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B Family Members in Control of Helminth Infection and Intestinal Inflammation1
* Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104;
Medical Research Council Center for Immune Regulation, School of Medicine, University of Birmingham, Birmingham, United Kingdom; and
Department of Medicine, Division of Immunology, Cornell University Medical College, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B family of transcription factors is critical in
controlling the expression of a wide range of immune response genes.
However, whether individual family members perform specific roles in
regulating immunity and inflammation remains unclear. Here we
investigated the requirement for NF-B1, NF-B2, and c-Rel in the
expression of Th2 cytokine responses, development of host protective
immunity, and regulation of intestinal inflammation following infection
with the gut-dwelling helminth parasite Trichuris muris.
While mice deficient in c-Rel mounted sufficient Th2 responses to expel
infection, NF-B1 knockout (KO) and NF-B2 KO mice developed
chronic infections associated with elevated production of Ag-specific
IFN-
. However, only infected NF-B1 KO mice exhibited polarized
IFN- responses associated with the loss of intestinal goblet cells
and the development of destructive colitis-like pathology. Furthermore,
blockade of IL-12 (previously shown to confer resistance in susceptible
strains) recovered Ag-specific IL-13 responses and resistance to
infection in NF-B2 KO, but not NF-B1 KO mice. Therefore, unique
infection, immunological, and pathological outcomes were observed in
different NF-B KO strains. Taken together, these results
provide direct evidence of nonoverlapping functions for NF-B
family members in the development of Th2 cytokine-mediated resistance
to T. muris and the control of infection-induced
intestinal inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B family of transcription factors (composed of NF-B1
(p105/p50), NF-B2 (p100/p52), c-Rel, RelA, and RelB) regulates the
expression of a wide range of immune response genes whose products play
a critical role in orchestrating inflammatory responses (1, 2). Homo- or heterodimeric forms of NF-B are sequestered in
the cytoplasm of resting cells by the IB family of proteins.
Following exposure of cells to infectious or inflammatory stimuli,
IB is phosphorylated and degraded, allowing NF-B to translocate
to the nucleus and influence the transcription of a wide range of
immune response genes (3, 4). In addition to controlling
the expression of adhesion molecules and chemokines, NF-B can
regulate the expression of cytokines that promote Th1 (IL-12 and
IFN-) and Th2 (IL-4, IL-13) responses (5, 6, 7, 8, 9).
Therefore, NF-B is a key transcription factor in regulating immune
responses and pathogenesis associated with asthma, autoimmunity, and
graft rejection (10, 11, 12). In addition, NF-B activation
is essential in initiating innate and adaptive immune responses to
intracellular pathogens (13), although its role in
immunity to helminth parasites has not been investigated.
NF-B is also critical in regulating inflammation in the intestine.
Activation of NF-B and expression of inflammatory cytokines has been
reported in human and murine models of inflammatory bowel disease
(14, 15). Furthermore, studies in models of colitis have
demonstrated that blockade NF-B1 enhanced
Helicobacter-induced intestinal inflammation, while
disruption of RelA has been shown to either ameliorate or exacerbate
colitis (16, 17). Together, these reports suggest NF-B
plays a complex role in regulating inflammation within the intestinal
microenvironment. Infection with the gut-dwelling helminth parasite
Trichuris muris is also associated with intestinal
inflammation, characterized by a mild inflammatory cell infiltrate and
the development of crypt hyperplasia (18, 19). T.
muris is a natural pathogen of mice, inhabiting the cecum and
large intestine, and provides a well-defined model of human infection
and disease (20, 21). Expulsion of infection requires the
initiation of Th2 cytokine responses (IL-4, IL-9, and IL-13), while
susceptibility to chronic infection is promoted by type 1 cytokines
(IL-12, IL-18, and IFN-) (22, 23, 24, 25). In this study we
have focused on the role of NF-B in transcriptional regulation of
resistance to infection and the control of helminth-induced gut
pathology.
Our results identify novel differential requirements for NF-B1,
NF-B2, and c-Rel in the regulation of Th cell responses, protective
immunity, and control of intestinal inflammation following T.
muris infection. Although Th2 cytokine responses were reduced in
all NF-B knockout
(KO)3 strains,
c-Rel-deficient mice mounted sufficient type 2 cytokine responses to
clear infection. In contrast, NF-B1 KO and NF-B2 KO mice failed
to clear infection, while only chronically infected NF-B1 KO mice
developed destructive colitis-like pathology. Moreover, blockade of
endogenous IL-12 recovered Ag-specific IL-13 responses and resistance
to infection in NF-B2 KO mice, but not NF-B1 KO mice, suggesting
that NF-B2 played an indirect role while NF-B1 played a direct
role in regulating Th2 cytokine responses and host resistance. The
nonoverlapping functions of individual family members suggest that
targeting specific NF-B transcription factors will be a useful
therapeutic approach in manipulating cytokine responses and
inflammation in infectious and other disease states.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following genetically manipulated mice were generated as
previously described and maintained in specific-pathogen free
conditions at University of Pennsylvania: NF-B1 KO (from Drs. D.
Baltimore and W. Sha, Berkeley, CA) (26), NF-B2 KO
(from Dr. J. Caamaño, University of Birmingham, Birmingham, U.K.)
(27), and c-Rel KO (from Dr. H. C. Liou, Cornell
University Medical College, New York, NY) (28). Unless
otherwise stated, all mice were backcrossed to a B6 background. In some
experiments NF-B1 KO mice maintained as random B6 x 129
hybrids were used (purchased from The Jackson Laboratory, Bar Harbor,
ME). Wild-type (WT) B6 and B6 x 129 F2 mice
were purchased from The Jackson Laboratory. No differences between
NF-B1 KO mice on a mixed or pure B6 background were observed in
these studies. In all experiments mice were infected between 6–10 wk
of age, and experimental groups contained four to six mice. Animals
were maintained under specific pathogen-free conditions, and no signs
of intestinal inflammation were observed in any animals before
infection. All experiments were performed under the regulations of the
University of Pennsylvania institutional animal care and use
committee.
Parasite
The maintenance, infection, and recovery of T. muris were described previously (29). Mice were infected on day 0 with 150–200 embryonated eggs, and equivalent establishment of infection in different mouse strains was determined on day 10 postinfection (p.i.). Assessment of worm burdens and preparation of T. muris excretory-secretory Ag for use in restimulation assays were previously described (23).
EMSAs
Mesenteric LN cells were isolated from naive and infected mice,
and purified T cells were obtained using murine
CD3+ T cell enrichment columns following the
manufacturer’s instructions (R&D Systems, Minneapolis, MN). Cell
purity was determined by staining with fluorochrome-labeled
anti-CD3 mAb (BD PharMingen, San Diego, CA). Cells were acquired on
a FACSCalibur flow cytometer and analyzed using CellQuest software (BD
Biosciences, San Jose, CA). CD3+ T cell purities
between 85 and 94% were routinely obtained using this protocol.
Nuclear extracts were prepared from purified CD3+
T cells as previously described (30) and used for EMSA. In
brief, double-stranded oligodeoxynucleotides corresponding to the
palindromic B site (5'-GGGAATTCCC-3') were labeled by filling the
overlapping ends with the Klenow fragment of DNA polymerase I and
[
-32P]dCTP. Following removal of
unincorporated nucleotides, labeled oligonucleotide (50,000 cpm) was
incubated with 6 µg protein extracts and 2 µg poly(dI-dC) in buffer
containing 20 mM HEPES (pH 7.9), 100 mM NaCl, 5 mM
MgCl2, 1 mM DTT, 0.7 mM PMSF, and 17% glycerol
in a final volume of 22 µl for 15 min at 20°C. Complexes were
separated on 5.5% polyacrylamide gels run on 0.25% Tris-borate-EDTA
buffer, dried, and exposed to Kodak X-OMAT AR film (Rochester, NY) at
-70°C.
In vivo depletions
One milligram of neutralizing anti-IL-12 mAb (C17.8; from Dr. G. Trinchieri, formerly at Wistar Institute, Philadelphia, PA) was administered i.p. on days 4, 8, 12, 16, and 20 p.i. This treatment regimen confers resistance against T. muris in mice genetically susceptible to infection (Dr. R. Grencis, University of Manchester, Manchester, U.K., personal communication). Control mice received equivalent amounts of purified rat IgG (Sigma, St. Louis, MO).
Cell culture and cytokine analysis
At necropsy, mesenteric LN were harvested, and single-cell suspensions were prepared in DMEM supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 25 mM HEPES, and 5 x 10-5 M 2-ME. Cells were plated at 5 x 106/ml in 24-well culture plates in medium alone or in the presence of T. muris ES Ag (50 µg/ml). In addition, 5 µg/ml anti-IL-4R mAb (BD PharMingen) was added to cultures to enhance detection of IL-4.
Cell-free supernatants were harvested after 24 h, and cytokine
analysis was conducted by sandwich ELISA using paired mAb to detect
IL-4 (11B11 and BVD6-24G2.3; cell line from Dr. T. Mormon, University
of Rochester Medical Center, Rochester, NY) and IL-13 (R&D Systems).
IFN- production was assessed using anti-IFN- mAb R46A2 (Dr.
T. Mosmann) and polyclonal rabbit anti-IFN-, followed by
peroxidase-conjugated donkey anti-rabbit polyclonal Ab (Jackson
ImmunoResearch Laboratories, West Grove, PA).
Histology
One-centimeter segments of mid-cecum were removed, washed in sterile PBS, and fixed for 24 h in 10% neutral buffered formalin. Tissues were processed routinely and were paraffin-embedded using standard histological techniques. Five-micrometer sections were cut and stained with H&E or Alcian blue-periodic acid for detection of intestinal goblet cells. The numbers of intestinal goblet cells per 20 crypt units were assessed under light microscopy.
Statistical analysis
Significant differences (p < 0.05) between experimental groups were determined using the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is activated following T. muris infection
A number of reports have demonstrated NF-B activation following
infection with viral, bacterial, and protozoan pathogens (reviewed in
Ref. 13). To determine whether NF-B is activated
following T. muris infection, WT B6 mice were infected, and
NF-B activation was assessed by EMSA using nuclear extracts prepared
from draining mesenteric LN cells at various times p.i. As shown in
Fig. 1
, NF-B activation was detected
as early as day 7 p.i. and was maintained throughout infection.
The two discrete bands (Fig. 1, black arrows) suggested that at least
two dominant species of NF-B were present following infection. To
our knowledge this is the first report of NF-B activation following
infection with a gut-dwelling helminth parasite.
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B1 KO and
NF-B2 KO mice develop chronic infections
To determine the roles of specific NF-B family members in the
development of Th2 cytokine-dependent resistance to T. muris
infection, B6 WT mice (genetically resistant to T. muris)
and mice deficient in the individual family members NF-B1, NF-B2,
or c-Rel were infected with T. muris, and infection outcome
was monitored. (The embryonic lethality of RelA KO and premature death
of RelB KO mice (31, 32) precluded their inclusion in
these studies.) Analysis of worm burdens
on day 10 p.i. confirmed that establishment of infection
was equivalent in different experimental groups (Table I). As shown in
Fig. 2, WT mice cleared infection by
35 p.i., confirming our previous results (33). Mice
deficient in c-Rel were also successful in expelling T.
muris, with no worms detected by day 35 p.i. In contrast,
NF-B1 KO and NF-B2 KO mice were unable to clear infection and
developed chronic infections with persistent parasites beyond day
35 p.i. (Fig. 2).
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B1 KO mice develop
destructive colitis-like intestinal pathology
While no differences in intestinal morphology or goblet cell
numbers were observed between naive KO and WT animals (Fig. 4 and data
not shown), histological evaluation of the ceca from infected mice
demonstrated distinct pathological outcomes in different KO mice. WT
and c-Rel KO mice showed morphology typical of mouse strains that had
successfully cleared the pathogen, with little or no inflammatory
infiltrate in the lamina propria or mucosa and no crypt hyperplasia
(Fig. 3, A and B).
Mice deficient in NF-B2 had moderate intestinal pathology, with mild
leukocytic inflammation visible in the mucosa and numerous parasites
present in the intestinal lumen (Fig. 3C, red arrow). Crypt
hyperplasia, a characteristic of chronic T. muris infection
previously observed in susceptible mouse strains (19), was
also evident in infected NF-B2 KO mice (Fig. 3C, black
arrow).
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B2 KO and c-Rel KO animals, the pathological
outcome of infection in NF-B1-deficient mice was notably more
severe. A profound inflammatory infiltrate (composed of lymphocytes and
granulocytes) was evident throughout the muscular layers, submucosa,
lamina propria, and mucosa and extended to the serosal surface. Cell
infiltration and edema resulted in significant thickening of these
layers (Fig. 3
D, black arrows). Foci of erosion and
ulceration were also present in the mucosa, associated with neutrophil
infiltration and fibrin deposition. Dramatic crypt hyperplasia was
evident (Fig. 3D), suggesting that marked dysregulation of
epithelial cell proliferation and differentiation was occurring in the
absence of NF-B1. Indeed, the lumenal surface adjacent to ulceration
was almost completely devoid of an epithelial layer (Fig. 3D, red arrows) and displayed pathology reminiscent of that
reported in murine models of inflammatory bowel disease
(34, 35, 36, 37). Therefore, although both NF-B1 KO and
NF-B2 KO mice developed chronic infections, these family members
performed distinct roles in regulating infection-induced intestinal
inflammation.
Significant differences in mucosal goblet cell responses in different
KO mice were also observed following infection. Goblet cell responses
are enhanced by Th2 cytokines (38, 39) and serve as useful
markers of the magnitude of Th2 responses in the intestinal
microenvironment. Intracellular staining of goblet cell mucins
demonstrated that both c-Rel KO and WT mice had significant numbers of
goblet cells in the mucosa, a characteristic of resistant mice that
have developed a protective Th2 response in the intestine and expelled
T. muris (Fig. 3, E and F, red arrows,
and Fig. 4). Goblet cell hyperplasia was
also observed in infected NF-B2 KO mice, although at a significantly
lower magnitude than in WT mice (Fig. 3G, red arrows, and
Fig. 4). In contrast, there was a complete absence of goblet cells
following infection of NF-B1 KO mice (Figs. 3H and 4),
identifying a critical role for NF-B1 in intestinal goblet cell
responses following infection.
NF-B1 KO, NF-B2 KO, and c-Rel KO mice demonstrate
altered Ag-specific cytokine responses following infection
As previously reported (33), Ag-specific
restimulation of mesenteric LN cells from infected B6 WT mice
demonstrated enhanced IL-4 and IL-13 responses with low IFN-
production (Fig. 5, A–C). In
contrast, all NF-B KO mice secreted low levels of IL-4 and IL-13
following restimulation (Fig. 5, A and B). While
low Th2 responses were expected in NF-B1 KO and NF-B2 KO mice due
to their susceptibility to infection, it was surprising that c-Rel KO
mice (that successfully cleared infection) developed poor IL-4 and
IL-13 responses. However, significant differences in production of
Ag-specific IFN- were observed between KO mice (Fig. 5C).
Mice deficient in c-Rel secreted low levels of IFN- (Fig. 5C). In contrast, NF-B1 KO mice developed polarized Th1
responses, with the production of significantly higher levels of
IFN- than observed in WT mice (Fig. 5C). Therefore, the
difference in infection outcome between different KO mice was likely to
reflect subtle differences in the balance of Th1 vs Th2 responses.
Indeed, expression of IL-4/IFN- production as a ratio demonstrated
that although the ratio of IL-4/IFN- was lower in c-Rel KO mice than
in WT animals (Fig. 5D), it was higher than in other KO mice
and was sufficient to mediate worm expulsion (Fig. 2). In contrast, the
lower level of IL-4 and IL-13 production in the context of elevated
IFN- responses observed in NF-B1 KO and NF-B2 KO mice was
reflected in decreased ratio of IL-4/IFN- and was not sufficient to
clear infection (Fig. 5D). A similar pattern in the ratio of
IL-13/IFN- responses was observed (data not shown). T cell
development is normal in the NF-B KO strains used in these studies
(26, 27, 28); in addition, no significant difference in cell
recovery or the ability of cells to proliferate in response to
mitogenic stimuli was observed between KO and WT mice (data not shown).
Therefore, the differences in infection outcome and cytokine responses
observed between different KO mice are unlikely to be due to
alterations in lymphocyte subsets or defects in lymphocyte
proliferation. Rather, differences in the balance of Th1 and Th2
cytokine responses between NF-B KO mice appear to be critical in
determining resistance and susceptibility to infection.
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B2 KO, but not NF-B1 KO, mice
Susceptibility to T. muris infection in NF-B1 KO and
NF-B2 KO mice was associated with enhanced IFN- production in the
absence of Th2 cytokine responses (Fig. 5). To determine whether
blockade of endogenous Th1 responses would allow the expansion of
protective Th2 cytokine responses and subsequent expulsion of T.
muris in these animals, mice deficient in NF-B1 or NF-B2
were infected with T. muris and treated systemically every 4
days with either control Ig or anti-IL-12 mAb. As shown in Fig. 6, anti-IL-12 treatment of NF-B1
KO mice significantly reduced the production of IFN- following
Ag-specific restimulation, but failed to enhance the production of
either IL-4 or IL-13. The failure of anti-IL-12 treatment to
promote Th2 cytokine responses in NF-B1 KO mice resulted in chronic
infections persisting in both control and anti-IL-12-treated
NF-B1 KO mice (Fig. 7A).
Increasing doses of anti-IL-12 and coadministration of
anti-IL-12/anti-IFN- mAb also failed to unmask Ag-specific
Th2 responses and host protective immunity in NF-B1 KO mice (data
not shown). In contrast, blockade of IL-12 activity in NF-B2 KO mice
led to a significant reduction in IFN- production and a concomitant
elevation in the production of Ag-specific IL-13 (Fig. 6). However,
anti-IL-12 treatment failed to enhance the production of IL-4 (Fig. 6). Critically, the enhanced IL-13 levels observed in supernatants from
NF-B2 KO mice, although still lower than levels observed in WT
animals, correlated with the expulsion of T. muris (Fig. 7B). The ability of IL-13 to mediate expulsion of T.
muris in the absence of IL-4 has been reported previously
(23). These results demonstrate that NF-B2 KO mice can
mount functional IL-13 responses if IL-12 responses are blocked,
suggesting that NF-B2 plays an indirect role in regulating the
production of Th2 cytokines. In contrast, the failure of
anti-IL-12-treated NF-B1 KO mice to develop Th2 responses
suggests an absolute requirement for NF-B1 in the induction of Th2
cytokine genes and resistance to helminth infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B in promoting proinflammatory responses has been
widely reported in murine models of inflammation and immunity to
intracellular pathogens (reviewed in Ref. 2). Indeed,
defective IL-12- and/or IFN--mediated immune responses have been
reported in mice deficient in NF-B1, NF-B2, or c-Rel (9, 40, 41, 42, 43). Our results provide new insights into the differential
requirements for these family members in regulating Th2
cytokine-mediated resistance to helminth infection and in controlling
infection-induced intestinal inflammation. Firstly, we found that in
contrast to c-Rel KO animals, mice deficient in NF-B1 or NF-B2
developed chronic infections. Critically, blockade of IL-12 (previously
shown to confer resistance to infection in susceptible mouse strains)
recovered protective IL-13 responses and resistance to infection in
NF-B2 KO, but not NF-B1 KO, mice. These results demonstrated that
individual NF-B KO mice differed in their ability to mount
protective Th2 cytokine responses and control infection. Secondly, we
showed that KO mice exhibited differential defects in their ability to
generate intestinal goblet cell responses following infection that
correlated with their ability to generate Th2 cytokine responses and
control infection. Lastly, severe infection-induced intestinal
pathology was only observed in infected NF-B1 KO mice, identifying a
unique role for this family member in controlling intestinal
inflammation. Taken together, these results identify nonoverlapping
functions for NF-B family members in controlling immunity and
inflammation following intestinal helminth infection.
The ability of c-Rel KO, but not NF-B1 KO or NF-B2 KO, mice to
successfully clear T. muris was reflected in the balance of
Ag-specific Th1 and Th2 cytokine responses following infection. Mice
deficient in NF-B1 or NF-B2 exhibited a low ratio of Th2:Th1
cytokine responses and developed chronic infections, while this ratio
was higher in c-Rel KO mice and was sufficient to clear infection.
However, the magnitude of IL-4 and IL-13 responses were significantly
lower in lymph node cell cultures from c-Rel KO mice than in those from
WT mice. The reduced Th2 cytokine responses in c-Rel KO mice may
reflect a lower frequency of IL-4- and IL-13-producing cells or
suboptimal cytokine responses in KO animals. These hypotheses are
supported by in vitro studies that demonstrated a requirement for c-Rel
in optimum IL-4 production by Jurkat T cells (44).
Alternatively, cytokine-producing cells may have been preferentially
located in the intestinal microenvironment rather than the lymph node
of KO mice. The equivalent goblet cell responses, a hallmark of mucosal
Th2 responses during intestinal and pulmonary inflammation (38, 39) (see below), coupled with the ability of c-Rel KO mice to
clear T. muris infection support this hypothesis.
Although both NF-B1 KO and NF-B2 KO mice had defective Th2
cytokine responses and developed chronic infections, anti-IL-12
treatment recovered protective IL-13 responses and resistance to
infection in NF-B2 KO, but not NF-B1 KO, mice. These results
demonstrated that if endogenous Th1 responses were blocked, NF-B2
was also dispensable for sufficient Th2 responses to control helminth
infection. The ability to recover IL-13 production in the absence of
NF-B2 is supported by in vitro studies demonstrating that Th2 cell
differentiation is intact following polyclonal stimulation of NF-B2
KO T cells under Th2-polarizing conditions (43).
Therefore, in vitro and in vivo studies have shown that NF-B2 KO T
cells can become Th2 cells. Taken together, these results suggested an
indirect role for NF-B2 in regulating Th2 cytokine responses. For
instance, NF-B2 may be important in accessory cell function, a
suggestion supported by the predominant expression of this family
member in the myeloid cell lineage (45) and reports of
overproduction of proinflammatory cytokines by NF-B2 KO accessory
cells following stimulation (46).
In contrast, blockade of endogenous Th1 responses failed to recover
resistance to T. muris in the absence of NF-B1,
demonstrating that NF-B1 (unlike NF-B2 or c-Rel) is essential in
regulating the expression of Th2 cytokine genes and host protective
immunity following T. muris infection. Evidence of a direct
requirement for NF-B1 in the expression of Th2 cytokine responses is
supported by in vitro polarization studies demonstrating that even
under conditions favoring Th2 cell polarization, NF-B1 KO T cells
are unable to produce Th2 cytokines (our unpublished results and Ref.
8). Furthermore, a recent report by Das et al.
(8) demonstrated that NF-B1 directly influences the
expression of GATA-3, a transcription factor known to control Th2 cell
differentiation (47, 48). Although the in vivo expression
of GATA-3 following T. muris infection has not been
investigated, it is likely that the absence of NF-B1 prevents
up-regulation of GATA-3, resulting in the failure of KO mice to develop
protective Th2 responses.
The generation of intestinal goblet cell responses following infection
also differed between mice deficient in individual NF-B family
members. Intestinal goblet cells, whose differentiation is controlled
by Th2 cytokines (38, 39), perform a number of functions,
including secretion of mucins and other anti-microbial peptides.
Indeed, goblet cells have been proposed as effector cells following
infection with intestinal helminth parasites (39, 49).
Consistent with their ability to mount sufficient Th2 responses to
clear T. muris infection, mice deficient in c-Rel had goblet
cell responses equivalent to those of WT mice following infection.
However, while the magnitude of goblet cell responses was reduced in
chronically infected NF-B2 KO compared with WT mice, there was a
complete absence of intestinal goblet cell responses in infected
NF-B1 KO mice. The loss of goblet cell responses following infection
of NF-B1 KO mice correlated with the inability of these KO mice to
develop any Th2 cytokine responses and their susceptibility to
infection. Goblet cell-derived mucins and other secreted products have
also been implicated in regulating intestinal epithelial cell function
and mucosal integrity following insult (50), and their
absence in infected NF-B1 KO mice may contribute to the colitis-like
pathology observed.
The severe destructive intestinal inflammation in chronically infected
NF-B1 KO mice was not observed in other NF-B KO strains and was
accompanied by the secretion of significantly higher levels of
Ag-specific IFN- than those in WT mice. This was a surprising
result, as NF-B1 KO mice have been reported to have defects in
IFN- production in response to other stimuli (40).
However, enhanced production of proinflammatory cytokines, including
IL-12, TNF, and IFN-, has been reported in the intestine of
NF-B1-deficient mice following enteric bacterial infection
(17). This observation suggested that NF-B1 (either
p105 or p50) might also be operating as a transcriptional repressor in
the intestinal microenvironment, inhibiting the production of
proinflammatory cytokines in response to enteric bacteria. Given that
Trichuris infection can promote the invasion of enteric
pathogenic bacteria into the intestinal mucosa (51), it is
possible that in the absence of NF-B1, levels of proinflammatory
cytokines such as IL-12 and TNF are elevated in the intestine of KO
mice. Enhanced production of these proinflammatory factors may, in
turn, augment the production of Ag-specific IFN- following T.
muris infection in NF-B1 KO mice. The production of
proinflammatory mediators in response to bacterial products coupled
with elevated T. muris-specific IFN- production are
likely to contribute to the intestinal inflammation observed.
There may also be a role for IL-10 in controlling intestinal
inflammation in NF-B1 KO mice. A recent report by Schopf et al.
(52) described the development of intestinal inflammation
associated with an inflammatory cell infiltrate and loss of goblet
cells following T. muris infection of IL-10 KO mice.
Although parallels exist between the pathology found in infected
NF-B1 KO and IL-10 KO mice, mice deficient in IL-10 developed weight
loss and premature death around day 20 postinfection, an outcome we
have never observed in NF-B1 KO mice. Therefore, while it is
possible that reduced IL-10 levels in infected NF-B1 KO mice
contribute to the development of intestinal inflammation, this is
clearly not the only reason for the pathology they exhibit. Rather,
defects in IL-10 coupled with the imbalance in Th1 and Th2 cytokine
responses we report here are likely to result in susceptibility to
infection and the severe pathological outcome observed. An interesting
hypothesis we are currently investigating is that in addition to
regulating protective Th2 cytokine responses, NF-B1 is required for
the development of IL-10-producing regulatory B and T cells, two
populations that have been shown to inhibit intestinal inflammation in
other systems (53, 54, 55, 56).
In summary, it is clear that nonredundant functions for NF-B family
members exist following intestinal helminth infection, with NF-B1
playing a unique and critical role in regulating protective Th2
cytokine responses and infection-induced inflammation. Key questions
now include defining the cell types and receptor-ligand interactions
that are required for NF-B1 activation and the subsequent control of
inflammation and induction of protective Th2 cytokines. An
attractive candidate is TNF-TNFR signaling, a potent activator of
NF-B (2) and a signaling pathway we have previously
shown to be required for optimum Th2 cytokine production and efficient
clearance of T. muris (33). Indeed, TNF
production and sustained NF-B activation have also been reported in
animal models of asthma (57). Identifying pathways of
NF-B1 activation and how this family member interacts with other
transcription factors will provide new insights into the molecular
regulation of Th2 responses and identify novel targets to specifically
manipulate Th cell responses and inflammation in vivo.
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Acknowledgments |
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B1 KO mice used in these experiments. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. David Artis or Dr. Phillip Scott, Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104. E-mail address: dartis{at}vet.upenn.edu
3 Abbreviations used in this paper: KO, knockout; LN, lymph node; p.i., postinfection; WT, wild type.
Received for publication June 20, 2002. Accepted for publication August 2, 2002.
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