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*
Department of Pathology, University of Utah, Salt Lake City, UT 84132;
Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 61801; and
Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
provides a mechanism for the initiation and modification of
inflammatory events associated with Lyme disease (3, 9, 10, 11, 12, 13). Toll-like receptors (TLR)4 are highly conserved throughout evolution and have been implicated in the innate defense to many pathogens; Drosophila toll is required for the anti-fungal response (14), while the related 18-wheeler is involved in antibacterial defenses (15). In mammals, TLR have been implicated in both inflammatory responses and innate host defense to pathogens (16). The best example is the C3H/HeJ mouse, which possess a nonactivating mutation in the gene encoding TLR4, the inflammatory signaling receptor for enterobacterial LPS (17, 18). C3H/HeJ mice are highly resistant to LPS-induced shock while being exquisitely susceptible to the lethal consequences of infection by the LPS-bearing pathogen Salmonella typhimurium (19). Interestingly, B. burgdorferi does not produce LPS (1, 20), and TLR4 appears to play no physiological role in the host defense to B. burgdorferi: C3H/HeJ mice display identical parameters of infection and disease as congenic C3H/HeN mice that possess a wild-type TLR4, including the kinetics of bacterial dissemination, the persistence of spirochetes in tissues, and the pathological severity of arthritis and carditis (21, 22). Furthermore, macrophages from LPS-hyporesponsive C3H/HeJ mice respond strongly to B. burgdorferi lipoproteins and sonicated bacteria (5). These findings suggest that TLR2 could play a unique role in the inflammatory response and host defense to infection with B. burgdorferi, analogous to that of TLR4 during infection with enterobacterial species. This hypothesis was tested by infecting mice possessing a targeted disruption in the tlr2 gene with B. burgdorferi.
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Materials and Methods |
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C3H/HeN mice were obtained from the National Cancer Institute,
and C57BL/6J mice were purchased from The Jackson Laboratory
(Bar Harbor, ME). TLR2-deficient mice were generated by replacing a
portion of the tlr2 gene with the neomycin resistance gene,
oriented in the opposite reading frame (23). These mice do
not produce TLR2 protein, and TLR2 transcripts are larger than wild
type due to the presence of the neomycin resistance gene
(23). Cells from these mice are functionally deficient in
TLR2-mediated signaling (see Fig. 1
) (23). The
TLR2-/- mutation was on a mixed 129SV x
C57BL/6 background (see Figs. 1 and 2A) or was backcrossed
four generations onto C57BL/6J mice, and heterozygous parents were used
to generate experimental TLR2-/-,
TLR2+/-, and TLR2+/+
littermates (see Figs. 2B and 3–5 and Tables I–III). Mice
were housed in the Animal Resource Center at the University of Utah
Medical Center, according to guidelines of the National Institutes of
Health for the care and use of laboratory animals.
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Bone marrow-derived macrophages from C3H/HeN, C57BL/6, TLR2-/-, and TLR2+/+ mice were prepared as previously described (24). Briefly, bone marrow cells were cultured in RPMI supplemented with L929-conditioned medium for 7 days at 37°C. Macrophages were recovered with ice-cold PBS and replated in 12-well culture dishes at a density of 5 x 105/well in serum-free medium containing 1% Nutridoma (Roche, Indianapolis, IN). After overnight incubation at 37°C, nonadherent cells were removed, and the indicated agonists were added. Supernatants were harvested from these cultures at the designated times and either assayed immediately (IL-10 and TNF) or stored at -20°C until assayed.
Infection of mice with B. burgdorferi
TLR2-/-, TLR2+/+, and TLR2+/- littermate controls were infected by intradermal injection with 2 x 103 organisms of a passage five culture of the N40 isolate of B. burgdorferi provided by S. Barthold (University of California, Davis, CA) (25).
Measurement of ankle joints
Ankle joint measurements were made as described previously (26). Rear ankle joints were measured at the time of infection and at 2, 4, or 8 wk following infection using a metric caliper, and data were reported as an increase in swelling. Measurements were performed on TLR2-/- mice and littermate controls in a blinded fashion on the thickest portion (anterior-posterior) of the ankle, with the joint extended.
Lesion assessment of ankle joints
Histological analyses were performed on the rear ankle joint that exhibited the greatest swelling from mice sacrificed at 2, 4, or 8 wk postinfection. Joints were fixed in 10% neutral-buffered formalin, decalcified, embedded in paraffin, and sectioned at 5 µm before staining with H&E. Sections were viewed in a blinded fashion and assessed for various lesions of disease, including edema, neutrophil and/or mononuclear inflammation, tendon sheath thickness, and reactive/reparative responses. Each lesion was issued a score ranging from 0 to 5, with 5 representing the most severe lesion and 0 indicating normal tissue. Scores for individual lesions were incorporated into the overall lesion score reported in this study.
Reagents
Sonicated B. burgdorferi was prepared from 10-day cultures as previously described (27). The protein content of this preparation was determined by Bradford assay. Recombinant outer surface protein A (OspA) was provided by J. Dunn (Brookhaven National Laboratories). The recombinant OspA and sonicated B. burgdorferi contained <0.3 endotoxin units/500 ng as determined by Limulus amebocyte assay (Associates of Cape Cod, Cape Cod, MA). Ab and standards for cytokine and Ig ELISAs were obtained from PharMingen (San Diego, CA), and polymyxin B was obtained from Sigma (St. Louis, MO). LPS from Escherichia coli D31 m4 (Re) was purchased from List Biological Laboratories (Campbell, CA) and was repurified according to our previous report (28).
Isolation of DNA from infected mouse tissues
One rear ankle joint, the heart, and ear tissues were harvested from experimental animals sacrificed at 2, 4, or 8 wk postinfection, and DNA was prepared as previously described (22). Briefly, individual tissues were incubated in 0.1% collagenase at 37°C overnight before adding an equal volume of 0.2 mg/ml proteinase K (Sigma). After overnight incubation at 55°C, DNA was recovered by phenol/chloroform extraction and ethanol precipitation. Following digestion with 1 mg/ml DNase-free RNase (Sigma), samples were again extracted, and DNA was recovered by precipitation. This precipitate was resuspended in 1.5 ml water, and DNA content was determined by absorbance at 260 nm.
Quantification of B. burgdorferi by continuous monitoring of PCR
PCR analyses were performed in a fluorescence temperature cycler (LightCycler LC24, Idaho Technology, Idaho Falls, ID) as previously described (29). Briefly, amplification was performed on 200 ng sample DNA in a 10-µl final volume containing 50 mM Tris (pH 8.3), 3 mM MgCl2, 4.5 µg BSA, 200 µM dNTP, a 1/30,000 dilution of SYBR Green I (Molecular Probes, Eugene, OR), 5 µM of each primer, 0.5 U Taq polymerase (Life Technologies, Gaithersburg, MD), and 110 ng TaqStart Ab (Clontech, Palo Alto, CA). Amplification was performed at 40 cycles, with each cycle comprised of heating at 20°C/s to 95°C with a 1-s hold, cooling at 20°C/s to 60°C with a 1-s hold, and heating at 1°C/s to 84°C. This technique continuously monitors the cycle-by-cycle accumulation of fluorescently labeled product. The cycle at which the product is first detected is used as an indicator of the relative starting copy numbers present in the sample. Copy numbers for mouse nidogen and B. burgdorferi recA were calculated using the LightCycler software, and recA values were corrected by normalization based on nidogen copies. The oligonucleotide primers used to detect mouse nidogen were nido.F (5'-CCA GCC ACA GAA TAC CAT CC-3') and nido.R (5'-GGA CAT ACT CTG CTG CCA TC-3'). The oligonucleotide primers used to detect B. burgdorferi recA were nTM17.F (5'-GTG GAT CTA TTG TAT TAG ATG AGG CTC TCG-3') and nTM17.R (5'-GCC AAA GTT CTG CAA CAT TAA CAC CTA AAG-3').
Ig quantification
Serum obtained by retro-orbital bleeding of experimental animals was assayed by ELISA to determine Ig content. Microtiter plates were coated with either sonicated B. burgdorferi or goat Ab to mouse IgG, IgM, and IgA (Life Technologies). Serum dilutions were added to plates for 90 min at 37°C and bound murine Ig was detected by addition of HRP-conjugated Abs to murine IgG or IgM (Zymed, San Francisco, CA). Ig content was estimated by comparing with standard curves using purified IgG or IgM. B. burgdorferi-specific Ig subclasses were assessed by titrating serum samples on plates coated with sonicated B. burgdorferi using isotype-specific HRP-conjugated secondary Abs (Zymed).
Western blot analysis
Western blots were prepared by separating 120 µg sonicated N40 isolate of B. burgdorferi on 12% SDS-PAGE and transferring to Immobilon (Millipore, Bedford, MA). Blots were incubated with a 1/50 dilution of infected or control mouse sera. OspC and OspA were detected with a 1/500 dilution of rabbit polyclonal anti-OspC or 2 µg/ml monoclonal anti-OspA, provided by T. Schwan (Rocky Mountain Laboratories, Hamilton, MT). Bands were detected with alkaline phosphatase-labeled goat anti-mouse or goat anti-rabbit IgG (Life Technologies).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A–C), while
responding strongly to repurified LPS (28, 30). The
OspA-stimulated production of the proinflammatory products, IL-6 (Fig. 1A), NO (Fig. 1B), and TNF-
(Fig. 1D), was abolished in TLR2-deficient macrophages, as was the
production of the anti-inflammatory cytokine IL-10 (Fig. 1C). Thus, lipoprotein signaling through TLR2 is required
for both pro- and anti-inflammatory signaling in response to
B. burgdorferi lipoproteins, as was previously found for the
facilitating molecule CD14 (13, 31, 32).
Because B. burgdorferi lacks LPS but abundantly produces
lipoproteins, it has been hypothesized that the majority of cytokine
stimulatory activities of the bacteria result from the lipoproteins
(33). Macrophages from TLR2-deficient mice were used to
test for the presence of additional, TLR2-independent, stimulatory
molecules in a sonicated preparation of B. burgdorferi.
These experiments demonstrated that the major stimulatory component of
B. burgdorferi acts through TLR2, with little response from
TLR2-deficient macrophages to 0.5 or 5 µg/ml sonicate (Fig. 2A). However, 50 µg/ml of
sonicated B. burgdorferi possessed strong stimulatory
activity for macrophages from TLR2-/- mice,
although a 10-fold greater concentration of sonicate was required to
elicit IL-6 production equivalent to that of macrophages from wild-type
C3H/HeN mice (Fig. 2A). The differences in the response of
macrophages from C3H/HeN and TLR2-deficient mice (derived from
129SV x C57BL6/J) to sonicated B. burgdorferi (Fig. 2A) could have been influenced by other variations besides
the specific disruption of the tlr2 gene. Therefore,
macrophages were prepared from TLR2-/- mice and
their wild-type littermates for comparison of responses to OspA and
sonicated B. burgdorferi. Macrophages from
TLR2-/- mice did respond to sonicated B.
burgdorferi, but again required 10- to 100-fold more sonicate for
equivalent NO production as macrophages from wild-type littermates
(Fig. 2B).
The ability of sonicated B. burgdorferi to stimulate
responses in macrophages from TLR2-deficient mice is not likely to be
due to a second receptor for lipoproteins, as these macrophages failed
to respond to 5 µg/ml OspA, a 10-fold higher concentration than the
500 ng/ml found to be optimal in our laboratory (Fig. 2B).
Failure of TLR2-deficient macrophages to respond to 5 µg/ml OspA is
shown for NO, the most sensitive indicator of macrophage activation,
and similar results were obtained with TNF-, IL-6, and IL-10. The
diminished response by wild-type macrophages to 5 µg/ml OspA relative
to the more standard maximum concentration of 500 ng/ml (Fig. 2B) was demonstrated to be due to the effects of detergent
in the OspA preparation (not shown). These results suggest that the
TLR2-independent stimulatory activity in sonicated B.
burgdorferi is not mediated by lipoproteins, but, rather, involves
additional, less potent microbial products. Numerous products
associated with Gram-negative bacteria have been found to signal
through various TLR, including peptidoglycan (34),
glycolipids (35), unmethylated CpG containing DNA
(36), and flagellin (36). Interestingly, the
contribution of these nonlipoprotein bacterial compounds to B.
burgdorferi stimulation of murine macrophages was only evident in
the absence of TLR2 signaling.
The contribution of TLR2 to the development of Lyme disease was
determined by infecting TLR2-/- mice with
B. burgdorferi and assessing arthritis severity at 4 wk
postinfection, the peak of arthritis development.
TLR2-/- mice displayed a striking increase in
rear ankle swelling compared with their TLR2+/+
and TLR2+/- littermates (Table I). Mice heterozygous for the TLR2
deficiency were similar to homozygous wild-type littermates. The
increase in ankle swelling in TLR2-/- mice was
also remarkable, as both rear ankle joints displayed uniformly
increased swelling, whereas infection of arthritis-susceptible
immunocompetent mice typically results in one rear ankle joint being
more severely affected (21). Ankle swelling gives only a
partial picture of joint pathology in murine Lyme disease, providing
information primarily on the degree of edema within the ankle tissue.
At sacrifice, the most severely swollen ankle joint was taken for
histological analysis to obtain a more complete picture of joint
pathology. The C57BL/6 mouse strain typically develops mild to moderate
arthritis when infected with B. burgdorferi, with
histopathology peaking at about 4 wk following infection. We expected
that TLR2 deficiency could result in less severe arthritis, and
therefore, its effect might not be dramatic on this mouse background.
Lesions within ankle joints were evaluated for disease parameters,
including neutrophil infiltration, mononuclear cell infiltration,
sheath thickness, reactive and reparative responses, and overall lesion
severity. Joints from TLR2-/- mice received
slightly higher scores for mononuclear cell infiltration and tendon
sheath thickness than did TLR2+/+ and
TLR2+/- littermates, However, the scores for
these individual parameters and for overall lesion severity for
TLR2-/- mice were not statistically different
from those for the other two genotypes (Table I). Therefore, on this
relatively arthritis-resistant background, TLR2 deficiency did not
significantly influence parameters of lesion severity. These findings
suggest that the ankle swelling measurements were primarily monitoring
edema. The finding that ankle swelling was more dramatically influenced
by deletion of TLR2 than was histopathologically scoring of arthritic
lesions is consistent with our previous observation that these two
traits are regulated independently by genetic loci mapping to distinct
locations (37).
The impact of TLR2 on host defense to B. burgdorferi was
determined by quantification of spirochetes in DNA prepared from
tissues of infected mice 2 and 4 wk postinfection. Hearts, rear ankle
joints, and ears were analyzed, as these tissues consistently harbor
the highest concentrations of persistent bacteria. All
TLR2-/- animals possessed elevated numbers of
spirochetes in tissues at both time points tested, ranging up to
100-fold higher in individual TLR2-/- mice than
in TLR2+/+ and TLR2+/-
littermates (Fig. 3). The impairment to
host defense was greatest in rear ankle joints of mice at 2 wk
postinfection, with TLR2-/- mice harboring an
average of 40-fold more spirochetes than TLR2+/+
mice (p < 0.000001). B. burgdorferi
rapidly colonize the skin of infected mice, exhibited by increased
numbers in ear tissues from TLR2+/+ and
TLR2+/- control mice at 2 wk compared with 4 wk.
In contrast, TLR2-/- mice failed to clear the
high concentrations of spirochetes from ear tissue by 4 wk, harboring
an average of 100-fold more bacteria than littermate controls.
Spirochete levels were also much greater in hearts of mice sacrificed
at 2 wk than at 4 wk postinfection regardless of the TLR2 genotype.
However, at both time points hearts from
TLR2-/- mice displayed 
10-fold more bacteria
than the TLR2+/+ or
TLR2+/- littermate controls. To determine
whether overwhelming systemic infection was underlying the increased
presence of tissue spirochetes, the levels of B. burgdorferi
in spleens of infected animals were determined. Spleen and blood
generally harbor very low numbers of B. burgdorferi, and
blood levels are difficult to quantify by PCR. The concentration of
B. burgdorferi in the spleens of wild-type mice was
extremely low even at the 2 wk peak of tissue spirochetes, (average,
0.05 B. burgdorferi recA genes/1000 copies of the mouse gene
nidogen). Although spirochete levels in TLR2-deficient mice
(average, 0.34 B. burgdorferi recA genes/1000
nidogen genes) were greater than those in wild-type mice,
they were much lower than those found in the three other tissues
assayed at 2 wk. Thus, the previous observation that blood and
blood-filtering tissues are not major reservoirs for this pathogen
(21) is true even in TLR2-deficient mice harboring
extremely high levels of bacteria in other tissues. These quantitative
differences in spirochete levels in numerous tissues from
TLR2-/- mice and littermate controls
unequivocally point to a crucial role for TLR2 in the host defense to
B. burgdorferi.
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). Quantitative PCR
revealed little change in spirochete levels in hearts, rear ankles, and
ears by 8 wk following infection. Thus, the extremely high levels of
B. burgdorferi found in tissues from TLR2-deficient mice at
4 wk following infection persisted at 8 wk following infection. Levels
in wild-type mice were also similar at 4 and 8 wk following infection.
These findings suggest that clearance of spirochetes primarily occurs
between 2 and 4 wk, and that spirochetes in wild-type mice may persist
at a relatively constant, low level after 4 wk of infection.
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for
TLR2+/- and TLR2+/+ mice)
has been attributed to the appearance of acquired immunologic defenses,
particularly Ab directed to surface exposed proteins of B.
burgdorferi. Importantly, in mice with the scid
mutation, spirochete numbers remain elevated at later time points, and
passive transfer of Abs can reduce spirochete concentrations in
tissues, supporting the role of acquired defenses in clearing infection
and resolving disease (38, 39, 40). We hypothesized that the
persistently elevated concentrations of spirochetes in tissues from
TLR2-/- mice could result from defective
development of an Ab response. Serum was collected from infected mice
at 2, 4, and 8 wk following infection, and the amount of B.
burgdorferi-specific IgM and IgG was determined by capture ELISA.
Surprisingly, TLR2-/- mice produced similar
levels of anti-B. burgdorferi IgM at 2 wk and of IgG at
4 and 8 wk as their littermates controls (Table II). Infection of mice with B.
burgdorferi has been associated with an increase in total
circulating IgG (26). Uninfected mice possess 1–2 mg/ml
circulating IgG, while in infected mice the level of total serum IgG
increased 5- to 10-fold, but was similar for all genotypes of infected
mice at both 4 and 8 wk (Table II).
The absence of a particular subclass of Ab could result in the poor
control of spirochetes in tissues. To assess this possibility, isotype
distribution of Borrelia-specific Ab was determined in sera
collected 4 wk following infection. IgG1, IgG2b, and IgG3 isotypes are
produced by C57BL/6 mice, while IgG2a is not (41). As
shown in Table III, there was no major
shift in the distribution of IgG isotypes in mice of different
genotypes, although IgG2b was somewhat under-represented in sera from
infected TLR2-/- mice. However, the
animal-to-animal variation within groups for
Borrelia-specific IgG2b was larger than the average
differences between genotypes, resulting in a lack of statistical
significance. This strongly argues against under-representation of a
particular subclass of anti-Borrelia IgG in
TLR2-/- mice that could be responsible for the
increased numbers of spirochetes in tissues. The lack of influence on
isotype is in sharp contrast to the dramatic effect of IL-4 gene
disruption on IgG1 production in B. burgdorferi-infected
mice, a finding detected using this assay (42).
The effect of TLR2 deficiency on the spectrum of B.
burgdorferi-specific Ags recognized by the humoral immune response
was assessed by Western blot analysis, using a preparation of the N40
culture. The complexity of Ags recognized by sera from
TLR2-/- mice was similar to that from infected
TLR2+/+ mice (Fig. 5). In fact, the number of bands detected
and the intensity of staining were actually greater with sera from
TLR-deficient mice than with those from wild-type littermates at both 4
and 8 wk postinfection (Fig. 5, A and B). This is
likely to reflect the much greater levels of spirochetes, and therefore
Ags, present in numerous tissues from TLR2-deficient mice. The presence
of Abs against two well-characterized Borrelia Ags, OspA and
OspC, was also assessed by Western blot analysis. Control lanes were
developed with monoclonal or polyclonal Abs directed against OspA or
OspC, respectively, and indicated that these Ags were present in the Ag
preparation (Fig. 5). Infected mouse sera from both genotypes of mice
contained Ig that reacted with proteins migrating in the same range as
OspA and OspC. However, when the control and infected mouse sera were
used in Western blots using recombinant OspA and OspC, reactivity was
detected with the control anti-OspA or anti-OspC Abs, but not
with the infected mouse sera (not shown). Although cultured bacteria do
not represent the totality of Ags expressed by spirochetes within the
mammal, this sampling of Ags is likely to reveal major alterations in
Ags recognized by TLR2-deficient mice. The Western blot results
indicate that the complexity of the humoral response is not reduced by
deficiency in TLR2, and that gross absence of Ab directed to
immunological proteins is not responsible for the observed failure to
clear the spirochetes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 4). A fourth tissue, the spleen, was assayed at 2 wk following
infection, and TLR2-/- mice were found to
harbor 7-fold more spirochetes than wild-type littermates. These
findings present a comprehensive picture of a host severely compromised
in its ability to regulate spirochete levels in tissues.
These results provide hints about the normal mechanism of host control
of spirochetes in tissues. In the infected wild-type mice, levels of
tissue spirochetes peak at about 2 wk following infection
(43), and drop in heart and ears by 4 wk following
infection. Spirochete levels in ankles of wild-type mice remained
relatively constant at all three time points: 8, 19, and 11
spirochetes/1000 host genomes. These findings are dramatically
demonstrated by the data presented in Figs. 3 and 4. In TLR2-deficient
mice, the 2 wk levels of tissue spirochetes were even more elevated
than in wild-type mice and remained abnormally high at 4 and 8 wk
following infection. Several studies have demonstrated a critical role
of Borrelia-specific Ig in the protective host defense to
B. burgdorferi (38, 39, 40). These results are
consistent with those findings, as the anti-B.
burgdorferi humoral response at 2 wk is characterized by low level
of IgM (26). By 4 wk following infection higher
concentrations of Borrelia-specific IgG appear
(21) (Table II). This suggests that the drop in spirochete
number seen in wild-type mice between 2 and 4 wk following infection is
due to the appearance of IgG Abs isotypes.
B. burgdorferi lipoproteins have been reported to possess
potent B cell mitogenic properties capable of stimulating polyclonal
activation of proliferation and Ig production in vitro
(5). These properties as well as the adjuvant properties
of lipoprotein vaccines are dependent on the
Pam3Cys modification (44, 45). We
hypothesized that the humoral response would be altered in
TLR2-/- mice, and that this was responsible for
the failure to control tissue spirochetes. A complete analysis of the
Ab response revealed virtually no difference between
TLR2-/- mice and TLR2+/+
littermates; the kinetics of appearance of IgM and class switching to
IgG appeared to be normal, the magnitude of IgM and IgG response to
B. burgdorferi Ags was similar, the relative distribution of
IgG isotypes was the same, and the complexity of Ags recognized was
similar. In fact, the findings presented in Tables II and III and Fig. 5 fail to support any compromise in the humoral response to B.
burgdorferi in TLR2-deficient mice and indicate that the increased
number of spirochetes found in tissues of TLR2-deficient mice cannot be
attributed to a defective humoral immune response.
The finding that the host defense to B. burgdorferi in
TLR2-deficient mice is severely compromised even though
anti-spirochete IgG levels are normal argues that effective
elimination of B. burgdorferi requires not only Ab, but also
a TLR2-expressing effector cell. Phagocytic cells, such as neutrophils
and macrophages, are probably TLR2-expressing cells types that could
mediate the Ab-dependent clearance of B. burgdorferi
(46). Neutrophils have been shown to play a critical role
in spirochete clearance (47), and they may be very
important in controlling numbers of Ab-opsonized B.
burgdorferi in TLR2-deficient mice. The complete lack of in vitro
response of cells from TLR2-/- mice to purified
lipoproteins (Figs. 1 and 2) (23) suggests that
TLR2-mediated activation of inflammatory cells is critical to the
control of spirochete numbers in tissues. Interestingly, there was some
clearance from the hearts between 2 and 4 wk in
TLR2-/- mice that was not observed in ear
tissues and ankle joints. Others have suggested that the inflammatory
infiltrate in hearts of infected mice is predominated by monocytic
cells, whereas the infiltrate in joints is predominately neutrophil
(48). Thus, understanding the TLR2-dependent control of
host defense could provide insight into the tissue-specific control of
B. burgdorferi.
An unexpected finding from this study was the ability of macrophages from TLR2-deficient mice to respond to nonlipoprotein components of B. burgdorferi. The TLR2-lipoprotein interaction clearly dominates the macrophage response to this spirochete; however, the unique situation of the TLR2-/- mouse allows detection of the effects of other stimulatory molecules. Although the significance during infection of TLR2-independent signaling events during infection remains to be determined, the fact that environmental regulation of lipoprotein gene expression is certainly involved in the persistence and pathogenesis of this organism suggest potential importance during selective stages of infection. The finding that the humoral response to B. burgdorferi was normal in TLR2-deficient mice argues that TLR2-independent signaling supports B cell activation in mice. This was surprising due to the complete dependence of the OspA vaccine on the Pam3Cys modification (44). Whether the humoral response requires signals from other TLR or reflects Ag uptake and processing pathways distinct from TLR cannot be determined from these studies. The possible involvement of other TLR is suggested by the wide variety of microbial products that are able to mediate macrophage activation. Several of these signal through TLR2 (12) and therefore should not contribute to the TLR2-independent activation. Some of these may signal through TLR4, such as cellular glycolipids (35), although the in vivo significance of TLR4 signaling is questionable, as C3H/HeJ mice develop disease that is indistinguishable from that in C3H/HeN mice (21, 22). One candidate for nonlipoprotein stimulatory activity in sonicated B. burgdorferi is unmethylated CpG DNA, which has recently been shown to activate murine cells through TLR9 (36). Additionally, flagella from two diverse bacteria, Listeria monoctyogenes and S. typhimurium, have recently been found to signal through TLR5, suggesting that Borrelia flagellin could also contribute to the TLR2-independent response (49). The identification of spirochetal products responsible for TLR2-independent signaling will require further studies, as will determination of the relative physiological importance of such host-pathogen interactions. The complexity of TLR involvement has been recently observed in the macrophage response to Staphylococcus aureus, suggesting that the integration of multiple signaling receptors may be a common theme in bacterial pathogenesis (50).
Although TLR2-independent responses require significantly higher concentrations of bacteria than do TLR2-dependent responses in cell cultures, these stimuli could be important in vivo, where localized concentrations of bacteria can be relatively high. For example, ankle joints from TLR2-/- mice not only harbor an average of 10-fold more spirochetes than joints from wild-type mice, but also display increased ankle swelling relative to littermate controls. This finding clearly demonstrates that a pattern recognition receptor independent of TLR2 can contribute to B. burgdorferi-induced lesions in vivo. A more complete picture of the involvement of TLR2 in inflammatory arthritis development will require studies in which the TLR2 deficiency has been crossed onto a mouse strain that develops more severe Lyme arthritis.
The results presented in this study give a clear picture of TLR2 involvement in control of spirochete levels in tissues, but not in the development of the acquired humoral response. This suggests that the lipoprotein-TLR2 interaction plays a crucial role in activating cells of the innate defense for infiltration, phagocytosis, or killing of spirochetes, and that spirochete ligands for additional TLRs cannot compensate for this interaction. In contrast, the Ab response appeared normal, suggesting that TLR2-independent activation of B lymphocytes functions efficiently in the absence of TLR2. It has been reported with human B lymphocytes that TLR2 is expressed by activated B lymphocytes within germinal centers, but is not expressed by resting B lymphocytes (46, 51). It has also been reported that accessory cells are required for lipoprotein-mediated polyclonal activation of B lymphocytes (52). These results suggests that B cell activation by lipoproteins may be subsequent to activation of accessory cells, which may express numerous TLR. Our results demonstrate that innate defenses to B. burgdorferi are more severely compromised by TLR2 deficiency than are acquired defenses and suggest a new paradigm for host-pathogen interactions.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Medical College of Ohio, Health Education Building, Room 254, 3055 Arlington Avenue, Toledo, OH 43614-5806.
3 Address correspondence and reprint requests to Dr. Janis J. Weis, Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, UT 84132. E-mail address: janis.weis{at}path.utah.edu
4 Abbreviations used in this paper: TLR, toll-like receptor; OspA, outer surface protein A.
Received for publication September 5, 2001. Accepted for publication October 22, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and inflammatory activation in human endothelial cells. J. Immunol. 157:4584.[Abstract]
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  M. M. Petzke, A. Brooks, M. A. Krupna, D. Mordue, and I. Schwartz Recognition of Borrelia burgdorferi, the Lyme Disease Spirochete, by TLR7 and TLR9 Induces a Type I IFN Response by Human Immune Cells J. Immunol., October 15, 2009; 183(8): 5279 - 5292. [Abstract] [Full Text] [PDF]
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 C. L. Koening, J. C. Miller, J. M. Nelson, D. M. Ward, J. P. Kushner, L. K. Bockenstedt, J. J. Weis, J. Kaplan, and I. De Domenico Toll-like receptors mediate induction of hepcidin in mice infected with Borrelia burgdorferi Blood, August 27, 2009; 114(9): 1913 - 1918. [Abstract] [Full Text] [PDF]
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 N. Liu, A. A. Belperron, C. J. Booth, and L. K. Bockenstedt The Caspase 1 Inflammasome Is Not Required for Control of Murine Lyme Borreliosis Infect. Immun., August 1, 2009; 77(8): 3320 - 3327. [Abstract] [Full Text] [PDF]
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F. Han, H. Yu, C. Tian, S. Li, M. R. Jacobs, C. Benedict-Alderfer, and Q. Y. Zheng Role for Toll-Like Receptor 2 in the Immune Response to Streptococcus pneumoniae Infection in Mouse Otitis Media Infect. Immun., July 1, 2009; 77(7): 3100 - 3108. [Abstract] [Full Text] [PDF]
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M. Puliti, S. Uematsu, S. Akira, F. Bistoni, and L. Tissi Toll-Like Receptor 2 Deficiency Is Associated with Enhanced Severity of Group B Streptococcal Disease Infect. Immun., April 1, 2009; 77(4): 1524 - 1531. [Abstract] [Full Text] [PDF]
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S. Malkiel, C. J. Kuhlow, P. Mena, and J. L. Benach The Loss and Gain of Marginal Zone and Peritoneal B Cells Is Different in Response to Relapsing Fever and Lyme Disease Borrelia J. Immunol., January 1, 2009; 182(1): 498 - 506. [Abstract] [Full Text] [PDF]
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J. C. Miller, Y. Ma, J. Bian, K. C. F. Sheehan, J. F. Zachary, J. H. Weis, R. D. Schreiber, and J. J. Weis A Critical Role for Type I IFN in Arthritis Development following Borrelia burgdorferi Infection of Mice J. Immunol., December 15, 2008; 181(12): 8492 - 8503. [Abstract] [Full Text] [PDF]
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O. S. Shin, R. R. Isberg, S. Akira, S. Uematsu, A. K. Behera, and L. T. Hu Distinct Roles for MyD88 and Toll-Like Receptors 2, 5, and 9 in Phagocytosis of Borrelia burgdorferi and Cytokine Induction Infect. Immun., June 1, 2008; 76(6): 2341 - 2351. [Abstract] [Full Text] [PDF]
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A. Woods, P. Soulas-Sprauel, B. Jaulhac, B. Arditi, A.-M. Knapp, J.-L. Pasquali, A.-S. Korganow, and T. Martin MyD88 Negatively Controls Hypergammaglobulinemia with Autoantibody Production during Bacterial Infection Infect. Immun., April 1, 2008; 76(4): 1657 - 1667. [Abstract] [Full Text] [PDF]
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J. J. Lazarus, M. A. Kay, A. L. McCarter, and R. M. Wooten Viable Borrelia burgdorferi Enhances Interleukin-10 Production and Suppresses Activation of Murine Macrophages Infect. Immun., March 1, 2008; 76(3): 1153 - 1162. [Abstract] [Full Text] [PDF]
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A. R. Cruz, M. W. Moore, C. J. La Vake, C. H. Eggers, J. C. Salazar, and J. D. Radolf Phagocytosis of Borrelia burgdorferi, the Lyme Disease Spirochete, Potentiates Innate Immune Activation and Induces Apoptosis in Human Monocytes Infect. Immun., January 1, 2008; 76(1): 56 - 70. [Abstract] [Full Text] [PDF]
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 D. T. Nardelli, S. M. Callister, and R. F. Schell Lyme Arthritis: Current Concepts and a Change in Paradigm Clin. Vaccine Immunol., January 1, 2008; 15(1): 21 - 34. [Full Text] [PDF]
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H. Izadi, A. T. Motameni, T. C. Bates, E. R. Olivera, V. Villar-Suarez, I. Joshi, R. Garg, B. A. Osborne, R. J. Davis, M. Rincon, et al. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages Infect. Immun., October 1, 2007; 75(10): 5027 - 5034. [Abstract] [Full Text] [PDF]
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T. A. Rupprecht, C. J. Kirschning, B. Popp, S. Kastenbauer, V. Fingerle, H.-W. Pfister, and U. Koedel Borrelia garinii Induces CXCL13 Production in Human Monocytes through Toll-Like Receptor 2 Infect. Immun., September 1, 2007; 75(9): 4351 - 4356. [Abstract] [Full Text] [PDF]
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M. E. Woodman, A. E. Cooley, J. C. Miller, J. J. Lazarus, K. Tucker, T. Bykowski, M. Botto, J. Hellwage, R. M. Wooten, and B. Stevenson Borrelia burgdorferi Binding of Host Complement Regulator Factor H Is Not Required for Efficient Mammalian Infection Infect. Immun., June 1, 2007; 75(6): 3131 - 3139. [Abstract] [Full Text] [PDF]
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O. Hoffmann, J. S. Braun, D. Becker, A. Halle, D. Freyer, E. Dagand, S. Lehnardt, and J. R. Weber TLR2 Mediates Neuroinflammation and Neuronal Damage J. Immunol., May 15, 2007; 178(10): 6476 - 6481. [Abstract] [Full Text] [PDF]
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S. Liang, M. Wang, K. Triantafilou, M. Triantafilou, H. F. Nawar, M. W. Russell, T. D. Connell, and G. Hajishengallis The A Subunit of Type IIb Enterotoxin (LT-IIb) Suppresses the Proinflammatory Potential of the B Subunit and Its Ability to Recruit and Interact with TLR2 J. Immunol., April 15, 2007; 178(8): 4811 - 4819. [Abstract] [Full Text] [PDF]
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K. R. Alugupalli, S. Akira, E. Lien, and J. M. Leong MyD88- and Bruton's Tyrosine Kinase-Mediated Signals Are Essential for T Cell-Independent Pathogen-Specific IgM Responses J. Immunol., March 15, 2007; 178(6): 3740 - 3749. [Abstract] [Full Text] [PDF]
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L. Krishnan, K. Gurnani, C. J. Dicaire, H. van Faassen, A. Zafer, C. J. Kirschning, S. Sad, and G. D. Sprott Rapid Clonal Expansion and Prolonged Maintenance of Memory CD8+ T Cells of the Effector (CD44highCD62Llow) and Central (CD44highCD62Lhigh) Phenotype by an Archaeosome Adjuvant Independent of TLR2 J. Immunol., February 15, 2007; 178(4): 2396 - 2406. [Abstract] [Full Text] [PDF]
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C. M. Olson, M. N. Hedrick, H. Izadi, T. C. Bates, E. R. Olivera, and J. Anguita p38 Mitogen-Activated Protein Kinase Controls NF-{kappa}B Transcriptional Activation and Tumor Necrosis Factor Alpha Production through RelA Phosphorylation Mediated by Mitogen- and Stress-Activated Protein Kinase 1 in Response to Borrelia burgdorferi Antigens Infect. Immun., January 1, 2007; 75(1): 270 - 277. [Abstract] [Full Text] [PDF]
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 M. Chieppa, M. Rescigno, A. Y.C. Huang, and R. N. Germain Dynamic imaging of dendritic cell extension into the small bowel lumen in response to epithelial cell TLR engagement J. Exp. Med., December 25, 2006; 203(13): 2841 - 2852. [Abstract] [Full Text] [PDF]
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D. D. Bolz, R. S. Sundsbak, Y. Ma, S. Akira, J. H. Weis, T. G. Schwan, and J. J. Weis Dual Role of MyD88 in Rapid Clearance of Relapsing Fever Borrelia spp. Infect. Immun., December 1, 2006; 74(12): 6750 - 6760. [Abstract] [Full Text] [PDF]
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H. Crandall, D. M. Dunn, Y. Ma, R. M. Wooten, J. F. Zachary, J. H. Weis, R. B. Weiss, and J. J. Weis Gene Expression Profiling Reveals Unique Pathways Associated with Differential Severity of Lyme Arthritis J. Immunol., December 1, 2006; 177(11): 7930 - 7942. [Abstract] [Full Text] [PDF]
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J. J. Lazarus, M. J. Meadows, R. E. Lintner, and R. M. Wooten IL-10 Deficiency Promotes Increased Borrelia burgdorferi Clearance Predominantly through Enhanced Innate Immune Responses J. Immunol., November 15, 2006; 177(10): 7076 - 7085. [Abstract] [Full Text] [PDF]
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 L. J. GLICKSTEIN and J. L. COBURN ASSOCIATION OF MACROPHAGE INFLAMMATORY RESPONSE AND CELL DEATH AFTER IN VITRO BORRELIA BURGDORFERI INFECTION WITH ARTHRITIS RESISTANCE Am J Trop Med Hyg, November 1, 2006; 75(5): 964 - 967. [Abstract] [Full Text] [PDF]
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Q. Xu, S. V. Seemanapalli, K. McShan, and F. T. Liang Constitutive Expression of Outer Surface Protein C Diminishes the Ability of Borrelia burgdorferi To Evade Specific Humoral Immunity Infect. Immun., September 1, 2006; 74(9): 5177 - 5184. [Abstract] [Full Text] [PDF]
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P. E. Stewart, X. Wang, D. M. Bueschel, D. R. Clifton, D. Grimm, K. Tilly, J. A. Carroll, J. J. Weis, and P. A. Rosa Delineating the Requirement for the Borrelia burgdorferi Virulence Factor OspC in the Mammalian Host. Infect. Immun., June 1, 2006; 74(6): 3547 - 3553. [Abstract] [Full Text] [PDF]
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 J. Sodek, A. P. Batista Da Silva, and R. Zohar Osteopontin and Mucosal Protection Journal of Dental Research, May 1, 2006; 85(5): 404 - 415. [Abstract] [Full Text] [PDF]
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A. K. Behera, E. Hildebrand, R. T. Bronson, G. Perides, S. Uematsu, S. Akira, and L. T. Hu MyD88 Deficiency Results in Tissue-Specific Changes in Cytokine Induction and Inflammation in Interleukin-18-Independent Mice Infected with Borrelia burgdorferi Infect. Immun., March 1, 2006; 74(3): 1462 - 1470. [Abstract] [Full Text] [PDF]
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 G. Hajishengallis, P. Ratti, and E. Harokopakis Peptide Mapping of Bacterial Fimbrial Epitopes Interacting with Pattern Recognition Receptors J. Biol. Chem., November 25, 2005; 280(47): 38902 - 38913. [Abstract] [Full Text] [PDF]
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Q. Xu, S. V. Seemanapalli, L. Lomax, K. McShan, X. Li, E. Fikrig, and F. T. Liang Association of Linear Plasmid 28-1 with an Arthritic Phenotype of Borrelia burgdorferi Infect. Immun., November 1, 2005; 73(11): 7208 - 7215. [Abstract] [Full Text] [PDF]
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A. M. C. van Rossum, E. S. Lysenko, and J. N. Weiser Host and Bacterial Factors Contributing to the Clearance of Colonization by Streptococcus pneumoniae in a Murine Model Infect. Immun., November 1, 2005; 73(11): 7718 - 7726. [Abstract] [Full Text] [PDF]
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N. W. J. Schroder, I. Diterich, A. Zinke, J. Eckert, C. Draing, V. v. Baehr, D. Hassler, S. Priem, K. Hahn, K. S. Michelsen, et al. Heterozygous Arg753Gln Polymorphism of Human TLR-2 Impairs Immune Activation by Borrelia burgdorferi and Protects from Late Stage Lyme Disease J. Immunol., August 15, 2005; 175(4): 2534 - 2540. [Abstract] [Full Text] [PDF]
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J. C. Salazar, C. D. Pope, M. W. Moore, J. Pope, T. G. Kiely, and J. D. Radolf Lipoprotein-Dependent and -Independent Immune Responses to Spirochetal Infection Clin. Vaccine Immunol., August 1, 2005; 12(8): 949 - 958. [Abstract] [Full Text] [PDF]
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 K. Craig-Mylius, G. F. Weber, J. Coburn, and L. Glickstein Borrelia burgdorferi, an extracellular pathogen, circumvents osteopontin in inducing an inflammatory cytokine response J. Leukoc. Biol., May 1, 2005; 77(5): 710 - 718. [Abstract] [Full Text] [PDF]
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M. R.-E.-I. Benhnia, D. Wroblewski, M. N. Akhtar, R. A. Patel, W. Lavezzi, S. C. Gangloff, S. M. Goyert, M. J. Caimano, J. D. Radolf, and T. J. Sellati Signaling through CD14 Attenuates the Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease J. Immunol., February 1, 2005; 174(3): 1539 - 1548. [Abstract] [Full Text] [PDF]
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A. Q. Khan, Q. Chen, Z.-Q. Wu, J. C. Paton, and C. M. Snapper Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2 Infect. Immun., January 1, 2005; 73(1): 298 - 307. [Abstract] [Full Text] [PDF]
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X. Wang, Y. Ma, J. H. Weis, J. F. Zachary, C. J. Kirschning, and J. J. Weis Relative Contributions of Innate and Acquired Host Responses to Bacterial Control and Arthritis Development in Lyme Disease Infect. Immun., January 1, 2005; 73(1): 657 - 660. [Abstract] [Full Text] [PDF]
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M. B. Lawrenz, R. M. Wooten, and S. J. Norris Effects of vlsE Complementation on the Infectivity of Borrelia burgdorferi Lacking the Linear Plasmid lp28-1 Infect. Immun., November 1, 2004; 72(11): 6577 - 6585. [Abstract] [Full Text] [PDF]
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F. T. Liang, J. Yan, M. L. Mbow, S. L. Sviat, R. D. Gilmore, M. Mamula, and E. Fikrig Borrelia burgdorferi Changes Its Surface Antigenic Expression in Response to Host Immune Responses Infect. Immun., October 1, 2004; 72(10): 5759 - 5767. [Abstract] [Full Text] [PDF]
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M. C. Dolan, J. Piesman, B. S. Schneider, M. Schriefer, K. Brandt, and N. S. Zeidner Comparison of Disseminated and Nondisseminated Strains of Borrelia burgdorferi Sensu Stricto in Mice Naturally Infected by Tick Bite Infect. Immun., September 1, 2004; 72(9): 5262 - 5266. [Abstract] [Full Text] [PDF]
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 F. T. Liang, E. L. Brown, T. Wang, R. V. Iozzo, and E. Fikrig Protective Niche for Borrelia burgdorferi to Evade Humoral Immunity Am. J. Pathol., September 1, 2004; 165(3): 977 - 985. [Abstract] [Full Text] [PDF]
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D. D. Bolz, R. S. Sundsbak, Y. Ma, S. Akira, C. J. Kirschning, J. F. Zachary, J. H. Weis, and J. J. Weis MyD88 Plays a Unique Role in Host Defense but Not Arthritis Development in Lyme Disease J. Immunol., August 1, 2004; 173(3): 2003 - 2010. [Abstract] [Full Text] [PDF]
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N. Liu, R. R. Montgomery, S. W. Barthold, and L. K. Bockenstedt Myeloid Differentiation Antigen 88 Deficiency Impairs Pathogen Clearance but Does Not Alter Inflammation in Borrelia burgdorferi-Infected Mice Infect. Immun., June 1, 2004; 72(6): 3195 - 3203. [Abstract] [Full Text] [PDF]
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 D. Grimm, K. Tilly, R. Byram, P. E. Stewart, J. G. Krum, D. M. Bueschel, T. G. Schwan, P. F. Policastro, A. F. Elias, and P. A. Rosa Outer-surface protein C of the Lyme disease spirochete: A protein induced in ticks for infection of mammals PNAS, March 2, 2004; 101(9): 3142 - 3147. [Abstract] [Full Text] [PDF]
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M. B. Drennan, D. Nicolle, V. J. F. Quesniaux, M. Jacobs, N. Allie, J. Mpagi, C. Fremond, H. Wagner, C. Kirschning, and B. Ryffel Toll-Like Receptor 2-Deficient Mice Succumb to Mycobacterium tuberculosis Infection Am. J. Pathol., January 1, 2004; 164(1): 49 - 57. [Abstract] [Full Text] [PDF]
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L. Glickstein, B. Moore, T. Bledsoe, N. Damle, V. Sikand, and A. C. Steere Inflammatory Cytokine Production Predominates in Early Lyme Disease in Patients with Erythema Migrans Infect. Immun., October 1, 2003; 71(10): 6051 - 6053. [Abstract] [Full Text] [PDF]
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S. S. Way, T. R. Kollmann, A. M. Hajjar, and C. B. Wilson Cutting Edge: Protective Cell-Mediated Immunity to Listeria monocytogenes in the Absence of Myeloid Differentiation Factor 88 J. Immunol., July 15, 2003; 171(2): 533 - 537. [Abstract] [Full Text] [PDF]
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A. Yoder, X. Wang, Y. Ma, M. T. Philipp, M. Heilbrun, J. H. Weis, C. J. Kirschning, R. M. Wooten, and J. J. Weis Tripalmitoyl-S-Glyceryl-Cysteine-Dependent OspA Vaccination of Toll-Like Receptor 2-Deficient Mice Results in Effective Protection from Borrelia burgdorferi Challenge Infect. Immun., July 1, 2003; 71(7): 3894 - 3900. [Abstract] [Full Text] [PDF]
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I. Diterich, C. Rauter, C. J. Kirschning, and T. Hartung Borrelia burgdorferi-Induced Tolerance as a Model of Persistence via Immunosuppression Infect. Immun., July 1, 2003; 71(7): 3979 - 3987. [Abstract] [Full Text] [PDF]
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R. B. Oliveira, M. T. Ochoa, P. A. Sieling, T. H. Rea, A. Rambukkana, E. N. Sarno, and R. L. Modlin Expression of Toll-Like Receptor 2 on Human Schwann Cells: a Mechanism of Nerve Damage in Leprosy Infect. Immun., March 1, 2003; 71(3): 1427 - 1433. [Abstract] [Full Text] [PDF]
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 R. Girard, T. Pedron, S. Uematsu, V. Balloy, M. Chignard, S. Akira, and R. Chaby Lipopolysaccharides from Legionella and Rhizobium stimulate mouse bone marrow granulocytes via Toll-like receptor 2 J. Cell Sci., January 15, 2003; 116(2): 293 - 302. [Abstract] [Full Text] [PDF]
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U. Koedel, B. Angele, T. Rupprecht, H. Wagner, A. Roggenkamp, H.-W. Pfister, and C. J. Kirschning Toll-Like Receptor 2 Participates in Mediation of Immune Response in Experimental Pneumococcal Meningitis J. Immunol., January 1, 2003; 170(1): 438 - 444. [Abstract] [Full Text] [PDF]
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A. Sing, D. Rost, N. Tvardovskaia, A. Roggenkamp, A. Wiedemann, C. J. Kirschning, M. Aepfelbacher, and J. Heesemann Yersinia V-Antigen Exploits Toll-like Receptor 2 and CD14 for Interleukin 10-mediated Immunosuppression J. Exp. Med., October 21, 2002; 196(8): 1017 - 1024. [Abstract] [Full Text] [PDF]
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B. T. Edelson and E. R. Unanue MyD88-Dependent but Toll-Like Receptor 2-Independent Innate Immunity to Listeria: No Role for Either in Macrophage Listericidal Activity J. Immunol., October 1, 2002; 169(7): 3869 - 3875. [Abstract] [Full Text] [PDF]
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H. Weighardt, S. Kaiser-Moore, R. M. Vabulas, C. J. Kirschning, H. Wagner, and B. Holzmann Cutting Edge: Myeloid Differentiation Factor 88 Deficiency Improves Resistance Against Sepsis Caused by Polymicrobial Infection J. Immunol., September 15, 2002; 169(6): 2823 - 2827. [Abstract] [Full Text] [PDF]
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 K. Bieback, E. Lien, I. M. Klagge, E. Avota, J. Schneider-Schaulies, W. P. Duprex, H. Wagner, C. J. Kirschning, V. ter Meulen, and S. Schneider-Schaulies Hemagglutinin Protein of Wild-Type Measles Virus Activates Toll-Like Receptor 2 Signaling J. Virol., July 29, 2002; 76(17): 8729 - 8736. [Abstract] [Full Text] [PDF]
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J. Anguita, S. W. Barthold, R. Persinski, M. N. Hedrick, C. A. Huy, R. J. Davis, R. A. Flavell, and E. Fikrig Murine Lyme Arthritis Development Mediated by p38 Mitogen-Activated Protein Kinase Activity J. Immunol., June 15, 2002; 168(12): 6352 - 6357. [Abstract] [Full Text] [PDF]
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