TLR2 Modulates Antibodies Required for
Intestinal Ischemia/Reperfusion-Induced
Damage and Inflammation
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J Immunol 2015; 194:1190-1198; Prepublished online 24
December 2014;
doi: 10.4049/jimmunol.1303124
http://www.jimmunol.org/content/194/3/1190
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References
Michael R. Pope and Sherry D. Fleming
The Journal of Immunology
TLR2 Modulates Antibodies Required for Intestinal
Ischemia/Reperfusion-Induced Damage and Inflammation
Michael R. Pope and Sherry D. Fleming
A
lthough the mortality rate for mesenteric ischemia/
reperfusion (IR) has decreased in recent years, it
remains at 40–60% (1, 2). Cellular damage induced by
the lack of blood flow to the intestine (mesenteric ischemia) is
significantly enhanced upon return of blood flow (reperfusion) and
frequently results in systemic inflammation. During reperfusion,
both a cellular and a humoral innate response are required, and
inhibition of either the humoral cascade or the cellular infiltrate
attenuates IR-induced tissue damage (3, 4). The inflammatory
infiltrate of neutrophils and macrophages releases significant
levels of free radicals, cytokines, and eicosanoids, including PGE2
and leukotriene B4 (LTB4) (5). Importantly, the release of PGE2 is
necessary, but not sufficient for intestinal IR-induced injury (6).
The humoral response includes naturally occurring Ab recognition of newly expressed neoantigens and generation of an excessive inflammatory response, including complement activation
[reviewed in (7)]. Multiple groups identified neoantigens by administering mAb to IR-resistant, Ab-deficient Rag-12/2 mice (8–
10). Using this model, several intracellular Ags, including DNA,
nonmuscle myosin (NMM), and annexin IV (Ann IV), have been
identified (9, 11–13). In conjunction with anti-phospholipid mAb,
Ab to the serum protein, b2-glycoprotein I (b2-GPI), also restored
tissue damage in Rag-12/2, IR-resistant mice (10). Although
Division of Biology, Kansas State University, Manhattan, KS 66506
Received for publication November 21, 2013. Accepted for publication November
20, 2014.
This work was supported by National Institutes of Health Grants R01 AI061691
(to S.D.F.), P20 GM103418, and RR016475; American Heart Association Grant
11GRNT6940000 (to S.D.F.); and Kansas State University.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National
Institutes of Health.
Address correspondence and reprint requests to Dr. Sherry D. Fleming, 18 Ackert
Hall, Kansas State University, Manhattan, KS 66506. E-mail address: sdflemin@
ksu.edu
Abbreviations used in this article: Ann IV, annexin IV; b2-GPI, b2-glycoprotein I; IR,
ischemia/reperfusion; LTB4, leukotriene B4; MBL, mannose-binding lectin; NMM,
nonmuscle myosin; SLE, systemic lupus erythematosus.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303124
multiple neoantigens have been identified, the mechanism of expression of these neoantigens remains unknown.
Recent studies also indicate a significant role for TLRs in IRinduced tissue damage and inflammation (6, 14). As pathogenic
receptors, TLRs recognize distinct components of the microbe,
with TLR2 recognizing Gram-positive bacterial lipoproteins and
lipoteichoic acid, although TLR4 recognizes LPS from Gramnegative bacteria (15). Although TLRs recognize commensal
microflora to maintain intestinal homeostasis (16), these pathogen
recognition receptors also induce inflammation after tissue damage (17). Upon activation, most TLRs, including TLR2 and TLR4,
signal through the common MyD88 pathway. Recently, we demonstrated that MyD88 has a critical role in intestinal IR-induced
tissue damage (6). As a regulator of complement activation, TLR4
is critical in IR-induced tissue injury, C3 production, and the
cellular response in the intestine, kidney, brain, lung, and heart (6,
18–23). Similarly, TLR2 plays a role in renal, cerebral, and
myocardial IR (18, 24, 25). A recent publication indicated that
TLR2 is required for the cellular response to intestinal IR (26).
However, the role of TLR2 in Ab deposition and complement
activation remains unclear.
As both TLR2 and TLR4 use a similar signal transduction pathway
through MyD88, we hypothesized that, similar to TLR4, TLR2 is
critical to initiation of IR-induced pathology. Using TLR22/2 mice,
we demonstrate that TLR2 is required for both the humoral and the
cellular response during IR-induced injury. TLR2 plays a role in
activation of the cellular infiltrate. Unlike TLR4- or TLR9-deficient
mice (27), TLR22/2 mice also lack the appropriate Ab repertoire to
initiate intestinal IR-induced damage or inflammation. In addition,
despite the presence of the proteins, TLR2, but not TLR4, is required
for neoantigen exposure, indicating a dual role for TLR2 in IRinduced injury and inflammation. Thus, although both TLRs are required, TLR2 has a unique role in intestinal IR compared with TLR4.
Materials and Methods
Mice
C57BL/6 (wild-type control), TLR22/2, and Rag12/2 mice were obtained
from Jackson ImmunoResearch Laboratories and bred in the Division of
Biology at Kansas State University with food and water access ad libitum.
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In multiple clinical conditions, including trauma and hemorrhage, reperfusion magnifies ischemic tissue damage. Ischemia induces
expression of multiple neoantigens, including lipid alterations that are recognized by the serum protein, b2-glycoprotein I (b2-GPI).
During reperfusion, binding of b2-GPI by naturally occurring Abs results in an excessive inflammatory response that may lead to
death. As b2-GPI is critical for intestinal ischemia/reperfusion (IR)-induced tissue damage and TLR2 is one of the proposed
receptors for b2-GPI, we hypothesized that IR-induced intestinal damage and inflammation require TLR2. Using TLR22/2 mice,
we demonstrate that TLR2 is required for IR-induced mucosal damage, as well as complement activation and proinflammatory
cytokine production. In response to IR, TLR22/2 mice have increased serum b2-GPI compared with wild-type mice, but b2-GPI is
not deposited on ischemic intestinal tissue. In addition, TLR22/2 mice also did not express other novel Ags, suggesting a sequential
response. Unlike other TLRs, TLR22/2 mice lacked the appropriate Ab repertoire to induce intestinal IR tissue damage or
inflammation. Together, these data suggest that, in addition to the inflammatory response, IR-induced injury requires TLR2
for naturally occurring Ab production. The Journal of Immunology, 2015, 194: 1190–1198.
The Journal of Immunology
The TLR22/2 mice were backcrossed to the C57BL/6 background for
at least nine generations and maintained as specific pathogen free
(Helicobacter species, mouse hepatitis virus, minute virus of mice,
mouse parvovirus, Sendai virus, murine norovirus, Mycoplasma pulmonis,
Theiler’s murine encephalomyelitis virus, and endo- and ectoparasites).
Research was conducted in compliance with the Animal Welfare Act and
other federal statutes and regulations relating to animals and experiments
involving animals and was approved by the Institutional Animal Care and
Use Committee at Kansas State University.
Ischemia/Reperfusion
1191
were minced, washed, and resuspended in 37˚C oxygenated Tyrode’s
buffer (Sigma-Aldrich, St. Louis, MO). After a 20-min incubation at 37˚C,
supernatants were collected and supernatants and tissue were stored at
280˚C until assayed. The concentration of LTB4 and PGE2 was determined
using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI).
Cytokine analysis of the same intestinal supernatants was determined using
a Milliplex mitogen-activated protein immunoassay kit (Millipore) and
read on a Milliplex Analyzer (Millipore). The tissue protein content was
determined using the bicinchoninic acid assay (Pierce, Rockford, IL)
adapted for use with microtiter plates. Eicosanoid and cytokine production
was expressed per mg protein per 20 min.
Quantitative real-time PCR
Histology and immunohistochemistry
Immediately after sham or IR treatment, blood was collected from wildtype and TLR22/2 mice and allowed to clot on ice at least 30 min to
collect whole serum after centrifugation. Sera (3 mL per lane) was loaded
in nonreducing SDS buffer and run on a 10% SDS-PAGE gel, followed
by transfer to a polyvinylidene difluoride membrane. The membrane was
blocked with 5% milk in TBS with 0.1% Tween 20 solution for 1 h,
followed by overnight incubation with a HRP goat anti-human b2-GPI
Ab (Bethyl Laboratories) diluted in block solution at 1:2500. The blots
were visualized with SuperSignal West Pico ECL Substrate (Thermo
Scientific) and developed with an ECOMAX film developer (Protec
Medical Devices).
Immediately after removal, midjejunal specimens were fixed in 10%
buffered formalin phosphate and embedded in paraffin, sectioned transversely (8 mm), and H&E stained. The mucosal injury score was graded on
a six-tiered scale similar to that of Chiu et al. (28). Briefly, the average
damage score of the intestinal section was determined by the average
scores of two blinded observers (trained in evaluating intestinal injury).
Each observer graded 75–150 villi on a scale of 0–6. Normal villi were
assigned a score of zero; villi with tip distortion were assigned a score of 1;
a score of 2 was assigned when Guggenheims’ spaces were present; villi
with small regions of disruption of the epithelial cells were assigned a score
of 3; a score of 4 was assigned to villi with large regions of exposed, but
intact lamina propria with epithelial sloughing; a score of 5 was assigned
when the lamina propria was exuding; last, villi that displayed hemorrhage
or were denuded were assigned a score of 6. Photomicrographs were obtained from H&E-stained slides using an original magnification 320, 0.5
Plan Fluor objective on Nikon 80i microscope and images acquired at room
temperature using a Nikon DS-5M camera with DS-L2 software.
An additional 2-cm intestinal section was immediately snap frozen in
O.C.T. freezing medium, and 8-mm sections were transversely cut and placed
on slides for immunohistochemistry. Following acetone fixation, the nonspecific binding was blocked for 30 min by incubating with 10% sera in
PBS. After washing in PBS, the tissues were incubated with Ab for 1 h at
room temperature or overnight at 4˚C. The C3, IgM, mannose-binding
lectin (MBL)-c, and b2-GPI deposition, and Ann IV and NMM expression on the tissue sections were detected by staining with a purified rat
anti-mouse anti-C3 (Hycult Biotechnologies) or anti-IgM Ab or anti–
MBL-C Ab, followed by a Texas Red–conjugated donkey anti-rat IgG
secondary Ab (Jackson ImmunoResearch Laboratories) or anti-Ann IV or
anti-NMM (AbCam), followed by a Texas Red–conjugated donkey antirabbit IgG secondary Ab or anti–b2-GPI Ab (Millipore), followed by
Texas Red–conjugated donkey anti-mouse IgG secondary Ab. The detection of b2-GPI was performed similarly, with the exception of all solutions
being made with 1% BSA in PBS and 2% Rag-12/2 sera used for blocking
with an additional 1 h of blocking in only 1% BSA in PBS prior to the sera
block. Each experiment contained serial sections stained with the appropriate isotype control Ab. All slides were mounted with ProLong Gold
(Invitrogen). A blinded observer obtained images at room temperature using
a Nikon eclipse 80i microscope equipped with a CoolSnap CF camera
(Photometrics) and analyzed using Metavue software (Molecular Devices).
Eicosanoid and cytokine determination
The ex vivo generation of eicsanoids in small intestine tissue was determined, as described previously (29). Briefly, fresh midjejunum sections
Total RNA was isolated from the jejunum using TRIzol reagent (Invitrogen),
according to manufacturer’s instructions. RNA integrity and genomic DNA
contamination were assessed using a BioAnalyzer (Agilent), and quantity
was determined by Nanodrop evaluation. Only samples with no DNA
contamination and RNA integrity values .7.0 were used for cDNA synthesis. Total RNA (1 mg) was reverse transcribed using qScript first-strand
cDNA synthesis kit (Quanta Biosciences) using random primers. Quantitative real-time PCR was performed in 25-ml vol using a Mini-Opticon
real-time thermal cycler (Bio-Rad) and Perfecta SYBR Green Fastmix
reagent (Quanta Biosciences) using the following protocol: 3 min at 95˚C;
50 cycles of 10 s at 95˚C, 20 s at annealing temperature (Table I), 10 s at
72˚C; melt curve starting at 65˚C, increasing 0.5˚C every 5 s up to 95˚C.
After amplification, Cox-2 and b2-GPI cycle threshold values were normalized to 18S rRNA, and then DD cycle threshold fold change relative to
Sham-treated wild-type mice was determined, as described previously
(30). Melt-curve analysis of the PCR products ensured amplification of
a single product.
b2-GPI Western blot analysis
Anti–b2-GPI ELISA
Blood was collected from untreated wild-type and TLR22/2 mice and
allowed to clot on ice at least 30 min, and whole serum collected by
centrifugation. Polystyrene microtiter plates (NUNC Immuno) were coated
overnight with 8 mg/ml mouse b2-GPI [purified as in (31)] in 0.02 M
carbonate buffer (pH 9.6). All wells were blocked for 1 h with 20% FBS in
PBS. Serum and standard samples were incubated for 1 h with shaking,
washed with PBS with 0.05% Tween 20, and then incubated with HRPconjugated donkey anti-mouse IgG Ab (Jackson ImmunoResearch Laboratories) and developed with TMB 1-Component substrate (KPL). Purified
anti-mouse b2-GPI mAb (FC1) (10) was used to obtain a standard curve.
Ab isotype analysis
Blood was collected from untreated wild-type and TLR22/2 mice and
allowed to clot on ice at least 30 min, and whole serum was collected by
centrifugation. A Milliplex Mouse Ig Isotyping Immunoassay (Millipore)
was used to determine serum Ab isotypes from each strain of mice,
according to manufacturer’s instructions; read on a Milliplex Analyzer
(Millipore); and analyzed using Milliplex Analyst software (Millipore).
Statistical analysis
Data are presented as mean 6 SEM and were compared by one-way
ANOVA with post hoc analysis using Newman–Kuels test (Graph Pad/
Instat Software, Philadelphia, PA). The difference between groups was
considered significant when p , 0.05.
Results
Previous studies indicated that complement and TLR4 interact to
mediate inflammation and tissue damage in response to mesenteric
IR (6, 14). In addition, intestinal damage required the adapter
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Animals were subjected to IR, as described previously (6). Briefly, ketamine
(16 mg/kg)- and xylazine (80 mg/kg)- or isofluorane (2%)-anesthetized
mice were subjected to a laparotomy 30 min prior to applying a small
vascular clamp (Roboz Surgical Instruments) to the superior mesenteric
artery. Ischemia was confirmed by blanching of the intestine and absence
of pulsations distal to the clamp. Covering the bowel with surgical gauze
moistened with warm normal saline prevented intestinal desiccation. After
30 min of ischemia, 2 h of reperfusion was induced by removing the clamp
and confirming the return of pulsatile flow to the superior mesenteric artery. All mice received buprenorphine (0.06 mg/kg) for pain. Some
experiments reconstituted Rag-12/2 or TLR22/2 mice by i.v. injection of
200 ml whole sera or 100 mg protein G–purified Ab from TLR22/2 or wildtype (C57BL/6) mice 30 min prior to ischemia. Sham-treated animals
underwent the same surgical intervention, except for vessel occlusion. All
procedures were performed with the animals breathing spontaneously and
body temperature maintained at 37˚C using a water-circulating heating
pad. Additional ketamine and xylazine or isofluorane were administered
immediately prior to sacrifice. After sacrifice, blood and 2-cm sections of
the small intestine 10 cm distal to the gastroduodenal junction were harvested for histological evaluation as well as eicosanoid and cytokine determination.
1192
ROLES OF TLR2 IN GUT IR-INDUCED INJURY AND INFLAMMATION
FIGURE 1. IR-induced intestinal
damage requires TLR2 expression. Wildtype and TLR22/2 mice were subjected
to sham or IR treatment, and midjejunal
sections were H&E stained (original
magnification 3200) and (A) scored for
mucosal damage (75–150 villi per section). (B–E) Representative microphotographs of H&E-stained sections. Each
bar is representative of 7–10 animals per
group. *p # 0.05 compared with sham
treatment, Фp # 0.05 compared with
wild-type IR treatment.
and TLR22/2 mice (data not shown). We examined complement
initiation on the IR-treated intestinal tissue by examining intestinal deposition of IgM and complement components, MBL-C and
C3, by immunohistochemistry. As indicated in Fig. 3, IgM, C3,
and MBL-C were not deposited in response to sham treatment.
However, significant deposits of IgM and both MBL-C and C3
were present in wild-type mice after mesenteric IR (Fig. 3). In
contrast, little to no IgM, C3, or MBL-C were deposited after
similar treatment of the TLR22/2 mice (Fig. 3). Although TLR2
does not regulate complement production, these data suggest that
the absence of TLR2 significantly decreases initiation of complement activation compared with wild-type mice.
Previous studies indicated that the appropriate Ab repertoire is
required for recognition of IR-induced neoantigens, complement
activation, and eicosanoid production (14, 37). Importantly, TLR4
was not required for production of the appropriate Ab repertoire to
induce damage in Ab-deficient, IR-resistant, Rag-12/2 mice (8,
14, 37). To determine whether TLR22/2 mice have the proper Ab
FIGURE 2. Eicosanoid and cytokine production is significantly decreased in TLR22/2 mice. Wild-type and TLR22/2 mice were subjected to
sham or IR treatment. (A) Midjejunal cox-2 transcription was determined
by quantitative RT-PCR analysis. Intestinal sections were analyzed ex vivo
for (B) PGE2 and LTB4 and (C) IL-6, IL-12p40, TNF-a, and CXCL1
(keratinocyte-derived chemokine) production. Each bar is representative of
4–10 animals per group. *p # 0.05 compared with sham treatment, Фp #
0.05 compared with wild-type IR treatment.
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protein, MyD88 (6). Therefore, we hypothesized that mesenteric
IR-induced damage would be attenuated in TLR22/2 mice. We
tested the hypothesis by subjecting wild-type and TLR22/2 mice
to intestinal sham or IR treatment. As sham treatment was not
significantly different between the strains of mice, the scores
were pooled (Fig. 1A, 1B, 1D). As expected, after IR, wild-type
C57BL/6 sustained significant tissue injury (Fig. 1A, 1C). In
contrast, TLR22/2 mice sustained minimal intestinal IR-induced
damage that was not significantly different from sham treatment
(Fig. 1A, 1D, 1E).
Previous studies indicated that TLR4 and MyD88 activation
results in Cox-2–mediated PGE2 production, which is required for
IR-induced tissue damage (6, 14). As TLR2 also signals through
MyD88, we examined the intestinal eicosanoid production in response to IR in TLR22/2 mice. Similar to intestinal damage, Cox-2
transcript was elevated in response to IR in wild-type mice, but
not in TLR22/2 mice (Fig. 2A, Table I). Correlating with increased Cox-2 expression, the intestines of wild-type, but not
TLR22/2 mice produced significant quantities of PGE2 (Fig. 2B).
As demonstrated previously, intestinal IR also increases the chemotactic eicosanoid, LTB4, in wild-type mice (32–35). Similar to
injury, IR did not induce LTB4 in TLR22/2 mice (Fig. 2B). These
data suggest that TLR2 is required for the IR-induced eicosanoid
response. Similar to eicosanoid production and previous studies
with TLR42/2 and MyD882/2 mice (14), intestinal production of
CXCL1 (keratinocyte-derived chemokine) increased in response
to IR in wild-type, but not TLR22/2 mice (Fig. 2B). Despite a lack
of CXCL1 and LTB4 production, immunohistochemistry demonstrated an IR-induced macrophage infiltration in TLR22/2 mice
similar to that found in intestines of wild-type mice after IR (data
not shown). However, the TLR22/2 inflammatory infiltrate was
not activated to produce cytokines IL-6, IL-12p40, and TNFa,
whereas these cytokines were significantly elevated in intestines
from wild-type mice (Fig. 2C). Together, these studies suggest
that, similar to TLR4, TLR2 is critical to the cellular innate immune response to intestinal IR.
Tissue damage in response to intestinal IR also requires complement activation and Ab recognition of ischemia-induced neoantigens (36). TLR4-deficient mice produce the appropriate Ab,
but decrease complement C3 production and deposition as
a method of regulating the inflammatory response (14). To determine whether TLR22/2 mice regulate tissue damage and inflammation in a similar manner, we initially found no change in
complement C3 transcription in response to IR between wild-type
The Journal of Immunology
1193
Table I. Real-time PCR primer sequences
Gene
Annealing Temperature (˚C)
b2-Glycoprotein I
57
Cox-2
55
Ribosomal 18Sa
58
Sequence
FWD:
REV:
FWD:
REV:
FWD:
REV:
59-CGGATGACCTACCATTTGCT-39
59-GGGACACATCTCAGGGTGTT-39
59-ATTCAACACACTCTATCACTGGC-39
59-TGGTCAAATCCTGTGCTCATACAT-39
59-CTGGTAATTCATCTCTCTGCTCTG-39
59-GCGACCAAAGGAACCATAAC-39
a
Housekeeping gene to which genes of interest were normalized.
FWD, forward; REV, reverse.
FIGURE 3. Complement components and IgM are not
deposited in response to IR in TLR22/2 mice. Wild-type
and TLR22/2 mice were subjected to sham or IR treatment. Intestinal sections were stained for (A) IgM, (B)
C3, and (C) MBL-C by immunohistochemistry. Photomicrographs (original magnification 3200) are representative of three to four animals stained in at least three
independent experiments.
To determine whether providing the appropriate Ab was sufficient to restore injury to the TLR22/2 mice, we injected Ab purified from wild-type mice into TLR22/2 mice. Similar to previous
results, wild-type mice, but not TLR22/2 mice, sustained injury in
response to IR. Surprisingly, administration of wild-type Ab was
not sufficient to restore IR-induced injury in TLR22/2 mice
(Fig. 6A). Similarly, PGE2 and LTB4 were not elevated in response to IR when wild-type Abs were injected into TLR22/2
mice prior to IR (Fig. 6B, 6C). This lack of response may be due
to a lack of neoantigen expression and/or may be due to TLR2induced eicosanoids, cytokines, or free radicals from the inflammatory cell response.
As Ags stimulate Ab production, we examined the ability of
TLR22/2 mice to locally express b2-GPI transcript within the
intestine and systemic b2-GPI protein expression. In response to
IR, local intestinal b2-GPI mRNA was not increased in wild-type
mice, but was significantly elevated in TLR22/2 mice with a
6-fold increase compared with wild-type sham levels (Fig. 7A,
Table I). Under normal conditions, b2-GPI protein is present in
C57BL/6 and TLR22/2 sera at similar levels (Fig. 7B). In response
to IR in C57BL/6 mice, b2-GPI sera levels do not change; however, IR induces a significant release of b2-GPI into the sera of
TLR22/2 mice. Thus, despite the presence of b2-GPI, production
of the injurious Ab repertoire and, in particular, anti–b2-GPI Ab
appears to require TLR2.
Finally, we examined intestinal deposition and surface expression of three of the known IR-induced neoantigens, b2-GPI, NMM,
and Ann IV. Intestinal sections from sham-treated mice of either
strain showed minimal staining for each of the neoantigens
(Fig. 8). In contrast, intestinal sections from IR-treated, wild-type,
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repertoire, we injected Rag-12/2 mice with sera or Ig purified
from C57BL/6 or TLR22/2 mice and subjected the mice to IR. As
indicated in Fig. 4A, after IR, Rag-12/2 mice administered sera or
Ab obtained from C57BL/6 mice sustained significant intestinal
damage with no difference between administering sera or purified
Ab. In contrast, no significant increase in IR-induced intestinal
damage was observed when Rag-12/2 mice were injected with
sera or Ab from TLR22/2 mice (Fig. 4A). In addition, Rag-12/2
mice reconstituted with Ab from C57BL/6, but not TLR22/2 mice
secreted significant PGE2 (Fig. 4B) and LTB4 (Fig. 4C) in response to IR. Importantly, eicosanoid production by Rag-12/2
mice treated with Ab from TLR22/2 mice was similar to unreconstituted Rag-12/2 mice after sham or IR treatment (Fig. 4B,
4C). As there was no significant difference between treatments
with either sera or purified Ab, subsequent studies were performed
with purified Ab.
Previous studies suggested that Abs produced by TLR22/2 mice
differ from wild-type mice. The plasma from wild-type and
TLR22/2 mice contained equal quantities of total IgM Ab isotype
(Fig. 5A). Isotypes IgG3 and IgG2b were significantly lower in the
TLR22/2 plasma as well (Fig. 5A). To examine the damaging Abs,
we evaluated the anti–b2-GPI IgG and IgM. Sera pooled from
wild-type mice contained 49.3 6 8.1 ng/ml anti–b2-GPI IgG,
whereas sera obtained from TLR22/2 mice contained only 22.1 6
8.5 ng/ml anti–b2-GPI IgG (Fig. 5B). Sera from Rag-12/2 mice
contained no detectable Ab and was used as a negative control.
Importantly, the TLR22/2 mice also had significantly less anti–b2GPI IgM compared with the wild-type C57BL/6 mice (Fig. 5B).
Together these data suggest that the TLR22/2 Ab repertoire is not
sufficient to induce damage in response to IR.
1194
ROLES OF TLR2 IN GUT IR-INDUCED INJURY AND INFLAMMATION
FIGURE 4. TLR22/2 mice lack the pathogenic Ab
required for IR-induced intestinal damage and eicosanoid production. Rag12/2 mice were subjected to sham
or IR treatment with or without administration of Abs
from either wild-type or TLR22/2 mice. (A) Midjejunal
sections were H&E stained and scored for mucosal
damage. (B) Intestinal sections were analyzed ex vivo
for (B) PGE2 and (C) LTB4. Each bar is representative
of 5–10 animals per group. *p # 0.05 compared with
Rag12/2 IR treatment, Фp # 0.05 compared with
Rag12/2 IR + C57BL/6 Ab treatment.
FIGURE 5. The Ab repertoire is altered in TLR22/2 mice. Whole
plasma or sera from wild-type and TLR22/2 mice was analyzed by ELISA
for (A) total Ab isotypes and (B) anti–b2-GPI–specific IgG and IgM. Each
bar is representative of three to five different lots of plasma or sera. Фp #
0.05 compared with wild-type plasma or sera.
tines. These data suggest that, despite significant levels of b2-GPI
present in the serum, intestinal deposition of b2-GPI and surface
expression of neoantigens, NMM and Ann IV, require TLR2.
Discussion
Together, these data suggest that the absence of TLR2 compromises the IR-induced cellular and humoral innate responses by
significantly decreasing the release of proinflammatory cytokines
and eicosanoids, decreasing deposition of complement initiators,
attenuating neoantigen surface expression, and altering the Ab
repertoire. We propose the following model, which will require
testing in the future. The process of IR requires b2-GPI and anti–b
2-GPI Ab to induce endothelial damage and inflammation (10,
31, 38). As expected, TLR2 plays a role in the inflammatory
process; however, we demonstrate that TLR2 is critical to multiple components of intestinal IR-induced injury and inflammation. Specifically, TLR2 is essential for the following: 1) production
of the pathogenic naturally occurring Ab; 2) inflammatory cytokine production and PGE2 production; and 3) expression of neoantigens that are recognized by the naturally occurring Ab. Then
in a TLR2-independent manner, Ab bind to the cell surface and
activate complement. Thus, TLR2 plays a critical role in multiple inflammatory processes that occur during IR-induced tissue
damage.
Our data demonstrate that TLR2 is required for IR-induced intestinal damage using a time course of 30-min ischemia and 2-h
reperfusion. Using a 45-min ischemic period, followed by 60-min
reperfusion, a recent study also demonstrated that the neutrophilic
infitration, Cox-2, and TNF mediated at least a portion of IRinduced intestinal damage in a TLR2-dependent manner (26).
These studies differ from those performed on young (4-wk) mice in
which wild-type mice sustained minimal intestinal injury (0.67
injury score on a 0–4 scale) and TLR22/2 mice exhibited increased
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or TLR4def mice expressed all three neoantigens, whereas the IRtreated, TLR22/2 intestines expressed little, if any, of the three
neoantigens on the cell surface (Fig. 8). When quantitated using
Image J, expression on TLR22/2 intestine was significantly lower
than that of the wild-type or TLR4def intestines for each neoantigen examined (Fig. 8). The staining of TLR4def intestines was
not significantly different when compared with wild-type intes-
The Journal of Immunology
1195
FIGURE 6. Pathogenic Abs are not sufficient to restore IR-induced injury in TLR22/2 mice. Wild-type
and TLR22/2 mice were subjected to sham or IR
treatment with or without administration of Abs from
wild-type mice. (A) Midjejunal sections were H&E
stained and scored for mucosal damage. (B) Intestinal
sections were analyzed ex vivo for (B) PGE2 and (C)
LTB4. Each bar is representative of 4–10 animals per
group. *p # 0.05 compared with sham treatment, Фp #
0.05 compared with wild-type IR treatment.
nificantly reduced production of inflammatory cytokines in
TLR22/2 mice compared with wild-type mice (39). Similar
results in renal and myocardial IR indicate that multiple cell
types require TLR2 for the inflammatory response and tissue
damage (42, 44).
Previous studies demonstrated that complement is critical to
intestinal, renal, and myocardial IR-induced damage, although
renal IR-induced injury appears to use the alternative pathway,
whereas intestinal and myocardial IR-induced damage is dependent
on the classical and MBL pathways of complement activation (45).
The current study demonstrates that, in response to IR, complement deposition within intestines of mice also requires TLR2
expression. To our knowledge, this is the first study in intestinal IR
that examined the interactions of TLR2 and complement. In renal
IR, the alternative complement component, factor B, was shown to
interact with TLR2 (46). The current study did not examine the
FIGURE 7. TLR2 deficiency increases IR-induced b2GPI transcription and serum levels. Wild-type and TLR22/2
mice were subjected to sham or IR treatment. (A) Midjejunal b2-GPI transcription was determined by quantitative RT-PCR analysis. *p # 0.05 compared with sham
treatment. Each bar is representative of four to six animals
per group. (B) b2-GPI serum protein production was determined by Western blot analysis after sham or IR treatment. (C) A representative blot is shown. Graph and blot
are representative of three total blots with three different
sets of serum.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
injury (39). The time course in the previous study (39) was 60 min
of ischemia, followed by 90-min reperfusion, which others have
indicated is a nonrecoverable surgery (40). The discrepancy in the
results may be due to animal age, time course, or technical differences of using a bulldog clamp with unknown pressure
versus a vascular clamp or may be explained by incomplete
clamping, which would correlate with the low injury score reported for wild-type mice. In contrast, our data are consistent
with IR experiments performed in heart (41, 42) and kidney
(25, 43, 44). Multiple studies have demonstrated that the absence or blockade of TLR2 protects against IR-induced myocardial damage and inflammation (41, 42). Similarly, TLR2 is
critical to IR-induced renal damage that is mediated by both
MyD88-dependent and independent mechanisms (25, 43, 44).
In contrast to injury, the data presented in this work agree with
the previous intestinal IR-induced study that demonstrates sig-
1196
ROLES OF TLR2 IN GUT IR-INDUCED INJURY AND INFLAMMATION
specific pathways involved, but IgM, C3, and MBL depositions
correlated with injury, suggesting the classical and MBL pathways
were activated. As Ab is required for IR-induced injury in the
intestine (8) and heart (47), but not kidney (48), it appears that
different mechanisms of complement activation are active in
reperfusion injury of different organs.
Previous studies indicate that TLR4- and TLR9-deficient mice
contain the appropriate Ab to induce IR injury in Rag-12/2 mice
(6, 27). In contrast, TLR22/2 mice do not contain the necessary
pathogenic Ab for IR-induced tissue damage, despite a similar
number of total Ab. A requirement for TLR stimulation to produce
autoreactive Abs has been demonstrated in autoimmune disease.
In the AM14 mouse models of systemic lupus erythematosus
(SLE), immune complexes ligate both IgM and TLR7 or TLR9 to
induce autoreactive B cells to produce high quantities of Ab (49,
50). Additional studies in the MRL/lpr mouse model of SLE
demonstrated that kidney mesangial cells require immune complexes containing high mobility group protein B1 and TLR2 for
increased cytokine production, and human blood cells require
TLR2 for increased autoreative Ab production (51, 52).
The specific subclass of Ab produced also varies in the absences
of TLR2. Similar to the results of others (53), our data demonstrate
that TLR22/2 mice contain fewer IgG Abs compared with IgG
levels in wild-type mice. Lartigue et al. (53) showed that, in
a mouse model of SLE, both TLR2- and TLR4-deficient mice had
fewer IgG, and these results correlated with less severe disease. In
addition, the overall natural IgM levels were not significantly
different (53). Importantly, the absence of either TLR in the SLE
model resulted in decreased self-reactive autoantibodies from
multiple subclasses of IgG (53). Whereas they did not examine
IgM, our data indicate that, although the overall IgM concentration was not significantly different, the anti–b2-GPI–specific IgM
Abs decreased in the absence of TLR2. It is possible that other
endogenous Ags induce Abs in a TLR2-dependent manner as
well.
Our data also correlate with clinical studies showing induction of
significantly more inflammatory cytokines following myocardial
infarction in patients who have increased levels of autoreactive
Ab (54). In vitro studies provided more cell type–specific data,
demonstrating that Ab induce human monocyte-derived macrophages to secrete inflammatory cytokines in a TLR2-dependent
manner (54). In addition, pathogenic anti-phospholipid Ab stimulate an inflammatory response by multiple cell types, including
monocytes, fibroblasts, and endothelial cells (55, 56). Importantly,
blocking of TLR2 inhibits the Ab-induced cytokine release (54).
Within the last 15 y, multiple IR-induced neoantigens have been
identified. Based on Ab recognition and peptide inhibition, at least
three neoantigens appear to be required for intestinal IR-induced
damage (9, 10, 13, 38, 57). However, the specific role of each
neoantigen is unclear at this time. Our data also indicate that,
despite increased serum b2-GPI, TLR22/2 mice contain significantly less anti–b2-GPI Ab and decreased neoantigen is expressed
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
FIGURE 8. IR does not induce neoantigen expression in TLR22/2 mice. Wild-type, TLR42/2 and TLR22/2 mice were subjected to sham or IR treatment.
Intestinal sections were stained for (A) b2-GPI, (B) nonmuscle myosin, and (C) annexin IV by immunohistochemistry (original magnification 3200), and
relative fluorescence was measured by ImageJ analysis. Microphotographs are representative of three to four animals stained in at least three independent
experiments. Each bar is representative of three to four animals per group. *p # 0.05 compared with sham treatment, Фp # 0.05 compared with wild-type
IR treatment.
The Journal of Immunology
Acknowledgments
We acknowledge Jiena Gu’s assistance with the immunohistochemistry
studies.
Disclosures
The authors have no financial conflicts of interest.
References
1. Aliosmanoglu, I., M. Gul, M. Kapan, Z. Arikanoglu, F. Taskesen, O. Basol, and
M. Aldemir. 2013. Risk factors affecting mortality in acute mesenteric ischemia
and mortality rates: a single center experience. Int. Surg. 98: 76–81.
2. Yanar, F., O. Agcaoglu, I. S. Sarici, E. Sivrikoz, A. Ucar, H. Yanar, M. Aksoy,
and M. Kurtoglu. 2013. Local thrombolytic therapy in acute mesenteric ischemia. World J. Emerg. Surg. 8: 8.
3. Granger, C. B., K. W. Mahaffey, W. D. Weaver, P. Theroux, J. S. Hochman,
T. G. Filloon, S. Rollins, T. G. Todaro, J. C. Nicolau, W. Ruzyllo, and
P. W. Armstrong, COMMA Investigators. 2003. Pexelizumab, an anti-C5 complement antibody, as adjunctive therapy to primary percutaneous coronary intervention in acute myocardial infarction: the COMplement inhibition in myocardial
infarction treated with angioplasty (COMMA) trial. Circulation 108: 1184–1190.
4. Rehrig, S., S. D. Fleming, J. Anderson, J. M. Guthridge, J. Rakstang,
C. E. McQueen, V. M. Holers, G. C. Tsokos, and T. Shea-Donohue. 2001.
Complement inhibitor, complement receptor 1-related gene/protein y-Ig
attenuates intestinal damage after the onset of mesenteric ischemia/reperfusion
injury in mice. J. Immunol. 167: 5921–5927.
5. Zimmerman, B. J., and D. N. Granger. 1994. Mechanisms of reperfusion injury.
Am. J. Med. Sci. 307: 284–292.
6. Moses, T., L. Wagner, and S. D. Fleming. 2009. TLR4-mediated Cox-2 expression increases intestinal ischemia/reperfusion-induced damage. J. Leukoc.
Biol. 86: 971–980.
7. Fleming, S. D. 2006. Natural antibodies, autoantibodies and complement activation in tissue injury. Autoimmunity 39: 379–386.
8. Williams, J. P., T. T. V. Pechet, M. R. Weiser, R. Reid, L. Kobzik, F. D. Moore,
Jr., M. C. Carroll, and H. B. Hechtman. 1999. Intestinal reperfusion injury is
mediated by IgM and complement. J. Appl. Physiol. 86: 938–942.
9. Zhang, M., W. G. Austen, Jr., I. Chiu, E. M. Alicot, R. Hung, M. Ma, N. Verna,
M. Xu, H. B. Hechtman, F. D. Moore, Jr., and M. C. Carroll. 2004. Identification
of a specific self-reactive IgM antibody that initiates intestinal ischemia/
reperfusion injury. Proc. Natl. Acad. Sci. USA 101: 3886–3891.
10. Fleming, S. D., R. P. Egan, C. Chai, G. Girardi, V. M. Holers, J. Salmon,
M. Monestier, and G. C. Tsokos. 2004. Anti-phospholipid antibodies restore
mesenteric ischemia/reperfusion-induced injury in complement receptor 2/
complement receptor 1-deficient mice. J. Immunol. 173: 7055–7061.
11. Zhang, M., and M. C. Carroll. 2007. Natural antibody mediated innate autoimmune response. Mol. Immunol. 44: 103–110.
12. Keith, M. P., C. Moratz, R. Egan, A. Zacharia, E. L. Greidinger, R. W. Hoffman,
and G. C. Tsokos. 2007. Anti-ribonucleoprotein antibodies mediate enhanced
lung injury following mesenteric ischemia/reperfusion in Rag-1(-/-) mice. Autoimmunity 40: 208–216.
13. Kulik, L., S. D. Fleming, C. Moratz, J. W. Reuter, A. Novikov, K. Chen,
K. A. Andrews, A. Markaryan, R. J. Quigg, G. J. Silverman, et al. 2009. Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury. J. Immunol. 182: 5363–5373.
14. Pope, M. R., S. M. Hoffman, S. Tomlinson, and S. D. Fleming. 2010. Complement regulates TLR4-mediated inflammatory responses during intestinal ischemia reperfusion. Mol. Immunol. 48: 356–364.
15. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda,
and S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of
Gram-negative and Gram-positive bacterial cell wall components. Immunity 11:
443–451.
16. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, and
R. Medzhitov. 2004. Recognition of commensal microflora by Toll-like receptors
is required for intestinal homeostasis. Cell 118: 229–241.
17. Mollen, K. P., R. J. Anand, A. Tsung, J. M. Prince, R. M. Levy, and T. R. Billiar.
2006. Emerging paradigm: Toll-like receptor 4-sentinel for the detection of
tissue damage. Shock 26: 430–437.
18. Takeishi, Y., and I. Kubota. 2009. Role of Toll-like receptor mediated signaling
pathway in ischemic heart. Front Biosci (Landmark Ed) 14: 2553–2558.
19. Wu, H., G. Chen, K. R. Wyburn, J. Yin, P. Bertolino, J. M. Eris, S. I. Alexander,
A. F. Sharland, and S. J. Chadban. 2007. TLR4 activation mediates kidney
ischemia/reperfusion injury. J. Clin. Invest. 117: 2847–2859.
20. Li, C., T. Ha, J. Kelley, X. Gao, Y. Qiu, R. L. Kao, W. Browder, and
D. L. Williams. 2004. Modulating Toll-like receptor mediated signaling by
(1—.3)-beta-D-glucan rapidly induces cardioprotection. Cardiovasc. Res.
61: 538–547.
21. Gao, Y., X. Fang, Y. Tong, Y. Liu, and B. Zhang. 2009. TLR4-mediated MyD88dependent signaling pathway is activated by cerebral ischemia-reperfusion in
cortex in mice. Biomed. Pharmacother. 63: 442–450.
22. Victoni, T., F. R. Coelho, A. L. Soares, A. de Freitas, T. Secher, R. Guabiraba,
F. Erard, R. M. de Oliveira-Filho, B. B. Vargaftig, G. Lauvaux, et al. 2010. Local
and remote tissue injury upon intestinal ischemia and reperfusion depends on the
TLR/MyD88 signaling pathway. Med. Microbiol. Immunol. 199: 35–42.
23. Yang, Q. W., J. C. Li, F. L. Lu, A. Q. Wen, J. Xiang, L. L. Zhang, Z. Y. Huang,
and J. Z. Wang. 2008. Upregulated expression of Toll-like receptor 4 in monocytes correlates with severity of acute cerebral infarction. J. Cereb. Blood Flow
Metab. 28: 1588–1596.
24. Hua, F., J. Wang, I. Sayeed, T. Ishrat, F. Atif, and D. G. Stein. 2009. The TRIFdependent signaling pathway is not required for acute cerebral ischemia/
reperfusion injury in mice. Biochem. Biophys. Res. Commun. 390: 678–683.
25. Shigeoka, A. A., T. D. Holscher, A. J. King, F. W. Hall, W. B. Kiosses, P. S. Tobias,
N. Mackman, and D. B. McKay. 2007. TLR2 is constitutively expressed within the
kidney and participates in ischemic renal injury through both MyD88-dependent
and -independent pathways. J. Immunol. 178: 6252–6258.
26. Watanabe, T., T. Tanigawa, A. Kobata, S. Takeda, Y. Nadatani, K. Otani,
H. Yamagami, M. Shiba, K. Tominaga, Y. Fujiwara, and T. Arakawa. 2014. Tolllike receptor 2 mediates ischemia-reperfusion injury of the small intestine in
adult mice. PLoS One 9: e110441.
27. Slone, E. A., M. R. Pope, M. Roth, R. Welti, and S. D. Fleming. 2012. TLR9 is
dispensable for intestinal ischemia/reperfusion-induced tissue damage. Am. J.
Clin. Exp. Immunol. 1: 124–135.
28. Chiu, C.-J., A. H. McArdle, R. Brown, H. J. Scott, and F. N. Gurd. 1970. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic,
and metabolic reappraisal. Arch. Surg. 101: 478–483.
29. Sjogren, R. W., C. Colleton, and T. Shea-Donohue. 1994. Intestinal myoelectric
response in two different models of acute enteric inflammation. Am. J. Physiol.
267: G329–G337.
30. Zhao, A., J. F. Urban, Jr., R. M. Anthony, R. Sun, J. Stiltz, N. van Rooijen,
T. A. Wynn, W. C. Gause, and T. Shea-Donohue. 2008. Th2 cytokine-induced
alterations in intestinal smooth muscle function depend on alternatively activated
macrophages. Gastroenterology 135: 217–225, e1.
31. Tomasi, M., Y. Hiromasa, M. R. Pope, S. Gudlur, J. M. Tomich, and
S. D. Fleming. 2012. Human b2-glycoprotein I attenuates mouse intestinal
ischemia/reperfusion induced injury and inflammation. Mol. Immunol. 52: 207–
216.
32. Anderson, J., S. D. Fleming, S. Rehrig, G. C. Tsokos, M. Basta, and T. SheaDonohue. 2005. Intravenous immunoglobulin attenuates mesenteric ischemiareperfusion injury. Clin. Immunol. 114: 137–146.
33. Eror, A. T., A. Stojadinovic, B. W. Starnes, S. C. Makrides, G. C. Tsokos, and
T. Shea-Donohue. 1999. Antiinflammatory effects of soluble complement receptor type 1 promote rapid recovery of ischemia/reperfusion injury in rat small
intestine. Clin. Immunol. 90: 266–275.
34. Fleming, S. D., D. Mastellos, G. Karpel-Massler, T. Shea-Donohue,
J. D. Lambris, and G. C. Tsokos. 2003. C5a causes limited, polymorphonuclear
cell-independent, mesenteric ischemia/reperfusion-induced injury. Clin. Immunol. 108: 263–273.
35. Fleming, S. D., T. Shea-Donohue, J. M. Guthridge, L. Kulik, T. J. Waldschmidt,
M. G. Gipson, G. C. Tsokos, and V. M. Holers. 2002. Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural
antibody repertoire. J. Immunol. 169: 2126–2133.
36. Carroll, M. C., and V. M. Holers. 2005. Innate autoimmunity. Adv. Immunol. 86:
137–157.
37. Sparkes, B. L., E. E. Slone, M. Roth, R. Welti, and S. D. Fleming. 2010. Intestinal lipid alterations occur prior to antibody-induced prostaglandin E2 production in a mouse model of ischemia/reperfusion. Biochim. Biophys. Acta 1801:
517–525.
38. Fleming, S. D., M. R. Pope, S. M. Hoffman, T. Moses, U. Bukovnik,
J. M. Tomich, L. M. Wagner, and K. M. Woods. 2010. Domain V peptides inhibit
beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue
damage and inflammation. J. Immunol. 185: 6168–6178.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
in response to IR. This may be accounted for by multiple different
hypotheses. As both TLRs have been proposed to be a receptor for
b2-GPI, it is possible that TLR2 is a primary receptor and b2-GPI
is required for other neoantigen expression. Another possibility is
that crosstalk between TLR2 and TLR4 results in the differential
neoantigen expression. In a LPS stimulation of endothelial cells,
stimulation of TLR4 increases activation of TLR2 (58). A similar
process may occur in IR and subsequently enhance neoantigen
expression. Similarly, it is possible that TLR2 and TLR4 are
critical on different tissues and processes, and, similar to myocardial IR, crosstalk occurs between multiple signaling pathways
(59). Finally, it is possible that TLR2 is required for recognition
of other endogenous Ags, which also results in complement activation. Among others, these endogenous Ags may include high
mobility group protein B1 (60) or the gut microbiome as antibiotic
treatment downregulates TLR2 and TLR4, resulting in attenuated
IR-induced injury (61). Future studies will be required to determine whether TLR2 is directly or indirectly critical for b2-GPI
deposition and exposure of other neoantigens and the specific
interactions between TLR2 and TLR4 during intestinal IR.
1197
1198
ROLES OF TLR2 IN GUT IR-INDUCED INJURY AND INFLAMMATION
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
A. Marshak-Rothstein. 2005. RNA-associated autoantigens activate B cells by
combined B cell antigen receptor/Toll-like receptor 7 engagement. J. Exp. Med.
202: 1171–1177.
Qing, X., M. Pitashny, D. B. Thomas, F. J. Barrat, M. P. Hogarth, and
C. Putterman. 2008. Pathogenic anti-DNA antibodies modulate gene expression
in mesangial cells: involvement of HMGB1 in anti-DNA antibody-induced renal
injury. Immunol. Lett. 121: 61–73.
Wen, Z., L. Xu, X. Chen, W. Xu, Z. Yin, X. Gao, and S. Xiong. 2013. Autoantibody induction by DNA-containing immune complexes requires HMGB1
with the TLR2/microRNA-155 pathway. J. Immunol. 190: 5411–5422.
Lartigue, A., N. Colliou, S. Calbo, A. François, S. Jacquot, C. Arnoult, F. Tron,
D. Gilbert, and P. Musette. 2009. Critical role of TLR2 and TLR4 in autoantibody production and glomerulonephritis in lpr mutation-induced mouse lupus. J.
Immunol. 183: 6207–6216.
Pagano, S., N. Satta, D. Werling, V. Offord, P. de Moerloose, E. Charbonney,
D. Hochstrasser, P. Roux-Lombard, and N. Vuilleumier. 2012. Antiapolipoprotein A-1 IgG in patients with myocardial infarction promotes inflammation through TLR2/CD14 complex. J. Intern. Med. 272: 344–357.
Satta, N., S. Dunoyer-Geindre, G. Reber, R. J. Fish, F. Boehlen, E. K. Kruithof,
and P. de Moerloose. 2007. The role of TLR2 in the inflammatory activation of
mouse fibroblasts by human antiphospholipid antibodies. Blood 109: 1507–1514.
Satta, N., E. K. Kruithof, C. Fickentscher, S. Dunoyer-Geindre, F. Boehlen,
G. Reber, D. Burger, and P. de Moerloose. 2011. Toll-like receptor 2 mediates
the activation of human monocytes and endothelial cells by antiphospholipid
antibodies. Blood 117: 5523–5531.
Zhang, M., E. M. Alicot, I. Chiu, J. Li, N. Verna, T. Vorup-Jensen, B. Kessler,
M. Shimaoka, R. Chan, D. Friend, et al. 2006. Identification of the target selfantigens in reperfusion injury. J. Exp. Med. 203: 141–152.
Fan, J., R. S. Frey, and A. B. Malik. 2003. TLR4 signaling induces TLR2 expression in endothelial cells via neutrophil NADPH oxidase. J. Clin. Invest. 112:
1234–1243.
Ha, T., L. Liu, J. Kelley, R. Kao, D. Williams, and C. Li. 2011. Toll-like
receptors: new players in myocardial ischemia/reperfusion injury. Antioxid.
Redox Signal. 15: 1875–1893.
Mersmann, J., F. Iskandar, K. Latsch, K. Habeck, V. Sprunck, R. Zimmermann,
R. R. Schumann, K. Zacharowski, and A. Koch. 2013. Attenuation of myocardial
injury by HMGB1 blockade during ischemia/reperfusion is Toll-like receptor
2-dependent. Mediators Inflamm. 2013: 174168.
Yoshiya, K., P. H. Lapchak, T. H. Thai, L. Kannan, P. Rani, J. J. Dalle Lucca, and
G. C. Tsokos. 2011. Depletion of gut commensal bacteria attenuates intestinal
ischemia/reperfusion injury. Am. J. Physiol. Gastrointest. Liver Physiol. 301:
G1020–G1030.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
39. Aprahamian, C. J., R. G. Lorenz, C. M. Harmon, and R. A. Dimmit. 2008. Tolllike receptor 2 is protective of ischemia-reperfusion-mediated small-bowel injury in a murine model. Pediatr. Crit. Care Med. 9: 105–109.
40. Stringa, P., N. Lausada, D. Romanin, M. Machuca, A. Cabanne, M. Rumbo, and
G. Gondolesi. 2012. Defining the nonreturn time for intestinal ischemia reperfusion injury in mice. Transplant. Proc. 44: 1214–1217.
41. Arslan, F., M. B. Smeets, L. A. O’Neill, B. Keogh, P. McGuirk, L. Timmers,
C. Tersteeg, I. E. Hoefer, P. A. Doevendans, G. Pasterkamp, and D. P. de Kleijn.
2010. Myocardial ischemia/reperfusion injury is mediated by leukocytic Tolllike receptor-2 and reduced by systemic administration of a novel anti-Toll-like
receptor-2 antibody. Circulation 121: 80–90.
42. Favre, J., P. Musette, V. Douin-Echinard, K. Laude, J. P. Henry, J. F. Arnal,
C. Thuillez, and V. Richard. 2007. Toll-like receptors 2-deficient mice are protected against postischemic coronary endothelial dysfunction. Arterioscler.
Thromb. Vasc. Biol. 27: 1064–1071.
43. Ben Mkaddem, S., E. Pedruzzi, C. Werts, N. Coant, M. Bens, F. Cluzeaud,
J. M. Goujon, E. Ogier-Denis, and A. Vandewalle. 2010. Heat shock protein
gp96 and NAD(P)H oxidase 4 play key roles in Toll-like receptor 4-activated
apoptosis during renal ischemia/reperfusion injury. Cell Death Differ. 17: 1474–
1485.
44. Leemans, J. C., G. Stokman, N. Claessen, K. M. Rouschop, G. J. Teske,
C. J. Kirschning, S. Akira, T. van der Poll, J. J. Weening, and S. Florquin. 2005.
Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney. J.
Clin. Invest. 115: 2894–2903.
45. Morrison, H., J. Frye, G. Davis-Gorman, J. Funk, P. McDonagh, G. Stahl, and
L. Ritter. 2011. The contribution of mannose binding lectin to reperfusion injury
after ischemic stroke. Curr. Neurovasc. Res. 8: 52–63.
46. Amura, C. R., B. Renner, T. Lyubchenko, S. Faubel, P. L. Simonian, and
J. M. Thurman. 2012. Complement activation and Toll-like receptor-2 signaling
contribute to cytokine production after renal ischemia/reperfusion. Mol. Immunol. 52: 249–257.
47. Haas, M. S., E. M. Alicot, F. Schuerpf, I. Chiu, J. Li, F. D. Moore, and
M. C. Carroll. 2010. Blockade of self-reactive IgM significantly reduces injury in
a murine model of acute myocardial infarction. Cardiovasc. Res. 87: 618–627.
48. Park, P., M. Haas, P. N. Cunningham, L. Bao, J. J. Alexander, and R. J. Quigg.
2002. Injury in renal ischemia-reperfusion is independent from immunoglobulins
and T lymphocytes. Am. J. Physiol. Renal Physiol. 282: F352–F357.
49. Leadbetter, E. A., I. R. Rifkin, A. M. Hohlbaum, B. C. Beaudette, M. J. Shlomchik,
and A. Marshak-Rothstein. 2002. Chromatin-IgG complexes activate B cells by
dual engagement of IgM and Toll-like receptors. Nature 416: 603–607.
50. Lau, C. M., C. Broughton, A. S. Tabor, S. Akira, R. A. Flavell, M. J. Mamula,
S. R. Christensen, M. J. Shlomchik, G. A. Viglianti, I. R. Rifkin, and