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Infect Immun, January 1998, p. 197-202, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of Tumor Necrosis Factor Alpha in Gnotobiotic
Mice Infected with an Escherichia coli O157:H7
Strain
Emiko
Isogai,1,*
Hiroshi
Isogai,2
Koichi
Kimura,3
Shunji
Hayashi,3
Toru
Kubota,3
Nobuhiro
Fujii,3 and
Koichi
Takeshi4
Department of Preventive Dentistry, School of
Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu
1757, Hokkaido 061-02,1 and
Division of
Animal Experimentation2 and
Department
of Microbiology,3 Sapporo Medical University,
and
Hokkaido Institute of Public
Health,4 Sapporo 060, Japan
Received 11 August 1997/Returned for modification 11 September
1997/Accepted 27 October 1997
 |
ABSTRACT |
Gnotobiotic mice inoculated with an enterohemorrhagic
Escherichia coli (EHEC) O157:H7 strain developed a flaccid
paresis, usually culminating in death. The bacteria colonized feces at 109 to 1010 CFU per g (inoculum size: 2.0 × 109 CFU/mouse), and Shiga-like toxins (SLTs) were
detected in the feces. A microscopic examination of colons showed mild
inflammatory cell infiltration, thinning of the intestinal wall, or
necrotic foci. Necrosis of tubular cells was noted in these symptomatic mice. Microhemorrhage, thrombosis, and edematous changes of the brain
were also seen. Inflammatory cytokines, tumor necrosis factor alpha
(TNF-
), interleukin 1 (IL-1), and IL-6, were detected in the
kidney after EHEC infection, but not in the serum. In the brain, only
TNF- was detected. When 2.0 × 102 CFU of EHEC
O157:H7 was fed to germ-free mice, the number of bacteria began to rise
rapidly on day 1 and was maintained at 108 to
109 CFU/g of feces. SLTs were detected in the feces of the
mice. However, the mice showed no histological changes and no cytokine responses, similar to what was found for controls. Treatment with TNF- modified the clinical neural signs, histopathological changes, and cytokine responses; mice treated with TNF- developed severe neurotoxic symptoms and had higher frequencies of systemic symptoms and
glomerular pathology. Strong cytokine responses were seen in the kidney
and brain. Serum cytokines were also detected in this group. In
contrast, a TNF- inhibitor (protease inhibitor) inhibited these
responses, especially in the brain. However, local synthesis of the
cytokines was observed in the kidney. Thus, TNF- and the other
proinflammatory cytokines could be important in modifying the disease
caused by EHEC.
| |
INTRODUCTION |
Enterohemorrhagic Escherichia
coli (EHEC) strains are important causes of human hemorrhagic
colitis, hemolytic uremic syndrome (HUS), and encephalopathy (7,
13). A common histopathological finding in patients is the
destruction of endothelial cells lining small blood vessels in the
colon, kidneys, and central nervous system (20). The
virulence of EHEC has been linked to the production of Shiga-like
toxins (SLTs) (12, 14, 16). The SLTs act as an inhibitor of
protein synthesis, enzymatically modifying the translational machinery
of the host cell (3). Recently, Fujii et al. suggested that
SLT-II is toxic to both endothelial cells and neurons in the central
nervous system (5, 6). However, the precise contribution of
SLT-mediated inhibition of protein synthesis to the development of HUS
and encephalopathy remains mysterious.
A variety of animal models have been used to study the symptoms and
histopathologic changes associated with human EHEC infection. EHEC
strains caused gastrointestinal, neurologic, or systemic symptoms and
death in gnotobiotic piglets (4), rabbits (18), and mice (15, 26, 27). Acute tubular necrosis of the kidneys was found in inoculated animals, but glomerular pathology was not
observed (26, 27). Recently, Karpman et al. observed that mice inoculated with SLT-II-positive strains developed severe neurotoxic symptoms and a higher frequency of systemic symptoms, glomerular mesangial hypertrophy, and mesangial deposition than did
mice inoculated with SLT-II-negative strains (14). However, they did not observe the glomerular vascular lesions characteristic of
HUS in humans.
It has been suggested that cooperation between SLT and tumor necrosis
factor (TNF) may be important in producing the pathologic changes
observed in HUS (2). TNF- and SLT exhibited synergistic cytotoxic activity toward human endothelial cells (16).
Harel et al. suggested that local synthesis of TNF within the kidney may contribute to renal injury induced by SLT (8).
The aim of this study was to assess the relative importance of TNF-
in EHEC infection by using germ-free mice. EHEC infection induced TNF
synthesis within the kidney and brain. Treatment with TNF- or its
inhibitor modified the disease for experimental animals.
| |
MATERIALS AND METHODS |
Bacterial strains.
EHEC O157:H7 strain EDL 931 (22), which produces both SLT-I and SLT-II, was used for our
experiments. Nonpathogenic E. coli MV1184 was also used. The
organisms were incubated in brain heart infusion (BHI) medium for
24 h at 37°C. After one passage (incubation for 6 h at
37°C), viable counts were determined by plating on agar media.
Mice.
Germ-free IQI mice, bred from ICR mice, were obtained
from Japan Clea Co. Ltd. (Tokyo, Japan). Female and male mice were used at 4 to 5 weeks of age. Specific-pathogen-free ICR mice (male, 4 weeks
of age) were used as flora-positive controls.
Infection protocol.
Each E. coli strain was
prepared by washing the bacterial pellet twice in phosphate-buffered
saline (PBS; pH 7.4). Bacterial suspensions (0.1 ml in PBS; EHEC EDL
931: 2.0 × 103/ml or 2.0 × 1010/ml;
E. coli MV1184: 2.4 × 1010/ml) were
deposited intragastrically through a soft polyethylene catheter.
Immediately after inoculation, the catheter was removed, and no further
manipulations were performed. The controls received 0.1 ml of PBS.
Mouse colonization experiments.
Mice were maintained in a
level 3 environment. Food and drinking water for mice were autoclaved
before use. After bacterial inoculation, fecal samples were collected
from each mouse. They were suspended at a concentration of 100 mg/ml in
BHI medium, homogenized, and plated on Chromagar O157 (chromogenic
medium for the isolation and differentiation of EHEC O157; CHROMagar Microbiology, Paris, France) and BHI agar. In this investigation, colonizing ability was assessed by determining the level at which a
strain persisted in mouse feces.
SLT antigen level determination.
SLT antigen levels were
determined with an enzyme-linked immunosorbent assay (ELISA) kit
(Novapath EHEC; Japan Bio-Rad Laboratories, Tokyo, Japan). This
immunoassay is for the detection of SLT-I and -II in stool specimens
and cultures. An E. coli verotoxin detection kit for
reversed passive latex agglutination (RPLA; Denka Seiken Co. Ltd.,
Tokyo, Japan) was also used.
TNF- and protease inhibitor.
To determine the role of
TNF- in the pathogenesis of EHEC O157:H7, a TNF--treated mouse
group and a TNF--inhibited group were prepared. We used recombinant
TNF- (R & D Systems Europe Ltd., Abingdon, Oxon, England) for
intraperitoneal injections (10 ng per mouse). The dose was determined
in a study previously reported (9). The first injection of
TNF- was done 3 h before EHEC feeding. TNF- was injected at
2, 4, and 6 days postinfection. Nafamostat mesilate (NM;
6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate)
was provided by Torii Pharmaceutical Co. Ltd. (Tokyo, Japan) and was
used as a TNF- inhibitor (0.02 mg/mouse; intraperitoneal injections
3 h before and every day after EHEC feeding). NM is a synthetic
protease inhibitor that inhibits the various serine proteases during
the coagulation cascade as well as during the inflammatory process
(1). NM, at a concentration of 10
5 M,
inhibited the production of TNF- by lipopolysaccharide-stimulated monocytes in vitro (24).
Cytokine assay.
Cytokine assays were done by a method
previously described (10). TNF-, interleukin 1
(IL-1), and IL-6 levels were quantified with ELISA kits (Genzyme,
Cambridge, Mass.).
Hematology.
Blood was obtained from mice 1 and 7 days after
inoculation. Blood cell counts and hemoglobin concentrations were
determined by using a Sysmex microcell counter (model F-800; Toa
Medical Electronics Co. Ltd., Kobe, Japan).
Histological examinations.
Seven days postfeeding or when
signs of disease were first evident, animals colonized with E. coli O157:H7 were sacrificed and subjected to full necropsy.
Tissue specimens were collected for histological examination. Specimens
were fixed in 10% buffered neutral Formalin and processed by standard
procedures. Sections of paraffin-embedded tissue were stained with
hematoxylin and eosin.
SEM.
Intestinal tissue was obtained from mice after 1 and 7 days of inoculation. Specimens were treated by a routine method
described previously (11), dried to the critical point in an
HPC-2 critical point dryer (Hitachi Co. Ltd., Ibaragi, Japan), coated
with gold-palladium metal with an Eiko IB-3 coating machine, and
examined in a Hitachi X-650 scanning electron microscope (SEM).
Statistics.
Geometric mean CFUs from each group of three or
more animals were calculated, and the significance of differences
between means of the groups was determined by an unpaired t
test. Probability values <0.05 were considered significant.
| |
RESULTS |
Symptoms of disease in gnotobiotic mice inoculated with EHEC
O157:H7.
IQI mice fed the strain of EHEC (2.0 × 109 CFU) became lethargic, occasionally exhibited hind-limb
paralysis, stopped eating, retained urine, and died within 7 days of
infection (Table 1). The lethality was
60%. In TNF-treated mice, severe neurologic symptoms developed within
4 to 7 days after inoculation; all of them showed convulsions and died.
The clinical features of TNF-treated mice were similar to those
observed in humans except for the absence of hemorrhagic diarrhea. NM
treatment decreased lethality after EHEC infection. However, IQI mice
treated with both TNF- and NM showed neurologic symptoms and died.
Retention of urine was associated with renal failure (renal tubular
necrosis).
IQI mice inoculated intragastically with 2.0 × 102
CFU of either EHEC or control strain MV1184 did not develop
gastrointestinal, neurologic, and systemic symptoms. ICR mice
(specific-pathogen-free, flora-positive controls) also did not develop
any symptoms. Control mice receiving 0.1 ml of PBS intragastically did
not develop any symptoms.
Hematology.
The hematology results showed significant
alterations in mean platelet and leukocyte counts on day 1 after EHEC
infection (P < 0.01). Platelet and leukocyte counts
were (76.5 ± 24.1) × 104/mm3 and
(53.0 ± 3.6) × 102/mm3, respectively, in
the group subjected to EHEC infection, whereas they were (129.5 ± 26.4) × 104/mm3 and (22.5 ± 2.1) × 102/mm3, respectively, in the controls. TNF
treatment was effective in increasing the leukocyte count ([85.3 ± 14.4] × 102/mm3; P < 0.01; significantly higher than controls). The majority of the other
hematologic parameters were not significantly different for the three
treatments.
Concerning the hematology of surviving asymptomatic mice, there were no
significant alterations with only one exception: leukocyte
count. The
mean leukocyte count for mice subjected to EHEC infection
and not
treated with NM ([52.0 ± 2.8] × 10
2/mm
3) was two times higher than that of
controls; for mice infected
with EHEC and treated with NM, the mean
leukocyte count ([80.7
± 28.3] × 10
2/mm
3) was three times higher than that of
controls.
Histology.
A macroscopic examination of colons showed mild
edemas in all sick mice. A microscopic examination revealed mild
polymorphonuclear leukocyte infiltration, thinning of the intestinal
wall, or necrotic foci involving the entire intestinal wall. The
destruction of the mucous layer was observed in these mice by SEM
examinations.
Microhemorrhages, thrombosis, and edematous changes of the capillary
endothelia were observed in the brains of moribund mice
which were
showing neurological symptoms, especially in the group
subjected to
EHEC plus TNF-
(Fig.
1A). Several
neural cells looked
globular, and a strong degenerative change was
observed in these
mice. Sometimes, endothelial-like cell proliferation
was observed
in the brains of mice in this group (Fig.
1B). The
pathological
changes were mild in symptomatic mice subjected to EHEC
infection
only. Edemas of the brain were seen in these mice. It seemed
that
NM treatment was effective in inhibiting neural pathological
changes
after EHEC infection.
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FIG. 1.
Histopathological changes in a symptomatic mouse with
EHEC infection and TNF- treatment. Bars: 10.0 µm. (A) Edematous
change of the brain with microthrombosis at 7 days after infection. The
capillary endothelial cells also show edematous changes. In several
neural cells, globular, spindled, and degenerative changes are
observed. (B) Proliferation of endothelial-like cells in the brain. (C)
Microhemorrhages, thrombosis, and proliferation of glomerular mesangial
cells. The necrosis of tubular cells seen in this mouse is a common
histological change after EHEC infection, regardless of treatment.
|
|
The kidneys of symptomatic mice with EHEC infection were pale and
swollen. Histopathologic changes in the kidney sections
from
TNF-treated mice included focal proliferation of glomerular
mesangial
cells and increased deposition of mesangial matrix.
The changes of the
kidney in these experiments were similar to
those described by Karpman
et al. (
14). Slight proliferations
of glomerular mesangial
cells were seen in mice before the appearance
of clinical signs (day 1 after infection). The proliferation of
glomerular mesangial cells,
hemorrhages, and microthrombosis in
the TNF-treated mice was observed
on day 7 after infection (Fig.
1C). Focal proliferation of glomerular
mesangial cells was mild
in symptomatic mice subjected to EHEC
infection only. In NM-treated
mice, glomerular pathology was not
observed. Necrosis of tubular
cells was noted in all symptomatic mice.
Histopathologic changes were not observed in the colon, kidney, and
brain tissue from asymptomatic mice inoculated with the
strain or from
controls (PBS-treated mice and
E. coli MV1184-inoculated
mice).
In vivo colonization.
When 2.0 × 109 CFU of
EHEC strain EDL 931 was fed individually to germ-free mice, the
bacterium colonized the feces of the groups equally well
(109 to 1010 CFU/g of feces) for the
experimental period (Fig. 2). There were no significant differences in colonization among the three groups: EHEC
infection only, EHEC infection plus TNF treatment, and EHEC infection
plus NM treatment (data not shown). In contrast, the bacteria dropped
to undetectable numbers (<102 CFU/g of feces) by 3 days
postinoculation in flora-positive ICR mice. When 2.0 × 102 CFU of strain EDL 931 was fed to germ-free IQI mice,
the number of CFU of the strain in feces began to rise rapidly on day 1 until a stable level of colonization (108 to
109 CFU/g of feces) was maintained. Intestinal colonization
was examined by SEM. EHEC O157:H7 was randomly seen in the feces at
various sites. No aggregation of EHEC was observed.
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FIG. 2.
Pattern of colonization of germ-free mice by EHEC
O157:H7 strain EDL 931. At the times indicated, fecal samples were
plated on Chromagar. The bacterial detection limit for assessment of
initial colonization was 102 CFU/g of feces. Each point
represents the mean CFU per gram ± the standard deviation.  ,
IQI mice inoculated with EHEC 157:H7 (2.0 × 109
CFU/mouse);  , IQI mice inoculated with EHEC 157:H7 (2.0 × 102 CFU/mouse);  , ICR mice inoculated with EHEC 157:H7
(2.0 × 109 CFU/mouse);  , IQI mice inoculated with
E. coli MV1184 (2.4 × 109 CFU/mouse).
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Positive cultures were obtained from the stomach, small intestines, and
large intestines of infected mice. Blood cultures
were negative for
both symptomatic and asymptomatic mice. Cultures
from the kidney and
brain were also negative for these mice.
SLT level in the feces.
SLT was detected in the feces but not
in the serum. As shown in Fig. 3, SLT was
detected by ELISA at levels of more than 10 ng/g of feces, because
optical densities were 0.409 and 0.947 for standard SLT-I (1,000 pg/ml)
and SLT-II (1,000 pg/ml), respectively. SLT levels in feces of mice
inoculated with 2.0 × 109 CFU of EHEC were
significantly higher than those in the feces of mice inoculated with
2.0 × 102 CFU of EHEC on days 1 and 7 after
inoculation. SLT levels in the feces of IQI mice at 5 to 7 days after
inoculation with EHEC were similar to those in the feces of TNF-treated
mice (Table 2). Lower SLT levels were
detected in NM-treated IQI mice and IQI mice inoculated with 2.0 × 102 CFU of EHEC than in IQI mice inoculated with
2.0 × 109 CFU of EHEC. There were no significant
differences in SLT level between symptomatic and asymptomatic mice in
each group. Both SLT-I and SLT-II were detected by RPLA when the
samples were positive by ELISA. There was no detection of SLT in
control groups.
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FIG. 3.
Changes of SLT level in the feces of experimental IQI
mice. , EHEC O157:H7 inoculation at 2.0 × 109
CFU/mouse; , EHEC O157:H7 inoculation at 2.0 × 102
CFU/mouse.
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|
Cytokine responses in the serum, kidneys, and brains.
Serum
cytokines TNF- and IL-6 were detected in TNF-treated mice at 7 days,
but not in the mice at 1 day, after inoculation of EHEC. The levels of
TNF- and IL-6 were 179 ± 53 and 240 ± 90 pg/ml,
respectively. They were significantly higher than those for negative
controls. In the EHEC-only group, the EHEC plus NM group, and the EHEC
plus TNF- plus NM group, no proinflammatory cytokines were
detectable in the serum at days 1 and 7 after inoculation of EHEC.
Table
3 shows cytokine levels of the
kidneys on days 1 and 7 after infection. A TNF-
response, but not
IL-1
and IL-6 responses,
was seen at 1 day after EHEC infection. At
7 days after infection,
TNF-
, IL-1
, and IL-6 were detected in the
kidneys of mice with
systemic clinical signs. In the EHEC plus TNF-
group, proinflammatory
cytokines were clearly detected in the kidneys
at 1 and 7 days
after infection. In the EHEC plus NM group, cytokine
induction
was different than it was in the other two groups. The EHEC
plus
TNF-
plus NM group showed lower cytokine levels than the EHEC
plus TNF-
group, although mice in this group had severe clinical
signs.
As shown in Table
4, brain cytokines were
detected in IQI mice with or without TNF-
after EHEC infection but
not in NM-treated
mice. In the EHEC-only group, only TNF-
was
detectable in the
brain. Levels of TNF-
, IL-1
, and IL-6 in the
EHEC plus TNF-
group were significantly higher than those in other
groups at
1 day after EHEC infection (
P < 0.01).
TNF-
and IL-6 were detected
in the brains of mice in the EHEC plus
TNF-
group at 7 days after
EHEC infection.
No cytokines were detected in the serum, kidneys, and brains of MV1184
controls and flora-positive controls. IQI mice inoculated
with 2.0 × 10
2 CFU of EHEC also showed no cytokine responses,
similar to the
controls.
| |
DISCUSSION |
TNF- treatment causes severe damage to the target organs in the
EHEC mouse model. In contrast, a protease inhibitor had an inhibitory
effect on pathological symptoms in the model. Therefore, the inhibition
of TNF processing could act as an effective therapeutic agent in vivo
but did not eliminate EHEC and the organ failure associated with EHEC
infection (direct action of SLTs). The combination of an SLT-specific
inhibitor with a TNF inhibitor will be required to prevent HUS and
severe neurologic symptoms.
It has been reported that TNF treatment of human vascular endothelial
cells leads to enhanced biosynthesis of the SLT receptor, thereby
sensitizing cells to the cytotoxic action of SLT (25). Several hours' exposure to TNF- was enough to enhance the number of
SLT receptors (10- to 100-fold) on the endothelial cells
(25). NM treatment was not effective in decreasing the
severity of pathological changes in the target organs and lethality for
IQI mice treated with TNF-. NM could inhibit the synthesis of
TNF- but could not inhibit the action of inoculated TNF-.
Purified SLTs induced expression of proinflammatory cytokines from
peritoneal macrophages (23). Harel et al. showed that SLT
acts to induce TNF synthesis within the kidney and at the same time
increases renal sensitivity to the toxic effects of TNF (8).
We agree that local synthesis of TNF within the kidney may contribute
to renal injury. These observations would suggest an interaction
between SLT and cells capable of responding to the toxins by
synthesizing TNF-.
When EHEC was inoculated into gnotobiotic mice, no circulating TNF
could be detected by ELISA of serum samples. In contrast, TNF
production occurred within specific tissues such as the kidney and
brain. In the mice treated with TNF, strong cytokine responses were
recognized in the kidney before and after symptoms.
Cytokine levels in the brains of TNF-treated mice were significantly
higher than those in the brains of mice of other groups at day 1. At 7 days after EHEC infection, all of the groups showed local TNF
responses. Brain lesions were severe in TNF-treated mice but mild in
mice not treated with TNF. It has been suggested that the state of cell
differentiation or activation (including the cell type) is important in
determining the cellular response to SLT (19). In the
response to inflammatory stimuli, the presence of microbial products
such as SLT and host factors resulted in further proinflammatory
cytokine synthesis and tissue injury in the central nervous systems.
The results of this study suggest the following pathogenesis. The
bacteria are established in the alimentary tract. The destruction of
the mucous layer and/or focal necrosis of the gut wall may allow
bacterial components to cross the damaged intestinal wall and reach the
bloodstream. Whether EHEC O157:H7 strains can invade through an intact
intestinal mucosal barrier has been discussed (17). However,
we could not observe bacteremia in our mouse model. Actually, positive
blood cultures are rarely found for patients with HUS. Furthermore, IQI
mice with intact mucous layers were healthy after inoculation with
2.0 × 102 CFU of EHEC. Thus, bacteremia did not
appear to be essential for the development of HUS and neurologic
symptoms, and the destruction of the mucous layer could be important
for the entry of SLTs and bacterial components.
Symptoms and pathology in the target organs may be caused by the spread
of bacterial components. SLT is most important because pathological
events similar to those in experimental animals inoculated with EHEC
can occur in animals to which the toxin is administered (5, 15,
21). We found the highest frequency of symptoms in TNF-treated
mice infected with EHEC. This group of mice also developed the most
severe combination of renal and neural symptoms, as in human cases,
suggesting that SLT and TNF have an additive or synergistic effect.
Presumably, in HUS and encephalopathy, TNF levels are elevated in
response to circulating SLT and/or endotoxin. TNF produced in response
to toxins may then act to upregulate toxin receptor levels in target
(vascular endothelial) cells.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Preventive Dentistry, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu 1757, Hokkaido 061-02, Japan. Phone: 81-13-323-1404. Fax: 81-11-884-0184. E-mail:
emikoisogai{at}msn.com.
Editor: R. E. McCallum
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Infect Immun, January 1998, p. 197-202, Vol. 66, No. 1
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
