by an OUHSC College of
Medicine Alumni grant, an American Heart Association Scientist
Development grant (14SDG18700043), Oklahoma Center for
Advancement for Science and Technology grant (HR13-003), the
National Institute of General Medical Sciences of the National Insti-
tutes of Health (P20GM103447) and funding from the Presbyterian
Health Foundation and the Stephenson Cancer Center.
Declarations of interest
None.
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