Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology

and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

A role for Toll-like receptor 4 in the host response to the lung

infection of Yersinia pseudotuberculosis in mice
Jin-A Choi a , Yu-Jin Jeong a , Jae-Eun Kim a , Min-Jung Kang b , Jee-Cheon Kim c ,
Sang-Muk Oh a , Kyung-Bok Lee a , Dong-Hyun Kim d , Dong-Jae Kim e,∗ , Jong-Hwan Park b,∗∗
a
Department of Biochemistry, College of Medicine, Konyang University, Daejeon 302-718, Republic of Korea
b
Laboratory Animal Medicine, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea
c
CBD 5th-3, Agency for Defense Development, Daejeon 305-152, Republic of Korea
d
University of Michigan Medical School, Department of Pathology and Comprehensive Cancer Center, Ann Arbor, MI 48109, USA
e
Laboratory Animal Resource Center, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu 42988, South Korea

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

Article history: Although a Yersinia pseudotuberculosis (Yptb) lung infection model has been developed to study Y. pestis
Received 22 May 2015 pathogenesis, it is still necessary to establish a new animal model to mimic the pathophysiological fea-
Received in revised form tures induced by Y. pestis infection. Here, we provide a new lung infection model using the Yptb strain,
24 December 2015
IP2777, which displayed rapid spread of bacteria to the liver, spleen, and blood. In addition, we examined
Accepted 4 January 2016
whether TLR4 is involved in Yptb-induced pathogenesis in the lung infection model of mice we gener-
ated. Following lung infection of WT and TLR4-deficient mice with the Yptb strain IP2777, the survival
Keywords:
rate, bacterial colonization, histopathology, and level of cytokines and chemokines in the lung, spleen,
Yersinia pseudotuberculosis
Lung infection
liver, and blood were analyzed. TLR4-deficient mice had a lower survival rate than WT mice in response
Systemic dissemination to Yptb lung infection. Although the bacterial colonization and pathology of the lung were comparable
Toll-like receptor 4 between WT and TLR4-deficient mice, those of the spleen and liver were more severe in TLR4-deficient
mice. In addition, the levels of TNF-␣ and CXCL2 in the liver and IL-6 and CXCL2 in the blood were higher
in TLR4-deficient mice than in WT mice. Our results demonstrate that TLR4 is necessary for optimal host
protection against Yptb lung infection and TLR4-deficient mice may serve as a better genetic model of
Yptb infection for mimicking Y. pestis infection.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction
Yersinia is a genus of Gram-negative, rod-shaped bacteria that
belong to the Enterobacteriaceae family. Among Yersinia species,
Y. pestis, Y. pseudotuberculosis (Yptb), and Y. enterocolitica (Yec) are
known pathogenic strains for humans and rodents [1]. Y. pestis is
the etiologic agent of plague, which has been recorded as the most
dangerous contagion in human history [2]. Plague is classified into
three forms: bubonic, septicemic, and pneumonic [2,3]. Bubonic
plague is caused by the bite of an infected flea. The bacteria local-
ize to inflamed lymph nodes causing lymphadenitis, also known
as buboes. Septicemic or pneumonic plague is transmitted directly
from person to person through aerosols. The transmitted bacteria
∗ Corresponding author. Tel.: +82 53 785 0601.
∗∗ Corresponding author. Tel.: +82 62 530 2834.
E-mail addresses: Kimdj77@gmail.com (D.-J. Kim), jonpark@jnu.ac.kr
(J.-H. Park).
spread to multiple organs such as the lungs via the bloodstream,
which results in a highly lethal pneumonia. In contrast, Yptb and
Yec infect hosts via the oral route and cause typical enteric dis-
eases such as diarrhea, enteritis, and mesenteric lymphadenitis in
humans and rodents [4,5].
Yptb is the direct ancestor of Y. pestis. Due to their genetic sim-
ilarity, the Yptb lung infection model has been developed to study
the efficacy of new therapeutics and vaccines [6,7] as well as to
determine the function of virulence factors shared by both strains
[8,9]. Although mice infected intranasally with Yptb exhibited ful-
minant pneumonia, these models did not mimic the phenotype
induced by Y. pestis since bacterial spreading into systemic organs
was delayed [9]. It is therefore necessary to establish a new animal
model for lung infection of Yptb that mimics the pathophysiological
features induced by Y. pestis infection.
Toll-like receptors (TLRs) are a family of transmembrane
receptors, and are the most extensively studied pattern recog-
nition receptors (PRRs) [10]. They recognize various microbial
and endogenous molecules and initiate host innate immune

http://dx.doi.org/10.1016/j.cimid.2016.01.001
0147-9571/© 2016 Elsevier Ltd. All rights reserved.
 J.-A. Choi et al. / Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60
responses. Among the TLRs, TLR4 senses lipopolysaccharide (LPS)
of Gram-negative bacteria and induces various cellular signaling
pathways in host cells via TIR-domain-containing adapter-inducing
interferon-␤ (TRIF)- and myeloid differentiation primary response
gene 88 (MyD88)-dependent signaling.
TLR4 mediates Yersinia-induced cell death of immune cells
[11,12] and its deficiency impairs IL-6 production in macrophages
infected with Yec [13]. Although the role of TLR4 in the in vivo
pathogenesis of Yptb lung infection remains obscure, TLR4 is known
to play protective roles in lung infection by various bacteria. Air-
way epithelial TLR4 contributed to the sensing of Haemophilus
influenza and its signaling induced innate immune responses [14].
TLR4 mutant mice demonstrated a reduced capacity to elimi-
nate Mycobacterium tuberculosis, and bacterial dissemination to the
spleen and liver was induced 10–100 times compared to wild-type
mice [15]. In cases of Pneumococcal pneumonia and Klebsiella pneu-
monia, reduced survival of TLR4 mutant mice was associated with
higher bacterial burden in the lung [16]. TLR4 also plays a cen-
tral role in initial neutrophil recruitment and orchestration of early
innate defense against Bordetella pertussis [17].
In the present study, we sought to determine the role of TLR4 in
the pathogenesis of Yptb in the lung infection of mice. Our results
showed that TLR4 plays an important role in host defense against
Yptb lung infection. Therefore, TLR4 deficient mice infected with
Yptb may be a more appropriate model for mimicking Y. pestis
infection.
2. Materials and methods
2.1. Mice
TLR4-deficient mice on a C57BL/6 background were purchased
from Jackson Laboratory (Bar Harbor, ME, USA) and wild-type (WT)
C57BL/6 mice were purchased from Koatech (Pyeongtaek, Korea).
The Institutional Animal Care and Use Committee of Konyang
University approved all animal studies, and experiments were con-
ducted under approved protocols (Daejeon, Korea).
2.2. Preparation of Y. pseudotuberculosis (Yptb) cultures
The Yptb strain IP2777 that can highly produce invasion [18]
was used in this study. Single colonies were inoculated into 5 ml
of Luria–Bertani (LB) broth and grown overnight at 26 ◦ C in a
shaking incubator. A 1:5 dilution of the overnight culture in
fresh medium was allowed to grow at 26 ◦ C with shaking for a
further 2 h. Bacteria were measured as OD600 nm = 0.6 in sterile
phosphate-buffered saline (PBS; pH 7.4), which corresponded to
approximately 109 CFU/ml. After the bacteria were washed twice
with PBS, they were diluted with PBS to the desired concentrations.
2.3. In vivo experiment
WT (8- to 9-week-old) and TLR4-deficient mice were
anesthetized by intraperitoneal injection of 10 mg/kg rompun
(xylazine) (Bayer Korea Ltd, Seoul, Korea) and 10 mg/kg zoletil (Vir-
bac Korea Co, Ltd, Seoul, Korea), followed by intranasal infection
with 30 ␮l of prepared bacteria. Infected mice were monitored over
20 days for body weight changes and clinical signs. Mice were sac-
rificed 3, 5, and 7 days post inoculation, and the lungs, spleens, and
livers were collected in sterile PBS for bacterial counting. The col-
lected tissues were weighed, homogenized, and serially diluted.
The homogenate (50 ␮l) was then spread onto LB and Yersinia
Selective agar plates. After two days of culture in a 26 ◦ C incuba-
tor, the colony was counted and the number of bacteria (measured
as CFU/g tissue) was calculated.
55
2.4. Measurement of cytokines and chemokines
Blood was collected from the retro-orbital plexus of mice
using heparinized capillary tubes (Kimble Chase Life Science and
Research Products, Vineland, NJ, USA). It was then centrifuged in a
micro high speed centrifuge (Micro 17TR; Hanil Science Industrial,
Incheon, Korea) at 6300 × g for 20 min and tissue lysates were cen-
trifuged at 16,600 × g for 5 min. Supernatants were collected and
stored at −80 ◦ C for analysis. The levels of IL-6, IL-1␤, tumor necro-
sis factor-␣ (TNF-␣), chemokine (C-X-C motif) ligand 1 (CXCL1),
chemokine (C-X-C motif) ligand 2 (CXCL2), and chemokine (C-C
motif) ligand 2 (CCL2) in the supernatant of tissue lysates from
Yptb-infected mice were determined using commercial ELISA kits
(R&D Systems, Minneapolis, MN, USA).
2.5. Histopathology
Lungs, spleens, and livers were harvested and fixed in 10% neu-
tral formalin for histopathological observation. The tissues were
routinely processed with an alcohol and xylene series and embed-
ded in paraffin. Subsequently, 2 ␮m sections were prepared, stained
with hematoxylin (Sigma-Aldrich, California, USA) and eosin (BBC
Biochemical, Mount Vernon, WA, USA) (H&E), and examined by
microscopy. The number of microabscesses in the H&E-stained sec-
tions of livers collected 3 and 5 days after infection (5–6 sections
from each liver tissue) was counted and the average was deter-
mined. The pathology score was determined by grading neutrophil
infiltration and necrosis in H&E-stained sections of the spleen (2–3
sections from each spleen tissue) (0: not shown, 1: mild neu-
trophil infiltration, 2: neutrophil infiltration with necrosis in <50%
of spleen section, 3: neutrophil infiltration with necrosis in >50% of
spleen section) and average score of each group was shown.
2.6. Statistical analysis
Survival of mice was analyzed by Kaplan Meier survival curves.
The statistical comparison between WT and TLR4-deficient mice
was calculated using the log-rank test. The differences in mean val-
ues among different groups were determined, and the values were
expressed as mean ± SD. Statistical analyses were performed using
an unpaired Student’s test with the GraphPad Prism 5.0 software
(GraphPad Software, San Diego, California, USA). Values of p < 0.05
were considered statistically significant.
3. Results
3.1. TLR4 deficiency promotes lethality of mice infected
intranasally with Yptb
We first examined whether TLR4 is essential for host protec-
tion against lethal lung infection of Yptb. When infected with
1 × 104 CFU, all TLR4-deficient mice died by 5 days post infection
(Fig. 1A). In contrast, WT mice survived longer and died by 9 days
post infection (Fig. 1A). Moreover, infection with a lower Yptb dose
(1 × 103 CFU) led to the death of all TLR4-deficient mice by 9 days
post infection, whereas more than 85% of WT mice survived dur-
ing the experimental period (Fig. 1B). These findings indicate that
TLR4 may play a role in protecting mice from lethality caused by
Yptb lung infection.
3.2. TLR4 delays bacterial spreading to systemic organs in mice
infected intranasally with Yptb
We next determined the role of TLR4 on bacterial growth and
translocation in mice with Yptb lung infection. Bacterial burden was
56 J.-A. Choi et al. / Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60

A TLR4-mediated signaling plays a role in the inhibition of bacterial

WT (n=8) dissemination to systemic organs in Yptb-infected mice, although
100 TLR4 KO (n=12)
Survival (%)

it is dispensable for bacterial burden in the lungs.

50 3.3. TLR4 deficiency enhances the severity of histological lesions

in the spleen and liver of Yptb-infected mice
p=0.0303
0 Histological lesions in the lung, spleen, and liver of mice were
0 2 4 6 8 10
analyzed. Uninfected WT and TLR4-deficient mice showed normal
Days post infection
histology in the lung (Fig. 3A and B), liver (Fig. 4A and B), and spleen
(Fig. 5A and B). Multifocal inflammatory foci, consisting mostly of
B neutrophils, were observed in the lungs of Yptb-infected mice at
WT (n=7)
100 TLR4 KO (n=7) day 3 post infection (Fig. 3C and D). Large bacterial colonies and
Survival (%)

occasionally necrotic lesions were found within the foci (Fig. 3C

50
p=0.0004
0 cytes as well as neutrophils also increased. Necrotic and edematous
0 5 10 15 20
Days post infection
Fig. 1. TLR4 increases the survival rate of mice infected intranasally with Yptb. WT
and TLR4-deficient mice were anesthetized and infected intranasally with (A)
1 × 104 CFU or (B) 1 × 103 CFU of Yptb. The survival rate of infected mice was mon-
itored over 20 days. A statistical comparison between WT and TLR4-deficient mice
was calculated using the log-rank test.
maintained at a high number of colonies (about 1 × 106 CFU/g tis-
sue) in the lungs of both WT and TLR4-deficient mice and there was
no significant difference in bacterial CFU between the two groups
(Fig. 2A). However, bacterial dissemination into the spleen and liver
gradually increased and the number of bacteria was significantly
higher in the spleen and liver of TLR4-deficient mice than in WT
mice at 5 or 7 days post infection (Fig. 2B and C). The bacterial CFU
in the blood of TLR4-deficient mice was also increased at day 7 post
infection compared to WT mice (Fig. 2D). These data suggest that
and D). Neutrophilic infiltration was also observed in the lumen
of bronchioles and edematous lesions around the bronchus and in
the alveolar septa (Fig. 3C and D). At day 5 and 7, the number and
size of the foci increased in Yptb-infected mice. Infiltration of mono-
lesions were also expanded and became more severe (Fig. 3E–H).
The severity of such histological lesions at all-time points was sim-
ilar between WT and TLR4-deficient mice (Fig. 3C–H). In the liver,
microabscess (Fig. 4C–H) formation represented a distinct lesion.
Remarkably, at 5 days post infection, the number of microabscesses
was higher in the liver of TLR4-deficient mice than that in WT mice
(Fig. 4E, F, and I). At day 7, massive necrosis was found in the liver
of both WT and TLR4-deficient mice, making it difficult to count
the microabscesses (Fig. 4G and H). The necrotic lesion was sig-
nificantly expanded in TLR4-deficient mice compared to WT mice
(Fig. 4G and H). In the spleen, only mild neutrophilic infiltration
was observed in Yptb-infected mice at day 3 (Fig. 5C and D). How-
ever, multifocal necrosis with neutrophilic infiltration was found
in the red pulp and white pulp margin at day 5 and 7, and was
more severe in TLR4-deficient mice (Fig. 5E–I). These results corre-
late with the findings related to the bacterial number in systemic
organs (Fig. 2).

A B
8 Lung 10 Liver
WT ***
TLR4 KO *
8
7
log CFU/g
log CFU/g

6
6
4
5
2

4 0
3d 5d 7d 3d 5d 7d

C D
10 Spleen 5 Blood
* ***
8 4
log CFU/ml
log CFU/g

6 3

4 2

2 1

0 0
3d 5d 7d 3d 5d 7d

Fig. 2. TLR4 delays systemic dissemination of Yptb in mice. WT and TLR4-deficient mice were anesthetized and infected intranasally with 1 × 103 CFU of Yptb. At 3, 5, and 7 days
post infection, mice were sacrificed and the lungs (A), livers (B), spleens (C), and blood (D) were collected in sterile PBS. The collected tissues were weighed, homogenized,
and spread onto LB or Yersinia selective agar plates. After 2 days of incubation, bacterial colonies were counted and the bacterial number was calculated. The results are
representative of two independent experiments. Each dot represents an individual mouse (* p < 0.05 and *** p < 0.001).
 J.-A. Choi et al. / Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60 57

Fig. 3. TLR4 does not modulate the histopathology of the lungs of mice infected intranasally with Yptb. WT (A, C, E, and G) and TLR4-deficient (B, D, F, and H) mice were anesthetized
and infected intranasally with 1 × 103 CFU of Yptb. At 3, 5, and 7 days post infection, mice were sacrificed and the lungs were isolated, formalin-fixed, sectioned, and stained
with hematoxylin and eosin. A representative histological image of lungs from each group is depicted (Magnification ×40).

3.4. TLR4 influences the production of cytokines and chemokines
in multiple organs and the serum of Yptb-infected mice
Finally, we examined cytokine (IL-6, TNF-␣, and IL-1␤) and
chemokine (CXCL1, CXCL2, and CCL2) levels in the lung, liver,
spleen, and serum of Yptb-infected mice. The levels of IL-6, TNF-␣,
CXCL2, and CCL2 in the lung were significantly higher in TLR4-
deficient mice than in WT mice at day 5 or 7 post infection (Fig. 6Ai,
ii, v, and vi). In contrast, CXCL1 levels were higher in WT mice at
day 5 and were not different between WT and TLR4-deficient mice
at day 7 (Fig. 6Aiv). There was no significant difference between
IL-1␤ levels in the lungs of the two groups (Fig. 6Aiii). At day 7
post infection, the levels of TNF-␣ and CXCL2 were higher in the
liver of TLR4-deficient mice than in WT mice, whereas the levels
of other cytokines and chemokines were not different between the
two groups at any of the time points evaluated (Fig. 6Bi–vi). There
was no significant difference between the levels of all cytokines
and chemokines tested in the spleens of WT and TLR4-deficient
mice (Fig. 6Ci–vi). Slightly higher IL-6 levels were detected in the
liver of TLR4-deficient mice at all-time points tested (Fig. 6Di). TNF-
␣ was undetectable in the serum of both groups of mice (Fig. 6Dii).
IL-1␤ and CXCL2 levels were significantly higher in WT mice at day
3 or 7 (Fig. 6Diii and v). The levels of CXCL1 and CCL2 in the serum
were similar between the two groups (Fig. 6Div and vi).
4. Discussion
Our Yptb strain IP2777 lung infection model showed rapid
spread of bacteria to the liver, spleen, and blood compared to the
previously reported model using the Yptb strain IP2666 [9]. Bacte-
ria colonized the spleen and liver at about 106 and 104 CFU/g,
respectively, 3 days post infection, and this bacterial colonization
gradually increased over time. Recently, Paczosa et al. reported
a mouse model of Yersinia lung infection using the Yptb strain

Fig. 4. TLR4 attenuates the histopathology of livers of mice infected intranasally with Yptb. WT (A, C, E, and G) and TLR4-deficient (B, D, F, and H) mice were anesthetized and
infected intranasally with 1 × 103 CFU of Yptb. At 3, 5, and 7 days post infection, mice were sacrificed and sections from the liver were prepared for histological examination
as described in Fig. 3. A representative histological image of livers from each group is depicted (magnification: 100×). The number of microabscesses in the liver at 3 and 5
days post infection was determined by counting (I). Microabscesses are indicated by arrows and necrotic areas (N) were separated by a broken line. The statistical comparison
between WT and TLR4 mice was calculated using the Student’s t-test (* p < 0.05). (Magnification ×40).
58 J.-A. Choi et al. / Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60

Fig. 5. TLR4 attenuates the histopathology of the spleens of mice infected intranasally with Yptb. WT (A, C, E, and G) and TLR4-deficient (B, D, F, and H) mice were anesthetized
and infected intranasally with 1 × 103 CFU of Yptb. At 3, 5, and 7 days post infection, mice were sacrificed and spleens were isolated, formalin-fixed, sectioned, and stained
with hematoxylin and eosin. A representative histological image of the lungs from each group is depicted (magnification: 100×). Pathology scores were evaluated in spleens
3, 5 and 7 days post infection (I). The statistical comparison between WT and TLR4 mice was calculated using the Student’s t-test (* p < 0.05). (Magnification ×40).

Fig. 6. TLR4 influences the production of cytokines and chemokines in multiple organs and the serum of mice infected intranasally with Yptb. WT and TLR4-deficient mice were
anesthetized and infected intranasally with 1 × 103 CFU of Yptb. At 3, 5, and 7 days post infection, mice were sacrificed and lungs, livers, spleens, and blood were collected. The
levels of IL-6, IL-1␤, TNF-␣, CXCL1, CXCL2, and CCL2 in the supernatant of lung, liver, and spleen lysates and serum were determined using commercial ELISA kits. Data are
representative of two independent experiments. The statistical comparison between WT and TLR4 mice was calculated using the Student’s t-test (** p < 0.01 and *** p < 0.001).
 J.-A. Choi et al. / Comparative Immunology, Microbiology and Infectious Diseases 44 (2016) 54–60
IP32953 in BALB/c mice [8]. That model demonstrated more aggres-
sive infection compared to the IP2666 model, as measured by
increased lung colonization and systemic spread [8]. Similar to the
Yptb strain IP32953 model, our lung infection model using the Yptb
strain IP2777 in C57BL/6 mice mimics the quick systemic dissemi-
nation of Y. pestis.
In the present study, TLR4-deficient mice exhibited decreased
survival compared to WT mice after lung infection with Yptb. In
order to define the mechanism of the decreased survival rate of
TLR4-deficient mice, we analyzed the lungs, which is the primary
infection site. However, the bacterial colonization and pathology of
the lung were comparable between WT and TLR4-deficient mice.
Next, we analyzed systemic organs, particularly the spleen and
liver. Surprisingly, the bacterial colonization and pathology of the
spleen and liver were more severe in TLR4-deficient mice. Although
TLR4 has no effect on infection at the primary site, TLR4 could play
an important role in bacterial dissemination to the spleen, liver,
and blood after lung infection with Yptb. Following lung infection,
bacteria migrate from the primary infection site to the lymph nodes
and blood stream [19]. Bacteria can cross the alveolar epithelial bar-
rier by direct invasion and lysis of epithelial cells or by spreading
within phagocytes [19]. In contrast to our findings with intranasal
Yptb lung infection, mice intravenously infected with Yec displayed
a similar bacterial burden in the spleen between WT and TLR2/4-,
MyD88-, or TRIF-deficient mice [20]. Therefore, TLR4 may inhibit
bacterial penetration across the alveolar epithelial barrier during
Yptb lung infection.
We observed that the levels of pro-inflammatory cytokines,
such as TNF-␣ and IL-6, and the CXCL2 chemokine increased in
the systemic organs or blood of TLR4-deficient mice compared to
WT mice. When mice were challenged with Yec, CXCL2 expression
was observed in all infected organs and was strongly associated
with rapid neutrophil recruitment [21]. The induced expression of
CXCL2 in the systemic organs of TLR4 deficient mice corresponds
with severe neutrophilic infiltration in the same tissue. Neutrophils
play an important role in Yersinia clearance, and IL-6 is known to
play a protective role during Yersinia infection [22,23]. However,
the increase in cytokine and chemokine expression observed may
be due to an increased bacterial number, and not necessarily caused
by the infection itself.
It is well known that Y. pestis grown at 37 ◦ C produces tetra-
acylated lipid A and has low TLR4 activity, whereas hexa-acylated
LPS-lipid A is made when grown at low temperature (26–27 ◦ C)
[24,25]. Y. pestis expressing acyltransferase LpxL makes hexa-
acylated structure of lipid A when grown at both 26 and 37 ◦ C and
it could induce IL-8 production in HEK293 cells expressing human
TLR4-MD-2 [25]. In fact, Y. pestis with such strong LPS could not pro-
duce bubonic plague in WT mice, whereas it led to mice lethality
in TLR4- and MD2-deficient mice [25]. Moreover, in liver pathol-
ogy, massive bacterial burden without distinct local inflammation
was observed in mice infected with Y. pestis KIM1001 (low TLR4
activity), whereas Y. pestis expressing LpxL induced microabscess
formation in WT mice [25]. These observations suggest that Y. pestis
can survive and spreads into systemic organs and causes disease in
host by evading TLR4-mediated immune response. When grown
at 37 ◦ C, Yptb also synthesized two major form of lipid A, tetra-
acylated lipid A and penta structure containing an additional C16:0
fatty acid [26]. However, it still possessed hexa-acylated lipid A and
mass spectrometer spectra generated by Yptb were more complex
than those of Y. pestis [26]. In addition, Therisod et al. also revealed
that Yptb ATCC 29833 strain contains hexa-acylated lipd A when
grown at 37 ◦ C [27]. In the present study, TLR4-deficient mice died
much quickly and their systemic organs (the liver and spleen) con-
tained much higher bacterial burden, as compared with WT mice. In
aspect of that Y. pestis can evade TLR4-mediated immune response,
the lung infection model of Yptb using TLR4-deficient mice seem to
59
mimic Y. pestis infection much better. Although this model is use-
ful to test new antimicrobial agents including antibiotics, there is
still a disadvantage. TLR signalings have been targeted to develop
new therapeutics and vaccines against various diseases including
infection [28,29]. Accordingly, TLR4-deficient mice model has a
restriction in efficacy tests for candidates with such immunological
characteristics.
TLR2 was shown to play an important role in limiting bacte-
rial dissemination from Peyer’s patches and sepsis in mice orally
infected with Yptb [30]. TLR2-mediated Reg3beta expression in
Peyer’s patches with Yptb infection is required to eliminate bacte-
ria, while TLR2 was dispensable for systemic infection [30]. Further
studies are needed to determine the role of TLR2 in host defense
against Yptb lung infection.
5. Conclusion
TLR4 is necessary for optimal host protection against Yptb lung
infection. Furthermore, TLR4-deficient mice may serve as a better
genetic model of Yptb infection for mimicking Y. pestis infection
since it exhibits more rapid spreading of bacteria to systemic organs
and severe lethality even with low infection doses.
Conflict of interest statement
The authors declare no financial or commercial conflicts of inter-
est.
Acknowledgements
This study was supported by grants from the Agency for Defense
Development (No. ADD-12-70-06-01).
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