ORIGINAL RESEARCH
The Microbiome Activates CD4 T-cell–mediated Immunity
to Compensate for Increased Intestinal Permeability
Karen L. Edelblum,1,2 Gil Sharon,3 Gurminder Singh,2,4 Matthew A. Odenwald,2
Anne Sailer,2 Severine Cao,2 Sarina Ravens,5 Irene Thomsen,5 Kamal El Bissati,6,7
Rima McLeod,6,7 Chen Dong,8 Sandeep Gurbuxani,2 Immo Prinz,5
Sarkis K. Mazmanian,3 and Jerrold R. Turner2,4
1
Department of Pathology and Laboratory Medicine, Rutgers New Jersey Medical School, Newark, New Jersey;
Departments of Pathology, 6Ophthalmology and Visual Sciences, and 7Pediatrics, University of Chicago, Chicago, Illinois;
3
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California; 5Institute of
Immunology, Hannover Medical School, Hannover, Germany; 8Institute for Immunology, Tsinghua University, Beijing, China;
and 4Departments of Pathology and Medicine, Brigham and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts
2
SUMMARY
Despite common perceptions, studies of human subjects and
experimental animals show that intestinal barrier loss is
insufficient to induce disease in healthy individuals. Here we
define a compensatory immune response that prevents
pathogen invasion in mice with chronic barrier loss.
BACKGROUND & AIMS: Despite a prominent association,
chronic intestinal barrier loss is insufficient to induce disease in
human subjects or experimental animals. We hypothesized that
compensatory mucosal immune activation might protect
individuals with increased intestinal permeability from disease.
We used a model in which intestinal barrier loss is triggered by
intestinal epithelial-specific expression of constitutively active
myosin light chain kinase (CA-MLCK). Here we asked whether
constitutive tight junction barrier loss impacts susceptibility to
enteric pathogens.
METHODS: Acute or chronic Toxoplasma gondii or Salmonella
typhimurium infection was assessed in CA-MLCK transgenic or
wild-type mice. Germ-free mice or those lacking specific immune cell populations were used to investigate the effect of
microbial-activated immunity on pathogen translocation in the
context of increased intestinal permeability.
RESULTS: Acute T gondii and S typhimurium translocation
across the epithelial barrier was reduced in CA-MLCK mice.
This protection was due to enhanced mucosal immune
activation that required CD4þ T cells and interleukin 17A
but not immunoglobulin A. The protective mucosal immune
activation in CA-MLCK mice depended on segmented filamentous bacteria (SFB), because protection against early
S typhimurium invasion was lost in germ-free CA-MLCK mice
but could be restored by conventionalization with SFBcontaining, not SFB-deficient, microbiota. In contrast,
chronic S typhimurium infection was more severe in
CA-MLCK mice, suggesting that despite activation of protective mucosal immunity, barrier defects ultimately result
in enhanced disease progression.
CONCLUSIONS: Increased epithelial tight junction permeability
synergizes with commensal bacteria to promote intestinal CD4þ
T-cell expansion and interleukin 17A production that limits
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Edelblum et al
enteric pathogen invasion. (Cell Mol Gastroenterol Hepatol
2017;4:285–297; http://dx.doi.org/10.1016/j.jcmgh.2017.06.001)
Keywords: Barrier Function; Tight Junction; Microbiota; CD4 T
Cell; Mucosal Immunity; Salmonella.
T
he intestinal epithelium supports nutrient absorption and secretion but also serves as a selectively
permeable barrier that separates the mucosal immune
system from the intestinal lumen.1–3 The association of
many inflammatory bowel disease risk alleles with mucosal
immunity and bacterial recognition4 suggests that intact
epithelial barrier, which limits microbial contact with the
immune system, may prevent a severe inflammatory
response in susceptible individuals. Consistent with this
idea, intestinal barrier function is compromised in Crohn’s
disease and has been identified as a marker of impending
disease reactivation.5,6 Nevertheless, modest barrier loss is
present in a subset of healthy first-degree relatives of
Crohn’s disease patients and has been associated with
specific polymorphisms of the muramyl dipeptide receptor
NOD2.7,8 These human data correlate with results from rodent models showing that epithelial barrier dysfunction
alone is not sufficient to initiate disease.9–11
The mechanisms that prevent disease in individuals with
increased intestinal permeability are poorly understood.
Although mouse models can be useful in dissecting these
processes, most display far greater intestinal permeability
increases than those seen in healthy individuals with modest
barrier loss7,12,13 and use mechanisms that may not be relevant to human disease. We have focused on myosin light
chain kinase, which is well-recognized as a physiologically
and pathophysiologically relevant regulator of tight junctions
and the intestinal barrier.3,14 Transgenic mice that express
constitutively active myosin light chain kinase
(CA-MLCK)14–16 within the intestinal epithelium exhibit targeted increases in intestinal tight junction permeability,9 but,
unlike most other rodent models, they do not display evidence
of epithelial damage such as increased epithelial proliferation
and migration.9–11 Therefore, CA-MLCK mice provide an
exquisitely targeted model of increased intestinal tight junction permeability that is not associated with epithelial damage
but is quantitatively and qualitatively similar to barrier
defects observed in healthy Crohn’s disease relatives.
It is now widely recognized that the intestinal microbiota
shape mucosal immunity through mechanisms including
stimulation of innate immune cells by commensal-derived
products and induction of T-cell polarization.17,18 These
data indicate the presence of a continuous feedback loop in
which commensal bacteria “instruct” host immunity to
induce tolerance to the local microenvironment or, alternatively, serve as a rheostat to modulate pathogen clearance
or autoimmunity. In addition to the epithelial barrier,
several mechanisms have evolved to limit pathogen invasion,19 including the synthesis of antimicrobial immunoglobulin (Ig) A and IgG by plasma cells in the lamina propria
(LP) that is in turn transported across the epithelium into
the intestinal lumen.20
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
Increased intestinal permeability is not sufficient to
promote spontaneous disease.9–11,21,22 However, constitutive barrier loss in CA-MLCK mice does induce subclinical
immune activation and increased susceptibility to experimental immune-mediated colitis.9 Notably, the barrier loss
in these mice is due to selective effects on tight junction
permeability and is insufficient to trigger epithelial damage.9,23 We hypothesized that the barrier defects resulting
from CA-MLCK expression would render these mice more
susceptible to enteric infection.
Surprisingly, our data show that CA-MLCK mice are protected against early pathogen invasion. This protection
depended on adaptive immunity via LP CD4þ T-cell expansion, interleukin 17A (IL17A) production, and enhanced
neutrophil recruitment in infected CA-MLCK mice relative to
infected wild-type (WT) mice. Protection was abrogated in
germ-free CA-MLCK mice, indicating that commensal microbes promote a mild Th17 response that limits early
pathogen translocation. Although increased tight junction
permeability protected against acute bacterial invasion, CAMLCK mice did develop more severe intestinal and systemic disease during chronic infection. These data provide
new insight into the mechanisms that mucosal immunity
adapts to physiological-range epithelial barrier defects and
how this compensation eventually fails in the context of
chronic inflammatory disease.
Materials and Methods
Animals
Mice were 8–12 weeks of age, maintained on a C57BL/6
background, and housed under specific pathogen–free (SPF)
conditions. WT, Rag1 KO, and Tcrd KO mice were obtained
from the Jackson Laboratories, Bar Harbor, ME. In our colony,
all mice were colonized with segmented filamentous bacteria
(SFB). CA-MLCK mice9 were crossed to TcrdH2BeGFP
(TcrdEGFP),24 IL17A KO,25 or IghA KO26 mice (provided by
Dennis Metzger, Albany Medical College, Albany, NY).
Germ-free (GF) transgenic CA-MLCK mice were produced by rederivation of SPF C57BL/6J females mated with
SPF CA-MLCK males. Briefly, uteri from donor mothers were
removed on E19.5 by cesarean section and transferred into
a GF isolator in aseptic Virkon solution (DuPont, Parlin, NJ).
Offspring were then extracted from the uterus and reared
by a GF foster Swiss-Webster mother. Healthy offspring
were genotyped, and males harboring the transgene were
subsequently used for breeding with GF C57BL/6J females.
Abbreviations used in this paper: CA-MLCK, constitutively active
myosin light chain kinase; CFU, colony-forming unit; GF, germ-free; Ig,
immunoglobulin; IL, interleukin; LP, lamina propria; SEM, standard
error of the mean; SFB, segmented filamentous bacteria; SPF, specific
pathogen–free; WT, wild-type.
Most current article
© 2017 The Authors. Published by Elsevier Inc. on behalf of the AGA
Institute. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2352-345X
http://dx.doi.org/10.1016/j.jcmgh.2017.06.001
September 2017
For colonization experiments, littermate WT and
CA-MLCK mice were removed from the GF isolator at 3–4
weeks of age and gavaged with 100 mL cecal contents from
C57BL/6NTac (Taconic Farms, Hudson, NY) or C57BL/6J
mice (Jackson Laboratories). Cecal contents were resuspended in sodium bicarbonate solution under anaerobic
conditions (1:2 v/v). Salmonella typhimurium exposure and
tissue sampling were performed 4 weeks after colonization
on age-matched GF and conventionalized mice. All studies
were conducted in an Association of the Assessment and
Accreditation of Laboratory Animal Care (AALAC)–
accredited facility according to protocols approved by
Rutgers New Jersey Medical School, the University of
Chicago, and the California Institute of Technology Institutional Animal Care and Use Committees.
Pathogen Infection
DsRed-labeled S typhimurium strain SL3201 were provided by Andrew Neish, Emory University, Atlanta, GA. Mice
were anesthetized by intraperitoneal injection of ketamine/
xylazine, a 3- to 4-cm loop of jejunum or ileum was exposed,
and the luminal surface of the intestine was exposed as
described previously.27 For 30 minutes 108 colony-forming
units (CFUs) DsRed-SL3201 were applied directly to the
exposed luminal surface, after which mice were killed, and
the loop of intestine was fixed for analysis by fluorescence
microscopy as previously described.28
Systemic infection of S typhimurium was assessed by oral
gavage with 108 CFUs DsRed-SL3201 after 24 hours of
pretreatment with 100 mg/mL streptomycin. Mice were
killed 9 days after infection on the basis of the severity of
clinical scores or if an individual mouse lost more than 20%
of its initial body weight. Organs were harvested and either
fixed in 10% neutral buffered formalin or embedded in
optimal cutting temperature (OCT; Sakura, Torrance, CA)
medium for further immunohistochemical analysis. Clinical
scores were determined on the basis of fur texture, posture,
and activity on a scale of 0–2 each, with a potential combined score of 6. Histologic scores of colitis were determined on a scale of 0–2 on the basis of the following criteria:
crypt dilation and elongation, presence of crypt abscesses,
extent of epithelial erosion, and regeneration as well as the
number of apoptotic cells and goblet cells, with a potential
combined score of 14. Histologic scores of the spleen were
determined on a scale of 0–3 on the basis of the immune cell
infiltrate and the presence of neutrophils.
The ME49 strain of Toxoplasma gondii was maintained
as tachyzoites by serial passage in human foreskin fibroblasts as previously described.29 The 10- to 12-week-old
female HLA-B*0702 transgenic mice29 were infected intraperitoneally with 1 104 ME49 type II tachyzoites. Tissue
cysts were isolated from the brains of these mice 22 days
after infection and quantified.30 Mice were gavaged with 10
cysts in 100 mL sterile phosphate-buffered saline and killed
after 1 hour to assess parasite translocation.
Immunofluorescence and Image Analysis
Mouse intestine was fixed in 1% paraformaldehyde for
2 hours, washed with 50 mmol/L NH4Cl, and cryoprotected
Immune Compensation for Barrier Loss
287
in 30% sucrose (w/v) at 4 C overnight. Tissue was then
embedded in OCT medium, snap-frozen, and stored at
–80 C. Alternatively, cryosections of tissue that was snapfrozen without prior fixation were fixed in 1:1 methanol:acetone for 10 minutes at –20 C. Frozen sections were
immunostained as previously described31 by using primary
antibodies, rabbit anti-myeloperoxidase, rabbit anti-CD3
(Abcam, Cambridge, MA), rabbit anti-LDH1 antiserum,32
rat anti-CD4, rat anti-IgA (BD Pharmingen, San Jose, CA),
followed by AlexaFluor-conjugated phalloidin or secondary
antibodies and Hoechst 33342 dye (Invitrogen, Carlsbad,
CA). Slides were mounted with Prolong Gold (Invitrogen)
and visualized on a DMI6000 inverted epifluorescence microscope equipped with a Rolera EMC2 CCD camera
(QImaging, Surrey, BC, Canada), 20/0.50 PH2, 40/
0.60CORR/PH, or 63/0.70CORR objectives, and Metamorph 7 acquisition software (Molecular Devices, Sunnyvale, CA). Four mm Z-stacks were acquired with 0.2 mm
spacing, and images were deconvolved for 10 iterations by
using Autodeblur (Media Cybernetics, Rockville, MD).
Morphometric analysis of S typhimurium and T gondii was
quantified as previously described.28 Briefly, invasion was
quantified as the number of organisms that had invaded into
an epithelial cell or crossed into the LP. Epithelial invasion
required the organism to be localized basal to the perijunctional actomyosin ring, as defined by phalloidin staining.
Unprocessed images were used for quantitative analyses to
avoid potential deconvolution artifact. For each mouse, 6–8
fields, each containing approximately 0.1 mm2 of epithelialcovered villus mucosa, were analyzed by a blinded observer.
Intestinal Permeability
Mice were fasted for 3 hours and then gavaged with 1
mg/mL fluorescein and 20 mg/mL 70 kDa rhodamine
dextran in water. Blood was collected 3 hours later via the
retro-orbital sinus. Fluorescence intensity was determined
by using a plate reader at 495 nm excitation/525 nm
emission and 555 nm excitation/585 nm emission. Intestinal permeability was reported as serum fluorescein recovery normalized to that of 70 kDa rhodamine dextran, which
cannot cross tight junctions.
CD4 Depletion and Adoptive Transfer
CD4þ T cells were depleted by injecting mice intraperitoneally with 0.2 mg GK1.5 (University of Chicago Monoclonal Antibody Core). Injection of 0.2 mg IgG isotype
control (F156-6B9.7) was used as a control. CD4þ T-cell
depletion was confirmed after 48 hours by either flow
cytometric analysis of retro-orbital blood (before infection)
or splenocytes (on death).
Eight-week-old Rag1 KO or Rag1 KO CA-MLCK mice
were injected with 2 106 CD4þ splenocytes from sexmatched 10-week-old C57BL/6 donor mice. CD4þ T cells
were enriched by negative selection (Miltenyi Biotech, San
Diego, CA), sorted to 98% purity by flow cytometry, and
injected retro-orbitally. Infections were performed 2 weeks
after adoptive transfer. Age-matched mice not receiving
adoptive transfer were used as controls.
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Edelblum et al
Immunoglobulin A Enzyme-linked
Immunosorbent Assay
Fecal samples were homogenized in 0.01% NaN3 in
phosphate-buffered saline and centrifuged, and the supernatant was used for analysis. Plates were coated with
2 mg/mL goat anti-mouse IgA (Southern Biotech, Birmingham, AL) in phosphate-buffered saline, and samples were
diluted in 1% bovine serum albumin and incubated overnight at 4 C. Mouse IgA (clone M18-254; BD Pharmingen,
San Diego, CA) was used to generate a standard curve. After
washes in phosphate-buffered saline containing 0.05%
Tween, anti-IgA-avidin phosphatase (Southern Biotech) was
added, and p-nitrophenyl phosphate was used to develop
the reaction. Samples were read by plate reader at 405 nm.
Immunoglobulin A Repertoire Analysis
454 sequencing. After RNA extraction from fecal pellets,
cDNA synthesis was performed with SuperScript III (Invitrogen) by using random hexamer primer. Template
libraries of rearranged IgA sequences were generated by
polymerase chain reaction using a primer specific for the
constant Ca (IgA) region (50 CGTATCGCCTCCCTCGCGCCAT
CAG(MID)GAGCTCGTGGAGTGTCAGTG-30 ) and a primer
binding at all Vh genes (50 -CTATGCGCCTTGCCAGCCC
GCTCAGGAGGTGCAGCTGCAGGAGTCTGG-30 ).33,34 MIDs of
4-nucleotides at constant Ca/Cm primers allowed to pool
samples within sequencing lanes. Following polymerase
chain reaction conditions were used: 95 C, 4 minutes; 25
(94 C, 30 seconds; 62 C, 30 seconds; 72 C, 35 seconds); and
72 C for 10 minutes. Amplicons were purified by Qiaquick
Gel Extraction kit (Qiagen, Germantown, MD) and prepared
for 454 sequencing using the GS FLX Titanium SV emPCR
Lib-A kit and Genome Sequencer FLX system as
recommended by the manufacturer.
Sequence analysis. Sequences were annotated and
compared with reference sequences of the ImMunoGeneTics
(IMGT) HighV-QUEST database.35,36 IMGT results were
sorted for productive sequences and further analyzed with
Excel and VBA (Microsoft Corp, Redmond, WA).33 To
compare between samples we randomly analyzed 5000
CDR3 sequences per sample. The Shannon index is used to
measure IgA repertoire diversity and was calculated with
R studio (version 0.94.110; http://www.r-project.org/,
http://rstudio.org/) and the library “vegan” (command:
diversity). For quantification of VDJ segment usage only
major chains were taken into consideration.
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
not normally distributed, Kruskal-Wallis one-way analysis
of variance on ranks was performed, and the Dunn method
was used for multiple pairwise comparisons.
Results
Epithelial Constitutively Active Myosin Light
Chain Kinase Expression Results in Increased
Intestinal Permeability
The epithelial tight junction is intimately associated with
a perijunctional actomyosin ring that modulates paracellular
permeability via MLCK. CA-MLCK expression9,14 within the
intestinal epithelium markedly increased perijunctional
myosin II regulatory light chain phosphorylation (Figure 1A)
and also enhanced small intestinal paracellular, ie, tight
junction, permeability in a size-selective manner
(Figure 1B). Despite this barrier defect, CA-MLCK mice did
not develop spontaneous colitis. On the basis of these data
and the potential link between enteric infection and subsequent development of inflammatory bowel disease,37,38
we hypothesized that constitutive barrier dysfunction may
predispose these animals to enteric infection.
Increased Intestinal Epithelial Permeability
Activates Processes That Limit Early
Pathogen Translocation
Intestinal permeability, the converse of barrier function,
is regulated through paracellular flux. Paracellular transport
via the tight junction is size-selective and precisely regulated. In contrast, unrestricted flux, which represents flux
across damaged epithelia,3,39,40 allows passage of large
molecules and whole bacteria. CA-MLCK mice exhibit
increases in paracellular permeability that are similar to
those seen in Crohn’s disease patients during remission as
well as in a subset of their healthy relatives.9 Therefore, we
Statistical Analysis
All data are presented as either the mean ± standard
error of the mean (SEM) or with a 95% confidence interval.
P values of direct comparisons between 2 independent
samples were determined by a two-tailed Student t test and
considered to be significant if P .05. Mann-Whitney rank
sum tests were performed in cases in which the data were
not normally distributed. Comparisons between multiple
independent variables were determined by one-way analysis of variance, and Holm-Sidak method was used for
pairwise comparisons. In instances in which the data were
Figure 1. Constitutive epithelial expression of CA-MLCK
increases intestinal permeability. (A) MLC phosphorylation (green) at the epithelial perijunctional actomyosin ring
(f-actin, red) in WT and CA-MLCK mice. Nuclei are shown in
blue. Scale bar, 20 mm. (B) Small intestinal permeability is
shown as ratio of paracellular flux of fluorescein and 70 kDa
rhodamine dextran in WT and CA-MLCK mice. n ¼ 8–9 mice
per genotype; data from 2 independent experiments are
shown. *P < .05.
September 2017
chose this model to investigate the impact of physiologically
relevant tight junction barrier defects on host defense.
As previously reported in the colon,9 LP CD4þ T-cell
numbers were increased in the small intestine of CA-MLCK,
relative to WT, mice (Figure 2A and B). To investigate how
tight junction barrier loss and the associated LP immune
expansion affect susceptibility to enteric pathogens,
CA-MLCK and WT mice were infected orally with the
intracellular protozoan parasite T gondii (Figure 2C). Within
1 hour, the number of parasites that had transmigrated
across the epithelium and into the LP was reduced by 50%
± 8% in CA-MLCK mice relative to WT (Figure 2D). Paradoxically, this indicates that increased intestinal permeability limits parasite invasion. To determine whether this
barrier defect also conferred protection against other
Immune Compensation for Barrier Loss
289
pathogens, mice were exposed to S typhimurium. As with the
protozoan, bacterial translocation was reduced by
76% ± 6% in CA-MLCK relative to WT mice (Figure 2E and F).
To determine whether the increased LP infiltration of
adaptive immune cells was required for CA-MLCK–mediated
protection against bacterial invasion, Rag1 knockout (KO);
CA-MLCK mice lacking both B and T cells were challenged
with S typhimurium. Bacterial translocation in Rag1 KO;
CA-MLCK mice was similar to that in Rag1 KO mice alone
(Figure 2E and F), indicating that adaptive immunity is
required for the protective effect of CA-MLCK expression.
gd T cells also contribute significantly to protection from
early pathogen invasion.28,41 This was confirmed in our
experiments, which showed greater S typhimurium invasion
in Tcrd KO mice, which lack gd T cells (Figure 2E and F).
Figure 2. Increased intestinal permeability limits early enteric pathogen invasion. (A) Fluorescence micrographs of CD3þ
CD4þ LP T cells in WT and CA-MLCK mice. CD3, red; CD4, green; nuclei, blue. White arrowheads indicate CD4þ T cells. Scale
bar, 20 mm. (B) Morphometric analysis of LP CD4þ T cells. n ¼ 4–5 mice. **P < .01. (C) WT and CA-MLCK mice were orally
administered T gondii cysts and killed after 1 hour (SAC). Alternatively, mice were exposed to S typhimurium through the
addition of 108 CFUs directly to the mucosal surface for 30 minutes. (D) Morphometric analysis of parasite translocation 1 hour
after infection. n ¼ 5–6 mice from 2 independent experiments. *P < .05. (E) Fluorescence micrographs of S typhimurium (red,
arrows)–infected WT and CA-MLCK mice crossed to Rag1- or gd T-cell (Tcrd)–deficient backgrounds. Nuclei, blue; f-actin,
green. White arrowheads indicate translocation of S typhimurium, whereas yellow arrowheads are bacteria not counted. Scale
bar, 20 mm. (F) Morphometric analysis of S typhimurium invasion. n ¼ 5–7 mice from at least 2 independent experiments.
Approximately 200 villi were counted for each condition. *P < .05, **P < .01. (G) Morphometric analysis of gd T cells in WT and
CA-MLCK mice. n ¼ 4 mice. Mean ± SEM are shown.
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However, CA-MLCK expression within Tcrd KO mice
markedly reduced bacterial invasion, indicating that although
important to overall host defense, gd T cells did not account
for the observed effects of CA-MLCK expression (Figure 2E
and F). Consistent with this, gd T-cell numbers were similar in
CA-MLCK and WT mice (Figure 2G). Taken together, these
data demonstrate that increased epithelial permeability
promotes immune activation to markedly reduce early
invasion of parasites and bacterial pathogens during the
initial stages of infection. This protection requires adaptive
immunity but is gd T-cell–independent.
CD4þ T Cells Are Required for Constitutively
Active Myosin Light Chain Kinase–induced
Protection Against Bacterial Invasion
To better elucidate the immune cell populations
contributing to the invasion-resistant phenotype of
CA-MLCK mice, we depleted CD4þ T cells and challenged
mice with luminal S typhimurium (Figure 3A and B). CD4þ
T-cell depletion had no effect on early bacterial translocation in WT mice but increased bacterial invasion more
than 5-fold in CA-MLCK mice, relative to mice receiving
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
control IgG (Figure 3C). The disparity between CA-MLCK
and WT mice suggests that our initial hypothesis that
increased tight junction permeability would enhance pathogen invasion was correct. However, the data also indicate
that constitutive barrier dysfunction also elicits a CD4þ
T-cell response that confers protection against acute pathogen invasion.
The data above indicate that CD4þ T cells are necessary
for the protection observed in CA-MLCK mice. To determine
whether these cells were sufficient to confer protection, WT
CD4þ T cells were adoptively transferred into Rag1 KO or
Rag1 KO; CA-MLCK recipients (Figure 3D). Within 2 weeks,
CD4þ T cells were effectively reconstituted in the LP of both
CA-MLCK and WT mice (Figure 3E). Notably, 73% more LP
CD4þ T cells were present in the LP of Rag1 KO; CA-MLCK,
relative to Rag1 KO, recipients (Figure 3E). Whereas CD4þ
T-cell transfer into Rag1 KO mice had no effect on bacterial
invasion, CD4þ T-cell transfer into Rag1 KO; CA-MLCK
mice reduced invasion by 43% (Figure 3F). These data
demonstrate that CD4þ T cells are sufficient to limit
early S typhimurium invasion. Because CD4þ T cells from
WT mice were protective in Rag1 KO; CA-MLCK mice,
the data indicate that changes in the local mucosal
Figure 3. CD4D T cells are required to confer protection against bacterial invasion in CA-MLCK mice. (A) After
administration of anti-CD4 (GK1.5) or control IgG intraperitoneally, WT and CA-MLCK mice were exposed to S typhimurium
through direct application of bacteria to small intestinal mucosal surface for 30 minutes and then killed (SAC). (B) Percentage of
CD3þ CD4þ T splenocytes 48 hours after anti-CD4 administration. #P < .001. (C) Morphometric analysis of S typhimurium
invasion after administration of anti-CD4 or control IgG. n ¼ 6–9 mice from 2 independent experiments. *P < .05, #P < .001.
(D) Rag1 KO mice and those also expressing the CA-MLCK Tg received CD4þ T-cell adoptive transfer. After 2 weeks of
engraftment, mice were exposed to S typhimurium for 30 minutes. (E) Morphometric analysis of CD4þ LP T cells in Rag1 KO
and Rag1 KO CA-MLCK mice after CD4þ T-cell adoptive transfer. n ¼ 7–8 mice from 2 independent experiments. *P < .05.
(F) Analysis of S typhimurium translocation in Rag1 KO and Rag1 KO; CA-MLCK mice receiving CD4þ T-cell adoptive transfer.
n ¼ 7–11 mice from 2 independent experiments. *P < .05.
September 2017
microenvironment resulting from constitutive barrier loss
are sufficient to impart this phenotype. This may reflect
expansion or enhanced recruitment of CD4þ T cells to the
LP of Rag1 KO; CA-MLCK mice. In either case, it is clear that
the compensatory response to epithelial barrier loss
includes promotion of a new CD4þ T-cell function that is not
otherwise present, as indicated by the failure of CD4þ T cells
to reduce bacterial invasion in Rag1 KO mice.
We also asked whether other components of adaptive
immunity might contribute to the protective phenotype
observed in CA-MLCK mice. Plasma cell production of
commensal-specific IgA interferes with commensal antigen
expression and promotes immune exclusion by limiting
bacterial association with the mucosal surface, and this has
been implicated in the mucosal immune response to loss of
JAM-A expression.22 Morphometric analysis showed that
CA-MLCK mice exhibited an increase in IgAþ plasma cells
within the LP compared with WT (Figure 4A). However,
there were no differences in mucosal transforming growth
factor beta transcripts, expression or localization of the
polymeric Ig receptor (pIgR), or the amount of secreted
Immune Compensation for Barrier Loss
291
(luminal) IgA between CA-MLCK and WT mice
(Figure 4B).9
Despite the lack of quantitative increases in luminal IgA,
we hypothesized that increased exposure to bacterial products as a result of enhanced paracellular permeability could
promote expansion of plasma cells expressing microbespecific IgA. To test this, complementary determining region 3 region sequences were analyzed from productive IgA
VDJ rearrangements. Although the values indicate a highly
diverse IgA repertoire in all samples (Figure 4C), no differences were observed between WT and CA-MLCK mice.
Furthermore, analysis of the frequency of somatic hypermutations within IgA-producing plasma cells during affinity
maturation also failed to identify significant differences between the CA-MLCK and WT mice (Figure 4D). Therefore,
barrier loss does not affect the quality of mucosal IgA.
Previous studies have linked barrier loss to IgA production. Therefore, we asked whether, despite the negative
data above, increases in LP IgA-producing plasma cells
could contribute to protection against early pathogen invasion. CA-MLCK mice were bred onto an IgA-deficient
Figure 4. IgAD plasma cell number is increased, yet not required for CA-MLCK–mediated protection against bacterial
translocation. (A) Morphometric analysis of IgAþ plasma cells in the LP. n ¼ 4–6 mice. Mean ± SEM, *P ¼ .05. (B) Enzymelinked immunosorbent assay for fecal IgA. n ¼ 7–10 mice. Mean ± SEM. (C) Shannon Index was calculated from CDR3
sequence pools of given samples. The mean is shown. (D) Mutation frequencies in CDR1/2 and FR sequences calculated from
the average number of SHM within the Vh region. Frequencies were calculated through division of mutation numbers by the
number of nucleotides within the given FR or CDR regions. Mean ± standard deviation. (E) Morphometric analysis of
S typhimurium invasion in WT and CA-MLCK mice on an IghA KO background. n ¼ 5–7 mice from 2 independent experiments.
Mean is shown, *P ¼ .05. (F) Morphometric analysis of LP IgAþ plasma cells 48 hours after administration of anti-CD4 or
control IgG. Mean ± SEM, **P < .01. IghA, immunoglobulin A; SHM, somatic hypermutations.
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Edelblum et al
background (IghA KO). Bacterial invasion was similar to
CA-MLCK mice regardless of IgA expression (Figure 4E),
indicating that IgA was not required for the CA-MLCK–mediated protective effect. Because IgA is generated through
both T-cell–dependent and –independent mechanisms,42 we
assessed the role of CD4þ T cells in generating increased
numbers of LP IgAþ plasma cells in CA-MLCK mice. CD4þ
T-cell depletion did not affect IgA-producing plasma cell
number in the LP of CA-MLCK or WT mice (Figure 4F).
Because of the requirement for CD4þ T cells in the enhanced
immunity of CA-MLCK mice, these data lend further support
to the conclusion that IgA is not required for the protection
observed in CA-MLCK mice.
Interleukin 17A Expression Promotes
Neutrophil Recruitment to Prevent Early Bacterial
Invasion in Constitutively Active Myosin Light
Chain Kinase Mice
Certain strains of commensal bacteria modulate specific
immune responses. For example, SFB are known to promote
Th17 differentiation in the small intestine,18,43 and activation
of these Th17 cells is critical to pathogen clearance and
maintenance of host defense.44,45 To determine the role of
IL17 in CD4þ T-cell–mediated protection against bacterial
invasion, CA-MLCK mice were generated on an IL17Adeficient background. IL17A deficiency significantly
increased acute S typhimurium invasion in CA-MLCK transgenic mice (Figure 5A). More strikingly, IL17A knockout
eliminated CD4þ T-cell expansion within the LP of CA-MLCK
mice (Figure 5B). These data show that IL17A is required to
generate the increased numbers of LP CD4þ T cells seen in CAMLCK mice and that both IL17A and CD4þ T cells are necessary for early protection against S typhimurium translocation.
One mechanism by which Th17 cells contribute to host
defense is induction of granulopoiesis and neutrophil
recruitment via CXCL1 and CXCL2 to promote bacterial
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
clearance.46,47 S typhimurium exposure induced rapid
neutrophil recruitment into the LP of CA-MLCK but not WT
mice (Figure 5C). This acute recruitment was eliminated in
IL17A KO mice, indicating that IL17A may prime innate immunity for immediate response to initial pathogen exposure.
Commensal Bacteria Are Essential for
Activation of Host Defense in Response to
Intestinal Epithelial Barrier Dysfunction
The mucosal immune system and microbiota regulate one
another during differentiation of the immune response.2 To
test the hypothesis that commensal bacteria drive the
mucosal CD4þ T-cell expansion that limits early pathogen
invasion, CA-MLCK mice were rederived under GF conditions.
Consistent with previous reports, GF mice exhibited significantly diminished numbers of LP lymphocytes. Furthermore,
LP CD4þ T-cell and IgAþ plasma cell numbers were similar in
GF CA-MLCK and GF WT mice, indicating that the differences
induced by CA-MLCK expression in conventional mice are
dependent on the microbiota (Figure 6A and B). When challenged with S typhimurium, invasion was similar in GF
CA-MLCK and GF WT mice (Figure 6C).
SFB are commensal bacteria known to promote Th17
differentiation in the small intestine and, although abundant
in C57BL/6NTac mice from Taconic Farms, are undetectable
in C57BL/6J from Jackson Laboratories.48 Previous studies
have shown that in addition to driving Th17 differentiation,
SFB colonization of C57BL/6J from Jackson Laboratories
promotes Th17-dependent neutrophil recruitment47 similar
to that observed here (Figure 5C). To compare the effects of
SFB-containing and SFB-deficient microbiota, GF CA-MLCK
and WT mice were conventionalized with cecal contents
from Taconic Farms (TAC) and Jackson Laboratories
(JAX) mice. The enhanced resistance to acute bacterial
translocation observed in CA-MLCK mice was restored by
SFB-containing (TAC) microbiota, but not by SFB-deficient
Figure 5. IL17A is required to limit bacterial translocation through promotion of neutrophil recruitment. (A) Morphometric
analysis of S typhimurium invasion in WT and CA-MLCK mice on an IL17A-deficient background. n ¼ 6–7 mice from 2
independent experiments. *P < .05, **P < .01. (B) Morphometric analysis of CD4þ LP T cells in WT and CA-MLCK mice
crossed to IL17A-deficient mice. **P < .01. (C) Morphometric analysis of neutrophils within the small intestinal LP in uninfected
and infected (30 minutes) WT, CA-MLCK, IL17A KO or IL17A KO; CA-MLCK mice. n ¼ 3–5 mice. #P < .001.
September 2017
Immune Compensation for Barrier Loss
293
Constitutive Barrier Dysfunction Increases
Susceptibility to Chronic Salmonella Infection
The data above demonstrate that tight junction barrier
loss results in markedly enhanced pathogen invasion in CD4
T-cell–depleted mice. In immunocompetent mice, tight
junction barrier loss also activates mucosal immune
responses that limit acute S typhimurium invasion. We asked
whether these compensatory immune responses were sufficient to prevent chronic infection and systemic salmonellosis. Nine days after infection, CA-MLCK mice exhibited
more severe disease, as assessed clinically by reduced
activity and hunched posture (Figure 7A and B), as well as
greater mucosal damage and LP lymphoplasmacytic
infiltrates (Figure 7C and D). Differences between CA-MLCK
and WT mice were also apparent within the spleen.
CA-MLCK mice had sinus thrombi and dense lymphohistiocytic infiltrates that were reminiscent of, but more diffuse
than, typhoid nodules, whereas WT mice displayed
neutrophil-rich intrasplenic abscesses (Figure 7E and F).
Thus, although increased intestinal epithelial tight junction
permeability activates protective immune activation that
limits early invasion, this is ultimately insufficient to
compensate for barrier loss in the context of chronic
disease.
Discussion
Figure 6. Commensal bacteria are essential for promoting
mucosal immune responses to early Salmonella invasion
in response to increased intestinal permeability. (A) Fluorescence micrographs of CD3þ CD4þ LP T cells in WT and
CA-MLCK mice raised under either SPF or GF conditions.
Scale bar, 20 mm. Morphometric analysis of CD4þ LP T cells
in GF WT and CA-MLCK mice are shown. n ¼ 6–8 mice from
2 independent experiments. (B) Fluorescence micrographs of
IgAþ plasma cells in the LP in WT and CA-MLCK mice raised
under either SPF or GF conditions. Scale bar, 20 mm.
Morphometric analysis of IgAþ plasma cells in GF WT and
CA-MLCK mice is shown. n ¼ 5–8 mice from 2 independent
experiments. (C) Morphometric analysis of S typhimurium
invasion (30 minutes) in WT and CA-MLCK mice maintained
under GF conditions or conventionalized with Taconic (TAC)
or Jackson (JAX) microbiota. n ¼ 5–8 mice from 2 independent experiments. **P < .01.
(JAX) microbiota (Figure 6C). Together with the aforementioned results, these data suggest that SFB-induced Th17
differentiation is central to the protection against acute
S typhimurium invasion in CA-MLCK mice.
Targeted increases in paracellular flux through the
epithelial tight junction enhance susceptibility to experimental, immune-mediated colitis.9 Although tight junction–
specific barrier loss is insufficient to allow translocation of
intact microbes, we hypothesized that it would increase
susceptibility to enteric infection. Our data support this
hypothesis in the absence of adaptive immunity but also
demonstrate that chronic tight junction barrier loss
stimulates a subclinical immune response that limits acute
invasion of enteric pathogens. This early protection requires
CD4þ T cells and IL17A, but not IgA.
Consistent with previous studies by us and others,9,21,22
we found that CD4þ LP T-cell number is increased in
response to constitutive barrier loss. Notably, the physiologically relevant, tight junction–specific barrier loss studied
here is not associated with chronic epithelial damage or
homeostatic increases in epithelial proliferation. Furthermore, unlike transient, damage-associated barrier loss,21
regulatory T-cell populations are not expanded in
CA-MLCK mice.9,21 The increased paracellular permeability
resulting from MLCK activation induced an immune
response that limited not only early bacterial invasion but
also the transmigration of T gondii. We found that CD4þ T
cells, and in particular Th17 cells, were necessary to limit
acute S typhimurium invasion in CA-MLCK mice. The data
further suggest that IL17A-induced neutrophil recruitment
contributes to this enhanced immunity. Studies in GF
CA-MLCK mice showed that commensal bacteria are
required for both CD4þ LP T-cell expansion and early protection against bacterial invasion. Finally, after adoptive
transfer to T and B cell–deficient (Rag1 KO) CA-MLCK mice,
increased numbers of CD4þ T cells from WT mice were
294
Edelblum et al
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
Figure
7. Constitutive
barrier dysfunction enhances susceptibility to
Salmonella-induced colitis. (A) WT and CA-MLCK
gavaged with streptomycin 24 hours before oral
infection with 108 CFU S
typhimurium. (B) Clinical
scores are shown. n ¼ 17–
18 mice from at least 3
independent experiments,
*P < .05. Hematoxylineosin micrographs and
histologic scores of colon
(C and D) and spleen (E and
F) for infected mice are
shown. n ¼ 8–10 mice from
at least 3 independent
experiments are shown.
#
P < .001. SAC, sacrifice;
SL, S. typhimurium, strain
SL3201.
recruited to the LP of CA-MLCK mice where they contributed to enhanced protection against early bacterial translocation. Taken together, these findings demonstrate that
the continuous low-level exposure of LP cells to commensal
products as a result of physiologically relevant tight junction
barrier loss drives a subclinical immune response that
functions as a compensatory mechanism that limits invasion
of bacterial and protozoan pathogens.
Careful consideration must be given to models of barrier
dysfunction to better understand the effect of permeability
in disease states because of the broad definition of “barrier
defect” in the current literature.1 For example, although
intrarectal ethanol administration has been used to transiently disrupt barrier function,21 the mechanism involves
epithelial damage that likely enhances flux across the unrestricted pathway. Similarly, although JAM-A–deficient
mice exhibit markedly increased intestinal permeability in
addition to mucosal immune activation and inflammatory
cell recruitment,10,11 the cause of the barrier loss is likely
multifactorial. Although claudins-10 and -15 are upregulated in JAM-A KO mice,11 these claudins function as paracellular channels with diameters of 5–6 Å3,39,40 and,
therefore, cannot be responsible for the marked increases in
large molecule flux reported in these mice. Furthermore,
in vitro and in vivo studies have demonstrated that loss of
JAM-A increases intestinal epithelial apoptosis and results in
compensatory increases in epithelial proliferation
in vivo.10,11 Neither of these changes are present in
CA-MLCK transgenic mice.
As expected for tight junction–specific barrier loss, the
increased permeability induced by intestinal epithelial
CA-MLCK expression is substantially lower than that
observed in JAM-A knockout mice. However, the degree of
barrier loss in CA-MLCK mice is quantitatively and qualitatively similar to the barrier defects reported in healthy
first-degree relatives of Crohn’s disease patients.7,8 The
subclinical immune activation exhibited in CA-MLCK mice
confers a protective phenotype in response to acute exposure to S typhimurium or T gondii. It will be interesting to
determine whether similar mucosal immune activation is
present in the subset of healthy first-degree relatives of
Crohn’s disease patients who manifest barrier loss and
whether this confers increased risk of developing disease.
The studies in GF mice indicate that mucosal immune
priming in CA-MLCK mice is driven by the microbiota, which
is supported by studies showing that SFB colonization
induces IL17A production by CD4þ T cells to promote
neutrophil recruitment.47 Consistent with this, colonization
of GF CA-MLCK mice with SFB-deficient microbiota failed to
restore the protective phenotype. In contrast, colonization
with microbiota containing SFB was able to reduce
Salmonella invasion in GF mice. Thus, although GF mice fail
to develop a mature mucosal immune compartment and
exhibit reduced mucus thickness,49 it is not the absence of
all microbes that abrogates the protection. Rather, it is the
difference between TAC and JAX microbiota, most likely
SFB, that explains CA-MLCK–induced microbial resistance. It
is tempting to speculate that loss of these beneficial
September 2017
commensals may also, in part, explain the association between acute enteritis-driven dysbiosis and the onset of
chronic inflammatory bowel disease in a subset of
patients.37,38
Similar to our initial observations demonstrating
increased susceptibility of CA-MLCK mice to immunemediated colitis,9 we found that CA-MLCK expression in
the intestinal epithelium resulted in more severe
Salmonella-induced colitis after prolonged bacterial infection. This contrasts sharply with the protective phenotype
exhibited in response to acute exposure to S typhimurium or
T gondii. Due to the critical roles for IL17-dependent
neutrophil recruitment in early protection against microbial invasion, it would be reasonable to hypothesize that
although this early neutrophil influx clears bacteria to
confer acute protection, sustained neutrophil recruitment
leads to tissue destruction and more severe chronic disease.
In the context of systemic infection, these data support the
potential link between enteric infection and subsequent
development of inflammatory bowel disease.37,38
Overall, these data indicate that increased intestinal
epithelial tight junction permeability results in a shift in the
symbiotic relationship between mucosal immunity and
commensal microbes by increasing the frequency of sampling or the diversity of luminal antigens available to prime
mucosal surveillance. This increased sampling may allow for
constant modulation of tolerance to commensal bacteria and
provide immediate protection to limit early pathogen invasion. Finally, the mechanisms identified here may explain
both the absence of disease in relatives of Crohn’s disease
patients, despite barrier loss, as well as the increased disease susceptibility induced by barrier dysfunction.
Immune Compensation for Barrier Loss
5.
6.
7.
8.
9.
10.
References
1. Kiesler P, Fuss IJ, Strober W. Experimental models of
inflammatory bowel diseases. Cell Mol Gastroenterol
Hepatol 2015;1:154–170.
2. Ohland CL, Jobin C. Microbial activities and intestinal
homeostasis: a delicate balance between health and
disease. Cell Mol Gastroenterol Hepatol 2015;1:28–40.
3. Turner JR. Intestinal mucosal barrier function in health
and disease. Nat Rev Immunol 2009;9:799–809.
4. Jostins L, Ripke S, Weersma RK, Duerr RH,
McGovern DP, Hui KY, Lee JC, Schumm LP, Sharma Y,
Anderson CA, Essers J, Mitrovic M, Ning K, Cleynen I,
Theatre E, Spain SL, Raychaudhuri S, Goyette P, Wei Z,
Abraham C, Achkar JP, Ahmad T, Amininejad L,
Ananthakrishnan AN, Andersen V, Andrews JM,
Baidoo L, Balschun T, Bampton PA, Bitton A, Boucher G,
Brand S, Buning C, Cohain A, Cichon S, D’Amato M, De
Jong D, Devaney KL, Dubinsky M, Edwards C,
Ellinghaus D, Ferguson LR, Franchimont D, Fransen K,
Gearry R, Georges M, Gieger C, Glas J, Haritunians T,
Hart A, Hawkey C, Hedl M, Hu X, Karlsen TH,
Kupcinskas L, Kugathasan S, Latiano A, Laukens D,
Lawrance IC, Lees CW, Louis E, Mahy G, Mansfield J,
Morgan AR, Mowat C, Newman W, Palmieri O,
Ponsioen CY, Potocnik U, Prescott NJ, Regueiro M,
Rotter JI, Russell RK, Sanderson JD, Sans M, Satsangi J,
11.
12.
13.
14.
15.
295
Schreiber S, Simms LA, Sventoraityte J, Targan SR,
Taylor KD, Tremelling M, Verspaget HW, De Vos M,
Wijmenga C, Wilson DC, Winkelmann J, Xavier RJ,
Zeissig S, Zhang B, Zhang CK, Zhao H, International
IBDGCSilverberg MS, Annese V, Hakonarson H,
Brant SR, Radford-Smith G, Mathew CG, Rioux JD,
Schadt EE, Daly MJ, Franke A, Parkes M, Vermeire S,
Barrett JC, Cho JH. Host-microbe interactions have
shaped the genetic architecture of inflammatory bowel
disease. Nature 2012;491:119–124.
D’Inca R, Di Leo V, Corrao G, Martines D, D’Odorico A,
Mestriner C, Venturi C, Longo G, Sturniolo GC. Intestinal
permeability test as a predictor of clinical course in
Crohn’s disease. Am J Gastroenterol 1999;94:
2956–2960.
Wyatt J, Vogelsang H, Hubl W, Waldhoer T, Lochs H.
Intestinal permeability and the prediction of relapse in
Crohn’s disease. Lancet 1993;341:1437–1439.
Hollander D, Vadheim CM, Brettholz E, Petersen GM,
Delahunty T, Rotter JI. Increased intestinal permeability
in patients with Crohn’s disease and their relatives: a
possible etiologic factor. Ann Intern Med 1986;
105:883–885.
Buhner S, Buning C, Genschel J, Kling K, Herrmann D,
Dignass A, Kuechler I, Krueger S, Schmidt HH, Lochs H.
Genetic basis for increased intestinal permeability in
families with Crohn’s disease: role of CARD15 3020insC
mutation? Gut 2006;55:342–347.
Su L, Shen L, Clayburgh DR, Nalle SC, Sullivan EA,
Meddings JB, Abraham C, Turner JR. Targeted epithelial
tight junction dysfunction causes immune activation and
contributes to development of experimental colitis.
Gastroenterology 2009;136:551–563.
Vetrano S, Rescigno M, Cera MR, Correale C, Rumio C,
Doni A, Fantini M, Sturm A, Borroni E, Repici A, Locati M,
Malesci A, Dejana E, Danese S. Unique role of junctional
adhesion molecule-a in maintaining mucosal homeostasis in inflammatory bowel disease. Gastroenterology
2008;135:173–184.
Laukoetter MG, Nava P, Lee WY, Severson EA,
Capaldo CT, Babbin BA, Williams IR, Koval M,
Peatman E, Campbell JA, Dermody TS, Nusrat A,
Parkos CA. JAM-A regulates permeability and inflammation in the intestine in vivo. J Exp Med 2007;
204:3067–3076.
Nalle SC, Kwak HA, Edelblum KL, Joseph NE, Singh G,
Khramtsova GF, Mortenson ED, Savage PA, Turner JR.
Recipient NK cell inactivation and intestinal barrier loss
are required for MHC-matched graft-versus-host disease. Sci Transl Med 2014;6:243ra87.
Gerlach K, McKenzie AN, Neurath MF, Weigmann B. IL-9
regulates intestinal barrier function in experimental T cellmediated colitis. Tissue Barriers 2015;3:e983777.
Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V,
Schneeberger EE, Turner JR. Myosin light chain phosphorylation regulates barrier function by remodeling tight
junction structure. J Cell Sci 2006;119:2095–2106.
Clayburgh DR, Barrett TA, Tang Y, Meddings JB, Van
Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ, Turner JR.
Epithelial myosin light chain kinase-dependent barrier
296
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
Edelblum et al
dysfunction mediates T cell activation-induced diarrhea
in vivo. J Clin Invest 2005;115:2702–2715.
Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R,
Mrsny RJ, Madara JL. Physiological regulation of
epithelial tight junctions is associated with myosin lightchain phosphorylation. Am J Physiol 1997;273:
C1378–C1385.
Shih VF, Cox J, Kljavin NM, Dengler HS, Reichelt M,
Kumar P, Rangell L, Kolls JK, Diehl L, Ouyang W,
Ghilardi N. Homeostatic IL-23 receptor signaling limits
Th17 response through IL-22-mediated containment of
commensal microbiota. Proc Natl Acad Sci U S A 2014;
111:13942–13947.
Ivanov II, Frutos Rde L, Manel N, Yoshinaga K,
Rifkin DB, Sartor RB, Finlay BB, Littman DR. Specific
microbiota direct the differentiation of IL-17-producing
T-helper cells in the mucosa of the small intestine. Cell
Host Microbe 2008;4:337–349.
Hooper LV, Littman DR, Macpherson AJ. Interactions
between the microbiota and the immune system.
Science 2012;336:1268–1273.
Cao AT, Yao S, Gong B, Elson CO, Cong Y. Th17 cells
upregulate polymeric Ig receptor and intestinal IgA and
contribute to intestinal homeostasis. J Immunol 2012;
189:4666–4673.
Boirivant M, Amendola A, Butera A, Sanchez M, Xu L,
Marinaro M, Kitani A, Di Giacinto C, Strober W, Fuss IJ.
A transient breach in the epithelial barrier leads to regulatory T-cell generation and resistance to experimental
colitis. Gastroenterology 2008;135:1612–1623 e5.
Khounlotham M, Kim W, Peatman E, Nava P, MedinaContreras O, Addis C, Koch S, Fournier B, Nusrat A,
Denning TL, Parkos CA. Compromised intestinal
epithelial barrier induces adaptive immune compensation
that protects from colitis. Immunity 2012;37:563–573.
Weber CR, Raleigh DR, Su L, Shen L, Sullivan EA,
Wang Y, Turner JR. Epithelial myosin light chain kinase
activation induces mucosal interleukin-13 expression to
alter tight junction ion selectivity. J Biol Chem 2010;
285:12037–12046.
Prinz I, Sansoni A, Kissenpfennig A, Ardouin L,
Malissen M, Malissen B. Visualization of the earliest
steps of gammadelta T cell development in the adult
thymus. Nat Immunol 2006;7:995–1003.
Yang H, Madison B, Gumucio DL, Teitelbaum DH. Specific overexpression of IL-7 in the intestinal mucosa: the
role in intestinal intraepithelial lymphocyte development.
Am J Physiol Gastrointest Liver Physiol 2008;
294:G1421–G1430.
Harriman GR, Bogue M, Rogers P, Finegold M,
Pacheco S, Bradley A, Zhang Y, Mbawuike IN. Targeted
deletion of the IgA constant region in mice leads to IgA
deficiency with alterations in expression of other Ig
isotypes. J Immunol 1999;162:2521–2529.
Marchiando AM, Shen L, Graham WV, Weber CR,
Schwarz BT, Austin JR 2nd, Raleigh DR, Guan Y,
Watson AJ, Montrose MH, Turner JR. Caveolin-1dependent occludin endocytosis is required for
TNF-induced tight junction regulation in vivo. J Cell Biol
2010;189:111–126.
Cellular and Molecular Gastroenterology and Hepatology Vol. 4, No. 2
28. Edelblum KL, Singh G, Odenwald MA, Lingaraju A,
El Bissati K, McLeod R, Sperling AI, Turner JR.
Gammadelta intraepithelial lymphocyte migration limits
transepithelial pathogen invasion and systemic disease
in mice. Gastroenterology 2015;148:1417–1426.
29. Cong H, Mui EJ, Witola WH, Sidney J, Alexander J,
Sette A, Maewal A, El Bissati K, Zhou Y, Suzuki Y, Lee D,
Woods S, Sommerville C, Henriquez FL, Roberts CW,
McLeod R. Toxoplasma gondii HLA-B*0702-restricted
GRA7(20-28) peptide with adjuvants and a universal
helper T cell epitope elicits CD8(þ) T cells producing
interferon-gamma and reduces parasite burden in
HLA-B*0702 mice. Hum Immunol 2012;73:1–10.
30. Hutson SL, Mui E, Kinsley K, Witola WH, Behnke MS,
El Bissati K, Muench SP, Rohrman B, Liu SR,
Wollmann R, Ogata Y, Sarkeshik A, Yates JR 3rd,
McLeod R. T. gondii RP promoters & knockdown reveal
molecular pathways associated with proliferation and
cell-cycle arrest. PLoS One 2010;5:e14057.
31. Edelblum KL, Shen L, Weber CR, Marchiando AM,
Clay BS, Wang Y, Prinz I, Malissen B, Sperling AI,
Turner JR. Dynamic migration of gammadelta intraepithelial lymphocytes requires occludin. Proc Natl Acad
Sci U S A 2012;109:7097–7102.
32. Al-Anouti F, Tomavo S, Parmley S, Ananvoranich S. The
expression of lactate dehydrogenase is important for the
cell cycle of Toxoplasma gondii. J Biol Chem 2004;
279:52300–52311.
33. Lindner C, Wahl B, Fohse L, Suerbaum S,
Macpherson AJ, Prinz I, Pabst O. Age, microbiota, and
T cells shape diverse individual IgA repertoires in the
intestine. J Exp Med 2012;209:365–377.
34. Lindner C, Thomsen I, Wahl B, Ugur M, Sethi MK,
Friedrichsen M, Smoczek A, Ott S, Baumann U,
Suerbaum S, Schreiber S, Bleich A, Gaboriau-Routhiau V,
Cerf-Bensussan N, Hazanov H, Mehr R, Boysen P,
Rosenstiel P, Pabst O. Diversification of memory B cells
drives the continuous adaptation of secretory antibodies to
gut microbiota. Nat Immunol 2015;16:880–888.
35. Brochet X, Lefranc MP, Giudicelli V. IMGT/V-QUEST: the
highly customized and integrated system for IG and TR
standardized V-J and V-D-J sequence analysis. Nucleic
Acids Res 2008;36:W503–W508.
MP,
Giudicelli
V,
Ginestoux
C,
36. Lefranc
Jabado-Michaloud J, Folch G, Bellahcene F, Wu Y,
Gemrot E, Brochet X, Lane J, Regnier L, Ehrenmann F,
Lefranc G, Duroux P. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2009;
37:D1006–D1012.
37. Gradel KO, Nielsen HL, Schonheyder HC, Ejlertsen T,
Kristensen B, Nielsen H. Increased short- and long-term
risk of inflammatory bowel disease after salmonella or
campylobacter gastroenteritis. Gastroenterology 2009;
137:495–501.
38. Garcia Rodriguez LA, Ruigomez A, Panes J. Acute
gastroenteritis is followed by an increased risk of inflammatory bowel disease. Gastroenterology 2006;
130:1588–1594.
39. Nalle SC, Turner JR. Intestinal barrier loss as a critical
pathogenic link between inflammatory bowel disease
September 2017
40.
41.
42.
43.
44.
45.
46.
47.
and graft-versus-host disease. Mucosal Immunol 2015;
8:720–730.
Anderson JM, Van Itallie CM. Physiology and function of
the tight junction. Cold Spring Harb Perspect Biol 2009;
1:a002584.
Ismail AS, Severson KM, Vaishnava S, Behrendt CL,
Yu X, Benjamin JL, Ruhn KA, Hou B, DeFranco AL,
Yarovinsky F, Hooper LV. Gammadelta intraepithelial
lymphocytes are essential mediators of host-microbial
homeostasis at the intestinal mucosal surface. Proc
Natl Acad Sci U S A 2011;108:8743–8748.
Fagarasan S, Kawamoto S, Kanagawa O, Suzuki K.
Adaptive immune regulation in the gut: T cell-dependent
and T cell-independent IgA synthesis. Annu Rev Immunol
2010;28:243–273.
Goto Y, Obata T, Kunisawa J, Sato S, Ivanov II,
Lamichhane A, Takeyama N, Kamioka M, Sakamoto M,
Matsuki T, Setoyama H, Imaoka A, Uematsu S, Akira S,
Domino SE, Kulig P, Becher B, Renauld JC, Sasakawa C,
Umesaki Y, Benno Y, Kiyono H. Innate lymphoid cells
regulate intestinal epithelial cell glycosylation. Science
2014;345:1254009.
Ouyang W, Kolls JK, Zheng Y. The biological functions of
T helper 17 cell effector cytokines in inflammation.
Immunity 2008;28:454–467.
Geddes K, Rubino SJ, Magalhaes JG, Streutker C, Le
Bourhis L, Cho JH, Robertson SJ, Kim CJ, Kaul R,
Philpott DJ, Girardin SE. Identification of an innate
T helper type 17 response to intestinal bacterial pathogens. Nat Med 2011;17:837–844.
Fossiez F, Djossou O, Chomarat P, Flores-Romo L, AitYahia S, Maat C, Pin JJ, Garrone P, Garcia E, Saeland S,
Blanchard D, Gaillard C, Das Mahapatra B, Rouvier E,
Golstein P, Banchereau J, Lebecque S. T cell interleukin-17
induces stromal cells to produce proinflammatory and hematopoietic cytokines. J Exp Med 1996;183:2593–2603.
Flannigan KL, Ngo VL, Geem D, Harusato A, Hirota SA,
Parkos CA, Lukacs NW, Nusrat A, Gaboriau-Routhiau V,
Cerf-Bensussan N, Gewirtz AT, Denning TL.
IL-17A-mediated neutrophil recruitment limits expansion
of segmented filamentous bacteria. Mucosal Immunol
2017;10:673–684.
Immune Compensation for Barrier Loss
297
48. Ivanov II, Atarashi K, Manel N, Brodie EL, Shima T,
Karaoz U, Wei D, Goldfarb KC, Santee CA, Lynch SV,
Tanoue T, Imaoka A, Itoh K, Takeda K, Umesaki Y,
Honda K, Littman DR. Induction of intestinal Th17 cells
by segmented filamentous bacteria. Cell 2009;139:
485–498.
49. Johansson ME, Jakobsson HE, Holmen-Larsson J,
Schutte A, Ermund A, Rodriguez-Pineiro AM, Arike L,
Wising C, Svensson F, Backhed F, Hansson GC.
Normalization of host intestinal mucus layers requires
long-term microbial colonization. Cell Host Microbe
2015;18:582–592.
Received February 22, 2017. Accepted June 5, 2017.
Correspondence
Address correspondence to: Karen L. Edelblum, PhD, Rutgers New Jersey Medical
School, 205 South Orange Avenue, Cancer Center G1228, Newark, New Jersey
07103. e-mail: ke163@njms.rutgers.edu; or Jerrold R. Turner, MD, PhD, Brigham
and Women’s Hospital, 20 Shattuck Street, Thorn 1428, Boston, Massachusetts
02115. e-mail: jrturner@bwh.harvard.edu.
Acknowledgments
The authors thank Sara McBride for help with mouse rederivation, Taren Thorn
and Kimberly Ly for animal care, and the Frank W. Fitch Monoclonal Antibody
Core Facility at the University of Chicago for monoclonal antibody production.
Author contributions
Conceptualization, K.L.E and J.R.T.; methodology, K.L.E., G. Sharon, and
S.K.M.; formal analysis, S.R. and I.T.; investigation, K.L.E., G. Sharon, G.
Singh, A.S., S.C., S.G., S.R., K.E.B., and J.R.T.; resources, S.K.M., R.M.,
C.D., I.P., and J.R.T.; writing-original draft, K.L.E.; writing-review and editing,
K.L.E., G. Sharon, S.K.M., R.M., K.E.B., I.P., S.R., and J.R.T.; visualization,
K.L.E., S.R, and I.P.; supervision, K.L.E., S.K.M., and J.R.T.; funding, K.L.E.,
S.K.M., and J.R.T.
Conflicts of interest
The authors disclose no conflicts.
One author (J.R.T.) is a member of the CMGH Board of Editors (BOE). In
accordance with journal policy, the review process for this manuscript was
managed by a Guest Associate Editor, and the author BOE member was
blinded to the entire process.
Funding
Supported by National Institute of Health grants K01DK093627 and
F32DK084859 (K.L.E.), R01DK078938, R01GM099535 (S.K.M.), R01DK61931
and R01DK68271 (J.R.T.); Department of Defense W81XWH-09-1-0341
(J.R.T.); Crohn’s and Colitis Foundation of America (S.K.M. and J.R.T.) and
Broad Medical Research Foundation (J.R.T.); The Human Frontiers Science
Program - Long Term Fellowship (G. Sharon); and the Heritage Medical
Research Institute (S.K.M.)