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CUTTING EDGE |
B-Deficient Mice1
,
*
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139;
Department of Pathology, Immunology Research Division, Brigham and Women’s Hospital, Boston, MA 02115; and
Division of Emergency Medicine, Children’s Hospital, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a central pathway in many inflammatory disorders,
including colitis. Increased NF-B activity has been linked with
development of colitis in humans and animal models, thus it was
unexpected when NF-B-deficient mice developed spontaneous
typhlocolitis. To further characterize this finding, we induced
typhlocolitis in rederived NF-B-deficient mice using intragastric
infection with Helicobacter hepaticus. At 6 wk
postinfection (PI), severe colitis with increased type 1 cytokine
expression was seen in infected mice that lacked the p50 subunit of
NF-B and were also heterozygous for the p65 subunit of
NF-B(p50-/-p65+/-). Mice lacking the p50
subunit alone (p50-/-) were less severely affected, and
wild-type mice and p65+/- mice were unaffected. T cell
development in NF-B-deficient mice was normal. These data indicate
that p50 and p65 subunits of NF-B have an unexpected role in
inhibiting the development of colitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that the transcriptional regulator NF-B may
play a key role in regulating inflammatory gene expression in the
colon. NF-B is a family of five proteins related by the Rel homology
domain that have been implicated as a central regulator of
proinflammatory gene expression (6). These proteins are
found as homo- or heterodimeric complexes in the cytoplasm of most
cells, bound to members of the IB family that inhibit nuclear
translocation of NF-B. Many inflammatory signals activate a set of
IB kinases which phosphorylate IB molecules, leading
to their targeted degradation (7). This process frees
NF-B complexes to translocate to the nucleus and influence
transcription of key inflammatory cytokines including TNF-
, IL-1,
and IL-12 (6).
Increased NF-B activity and cytokine expression have been observed
in macrophages from human Crohn’s disease patients and in several
mouse models of colitis (8, 9). Increased NF-B
DNA-binding activity in macrophages from mice with colitis has been
attributed predominantly to increased binding of the p50 and p65
subunits. Indeed, increased cytokine expression in mice with
2,4,6-trinitrobenzene sulfonic acid-induced colitis was inhibited by
antisense oligonucleotides that inhibit p65 expression
(10).These studies suggest a central role for NF-B in
mediating expression of proinflammatory cytokines, which is an
important component of the pathology associated with IBD.
Recently, several mouse models lacking NF-B family members have been
developed (11, 12). Although mice lacking p50 are viable,
mice lacking p65 die during embryogenesis. However, mice homozygously
deficient for p50 (p50-/-) and also
heterozygous for p65
(p50-/-p65+/-) are
viable. We have been studying these mice and were surprised to find
that both p50-/- and
p50-/-p65+/- mice
developed spontaneous typhlocolitis when housed in a virus Ab-free
(VAF) colony. These mice were rederived into a helicobacter-free colony
because some mouse models develop spontaneous enterocolitis that is
reduced under defined microflora conditions and is inducible with
targeted reinfection with intestinal microorganisms
(2, 3, 4, 5). Mice were then infected with an enteric
helicobacter to provide a reproducible model of colitis. In this study,
we examine the development of typhlocolitis in rederived
NF-B-deficient mice experimentally infected with Helicobacter
hepaticus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All mice were housed in Association for the Assessment and Accreditation of Laboratory Animal Care approved facilities. Mice that developed spontaneous colitis were murine VAF, were naturally infected with opportunistic pathogens including Pasteurella pneumotropica and Helicobacter rodentium, but were not infected with H. hepaticus or Helicobacter bilis.
Rederivation of NF-B mutant mice
For experimental infection, mice were rederived using uterine
transfer of 3.5-day embryos of p50 and p65 mutants on a mixed (129
x C57BL/6) background. NF-B mutant and wild-type (WT) mice were
maintained under conditions free of known Helicobacter
species.
Experimental infection
H. hepaticus (type strain 3B1) was grown as described elsewhere (13). Cultures were examined by Gram stain and phase microscopy for contaminants and subcultured on blood agar to confirm purity. Bacteria were resuspended in Brucella broth with 30% glycerol at 108 bacteria/ml as confirmed by spectrophotometry (2). Experimental mice received 0.2 ml of fresh inoculum by gastric gavage every other day for three doses.
At age 6–8 wk, 64 helicobacter-free mice were dosed by gastric gavage with H. hepaticus suspended in broth and 40 were dosed with broth only. The infected and sham mice were housed in microisolator caging in different areas within the animal facility. These data were compiled from four replicate experiments using similar numbers of mice with comparable results for each group.
Confirmation of H. hepaticus infection
Cecal tissue was collected at necropsy and analyzed by PCR to confirm experimental infection using H. hepaticus-specific primers (14). Helicobacter-free status was confirmed in controls using PCR with Helicobacter genus-specific primers.
Histological evaluation
Formalin-fixed tissues were embedded in paraffin, cut at 5-µm sections, and stained with hematoxylin and eosin for assessment of morphology. A board-certified veterinary pathologist blinded to sample identity evaluated tissues. The cecal and colonic lesions were scored on the basis of size and frequency of hyperplastic and inflammatory lesions on a scale of 0–4, with ascending severity modified from Berg et al. (15).
Immunophenotypic characterization
Tissues were frozen in OCT in liquid nitrogen and cut into 5-µm sections using a Leica CM3050 cryostat. Slides were incubated with methanol and 3% hydrogen peroxide for 30 min, followed by 20% goat serum for 30 min and then washed with PBS. Primary Ab (rat anti-CD3 for T cells or rat anti-F4/80 for macrophages; Vector Laboratories, Burlingame, CA) or nonimmune rat serum was used at 1:10 for 60 min. For F4/80 staining, slides were incubated in HRP-conjugated goat anti-rat Ab at 1:25 for 30 min and diaminobenzidine was used as a color substrate. For anti-CD3 staining, slides were incubated with rabbit anti-rat Ab (1:100) for 60 min, followed by AP-conjugated goat anti-rabbit Ab at 1:500 for 60 min. Vector red was used as a color substrate.
Detection of cytokine mRNA expression in colon
One-centimeter segments of colon were harvested immediately upon euthanasia and snap frozen in liquid nitrogen. Frozen specimens were homogenized into Tri-reagent (Molecular Research Center, Cincinnati, OH) and RNA was prepared per the manufacturer’s instructions. RNase protection analyses were performed on 20 µg of total RNA using RiboQuant MultiProbe Template Sets (PharMingen, San Diego, CA). Intensities of the protected fragments were quantitated by phosphor imager analysis and normalized to internal controls.
Statistical analyses
Analysis of cecal and colonic lesion scores was performed using a Mann-Whitney U nonparametric test for categorical data. Data on RNase protection assays was analyzed using a two-tailed t test for nonequal sample size.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-deficient mice spontaneously develop typhlocolitis
Spontaneous typhlocolitis was observed in a colony of VAF
NF-B-deficient mice at age >6 mo. Severity of disease was assessed
histologically using a scale of 0–4 described in Materials and
Methods. Although WT mice (n = 4) cohoused with
NF-B-deficient mice were clinically normal,
p50-/-p65+/- mice had
severe diarrhea, colonic perforation, peritonitis, and typhlocolitis
(n = 9; cecum, p = 0.013; colon,
p = 0.032). Mice lacking p50
(p50-/-) alone had a more mild disease
(n = 9; cecum, p = 0.047; colon,
p = 0.623). Mice heterozygous for p65
(p65+/-) did not have significant clinical or
histological (n = 9; cecum, p = 0.215;
colon, p = 0.361) evidence of typhlocolitis. These
results suggested that the absence of certain subunits of NF-B could
predispose mice to the development of typhlocolitis.
Mice lacking NF-B are highly sensitive to typhlocolitis induced
by H. hepaticus
To determine whether typhlocolitis was experimentally reproducible
using infection with H. hepaticus as previously described
(2, 3, 4, 5), NF-B-deficient mice were embryo transfer
rederived into a helicobacter-free environment. Spontaneous
typhlocolitis was not observed in the helicobacter-free
NF-B-deficient colony. Mutant and control mice were then
experimentally infected with H. hepaticus. Two weeks after
intragastric inoculation with H. hepaticus,
p50-/-p65+/- mice
developed severe diarrhea. Six weeks after H. hepaticus
infection, approximately one-quarter of the
p50-/- mice also developed diarrhea. All
p65+/- and WT animals remained clinically
well.
To evaluate experimental animals for the presence and severity of
colitis, sections of cecum and colon were evaluated histologically.
Representative sections from infected and uninfected WT,
p50-/-, and
p50-/-p65+/- are shown
in Fig. 1
. The severity of typhlocolitis
was scored as described above. There was a correlation between
genotype, helicobacter status, and pathology score (Table I). The typhlocolitis was
significantly more severe in
p50-/-p65+/-
(n = 19; cecum, p < 0.00001; colon,
p < 0.00001) and p50-/-
(n = 15; cecum, p < 0.00001; colon,
p = 0.0001) compared with infected WT controls.
Furthermore, there was significantly more severe inflammation in
p50-/-p65+/- (cecum,
p = 0.0007; colon, p < 0.00001)
compared with p50-/- mice, suggesting that the
additional loss of a p65 allele exacerbated disease
severity. There was minimal inflammation in uninfected mice of all
genotypes.
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a). Three (3/19) mice had
colonic perforations indicated grossly by stricture and adhesions to
other viscera. Histologically, there was extensive, severe chronic
active transmural inflammation with marked distortion and replacement
of the normal architecture (Fig. 2b). Within these regions,
the thickened mucosa was characterized by extensive ulcerations,
elongated irregularly shaped glands, and crypt abscesses. The lamina
propria (LP) was expanded by fibrosis with effacing infiltrate of
granulocytes, macrophages, lymphocytes, and occasional microgranulomas.
Lymphofollicular hyperplasia and plasmacytes were infrequent in these
mice. The submucosa and serosa were frequently expanded and effaced by
dense inflammatory infiltrates, granulation tissue, dilated lymphatics,
and multifocal necrotizing vasculitis with thrombosis. Extensive areas
of necrosis and edema within these layers resulted in complete loss of
bowel wall integrity with extension of the lesions into adjacent
mesenteric lymph nodes and mesenteric vessels. These dysplastic regions
were interspersed with regions of mild to moderately severe colitis
with mixed inflammatory infiltrates (Fig. 2c). Although few
macrophages or T lymphocytes were seen in the LP of infected WT mice,
macrophages (F4/80+) were prevalent and T
lymphocytes (CD3+) could be identified in
scattered foci throughout the LP (Fig. 2, d–g) in infected
NF-B-deficient mice.
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To examine cytokine expression in NF-B-deficient mice with
helicobacter-induced typhlocolitis, total RNA was isolated from
specimens of colon and inflammatory gene expression was analyzed by
RNase protection analysis (Fig. 3). There
were statistically significant increases in the expression of IFN-
,
IL-12p40, TNF-, IL-1
, monocyte chemoattractant protein-1,
IFN--inducible protein-10, and macrophage-inflammatory protein-2 in
H. hepaticus-infected
p50-/-p65+/- and
p50-/- mice compared with infected WT mice.
Furthermore, there were significant increases in the expression of
TGF- and IL-1R antagonist (IL-1Ra) in infected
p50-/-p65+/-, but not
p50-/-, compared with infected WT mice. There
were statistically significant increases in the expression of TNF-,
IL-1, IFN-inducible protein-10, macrophage-inflammatory protein-2,
and IL-1Ra in infected
p50-/-p65+/- mice
compared with infected p50-/-. Little
inflammatory gene expression was observed in uninfected animals of any
group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B mutant
mice was rapidly reproducible using targeted infection with H.
hepaticus. Typhlocolitis involved extensive epithelial damage and
effacing inflammatory infiltrates. Increased expression of type 1
cytokines resembled findings in IL-10-deficient mice with colitis and
in humans with Crohn’s disease (4, 16). Colitis was
inducible using H. hepaticus in helicobacter-free mutant
mice, as described in IL-10-deficient mice by Kullberg et al.
(4), further supporting a role for intestinal microflora
in the induction of IBD. Inability to induce colitis with H.
hepaticus in some mouse models may be due to variations in
methodologies, host genotype, or constitutive intestinal microflora
(17).
Typhlocolitis in NF-B-deficient mice was unexpected because NF-B
subunits p50 and p65 have been previously implicated as promoters of
IBD (8, 9). Indeed, antisense p65 oligonucleotides have
been proposed to abrogate the severity of 2,4,6-trinitrobenzene
sulfonic acid-induced colitis (10). However, the findings
of increased inflammatory gene expression in this study indicate robust
inflammatory gene expression can occur in
p50-/- or
p50-/-p65+/- mice.
Thus, it appears that mice lacking p50 exhibit a defect in a pathway
that inhibits the development of colitis but this does not preclude the
possibility that other NF-B subunits could also play a reciprocal
proinflammatory role.
The mechanism by which p50 inhibits inflammation is not clear. Unlike
p65, p50 does not have intrinsic trans-activating potential.
It has been suggested that p50 homodimers have the ability to directly
inhibit gene expression. Also, p50 appears to suppress TNF-
expression after LPS challenge of peritoneal exudative macrophages
(18). Increased expression of TNF- or other
inflammatory cytokines after exposure of NF-B-deficient mice to
H. hepaticus could lead to excessive inflammation and the
development of typhlocolitis. However, it is not known whether
increased inflammatory gene expression is an initiating factor in the
development of disease. In contrast to a role as an inhibitor of
inflammatory gene expression, p50 could be required to promote
expression of a cytokine that inhibits inflammation such as IL-10,
TGF-, or IL-1Ra. However increased expression of IL-1Ra and TGF-
observed in the colons of NF-B-deficient mice with colitis make it
unlikely that p50 is necessary for regulation of these genes.
The p50-deficient mice used in this study also lack the p50 precursor
protein p105. The C-terminal domain of p105 has homology to IB
family members and has been shown to function as an IB in vivo
(19). Therefore, the proinflammatory phenotype observed in
p50-deficient mice could be related to defective p105 inhibitory
function. Indeed, mice lacking the C-terminal IB-like domain of
p105 but retaining p50 exhibit lymphoid infiltration into skin
and organs, although they have not been reported to develop colitis
(20).
Defects in T cell development in the absence of p50 could
predispose animals to the development of colitis (21, 22).
However, we found normal distributions of CD4 and CD8 lymphocytes in
the thymus and the spleen. Also, the percentage of
CD25+CD4+CD8-
T cells, which have been associated with repressive activity (23, 24)was normal in the thymus and the periphery (data not shown).
Furthermore, H. hepaticus does not appear to induce
generalized T cell activation, as the majority of
CD4+ T cells in the mesenteric lymph node of
infected mice were
CD62LhighCD69low (data not
shown). Thus, defects in the development of regulatory T cell
populations do not appear to be responsible for the susceptibility of
these mice to colitis, although function of regulatory T cells in
NF-B-deficient mice has not been examined.
In addition to certain regulatory T cell populations, B cells may also
play a role in the development of IBD (25, 26). Although
there are normal numbers of mature B cells in
p50-/- and
p50-/-p65+/- mice
(data not shown), it has previously been shown that
p50-/- mice lack marginal zone B cells
(27) and p50-deficient B cells have severe defects in
class switching and secretion of IgA in vivo and in vitro (11, 28). Indeed, lymphofollicular hyperplasia and plasmacytosis,
prominent features of colitis in other murine models (15),
were not features of typhlocolitis in the NF-B-deficient mice (data
not shown). These observations may represent a manifestation of
defective B cell function in NF-B mutants that is important in the
etiology of colitis.
It is unclear how the loss of one of the two alleles of p65 exacerbated colitis in this model. Mice heterozygous for p65 alone do not develop colitis and, unlike p50, inhibitory activity has not been demonstrated for the p65 subunit. The loss of one allele of p65 in addition to both alleles of p50 could lead to a gene dosage effect that increases susceptibility to colitis, or the loss of one allele of p65 could dysregulate an independent process that is only required in the absence of p50.
The findings reported here suggest that NF-B plays a critical role
in suppressing the development of IBD and have led us to hypothesize
that NF-B activation initiates a feedback loop that inhibits the
development of chronic inflammation in the colon. These observations
could lead to important insights into the pathophysiology of IBD in
humans and have implications for directed therapeutic strategies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Susan E. Erdman, Division of Comparative Medicine, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139.
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; VAF, virus Ab free; WT, wild type; PI, postinfection; LP, lamina propria; IL-1Ra, IL-1R antagonist.
Received for publication September 20, 2000. Accepted for publication November 21, 2000.
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 D. Haller, M. P. Russo, R. B. Sartor, and C. Jobin IKKbeta and Phosphatidylinositol 3-Kinase/Akt Participate in Non-pathogenic Gram-negative Enteric Bacteria-induced RelA Phosphorylation and NF-kappa B Activation in Both Primary and Intestinal Epithelial Cell Lines J. Biol. Chem., October 4, 2002; 277(41): 38168 - 38178. [Abstract] [Full Text] [PDF]
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),
p50-/- (
), or p50-/-p65+/-
(
) mice at 4–6 wk PI. Cytokine expression was analyzed by RNase
protection assays. Intensity of protected fragments were quantitated on
a phosphor imager and normalized to internal controls. The individual
cytokines are shown on the x-axis and the relative
expression compared with the internal controls is represented by the
y-axis, to the right of each group. The number at the
top of each y-axis indicates the maximal ratio
represented by that axis. Each group consists of four or five animals
and SD is shown.