1
CD14 MODULATES PI3K/AKT/p38-MAPK "LICENSING" OF NEGATIVE REGULATORS OF TLR
1
SIGNALING TO RESTRAIN CHRONIC INFLAMMATION
2
3
Bikash Sahay
1
, Rebeca L. Patsey
1
, Nicole Whatley
1
, Sasmita Nayak
1,2
, Christian H. Eggers
3,4
,
4
Justin D. Radolf
5,6
, and Timothy J. Sellati
1*
5
6
1
Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208
7
2
Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180
8
3
Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030
9
4
Present address: Department of Biomedical Sciences, Quinnipiac
University, Hamden, CT 06518
10
5
Departments of Medicine and
6
Genetics and Developmental Biology, University of Connecticut Health Center,
11
Farmington, CT 06030
12
13
Correspondence should be addressed to Dr. Timothy J. Sellati, Center for Immunology and Microbial Disease,
14
Albany Medical College, 47 New Scotland Avenue, MC151, ME205B, Albany, NY 12208-3479. Telephone:
15
16
17
Nonstandard abbreviations used: MEMs, microbe expressed structural molecules; MOI, multiplicity of
18
infection; p.i., post-infection; qRT-PCR, quantitative real-time PCR.
19
20
Key words: Bacterial, Monocytes/macrophages, Cell Surface Molecules, Signal Transduction
21
2
ABSTRACT
1
Current thinking emphasizes the primacy of CD14 in facilitating TLR recognition of microbes to initiate
2
proinflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein,
3
this paradigm is challenged by demonstrating that recognition of Borrelia burgdorferi not only triggers an
4
inflammatory response in the absence of CD14, but one that is uncontrolled as a consequence of impaired
5
PI3K/AKT/p38-MAPK signaling and negative regulation of TLR2. CD14 deficiency results in
6
hyperphosphorylation of AKT and reduced activation of p38. Such aberrant signaling leads to decreased
7
negative regulation by SOCS1, SOCS3, and CIS thereby engendering a more severe and persistent
8
inflammatory response to B. burgdorferi. Perturbation of this CD14/p38-MAPK-dependent mechanism of
9
immune regulation may underlie development of infectious chronic inflammatory syndromes.
10
3
Toll-like receptor (TLR) signaling orchestrates innate response to the microbe-expressed molecular
1
structures (MEMS) associated with pathogens. The principal proinflammatory MEMS of the human spirochetal
2
pathogen Borrelia burgdorferi, the causative agent of Lyme disease, are triacylated lipoproteins recognized by
3
heterodimers of TLR2 and TLR1
1
. Such recognition by TLR2 activates the NF-B, MAPK, and PI3K/AKT
4
pathways which coordinately regulate inflammation-associated gene activities responsible for host defense
2
.
5
Although many aspects of Lyme disease pathogenesis remain ill-defined, it generally is accepted that clinical
6
manifestations result primarily, perhaps entirely, from the host's local immune response to spirochetes
3
.
7
CD14, a GPI-anchored protein expressed by macrophages (M) and neutrophils, facilitates TLR-
8
dependent proinflammatory cytokine production. Mice deficient for CD14 and their M exhibit
9
hyporesponsiveness when exposed to MEMS in the form of a bacterial lysate or purified agonists such as LPS,
10
lipoproteins, and their synthetic analogs
4-6
. This hyporesponsiveness has been attributed to (i) the lower affinity
11
of non-CD14-complexed LPS for TLR4
7
, (ii) the requirement for CD14 in MyD88-independent signaling
8
,
12
and/or (iii) the inability of p38, a member of the serine/threonine MAPK family, to be induced in the absence of
13
CD14
9
. Reviewing the literature one might conclude that CD14 is indispensable for elaboration of an
14
inflammatory response to its cognate MEMs
4-6
. Surprisingly, however, both in vitro and in vivo recognition of
15
several pathogens (e.g., Borrelia burgdorferi, Staphylococcus aureus, Salmonella typhimurium, and
16
Streptococcus or Klebsiella pneumoniae) in the absence of CD14 leads to exaggerated proinflammatory
17
cytokine production and worsening disease
10-13
.
18
Following exposure of host cells to pathogens or their isolated constituents, p38 is activated through
19
phosphorylation
14
. The action of p38 drives maturation of the phagosome following microbial uptake
15
,
20
activates downstream kinases that result in the translocation of NF-B
16
, stabilizes mRNA encoding
21
cytokines
17
, and activates genes encoding suppressors of cytokine signaling 3 (SOCS3) which negatively
22
regulates pathogen-induced inflammation
18
. Because the pleiotropic action of p38 is thought to augment
23
inflammation, the pharmaceutical industry has actively pursued development of potent and specific p38
24
inhibitors for the treatment of various inflammatory disorders
14
. However, it has been reported that inhibition
25
of p38 both in vitro and in vivo results in higher cytokine production and more severe disease in a mouse model
26
4
of pneumococcal pneumonia and tuberculosis
19
. More recently, inhibition of p38 has been linked with
1
sustained expression of TNF receptor-1, which perpetuates TNF- induced NF-B signaling
20
.
2
Herein we advance a mechanistic explanation for these dichotomous findings which distinguishes
3
CD14-dependent from -independent signaling and recognition of live B. burgdorferi versus lysed spirochetes.
4
Using a murine model of Lyme borreliosis we show that CD14 deficiency results in (i) dysregulation of the
5
PI3K/AKT/p38-MAPK pathway, (ii) loss of negative regulation of TLR2 signaling, (iii) increased cytokine
6
production, and (iv) inefficient clearance of bacteria by M. In vitro studies of cytokine response of CD14
+/+
7
and CD14
-/-
cells to live spirochetes reveal that inhibition of p38 augments the TNF- response, and ablates the
8
response to lysed organisms. Collectively, our results support the rather provocative notion that CD14 signaling
9
is essential for prolonged p38 activation and thus clearance of bacteria as well as resolution of inflammation.
10
Importantly therapeutic benefit derived from p38 inhibition in certain models of inflammation (e.g., rheumatoid
11
arthritis)
14
may potentiate inflammatory processes under other circumstances (e.g. Lyme arthritis).
12
5
RESULTS
1
CD14 deficiency augments TLR-dependent gene activity. B. burgdorferi-induced activation of CD14
-/-
M
2
results in greater transcription, persistent surface expression of TLR2, and a concomitant increase in
3
proinflammatory cytokine production, particularly TNF-, compared to CD14
+/+
cells
12
. To more broadly
4
evaluate the impact of CD14 deficiency on the inflammatory transcriptome of M, we measured transcription
5
of 84 genes associated with TLR signaling. Compared to cells expressing CD14, greater transcription of
6
interleukins (i.e., IL-1, IL-1, IL-2, IL-6, IL-12, IFN-), chemokines (i.e., CCL-2), growth factors (i.e., G-
7
CSF), and proinflammatory lipid mediators (i.e., COX2) was observed in CD14
-/-
M at 24 h p.i. (Fig 1a).
8
TLR2 transcript levels, but not those of other TLRs, were elevated 50-fold in CD14
-/-
M compared to wild-
9
type cells. Also, much of the dysregulated gene activity in the former cells was associated with the NF-B
10
signaling pathway. In CD14
+/+
M gene induction peaked at 3 h and returned to baseline by 24 h p.i; in
11
contrast, general gene activity was greater in CD14
-/-
cells at 3 h and remained elevated throughout the course of
12
the experiment (data not shown) suggesting a critical role for CD14 in downmodulation of inflammatory
13
signaling.
14
15
B. burgdorferi fails to trigger the expression of negative regulators of TLR signaling in the absence of
16
CD14. Negative regulators of TLR signaling include soluble (s) molecules (e.g., sMyD88 and sTLRs),
17
competitive inhibitors (e.g., IRAKM), protein phosphatases [e.g., MAPK phosphatase 1, (MKP1)], E3 ligases
18
(e.g., SOCSs), etc.
21
. The above findings prompted an evaluation of the impact of CD14 deficiency on several
19
of these negative regulators. B. burgdorferi induced significantly higher transcription of socs1 (~3.5-fold) in
20
CD14
+/+
M than in their CD14
-/-
counterparts by 6 h p.i. (P < 0.05) (Fig. 1b). Similarly, increased expression
21
of SOCS-1 protein was observed in CD14
+/+
M, whereas no expression was detected in their CD14-deficient
22
counterparts (Fig. 1c). Socs3 transcription also was greater in CD14
+/+
M compared to CD14
-/-
cells (~3.5-
23
fold at three h and ~2.5-fold at six h) (P < 0.01) (Fig. 1b). Owing to rapid proteosome-mediated degradation of
24
SOCS3, this molecule only can be detected by Western blot analysis with addition of the proteosome inhibitor,
25
6
MG132 (10 M), for one h prior to collection of M co-incubated with B. burgdorferi for the specified time
1
periods. Under these conditions, SOCS3 expression peaked in CD14
+/+
cells at six h whereas no expression was
2
detected in M lacking CD14 (Fig. 1c). CD14
+/+
cells also exhibited cis gene activity ~5.6-fold above that seen
3
in CD14
-/-
M (P > 0.01) (Fig. 1b). At its peak, cis transcript levels in wild-type cells were ~400-fold over
4
mock controls. By 24 h p.i. transcription of these negative regulators in CD14
+/+
and CD14
-/-
M was
5
indistinguishable and transcription of mkp1 and irakm was not significantly different in these cells at any time
6
point studied (Supplementary Fig 1, online). Thus negative regulation of TLR signaling depends, at least in
7
part, on CD14.
8
9
CD14 signaling regulates the activation state of IRF and STAT molecules. To elucidate why transcription
10
of socs1 and socs3 is diminished in CD14
-/-
cells, we examined the expression of three transcription factors
11
known to either directly or indirectly regulate their transcription
22, 23
. B. burgdorferi induced ~13-fold higher
12
transcription of irf1 in CD14
+/+
M than in unstimulated controls (P < 0.05) (Fig. 2a). Transcription of both
13
irf7 and stat1 was significantly higher in CD14
-/-
M than in wild-type cells at 24 h (Fig 2a), whereas no
14
differences were seen for irf3, stat3, or stat4 (Supplementary Fig 1, online). Additionally, Western blot
15
analysis revealed that phospho-STAT1, but not phospho-STAT3, levels were higher in CD14
+/+
than in CD14
-/-
16
M (Fig. 2b). Diminished SOCS expression in CD14
-/-
M is consistent with the observed reduction in
17
transcriptional activation of irf1 and STAT1 phosphorylation.
18
19
The inflammatory response to B. burgdorferi is perpetuated in the absence of CD14. The mouse model of
20
Lyme borreliosis was used to elucidate the relationship between CD14 signaling, SOCS activity, and disease
21
progression. Arthritis, an inflammatory hallmark of Lyme disease, is a self-limiting process in mice that peaks
22
between 2 and 3 weeks p.i. and typically resolves by 6 weeks
24
. As previously reported for tick-mediated
23
infection
12
, syringe inoculation of mice with B. burgdorferi resulted in tibiotarsal joint swelling (reflective of
24
synovitis/arthritis with periarticular edema) in CD14
+/+
and CD14
-/-
mice which peaked at 2 weeks p.i..
25
Contrary to CD14
-/-
joints, joint inflammation in the wild-type mice resolved by 3 weeks p.i. (Fig. 3a).
26
7
Beginning at 2 weeks p.i. and continuing for the duration of the experiment, the joints of CD14
-/-
mice were
1
significantly more inflamed (P >0.001). Consistent with more severe and prolonged joint inflammation in
2
CD14
-/-
mice (Fig. 3a), transcription of socs1 and socs3 in joint tissue was greatly reduced compared to wild-
3
type mice (Fig. 3b), as was the transcription of cis and irakM (data not shown).
4
One might predict a superior capacity to clear spirochetes in B. burgdorferi-infected CD14
-/-
mice by
5
virtue of greater cytokine production (e.g., TNF- and IFN-) (Fig 7 and ref 12). Surprisingly, all organs
6
recovered from CD14
-/-
mice, with the exception of the heart, had heavier bacterial burdens than those of wild-
7
type animals (Fig. 3c). Taken together, these data suggest an indispensable role for CD14-dependent signaling
8
in negative regulation of inflammation, clearance of bacteria, and disease resolution.
9
10
Killing, but not internalization, of B. burgdorferi by M is significantly impaired in the absence of CD14.
11
A report that CD14 acts as a unique phagocytic receptor for Gram-negative bacteria
25
suggests this molecule
12
may serve a similar function in spirochetal uptake. In light of the inverse relationship between expression of
13
CD14 and spirochetal burden in infected tissues, we examined the impact of CD14 deficiency on phagocytosis
14
B. burgdorferi. GFP-expressing spirochetes were internalized as readily by CD14
-/-
M as by those expressing
15
CD14 (Fig. 4a). Furthermore, neither the kinetics of internalization (Fig. 4b), nor the number of bacteria
16
associated per cell, as measured by changes in mean fluorescence intensity, was influenced by CD14 deficiency
17
(data not shown).
18
Killing of bacteria within the phagolysosomal compartment depends, at least in part, on the production
19
of RNS/ROS
26
. Although differences in uptake were not observed, the ultimate fate of B. burgdorferi
20
internalized by CD14
+/+
and CD14
-/-
M might differ. Indeed, assessing gene activity both in vitro and in vivo
21
it was observed that inos transcript levels were significantly higher in the cells from and the joints of CD14
+/+
22
mice (Fig. 4c). These differences were reflected in two-fold more cultivable spirochetes being recovered from
23
6 h-infected CD14
-/-
(7,252 bacteria/ml) versus CD14
+/+
(3,567 bacteria/ml) M as measured using a modified
24
tissue culture infective dose method.
25
26
8
CD14 signaling regulates activation of the p38-MAPK pathway, inhibition of which leads to lower SOCS3
1
and higher cytokine production by
M. A p38-STAT1-IRF1 signaling axis negatively regulates TLR2
2
activity while positively regulating iNOS, SOCS1, and SOCS3 activity
27
. To determine whether CD14
3
signaling modulates this axis, the phosphorylation state of p38 was evaluated in CD14
-/-
M. In contrast to
4
cells expressing CD14, detectable levels of phospho-p38 were only observed transiently in B. burgdorferi-
5
stimulated CD14
-/-
M (Fig. 5a). This result was confirmed and extended through use of a phospho-CBA kit;
6
as seen in Fig. 5b, spirochetes stimulated CD14-dependent increases in the level of phospho-p38 in both a time-
7
and dose-dependent manner. In contrast, no such changes were observed in the phosphorylation state of two
8
other members of the MAPK family, ERK and JNK. Finally, it should be appreciated that given the known
9
transcriptional inhibition of tlr2 by p38
27
, the higher transcription of this gene seen in Fig. 1a and expression of
10
TLR2 on CD14
-/-
peritoneal M is entirely consistent with the decreased phospho-p38 levels observed in CD14
-
11
/-
M (Fig. 5a, 5b).
12
Pharmacological inhibition of p38 has implicated this molecule in proinflammatory responses to a
13
variety of stimuli of microbial, host, and environmental origin
14
. Thus, the coexistence of higher phospho-p38
14
and lower cytokine production by B. burgdorferi-stimulated CD14
+/+
M was counterintuitive. To clarify the
15
role of p38 in our model arctigenin, a general MAPK inhibitor, and SB202190, a p38-specific inhibitor, were
16
used. These inhibitors significantly lowered the transcription of socs3 and inos by CD14
+/+
M in reponse to B.
17
burgdorferi (Fig. 5c). As predicted based upon lower socs3 transcript levels, arctigenin and SB202190 relieved
18
the inhibition of TNF- release by wild-type cells (Fig. 5d) and did so in a dose-dependent fashion
19
(Supplementary Fig 2, online). In stark contrast, SB203580 decreased TNF- production by cells co-
20
incubated with borrelial lysate (10 g/ml), (Fig. 5e). This latter finding is entirely consistent with the widely-
21
reported observation that p38 enhances cellular inflammatory responses to isolated bacterial MEMS
16, 28
and
22
underscores the difference between stimulation of cells with live spirochetes as opposed to spirochetal lysates or
23
lipoproteins
12, 16, 29, 30
.
24
25
9
Suppression of the PI3K/AKT axis reimposes regulation of proinflammatory cytokine production by
1
CD14
-/-
M. Detection of phospho-p38 for only a brief period implied strict CD14-dependent modulation of
2
this signaling cascade (Fig. 5a). p38-MAPK is negatively regulated through activation of AKT, a serine-
3
threonine kinase activated via PI3K
31
. The levels of phospho-AKT in CD14
-/-
M were significantly increased
4
above baseline and above that seen in cells expressing CD14 (Fig. 6a). Of note, peak phosphorylation of AKT
5
in CD14-/- cells appeared to immediately precede disappearance of phospho-p38 (Fig. 5a, 10 min). The PI3K
6
inhibitors wortmannin and Ly294002 were used, to determine whether AKT activity contributing to decreased
7
phospho-p38 levels. As shown in Fig. 6B, exposure of cells to either inhibitor for 30 min prior to incubation
8
with B. burgdorferi eliminated the phospho-AKT pool (Fig. 6b). Blocking PI3K-dependent AKT function
9
should result in de-repression of p38 phosphorylation
31
. Ly294002, but not wortmannin, treatment of CD14
-/-
10
M resulted in a further ~2-fold enhancement of p38 phosphorylation (Fig. 6b) following co-incubation with
11
B. burgdorferi. The inability of wortmannin to have the same effect as Ly294002 may be attributable to its
12
known "off target" inhibition of the p38-MAPK pathway
32
. To explore further how signaling through the
13
PI3K/AKT axis influences B. burgdorferi-induced M activation, spirochetes were incubated with CD14
-/-
cells
14
that were untreated or treated with Ly294002. Enhanced phosphorylation of p38 correlates with a profound
15
reduction in B. burgdorferi-induced TNF- secretion by CD14
-/-
M whereas nearly 10-fold more TNF- is
16
secreted in the absence of the inhibitor (Fig. 6c), an effect that is dose-dependent (Supplementary Fig. 3,
17
online).
18
19
In the absence of CD14, B. burgdorferi triggers TNF- production in both a TLR2-dependent and -
20
independent fashion. Recognition that B. burgdorferi stimulates innate host responses primarily through
21
engagement of TLR2
33, 34
led us to consider whether the uncontrolled release of TNF- by CD14
-/-
M
22
correlates with their dramatically increased expression of TLR2 (Fig. 1a and Ref. 12). To answer this question,
23
mice deficient for CD14 or TLR2 (each backcrossed 10 generations onto a C3H/HeN background) were crossed
24
to establish animals homozygous recessive for both loci. M expressing CD14 and TLR2 and those deficient
25
for individual or both loci were co-incubated with B. burgdorferi and TNF- levels were measured (Fig 7). In
26
10
the absence of CD14, TNF- production was significantly higher compared to their wild-type counterparts at 24
1
hp.i.. TLR2 deficiency had the opposite effect insofar as TNF- release was significantly reduced compared to
2
wild-type M at 6 h and 24 h p.i.. Remarkably, the cell's capacity to secrete TNF- in the absence of both
3
CD14 and TLR2 was partially or fully restored to the levels released by CD14
-/-
and wild-type M,
4
respectively. This finding is very significant as it demonstrates that, despite similarities in the exaggerated
5
Lyme disease phenotype observed in CD14
-/-
and TLR2
-/-
mice, these two signaling pathways are not entirely
6
synonymous.
7
11
DISCUSSION
1
Regulation of the host response to B. burgdorferi begins with CD14 recognition of lipoproteins
12, 35
. In
2
the absence of such receptor engagement, Lyme borreliosis is typified by greater bacterial burden, more severe
3
histopathology, and dysregulated production of proinflammatory immunomodulators
12
. Given that binding of
4
borrelial lipoproteins to CD14 facilitates downstream signaling through TLR2 and the cytosolic adaptor protein
5
MyD88 it is perhaps not surprising that mice deficient for these molecules also harbor more bacteria in their
6
tissues and present with more severe arthritis
36-38
. Despite similar pathophysiological features in B.
7
burgdorferi-infected mice lacking CD14, TLR2, or MyD88, CD14 is distinct in its ability to tightly regulate
8
cytokine production both in vitro and in vivo
12
. CD14's unique role as an anti-inflammatory receptor is
9
supported further by the observation that signaling in its absence increases production of proinflammatory
10
cytokines in response to infection with Staphylococcus aureus
10
. A similar trend towards greater cytokine
11
production in response to E. coli bioparticles was seen with CD14
-/-
versus CD14
+/+
peritoneal M
39
. Finally,
12
blockade of CD14 by monoclonal antibody treatment increased tissue invasion and resulted in higher TNF-
13
production in rabbits infected with Shigella flexneri
40
.
14
Coordinate signaling through CD14 and TLR2 simultaneously activates NF-B, p38-MAPK, and PI3K
15
pathways
2, 41
to orchestrate both the initiation and resolution of inflammatory responses to B. burgdorferi.
16
CD14 deficiency, however, leads to excessive phosphorylation of AKT along with higher transcription and
17
surface expression of TLR2
12
. Given that TLR2 signaling regulates PI3K activity and leads to AKT activation,
18
we were led to consider that either elevated TLR2 expression and/or higher phospho-AKT levels in CD14
-/-
M
19
underlie the more severe and persistent course of Lyme borreliosis in these mice. Cytokine release by B.
20
burgdorferi-activated M deficient for both CD14 and TLR2 was comparable to that of wild-type cells, but was
21
significantly greater than that observed with TLR2
-/-
cells and less than that of CD14
-/-
cells. This finding
22
suggests only a partial role for TLR2 in recognition of B burgdorferi, one that is strictly proinflammatory, and
23
implies the existence of another receptor(s) for bacterial recognition in the absence of CD14.
24
One candidate non-TLR receptor is complement receptor 3 (CR3), a member of the 2-integrin family
25
of adhesion molecules
42
. Using CHO cells transfected with CD14, TLR2, and CR3 in various combinations it
26
12
was revealed that the "inside-out" signaling responsible for increasing the avidity of CR3 for its ligand requires
1
both CD14 and TLR2
43
. Similarly, antibodies directed against CD14 and/or TLR2 were shown to block
2
"inside-out" signaling required for CR3-mediated recognition of Porphyromonas gingivalis fimbriae
44
. Moore
3
and coworkers reported that inhibition of CR3 signaling reduced by 50% the E. coli-stimulated secretion of
4
TNF- by CD14
-/-
peritoneal M
39
. These various studies also implicate CR3 in uptake of bacteria
39, 43, 44
.
5
Like CD14, this phagocytic receptor resides within the lipid raft and clustering of CR3 precedes PI3K-mediated
6
phosphorylation and activation of AKT
42
. In light of the ability of CR3 to mediate internalization of B
7
burgdorferi (personal communication, Juan Anguita, Veterinary & Animal Sciences, University of
8
Massachussetts, Amherst) and its capacity to activate PI3K, it is intriguing to speculate on whether CR3 is a
9
phagocytic receptor in the absence of CD14 whose action may account for higher cytokine production during
10
infection. Such a role is supported by the fact that spirochetal uptake was not diminished in the absence of
11
CD14.
12
AKT has pleiotropic effects that include promotion of cell survival, NF-B activation, and inhibition of
13
ASK1, a MAPKKK responsible for p38 activity
31, 45
. p38 activity is associated with NF-B activation and
14
stabilization of mRNA encoding proinflammatory cytokines such as TNF-
17
. That inhibition of PI3K
15
prevented phosphorylation of AKT and thus increased p38 activity in B. burgdorferi-activated CD14
-/-
M is
16
consistent with the inverse relationship between AKT and p38 activity. Unexpectedly, however, PI3K
17
inhibition also resulted in decreased TNF- production, a finding inconsistent with higher p38 activity and its
18
accepted role as a proinflammatory mediator
14
. Nevertheless, inhibition of p38 in CD14
+/+
cells with either
19
general or specific antagonists resulted in lower inos and socs3 transcription and a dose-dependent increase in
20
TNF- production in response to B. burgdorferi. Contrarily, it has been reported that SB203580 inhibits TNF-
21
release by the mouse macrophage RAW264.7 cell line in response to borrelial lysates
16
. One potential
22
explanation for these divergent findings is that the "context" in which bacterial MEMS are recognized (i.e., live
23
spirochetes versus lysed organisms) influences the downstream signaling cascades initiated within the host cell.
24
Consistent with this idea, treatment of CD14
+/+
M with SB203580 inhibited TNF- production in response to
25
borrelial lysates as previously reported
16
. This suggests that, in conjunction with CD14 and TLR2 engagement,
26
13
phagocytic receptors with a differential capacity to internalize live spirochetes versus a lysate may orchestrate
1
intracellular signaling events, a notion supported by earlier studies
12, 29
. The anti-inflammatory capacity of p38
2
is further demonstrated in mouse models of pneumococcal pneumonia and tuberculosis where its inhibition
3
results in impaired bacterial clearance and increased TNF- production both in vitro and in vivo
19
. It also has
4
been established that p38 induces the shedding of TNF receptor-1 from activated cells thus dampening their
5
responsiveness to TNF-
20
. The combination of increased TNF- production and maintenance of its cognate
6
receptor on the cell surface likely contributes to the cytokine "surge" and exacerbates the pathology associated
7
with bacterial infection. Taken together, these results challenge the notion that isolated MEMS (e.g., purified
8
LPS, lipoproteins, etc.) are equivalent to the whole organisms from which they are extracted
12, 29
. They also
9
sound a cautionary note regarding application of p38 inhibitors to treat inflammatory disorders especially those
10
of infectious-origin. Development of immunotherapeutic strategies that enhance the action of SOCS may
11
represent a more fruitful avenue in pursuit of novel anti-inflammatory drugs
46-48
.
12
Finally, another set of intriguing observations was the persistent arthritis and increased spirochetal-
13
burden in the tissues of CD14
-/-
mice. Considering the importance of bacterial clearance as an element of
14
disease resolution, we evaluated CD14 as a potential phagocytic receptor for spirochetes. Consistent with the
15
findings of Moore et al. using E. coli bioparticles
39
, we report that CD14 deficiency does not impair
16
internalization of spirochetes. During the process of engulfment various receptors are engaged which initiate
17
kinase and phosphatase cascades that facilitate killing of phagocytosed bacteria. Despite the equivalent
18
capacity of CD14
+/+
and CD14
-/-
M to engulf spirochetes, activation of p38 in the CD14
-/-
cells was transient
19
and thus insufficient, either in vitro or in vivo, to drive the transcription of inos and irf1 whose gene products
20
participate in maturation of the phagosome and bacterial killing
49
. The lower transcript levels of inos in CD14
-/-
21
M likely are associated with decreased RNS/ROS production
50
and may contribute, at least in part, to the
22
higher bacterial burden observed in the infected tissues of these mice. In contrast to the association of IRF1
23
with early maturation of the phagosome, IRF7 induction occurs later in this process and often is linked to
24
persistence of exogenous material within the phagosomal compartment, particularly in plasmacytoid dendritic
25
cells (pDCs)
51
. Higher irf7 transcription is a hallmark of activated pDCs and is necessary for the induction of
26
14
Type I IFN, an important element of viral and bacterial clearance
52
. Specifically, at a point when the ifn locus
1
is transcriptionally silent in B. burgdorferi-activated CD14
-/-
M little IRF7 mRNA is being transcribed.
2
However, like activated pDCs, later in their activation program irf7 transcription is significantly greater in
3
CD14
-/-
cells than their wild-type counterparts. As in activated pDCs and RAW264.7 cells, where increased
4
IRF7 coincides with an accumulation of viral antigen or purified TLR agonist (i.e., CpG ODNs)
51
, we propose
5
that "frustrated" clearance of borrelial antigen from the phagosomal compartment of CD14
-/-
M may drive the
6
transcription of irf7. A corollary of this scenario is that persistence of borrelial TLR agonists in the phagosomal
7
compartment may perpetuate inflammatory signaling through TLR2 and other receptors of TLR or non-TLR
8
origin.
9
In toto, we detail a critical and unanticipated role for CD14 in downmodulating TLR2-dependent and -
10
independent pathways which regulate NF-B signaling events. CD14 exerts its influence on the intensity and
11
duration of inflammation through the PI3K/AKT/p38-MAPK axis which we propose serves as a "rheostat" to
12
provide fine regulation of NF-B activity, the "backbone" of the host's inflammatory response to pathogenic
13
challenge. As depicted in our model (Fig 8), B. burgdorferi-initiated inflammatory signaling in the absence of
14
CD14 stimulates greater AKT activity which enhances NF-B signaling while at the same time suppressing p38
15
and STAT1. p38 and STAT1 are responsible for inducing IRF1 and SOCS activity which ultimately facilitates
16
spirochete clearance and/or dampens cytokine production. Thus the more severe and persistent Lyme disease
17
phenotype observed in CD14
-/-
mice reflects the concomitant increase in NF-B-mediated proinflammatory
18
signaling and loss of negative regulation via diminished SOCS activity. To our knowledge this is the first
19
report implicating the CD14/p38-MAPK pathway as the driving force behind anti-inflammatory responses in a
20
mouse model of bacterial infection.
21
15
METHODS
1
Reagents. Great care was taken during the preparation of all buffers and reagents to minimize contamination
2
with environmental LPS by utilizing baked (180ºC for 4 h) and autoclaved glassware, disposable plasticware,
3
and pyrogen-free H
2
O that also is free of DNAse and RNAse activity.
4
5
Cultivation of B. burgdorferi. Low-passage B. burgdorferi strain 297 were maintained at 23°C in Barbour-
6
Stoenner-Kelley medium containing 6% normal rabbit serum (BSK
complete
) from Pel-Freez Biologicals (Rogers,
7
AR) and then temperature-shifted to 37°C. Increased expression of OspC was confirmed by silver staining of
8
whole borrelial lysates separated by SDS-PAGE. B. burgdorferi 297 expressing green fluorescent protein were
9
cultivated in BSK
complete
containing 400 g/ml kanamycin and were grown at 37°C until mid- to late-logarithmic
10
phase for subsequent use in phagocytosis experiments as previously described
29
.
11
12
Mice and infection protocol. Four to eight week-old C3H/HeN (CD14
+/+
) mice (Taconic, Germantown, NY)
13
were housed in the Animal Resources Facility at Albany Medical College. Food and water were provided ad
14
libitum and all animal procedures conformed to the Institutional Animal Care and Use Committee guidelines.
15
CD14
-/-
mice were generated as previously described
4
and subsequently backcrossed 10 generations onto a
16
C3H/HeN background
12
. TLR2
-/-
mice were provided by Tularik Inc. (now Amgen Inc., South San Francisco,
17
CA) and generated by Deltagen Inc. (Menlo Park, CA)
53
and were backcrossed 10 generations onto a C3H/HeN
18
background. C3H/HeN mice deficient for CD14 and TLR2 were crossed to establish animals homozygous
19
recessive for both alleles.
20
Mice were infected via intradermal administration of 5 × 10
5
spirochetes over the sternum. BSK
complete
21
was used as a mock-infection control. At one week intervals, tibiotarsal joint thickness was measured using
22
digital calipers and bacterial burden in infected tissues was determined using isolated genomic DNA as
23
previously described
12
. Total RNA also was isolated from infected tissues for qRT-PCR as described below.
24
25
16
Isolation and differentiation of M. M were isolated from the bone marrow of six to eight week-old mice.
1
Briefly, bone marrow cells recovered by flushing femurs and tibia with DMEM were incubated in tissue
2
culture-treated 25cm
2
-flasks (BD Falcon, BD Biosciences, San Jose, CA) overnight at 37°C with 5% CO
2
to
3
eliminate adherent fibroblasts, granulocytes, and any contaminating M
54
. The following day, 1 × 10
7
4
suspension cells were maintained in 10-cm
2
bacteriological Petri dishes (BD-Falcon) for three days with
5
DMEM supplemented with 10% fetal bovine serum, 20% L292-cell conditioned media, 0.01% HEPES, 0.01%
6
sodium pyruvate, and 0.01% L-glutamine. Cultures were supplemented with five ml of the above-described
7
medium and seven days after isolation cell monolayers were recovered using ice-cold PBS and scraping. Single
8
cell suspensions were used immediately or frozen in liquid nitrogen with 20% FBS and 10% DMSO for use in
9
future experiments.
10
11
B. burgdorferi-M co-incubation. M were seeded into 6-well tissue culture-treated plates at a concentration
12
of 1 × 10
6
cells/2 ml/well and allowed to adhere overnight. The following day, B. burgdorferi were enumerated
13
and re-suspended as described above. M were washed twice with serum-free DMEM to remove any traces of
14
FBS and spirochetes (resuspended in DMEM + 4% autologous serum) were added at an MOI of 10 and co-
15
incubated for different time intervals at 37°C in 5% CO
2
. Cells incubated with DMEM + 4% autologous serum
16
alone served as mock-infected controls.
17
18
Analysis of bacterial association with M: Fluorescence microscopic analysis. M were seeded into 8-well
19
chamber slides (Lab-Tek
TM
II CC2 Chamber Slide
TM
System) at a concentration of 1 × 10
5
cells/200l/well and
20
allowed to adhere overnight. The following day, GFP-expressing B. burgdorferi were enumerated and added to
21
cell monolayers at an MOI of 10 and co-incubated for different time intervals. After co-incubation, cells were
22
washed twice with serum-free DMEM and were fixed with 4% paraformaldehyde for 30 min. GFP-expressing
23
B. burgdorferi were visualized by fluorescence microscopy (Olympus UIS2Series: U-MNG2 Green excitation
24
mirror unit/NIB HIGHQ FITC). Flow cytometric analysis. M were aliquoted into five ml polystyrene round-
25
bottom tubes (BD Biosciences, Bedford, MA) at a concentration of 5 × 10
5
cells/600l/tube, centrifuged at 250
26
17
× g for 10 min followed by incubation with GFP-expressing B. burgdorferi resuspended in DMEM + 4%
1
autologous serum at an MOI of 10. Cells and bacteria were coincubated for different time intervals, washed
2
twice in FA buffer (BD Microbiological Systems, Sparks, MD) and fixed in FA buffer containing 4%
3
paraformaldehyde. Sample data was acquired on a BD FACSCanto
TM
Flow Cytometer (BD Immunocytometry
4
Systems) and results were analyzed using FlowJo software (Ashland, OR).
5
6
Quantitative real-time PCR. Total RNA was isolated from M using the RNeasy Mini Kit (Qiagen GmbH,
7
Hilden, Germany) as per the manufacturer. The amount and purity of RNA was quantified by Biophotometer
8
(Eppendorf AG, Hamburg, Germany) and 0.5µg were used for reverse transcription of cDNA using Superscript
9
II (Invitrogen Corporation, CA, USA). cDNA (20l) served as the template in quantitative real-time PCR (qrt-
10
PCR) analysis using Mouse TLR Signaling Pathway RT
2
ProfilerTM PCR Arrays (Superarray Bioscience).
11
These arrays contain primer pairs for 84 genes implicated in TLR signal transduction as well as housekeeping
12
genes and controls in a 96-well microtiter plate format. This qrt-PCR methodology directly quantifies transcript
13
levels based upon the 2
-Ct
method through measurement of SYBR green fluorescence using an iQ5 real-time
14
PCR detection system (Bio-Rad Laboratories, Hercules, CA).
15
For transcriptional analysis of genes not represented on the RT
2
ProfilerTM PCR Array, qrt-PCR was
16
performed in a final volume of 25l containing: 12.5l of 2X SYBR green master mix (Bio-Rad Laboratories),
17
25pMol of the specific forward and reverse primers, and 0.2l of cDNA. Primers were designed using Beacon
18
Designer version 7.0 software (PREMIER Biosoft Intl, Palo Alto, CA) and the sequences of specific primer sets
19
are provided in Supplemental Table 1 online. Amplification conditions were 95°C (3 min) and 40 cycles of
20
95°C (15s), 55°C (40s) and 72°C (30s). All the qrt-PCR reactions were run in triplicate with no-template
21
controls (NTC) and mean cT values were used for all the calculations using 18S rRNA as an internal control for
22
normalization. Effects greater than a two-fold change with respect to mock control were considered significant.
23
24
Western blot analysis. Antibodies directed against SOCS1, SOCS3 and CIS were obtained from Santa Cruz
25
Biotechnology, Inc. (Santa Cruz, CA), antibodies against AKT, STAT1, STAT3 and p38 were obtained from
26
18
Cell Signaling Technology, Inc. (Danvers, MA), and antibodies against -actin were obtained from Bethyl
1
Laboratories, Inc. (Montgomery, TX). Protein samples (25-100g, depending on the target) were resolved by
2
SDS-PAGE and transferred to nitrocellulose using semi-dry transblot (Bio-Rad Laboratories, Hercules, CA).
3
The membrane was blocked with 5% non-fat milk and then incubated overnight at 4°C with primary antibody
4
(1:1000, for AKT and STATs; 1:100 for SOCS; and 1:10,000 for -actin). Membranes were probed with HRP-
5
conjugated anti-rabbit IgG (Cell Signaling Technology) diluted 1:2000. Specific signal was developed using
6
the SuperSignal West-Dura chemiluminescent substrate (Pierce Endogen, Rockford, IL) and recorded either on
7
photographic film or using a FluorChem 7700 Chemiluminescence Imager (Alpha Innotech, San Leandro, CA).
8
9
Cytometric bead array (CBA) for cytokine and phospho-protein analysis. M were co-incubated with B.
10
burgdorferi at various MOI and cytokine levels were measured in the recovered culture supernatant using the
11
Mouse Inflammation CBA kit and a FACSArray flow cytometer [BD Immunocytometry Systems (BDIS), San
12
Jose, CA]. Data was acquired and analyzed using BD FACSArray software and FCAP Array software, version
13
1.0 (BDIS), respectively. For phospho-protein analysis, the protein content of the samples was normalized,
14
added to a phospho-specific CBA kit, and analyzed as described above.
15
16
Inhibition of signaling cascades by pharmacological inhibitors. M were treated with the inhibitors
17
arctigenin (1µM), SB202190 (0.5µM), SB203580 (10µM), wortmannin (100nM), and Ly294002 (100µM) for
18
30 min prior to addition of B. burgdorferi. In some experiments a range of inhibitor concentrations was used.
19
DMSO alone served as a control. Culture supernatants were collected for cytokine measurement and cells were
20
lysed for RNA isolation and Western blot analysis.
21
22
Statistical analysis. All results were expressed as mean
± SEM and comparisons between the groups were
23
made using one-way ANOVA followed by Bonferroni's correction. Differences between control and
24
experimental groups were considered significant using = 0.05.
25
19
ACKNOWLEDGMENTS
1
We thank Drs. James Drake, Jonathan Harton, Karsten Hazlett (Albany Medical College, NY), and Juan
2
Anguita (University of Massachusetts, Amherst, MA) for critical review of the manuscript. Special thanks to
3
Aaloki Shah and Sally Catlett (Albany Medical College, NY) for technical support and Daniel Desrosiers
4
(University of Connecticut Health Science Center, CT) for computer graphic design. Supported by the National
5
Institutes of Health (AI054546 to TJS; AI29735 and AI38894 to JDR) and an Arthritis Foundation Investigator
6
Award to TJS.
7
20
AUTHOR CONTRIBUTIONS
1
B.S. designed the study, did and analyzed experiments and contributed to manuscript preparation; R.L.P., N. W.
2
and S. N. did and analyzed experiments; C.H.E. generated the GFP-expressing B. burgdorferi; J.D.R. provided
3
GFP-expressing B. burgdorferi and contributed to manuscript preparation; T.J.S. designed and organized the
4
study and contributed to manuscript preparation.
5
21
FIGURE LEGENDS
1
2
Figure 1. Higher B. burgdorferi-induced inflammatory gene activity is associated with lower SOCS levels in
3
CD14
-/-
M. Total RNA from CD14
+/+
and CD14
-/-
M co-incubated with B. burgdorferi was analyzed by qrt-
4
PCR (a) for genes involved in the TLR signaling pathway or (b) for socs1, socs3, and cis. Results presented in
5
(a) reflect the ratio of fold changes in HPRT-normalized gene activity in CD14
-/-
versus CD14
+/+
M. All
6
results represent mean ± SEM. *P < 0.05, ** P < 0.01. (c) Equivalent protein amounts of lysed M were
7
separated by 12% SDS-PAGE, transferred to a PVDF membrane and probed with antibodies directed against
8
SOCS1, SOCS3, CIS, or -actin.
9
10
Figure 2. IRF1, IRF7 and STAT1 are differentially activated by B. burgdorferi in the absence of CD14. (a)
11
Total RNA from CD14
+/+
and CD14
-/-
M co-incubated with B. burgdorferi was analyzed by qrt-PCR for irf1,
12
irf7 and stat1. Results represent mean ± SEM. *P < 0.05, ** P < 0.01. (b) Equivalent protein amounts of
13
lysed M were separated by 12% SDS-PAGE, transferred to a nitrocellulose membrane and probed with
14
phospho-specific STAT1 or STAT3, or -actin antibodies.
15
16
Figure 3. Reduced socs transcription is associated with impaired bacterial clearance and more severe and
17
prolonged Lyme arthritis. (a) CD14
+/+
and CD14
-/-
mice were infected with 5 × 10
5
B. burgdorferi, and
18
tibiotarsal joint thickness was measured at 1-wk intervals. The horizontal bars indicate mean thickness for each
19
group and the data are representative of two independent experiments (n=24). (b) Total RNA was isolated from
20
the tibiotarsal joints of CD14
+/+
and CD14
-/-
mice (n=6) and an equal amount of RNA was pooled from each
21
joint and 0.5 g was used for preparing cDNA. The cDNA was analyzed by qRT-PCR to determine the in vivo
22
levels of socs1 and socs3 transcription. Data are representative of two independent experiments. (c) Organs
23
were collected six weeks p.i. and DNA was isolated for quantification of bacterial burden. Results represent
24
mean ± SEM. *P < 0.05, ** P < 0.01, *** P < 0.001.
25
22
1
Figure 4. B. burgdorferi-induced transcription of inos, but not phagocytic uptake of spirochetes, is impaired by
2
CD14 deficiency. GFP-expressing B. burgdorferi were co-incubated with CD14
+/+
and CD14
-/-
M for the
3
indicated times and phagocytosis was evaluated by (a) fluorescence microscopy and (b) flow cytometry. (c)
4
inos transcript levels were determined by qrt-PCR using RNA isolated from M co-incubated with B.
5
burgdorferi or using RNA pooled from joints isolated from infected mice as described in the legend for Fig. 3.
6
Results represent mean ± SEM. *P < 0.05, ** P < 0.01, *** P < 0.001.
7
8
Figure 5. CD14 deficiency results in dysregulated p38-MAPK signaling and cytokine production in response
9
to B. burgdorferi. (a) Equivalent protein amounts of lysed CD14
+/+
and CD14
-/-
M co-incubated with B.
10
burgdorferi were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with phopho-
11
p38 and -actin antibodies. (b) M were co-incubated with B. burgdorferi at different MOI and equal amounts
12
of total protein were used to estimate relative phosphorylated MAPK levels by phospho-specific CBA. The
13
data shown are representative of two independent experiments. (c) CD14
+/+
M were treated with DMSO
14
alone, SB202190 (0.5 M), or arctigenin (1 M), for 30 min prior to co-incubation with B. burgdorferi at an
15
MOI of 10 and total RNA was analyzed by qrt-PCR for inos and socs3. (d) CD14
+/+
M were treated as
16
described in (c), and culture supernatants were analyzed for the presence of TNF- by CBA. (e) CD14
+/+
M
17
were treated with DMSO alone or increasing concentrations of SB203580 for 30 min prior to co-incubation
18
with B. burgdorferi lysate (10g/ml). Cell-culture supernatants were collected and TNF- levels were
19
measured by CBA. Results in (c-e) represent mean ± SEM. *P < 0.05, ** P < 0.01, *** P < 0.001.
20
21
Figure 6. Inhibition of B. burgdorferi-induced AKT activation in CD14
-/-
M reestablishes p38 activity and
22
restores negative regulation of cytokine production. (a) Equivalent protein amounts of lysed CD14
+/+
and
23
CD14
-/-
M co-incubated with B. burgdorferi were separated by SDS-PAGE, transferred to a nitrocellulose
24
membrane and probed for phospho-AKT and -actin. The blot shown is representative of two independent
25
experiments. (b) CD14
-/-
M were treated with DMSO alone or PI3K inhibitors [wortmannin (100nM) or
26
23
Ly294002 (100µM)] for 30 min prior to co-incubation with B. burgdorferi. Western blots of cellular protein
1
were probed with phospho-specific AKT and p38 antibodies and then striped and reprobed for total AKT, p38,
2
and -actin. The data shown are representative of two independent experiments. (c) CD14
+/+
and CD14
-/-
M
3
were treated with Ly294002 (100µM) for 30 min prior to co-incubated with B. burgdorferi for three and six h.
4
Culture supernatants were collected and TNF- levels were measured by CBA. Results represent mean ± SEM.
5
*** P < 0.001.
6
7
Figure 7. TLR2 plays a partial role in CD14-independent cytokine production. M isolated from CD14
+/+
,
8
CD14
-/-
, TLR2
-/-
and CD14
-/-
/TR2
-/-
mice were co-incubated with B. burgdorferi for the indicated time points,
9
cell culture supernatants were collected and TNF- was measured by CBA. Results represent mean ± SEM.
10
*** P < 0.001.
11
12
Figure 8. Proposed model for CD14-dependent and -independent signaling in response to infection with B.
13
burgdorferi. In the absence of CD14, greater activation of AKT inhibits p38 activity resulting in increased
14
transcription of TLR2, reduced activation of STAT1 and IRF1, and decreased induction of SOCS necessary to
15
modulate the intensity and duration of Lyme borreliosis. The size of the red circles indicates relative
16
phosphorylation status of the indicated protein and the size of the individual protein(s) is reflective of relative
17
transcript/protein levels.
18
19
Supplementary Figure 1. mkp1, irakm irf3, stat3 and stat4 transcription is CD14-independent and unaltered by
20
its absence. Total RNA from CD14
+/+
and CD14
-/-
M co-incubated with B. burgdorferi was analyzed by qrt-
21
PCR for mkp1, irakm, irf3, stat3, and stat4. Results represent mean ± SEM.
22
23
Supplementary Figure 2. Inhibition of p38 or MAPK results in a dose-dependent increase of TNF-
24
production in CD14
+/+
cells. CD14
+/+
M were treated with DMSO or the indicated concentrations of
25
arctigenin, SB202190 or SB203580 for 30 min prior to co-incubation with B. burgdorferi. Cell culture
26
24
supernatants were collected 24 h post incubation and TNF- levels were measured by CBA. Results represent
1
mean ± SEM. *** P < 0.001.
2
3
Supplementary Figure 3. Inhibition of PI3K results in a dose-dependent decrease in TNF- production by
4
CD14
-/-
cells. CD14
-/-
M were treated with DMSO or the indicated concentration of LY294002 for 30 min
5
prior to co-incubation with B. burgdorferi. Cell-culture supernatants were collected 24 h post incubation and
6
TNF- levels were measured by CBA. Results represent mean ± SEM. *** P < 0.001.
7
8
Supplementary Table 1. Primer sequences used in qrt-PCR
9
25
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