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NF-B Is Required Within the Innate Immune System to Inhibit Microflora-Induced Colitis and Expression of IL-12 p40¹

Michal F. Tomczak^*, Susan E. Erdman, Theofilos Poutahidis, Arlin B. Rogers, Hilda Holcombe, Benjamin Plank^*, James G. Fox and Bruce H. Horwitz^2,*,

^* Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139; and Division of Emergency Medicine, Children’s Hospital, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously presented evidence demonstrating that mice deficient in NF-B subunits are susceptible to colitis induced by the pathogenic enterohepatic Helicobacter species, H. hepaticus. However, it has not been determined whether NF-B is required within inhibitory lymphocyte populations, within cells of the innate immune system, or both, to suppress inflammation. To examine these issues, we have performed a series of adoptive transfer experiments using recombination-activating gene (Rag)-2^-/- or p50^-/-p65^+/-Rag-2^-/- mice as hosts for wild-type (WT) and p50^-/-p65^+/- lymphocyte populations. We have shown that although the ability of H. hepaticus to induce colitis in Rag-2^-/- mice is inhibited by the presence of either WT or p50^-/-p65^+/- splenocytes, these splenocyte populations are unable to suppress H. hepaticus-induced colitis in p50^-/-p65^+/-Rag-2^-/- mice. Colitis in these animals is characterized by increased expression of inflammatory cytokines including IL-12 p40, and depletion of IL-12 p40 from p50^-/-p65^+/- mice ameliorates H. hepaticus-induced disease. Consistent with a primary defect in the regulation of IL-12 expression, H. hepaticus induced markedly higher levels of IL-12 p40 in p50^-/-p65^+/- macrophages than in WT macrophages. These results suggest that inhibition of H. hepaticus-induced IL-12 p40 expression by NF-B subunits is critical to preventing colonic inflammation in response to inflammatory microflora.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increasing evidence supports a prominent role for the involvement of inflammatory intestinal microflora in the development of inflammatory bowel disease (IBD)³. Several reports have suggested that patients are improved by treatment with antibiotics (1, 2). Recent data from mouse models demonstrate that experimental colitis is significantly ameliorated in mice raised in germfree condition (3, 4, 5, 6, 7). Furthermore, it has been shown that targeted introduction of proinflammatory microflora can induce colitis in several susceptible strains (8, 9, 10). One of the bacteria that is strongly associated with the induction of murine colitis is the enterohepatic Helicobacter species, H. hepaticus (11). H. hepaticus infection has been documented in several mouse models that develop spontaneous colitis, and targeted infection of IL-10-deficient or SCID mice induces the development of colitis (9, 10). Thus, evaluating lower bowel inflammation in response to H. hepaticus infection has become a valuable model for studying microflora-induced disease.

The observation that targeted infection of SCID mice or spontaneous infection of recombination-activating gene (Rag)-deficient mice with H. hepaticus leads to the development of colitis suggests that the innate immune response to H. hepaticus is sufficient to induce the clinical and histological manifestations of colitis. Observations demonstrating that adoptive transfer of CD4⁺CD45RB^high T cells into SCID mice exacerbates colitis induced by H. hepaticus suggest that it is likely that this innate response can be augmented by the presence of Th1 effector cells (9). However, while mice lacking lymphocytes or critical immunomodulatory molecules are susceptible to H. hepaticus-induced colitis, standard wild-type (WT) mouse strains are not (10), implying that despite the potential role for disease exacerbation by T lymphocytes, the predominant role of lymphocytes in the normal host is to inhibit the development of colitis.

These results suggest that interactions between suppressive lymphocyte populations and cells of the innate immune system are essential to preventing H. hepaticus-induced inflammatory responses. This suggestion is supported by recent observations that purified populations of CD4⁺CD25⁺ regulatory T cells are able to inhibit the ability of H. hepaticus to induce colitis in Rag-deficient mice (12, 13). However, little is known regarding the factors that regulate the inflammatory response to H. hepaticus infection. One factor that is thought to play a critical role in the host response to inflammatory microflora is the transcription factor NF-B (14). NF-B consists of a series of complexes derived from homo- and heterodimers of members of the Rel protein family, including p50, p65, c-Rel, RelB, and p52. NF-B is activated by a variety of inflammatory signals that lead to activation of the IK kinase complex, IB degradation, and nuclear translocation of NF-B (15). NF-B has been suggested to be involved in the induction of many inflammatory cytokines including IL-12 p40 (16, 17), which has been shown to play a central role in the development of H. hepaticus-induced colitis (10, 18).

Despite extensive evidence that NF-B is a critical mediator of proinflammatory gene expression, there are reports suggesting that in some situations NF-B can inhibit inflammatory gene expression. The p50 subunit of NF-B lacks a transactivation domain, and it has been recognized for some time that homodimers of this subunit have inhibitory activity when overexpressed in vitro (19, 20). Supporting an inhibitory role for p50, it has been reported that p50 is up-regulated during repetitive stimulation of macrophages with LPS and inhibits expression of TNF- (21, 22). Furthermore, a recent report suggests that NF-B activity may inhibit the development of inflammation in a model of irritant-induced pleurisy (23). Inhibitory activity may extend to other complexes, in addition to p50. Although there are both similarities and differences in the phenotypes of mice lacking p50 and p52 (24), a recent report has provided evidence that peritoneal macrophages isolated from p52-deficient mice secrete increased amounts of IL-12 in response to LPS challenge (25).

We have reported direct evidence of a role for NF-B in the inhibition of IBD, by demonstrating that mice lacking the p50 subunit of NF-B and heterozygous for the p65 subunit, p50^-/-p65^+/- (3X), are sensitized to the development of H. hepaticus-induced colitis (26). Colitis in these animals is characterized by elevated expression of IL-12 p40, TNF-, and a number of other inflammatory cytokines and chemokines. Although NF-B has previously been shown to play a prominent role in the development and activation of T cells, we have shown that CD4⁺CD25⁺ T cells, which have been associated with regulatory activity, are present in 3X mice (26). However, whether these NF-B-deficient T cells can exert regulatory function has not previously been evaluated.

These results suggests that NF-B has essential functions in inhibiting the inflammatory response to H. hepaticus within the lower bowel, possibly by limiting the expression of cytokines, such as IL-12 p40, that are critical to the development of disease. We hypothesized that a defect within either inhibitory lymphocyte populations or cells of the innate immune system, which respond to these inhibitory signals, could be responsible for disease in 3X animals. To address these issues, we performed a series of experiments in which WT or 3X lymphoid populations were adoptively transferred into Rag-2^-/- (RAG) or p50^-/-p65^+/- Rag-2^-/- (3X/RAG) hosts. The results of these experiments suggest that there is a defect within the innate immune system of 3X animals that abrogates the ability of lymphocytes to suppress the inflammatory response to H. hepaticus. Furthermore, we demonstrate that macrophages derived from 3X animals express dramatically higher levels of IL-12 p40 in response to H. hepaticus than macrophages derived from WT animals, and that expression of IL-12 p40 is necessary for H. hepaticus to induce colitis in 3X animals. Taken together, we believe that these results suggest that within the innate immune system, NF-B plays a critical role in preventing microflora-induced inflammation in the lower bowel, possibly by limiting the expression of IL-12 p40 after challenge with specific bacteria.

	Materials and Methods

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Experimental animals

All mice were housed in facilities approved by the Association for the Assessment and Accreditation of Laboratory Animal Care. All experiments were approved by the Massachusetts Institute of Technology Committee on Animal Care and the Harvard Medical Area Standing Committee on Animals. WT and 3X mice were maintained on a mixed 129 x C57BL/6 background. To generate RAG and 3X/RAG mice, 3X mice were back-crossed six generations onto the 129S6/SvEvTac-Rag2^tm1 background, and then littermates were intercrossed to generate RAG and 3X/RAG mice. All mice used in this study were maintained under conditions free of known Helicobacter species before targeted infection.

Experimental design

Splenocytes for adoptive transfer studies were harvested from Helicobacter-free WT and 3X mice. Single-cell suspensions were obtained by crushing spleens through cell strainers (BD Biosciences, Franklin Lakes, NJ). Red cell lysis was performed using a solution of tris ammonium chloride (pH 7.2). Cells were resuspended at 1.0 x 10⁸/ml. WT or 3X splenocytes (3 x 10⁷) were transferred to RAG or 3X/RAG hosts by i.v. injection in the retro-orbital sinus under anesthesia induced with tribromoethanol. Three days later, mice were infected with H. hepaticus or sham-infected. H. hepaticus-infected mice received 2 x 10⁷ bacteria by gastric gavage every other day for a total of three doses, as previously described (26). To analyze disease development, mice were euthanized with CO₂ 4 weeks post H. hepaticus infection.

For adoptive transfer of CD4⁺ cells, WT or 3X CD4⁺ cells were purified from single-cell suspension of splenocytes using L3T4 Dynabeads (Dynal Biotech, Oslo, Norway), and then detached from the beads using mouse CD4 Detachabeads (Dynal Biotech). Cells, which were >95% CD4⁺, were resuspended at 2.5 x 10⁷/ml, and then 5.0 x 10⁶ cells were transferred to anesthetized RAG hosts by i.v. injection into the retro-orbital sinus.

Analysis of cell populations present in spleen after adoptive transfer

At necropsy, spleens were harvested from mice and single-cell suspensions were obtained. After RBC lysis, the number of live cells per spleen was determined by counting trypan blue-negative cells with a hemocytometer. Splenocytes were stained with -CD3, -CD4, -CD8, -CD19, and -IgM (BD PharMingen, San Diego, CA). Flow cytometry was used to assess the fraction of cells in the live cell gate that had the surface phenotype of CD3⁺CD4⁺, CD3⁺CD8⁺, or CD19⁺IgM⁺.

Histopathologic evaluation

Formalin-fixed tissues were embedded in paraffin, cut at 5 µm, and stained with H&E. Colonic lesions were scored, by a comparative pathologist blinded to sample identity, using criteria previously described, on a 0–4 ascending scale (26, 27). Criteria assessed included mucosal/submucosal inflammation, transmural and mesenteric perivasculitis, hyperplasia, and dysplasia.

Analysis of cytokine mRNA expression in the colon

One centimeter segments of ascending colon were harvested immediately upon euthanasia and snap frozen in liquid nitrogen. Frozen specimens were homogenized into TriReagent (Molecular Research Center, Cincinnati, OH) and RNA was isolated according to manufacturer’s instruction. RNase protection analyses were performed on 10–20 µg total RNA using RiboQuant MultiProbe Template sets (BD PharMingen). Intensities of the protected fragments were quantified by phosphor imager analysis, and relative expression of individual genes was calculated as the ratio of the intensity of the fragment corresponding to the gene being analyzed to the intensity of the fragment corresponding to GAPDH, which is included as an internal control for each sample.

Bone marrow-derived macrophages (BMDM)

To isolate BMDM, bone marrow was flushed using 1 mM EDTA in PBS. Single-cell suspensions were incubated on tissue culture plates overnight in bone marrow macrophage medium, consisting of DMEM, 10% FBS, 5% horse serum, penicillin 100 IU/ml, streptomycin 100 µg/ml, 10 mM HEPES, 2 mM L-glutamine, and 10% L cell-conditioned medium, at 37°C in 5% CO₂. Nonadherent cells were removed 24 h later by washing with DMEM, and then fresh bone marrow macrophage medium was added. Four to five days later, macrophages were incubated in ice-cold 5 mM EDTA in PBS for 15 min and then removed from the plates by scraping. Approximately 98% of cells were CD11b⁺CD11c^-GR-1^-, as assessed by flow cytometry. Macrophages were replated in DMEM, 10% FBS, penicillin 100 IU/ml, streptomycin 100 µg/ml, 10 mM HEPES, 2 mM L-glutamine in six-well dishes, at concentrations of 3–4 x 10⁶/well. For stimulations with H. hepaticus, medium was changed to medium lacking antibiotics before stimulation.

To prepare L cell-conditioned medium, 2 x 10⁶ murine L-929 cells were cultured in 150 cm² T flasks in DMEM, 10% FBS, 2 mM L-glutamine, 10 mM HEPES, penicillin 100 IU/ml, and streptomycin 100 µg/ml. After 4 days the medium was removed and passed through a 0.22-µm filter and then stored frozen.

In vitro cell stimulation

H. hepaticus was grown on blood agar plates under microaerobic conditions at 37°C. Cultures were examined by Gram stain and phase microscopy for bacteria quality and purity. Bacteria were resuspended in cell culture medium and concentration was assessed by spectrophotometry as described (26). H. hepaticus was added to BMDM at a multiplicity of infection of 250:1, and then plates were centrifuged for 3 min at 600 g at 12°C. After 1 hour, bacteria were killed by adding gentamicin (100 µg/ml). To collect RNA at indicated time points, cells were lysed on the plates with 1 ml TriReagent. RNA was isolated according to the manufacturer’s instructions. Levels of gene expression were quantified as described for colonic samples.

EMSA

BMDM were washed and scraped in ice-cold PBS and lysed in 10 mM HEPES, 1 mM EDTA, 60 mM KCl, 1 mM DTT, 1 mM PMSF, 0.5% Nonidet P-40, and protease inhibitors (complete, mini, EDTA-free protease inhibitor tablets; Boehringer Mannheim, Mannheim, Germany). Nuclei were isolated by centrifugation and lysed in 20 mM Tris-HCl, 0.2 mM EDTA, 420 mM NaCl, 1.5 mM MgCl₂, 25% glycerol, 1 mM DTT, and 1 mM PMSF. Protein concentrations in these extracts were determined using the Bio-Rad assay kit (Bio-Rad, Richmond, CA). Oligonucleotides containing a consensus binding site for NF-B/c-rel homodimeric and heterodimeric complexes (Santa Cruz Biotechnology, Santa Cruz, CA) were ³²P end-labeled with T4 polynucleotide kinase (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. Nuclear extracts (3–4 µg) were incubated with 200,000 cpm of radiolabeled DNA probe in binding buffer (final volume 20 µl) containing 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1.5 µg poly(dIdC), 1.5 µg ssDNA, for 30 min at room temperature. After incubation, samples were loaded on a 5% polyacrylamide nondenaturing gel, and the DNA-protein complexes were resolved by electrophoresis. The gels were dried and exposed to film.

Immunoblotting

BMDM were washed twice with ice-cold PBS and lysed on the plate in 50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromphenol blue, 10% glycerol, and 100 mM DTT. Lysates were heated for 5 min at 95°C and sheared with a 25-gauge needle. After centrifugation for 10 min at 14,000 rpm at room temperature, supernatants were collected and protein concentrations were estimated using the Lowry method (Micro kit for total protein; Sigma-Aldrich, St. Louis, MO). Whole cell extracts (25 µg) were resolved on 10% SDS-polyacrylamide gel and transferred onto Immobilon-P membrane (Millipore, Bedford, MA) using a TransBlot Cell (Bio-Rad). Membranes were probed with polyclonal Abs against p50 and p105 (C-19), p65 (C-20), IB- (C-21), IB- (C-20) (Santa Cruz Biotechnology) and actin (Sigma-Aldrich). After incubation with HRP-conjugated anti-rabbit or anti-goat Abs as appropriate, reactive proteins were visualized with Supersignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL).

ELISA

After stimulation of BMDM with H. hepaticus as previously described, medium was collected at indicated time points. Cytokines were analyzed using sandwich ELISA with -IL-12 p40 (C15.6; Caltag Laboratories, Burlingame, CA) or with -IL-10 (JES5-2A5; BD PharMingen) as a capture Ab, and biotin-labeled -IL-12 p40 (C17.8; Pierce) or -IL-10 (BVD6-24G2; BD PharMingen), respectively, as detecting Ab. Bound Ab was visualized with avidin-HRP (BD PharMingen) and 3,3',5,5'-tetramethyl benzidine liquid substrate system for ELISA (Sigma-Aldrich). Within 5–30 min, reactions were stopped with 0.5 M sulfuric acid, and concentrations of cytokines were determined by 450 nm absorbency read by an E-max microplate reader (Molecular Devices, Sunnyvale, CA).

IL-12 p40 depletion

3X mice were injected i.p. with either PBS or 0.75 mg -IL-12 p40 Ab (C17.8; Bio Express, West Lebanon, NH) three times a week for the duration of the experiment. On the day after the first injection, mice were infected with H. hepaticus as described. To assess development of disease, mice in these experiments were euthanized with CO₂ 3 wks after infection.

Statistical analyses

Colitis scores between experimental groups were compared using the Mann-Whitney U test for nonparametric data. Gene expression data were analyzed using the unpaired Student t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
3X lymphocytes inhibit H. hepaticus-induced colitis

We have previously found similar distribution of B lymphocytes, CD4⁺ lymphocytes, and CD8⁺ lymphocytes in the periphery of WT and 3X animals (26). In addition, the proportions of CD4⁺CD25⁺ T cells, which have been associated with inhibitory activity, are similar in both animals (26). However, the ability of 3X lymphocyte populations to inhibit H. hepaticus-induced colitis has not been previously tested. To compare the ability of WT and 3X lymphocyte populations to inhibit H. hepaticus-induced colitis, RAG mice received nothing, 3 x 10⁷ WT splenocytes, or 3 x 10⁷ 3X splenocytes by i.v. injection. Three days later mice were infected with H. hepaticus by gastric gavage. One month later animals were euthanized. Flow cytometric analysis of spleens from animals that received WT splenocytes demonstrated the presence of both CD4⁺ and CD8⁺ T cells as well as CD19⁺IgM⁺ B cells (Table I, column A). Although the absolute number of lymphocytes is considerably lower than that observed in normal spleens and there is a small decrease in the proportion of B lymphocytes compared with the starting WT splenocyte population, these data suggest successful reconstitution of the major lymphocyte compartments. Interestingly, although there were similar numbers of CD8⁺ T cells in animals that received 3X splenocytes, there were modest reductions in the number of CD4⁺ T cells, and more marked reduction in the number of IgM⁺ B cells compared with animals that received WT splenocytes (Table I, column B).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Adoptive transfer of splenocytes fails to suppress H. hepaticus-induced inflammatory gene expression in 3X/RAG mice. Groups A–H correspond to those described in Table II. RAG (groups A–D) or 3X/RAG (groups E–H) mice were adoptively transferred with nothing (groups A, B, E, and F), 3 x 107 WT splenocytes (groups C and G) or 3 x 107 3X splenocytes (groups D and H). Mice were infected with H. hepaticus (groups B–D and F–H) or sham infected (groups A and E) and RNA was isolated from colons at 4 wk postinfection. Levels of cytokines mRNA expression were analyzed by RNase protection assay and relative expression compared with internal controls is represented on the y-axis. Each group consists of at least seven animals and SEM is shown. There was no statistical difference between columns B and F, p < 0.01, for comparisons of groups B and C, B and D, C and G, and D and H for all cytokines.

View larger version (162K): [in this window] [in a new window]   FIGURE 2. Mucosal inflammation in RAG and 3X/RAG mice. Photomicrographs of cecocolic junctional mucosa. Sham inoculated RAG (a) and 3X/RAG (b) mice with no significant lesions. H. hepaticus-infected RAG (c) and 3X/RAG (d) mice with marked, predominantly granulocytic inflammation, moderate hyperplasia, and in the 3X/RAG mouse, dysplasia. H. hepaticus-infected RAG (e) and 3X/RAG (f) mice that received WT splenocytes before infection. Note protection from disease with well-formed lymphoid follicle in RAG cecocolic junction (e) but no protection from disease in 3X/RAG mouse (f). H&E bar = 100 µM.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. CD4+ lymphocytes inhibit H. hepaticus-induced inflammatory gene expression in the colon. RAG mice received nothing (), WT CD4+ cells (), or 3X CD4+ cells (). Three days later mice were infected with H. hepaticus. Four weeks later RNA was isolated from the colon and levels of gene expression were analyzed as described in Fig. 1. Groups consist of at least seven animals and SEM is shown. Significant difference p < 0.01 for comparison of animals that received WT or 3X CD4+ to those that received no cells, for all cytokines analyzed.

The observation that 3X lymphoid populations retain regulatory function suggested that the defect that sensitizes 3X animals to colitis may reside within nonlymphoid cells of the innate immune system. To assess this possibility, we examined the ability of adoptively transferred splenocytes to inhibit colitis in 3X/RAG hosts. After adoptive transfer, there were similar numbers of B and T cells present in the peripheral lymphoid compartment of both RAG or 3X/RAG hosts (see Table I). Colitis scores and inflammatory gene expression were of similar severity in H. hepaticus-infected RAG and 3X/RAG animals that had not received splenocytes (Table II and Fig. 1, compare group B with F). However, colitis scores and the magnitude of inflammatory gene expression were significantly higher in 3X/RAG animals that had received either WT or 3X splenocytes than in RAG animals that had received either splenocyte population (Table II and Fig. 1, compare groups G and H with C and D, respectively). Furthermore, we detected higher circulating levels of IL-12 p40 in 3X/RAG mice that received splenocytes than in RAG mice that received splenocytes (data not shown). Colitis scores and inflammatory gene expression in 3X/RAG animals that had received splenocytes were not significantly different from the levels observed in 3X/RAG animals that had not received splenocytes (Table II and Fig. 1, compare group G and H with F).

Lesions in 3X/RAG mice that did not receive splenocytes were similar in nature to those described for RAG mice that did not receive splenocytes, although dysplasia was more advanced (Fig. 2d). However, unlike the RAG mice that were protected from disease by splenocyte transfer, 3X/RAG mice that received either WT (Fig. 2f) or 3X splenocytes (data not shown) exhibited mucosal lesions as, or more, severe than lesions observed in 3X/RAG mice that did not receive splenocytes. Additionally, 3X/RAG mice that received splenocytes frequently developed moderate to severe transmural and mesenteric perivasculitis and perilymphangitis (Fig. 4), consisting of aggregates of neutrophils, eosinophils, and variable numbers of mononuclear cells surrounding arterioles and/or lymphatic vessels, with extension into adjacent interstitium. Similar leukocytic aggregates surrounded myenteric and mesenteric nerve ganglia and fibers, although it was unclear whether perineural tissue represented a separate inflammatory target or was merely an extension of the vessel-centered lesions. These perivascular lesions were usually in the vicinity of severe mucosal/submucosal lesions, suggesting that they represented either a direct extension of mucosal typhlocolitis or that disruption of surface integrity in regions of severe inflammation permitted transepithelial passage of microbes or soluble factors from the gut lumen with induction of regional perivasculitis. These results suggest that there is a defect within nonlymphoid cells of 3X animals that prevents suppression of H. hepaticus-induced inflammation by lymphocyte populations. This defect could reside in hemopoietic cells of the innate immune system such as professional APCs or NK cells, and/or nonhemopoietic cells such as colonic epithelial cells.

View larger version (57K): [in this window] [in a new window]   FIGURE 4. Submucosal inflammation in 3X/RAG mice that received splenocytes. Photomicrographs of proximal colon. Note absence of lesions in tissue from H. hepaticus-infected RAG mouse that received splenocytes (a). In H. hepaticus infected 3X/RAG mouse that received WT splenocytes (b) note diffuse submucosal inflammation (top) extending transmurally (arrow) through the tunicae muscularis and serosa into the mesentery with perivascular, perilymphangial, and perineurial orientation. Higher power magnification of 3X/RAG mouse that received WT splenocytes (c), demonstrating myenteric perivasculitis characterized by inflammatory cell dissection between histologically normal smooth muscle fibers, and perivascular leukocytic microaggregates. H&E bar = 250 µM (a and b) and 100 µM (c).

H. hepaticus induces NF-B activation

The previous results suggested that NF-B was playing a critical role in regulating the inflammatory response to H. hepaticus infection. However, the ability of H. hepaticus to activate NF-B and induce inflammatory responses within cells of the innate immune system has not been previously reported. Macrophages are thought to play a critical role in the initiation and maintenance of inflammatory responses in the colon (28), and have served as a model with which to examine innate immune responses. Therefore, we examined the ability of H. hepaticus to activate NF-B and induce inflammatory gene expression in BMDM from WT and 3X animals.

WT BMDM expressed p50, p105, and p65 proteins, whereas as expected p50 or p105 protein was not detected in BMDM from 3X animals (Fig. 5a). Interestingly, there was less p65 protein observed in 3X BMDM than in WT BMDM, suggesting that the absence of one allele of p65 results in decreased cellular levels of p65 (Fig. 5a). Treatment of either WT or 3X macrophages with H. hepaticus at multiplicity of infection of 250:1 lead to degradation of both IB- and IB- (Fig. 5b). IB-degradation was unlikely to be the result of residual contamination of bacterial products present in the medium used to culture H. hepaticus, because scrapings from blood agar plates used for culture were unable to independently induce IB degradation. In addition to degradation of IB- and IB-, H. hepaticus induced the nuclear translocation of B binding activity in WT BMDM (Fig. 5c). However, little B binding activity was observed in H. hepaticus-stimulated 3X macrophages, suggesting that in macrophages, as previously demonstrated in B cells (29), complexes containing p50 are the predominant species detected by the gel retardation assay.

View larger version (55K): [in this window] [in a new window]   FIGURE 5. H. hepaticus induces IB degradation and NF-B activation in BMDM. a, Whole cell extracts were prepared from WT or 3X BMDM, and NF-B proteins p105, p50, p65, as well as actin were detected by immunobloting. b, Whole cell extracts were prepared from WT and 3X cells at indicated times (hours) after stimulation with H. hepaticus. IB- and IB- were detected by immunoblotting. c, Nuclear extracts were prepared from WT and 3X cells at indicated times (hours) after stimulation with H. hepaticus. Gel retardation assays were performed with oligonucleotides containing a consensus NF-B/c-rel binding site.

We next compared the ability of H. hepaticus to induce inflammatory gene expression in WT and 3X BMDM. Surprisingly, despite the marked reduction in NF-B binding activity, the expression of a number of NF-B-dependent genes including TNF-, MIP-2, and MIP-1 was similar in WT and 3X BMDM (Fig. 6). However, H. hepaticus induced significantly higher levels of IL-12 p40 and IP-10 in 3X BMDM than in WT BMDM (Fig. 6). In four independent experiments, the increase in expression of IL-12 p40 12 h after stimulation ranged from 10- to 100-fold. In contrast, there was a small but reproducible decrease in the expression of IL-10 in 3X BMDM compared with WT BMDM (Fig. 6).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. H. hepaticus induces higher levels of IL-12 p40 mRNA in 3X macrophages. a, RNA was isolated from WT and 3X BMDM at indicated times (hours) after stimulation with H. hepaticus. Gene expression was analyzed by RNase protection assays and a representative gel is shown. b, Relative expression compared with internal controls of indicated genes in H. hepaticus-stimulated WT () and 3X () BMDM as a function of time (hours). Data represent one of four independent experiments with similar results.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. Comparison of IL-12 p40 expression in macrophages lacking NF-B. Normalized levels of IL-12 p40 mRNA in H. hepaticus-stimulated BMDM from WT (), p65+/- (), p50-/- () and 3X () mice (upper graph). Expanded y-axis compares expression of IL-12 p40 in WT and p65+/- BMDM (lower graph).

View larger version (21K): [in this window] [in a new window]   FIGURE 8. H. hepaticus induces higher levels of IL-12 p40 secretion in 3X macrophages. WT and 3X BMDM were stimulated with H. hepaticus and at indicated times in hours (x-axis) cell culture media were collected and amounts of IL-12 p40 and IL-10 were assessed by sandwich ELISA (y-axis). Each group was done in triplicate and SEM is shown. Significant difference p < 0.01 in IL-12 p40 secretion at all time points shown, p < 0.05 for differences between IL-10 secretion only at the 24-h time point.

Depletion of IL-12 p40 suppresses the development of colitis

The results we have reported suggested that there may be a defect within the innate immune system of 3X animals that leads to increased expression of IL-12 p40 in response to H. hepaticus. Expression of IL-12 p40 is essential for the development of colitis in several models, including colitis induced by H. hepaticus infection of IL-10-deficient animals. However, whether IL-12 p40 is involved in the development of H. hepaticus-induced disease in 3X animals has not been determined. To address this issue, groups of 3X mice received 0.75 mg of C17.8 -IL-12 p40 Ab (31) or PBS by i.p. injection. The following day all mice were infected with H. hepaticus. Mice then received either 0.75 mg of -IL-12 p40 Ab or PBS three times per week for the following 3 wk. Treatment with -IL-12 p40 Ab significantly reduced both colitis scores and the magnitude of inflammatory gene expression within the colon (Table IV and Fig. 9), although treatment did not suppress inflammation to background levels observed in infected WT mice (data not shown). This failure to completely suppress colitis is expected, as previous reports suggest that depletion of IL-12 p40 with Abs does not suppress inflammation to the same degree as that observed in IL-12 p40-deficient animals (18, 32). Nonetheless, we believe that these results indicate that expression of IL-12 p40 plays a key role in the development of colitis in H. hepaticus-infected 3X animals.

View this table: [in this window] [in a new window]   Table IV. Colitis scores in 3X mice treated with -IL-12 p40 antibodya

View larger version (19K): [in this window] [in a new window]   FIGURE 9. Depletion of IL-12 p40 reduces inflammatory gene expression in the colon of 3X mice. Levels of inflammatory gene expression in the colons of 3X mice treated with PBS (group A) or IL-12 p40 Ab (group B) 3 wk after infection with H. hepaticus. Significant difference p < 0.01 for comparison of all three cytokines.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous data has suggested that lymphocytes inhibit the ability of H. hepaticus to induce both innate and acquired inflammatory responses (9, 10, 33, 34). This has been verified by demonstrating that the adoptive transfer of total splenocytes or purified CD4⁺ T cells into RAG animals before infection with H. hepaticus prevents the development of colitis. The observation that 3X splenocytes and 3X CD4⁺ T cells were able to inhibit H. hepaticus-induced inflammation as efficiently as their WT counterparts, indicates that the inhibitory function of these lymphoid populations is intact, and that a defect in this function is unlikely to be responsible for the development of colitis in 3X animals. These observations focused our attention on the role of NF-B within the innate immune system. In the absence of adoptively transferred splenocytes, H. hepaticus-induced inflammation was of similar severity in RAG and 3X/RAG mice. However, although adoptive transfer of splenocytes potently inhibited H. hepaticus-induced inflammation in RAG mice, splenocytes could not inhibit inflammation after adoptive transfer into 3X/RAG mice. These results indicate that there is a defect with the innate immune system of 3X animals that interferes with the ability of T cells to inhibit colitis. Although it has not been determined whether this defect is intrinsic to the hemopoietic or nonhemopoietic compartments of the innate immune system, one explanation for this phenomenon could be that p50/p65 mediates the ability of regulatory T cells to suppress inflammatory gene expression within APCs of the innate immune system. It is also possible that a defect within the innate immune system of 3X mice leads to altered protective Ab or T cell responses to H. hepaticus infection. However, specific Ab and T cell responses were not examined in this study.

It has previously been shown that inhibition of IL-12 p40 expression is essential to prevent H. hepaticus-induced colitis (10, 18). However, while splenocytes strongly suppress H. hepaticus-induced expression of IL-12 p40 after adoptive transfer into RAG mice, they are unable to suppress IL-12 p40 expression after transfer into 3X/RAG mice. Furthermore, we observed large but selective increases in the expression of IL-12 p40 in H. hepaticus-stimulated 3X BMDM compared with WT BMDM. The increase in IL-12 p40 expression observed in 3X BMDM may extend to mucosally derived APC populations as well, as in a preliminary experiment we have observed higher H. hepaticus-induced expression of IL-12 p40 in dendritic cells derived from the colonic lamina propria of 3X mice than in dendritic cells similarly derived from WT mice (data not shown). In addition to the observed increases in expression of IL-12 p40, we confirmed that the development of colitis in H. hepaticus-infected 3X mice depends upon IL-12 p40 expression, by demonstrating that depletion of IL-12 p40 ameliorates disease. Thus, we postulate that NF-B has a role in inhibiting expression of IL-12 p40, and that the absence of NF-B-mediated inhibition of IL-12 p40 expression may sensitize 3X mice to the development of H. hepaticus-induced colitis.

Despite the marked increases in expression of H. hepaticus-induced IL-12 p40 observed within the colon, serum, and APCs, concomitant increases in the expression of IL-12 p35 by H. hepaticus stimulated BMDM, or secretion of IL-12 p70 were not observed. These observations suggest that p40 expressed in response to H. hepaticus infection is not participating in formation of p70 heterodimers. However, it has recently been shown that p40 also participates in the formation of the proinflammatory cytokine IL-23 (30). Thus, the exact consequences of increased p40 expression within this model system will require additional study.

It has been demonstrated that expression of IL-10 by inhibitory lymphocyte populations is essential to inhibit H. hepaticus-induced colitis and the expression of IL-12 (18, 33). Therefore, the observation that inhibitory T cells are unable to suppress colitis and IL-12 p40 expression after transfer into 3X/RAG mice raises the possibility that the inhibitory effects of IL-10 could require the function of NF-B subunits within the cells of the innate immune system. This hypothesis would suggest that differential expression of IL-12 p40 in WT and 3X cells may only be observed in the presence of sufficient quantities of IL-10 to inhibit IL-12 p40 expression in WT cells. This could explain the observation that there is little difference in the expression of IL-12 p40 in the colons of H. hepaticus-infected RAG and 3X/RAG mice in the absence of splenocytes, as there does not appear to be sufficient production of IL-10 by the innate immune system alone to prevent colitis or inhibit expression of IL-12 in RAG animals. Furthermore, as we have shown that H. hepaticus-induces considerable IL-10 expression in both WT and 3X BMDM, it is possible that the significantly higher levels of H. hepaticus-induced IL-12 p40 expression observed in 3X BMDM compared with WT BMDM could be explained by differential sensitivity of WT and 3X BMDM to this endogenously produced IL-10.

Previous reports have examined the effects of NF-B subunits on expression of IL-12 p40 (35, 36). Contrary to results reported in this study, these studies have suggested that transfection of p50 and p65 are able to induce endogenous IL-12 expression within macrophages, and that fetal liver-derived macrophages lacking either p50 or p65, express lower levels of p40 RNA and protein in response to the combination of IFN- (10 U/ml) and LPS (10 µg/ml) than WT macrophages. However, overexpression studies may not accurately reflect the function of NF-B proteins when expressed at endogenous levels (37). Furthermore, the response of NF-B-deficient macrophages to relatively high doses of IFN-/LPS, may not reflect the response of NF-B-deficient macrophages to all inflammatory stimuli. In fact, while 3X BMDM stimulated with 1 µg/ml of LPS also produced less p40 than similarly treated WT BMDM, when we used a 10,000 times lower dose of LPS (100 pg/ml), p40 expression was higher in 3X than in WT BMDM (data not shown). Thus, we suspect that differences in the expression of IL-12 p40 observed in this study and previous studies likely reflect differences in the signaling pathways employed by high-dose IFN-/LPS, and H. hepaticus or low-dose LPS, respectively.

Although the mechanisms by which p50 and p65 inhibit H. hepaticus-induced gene expression have not been delineated, this inhibitory activity is clearly selective, as not all NF-B-dependent genes are expressed at higher levels. Furthermore, the observation that most NF-B-dependent genes are expressed at normal or augmented levels in 3X macrophages suggests that despite the marked reduction in gel shift activity observed in these cells, residual NF-B activity is sufficient to drive expression of these genes. It has recently been demonstrated that both p50 and p65 can recruit histone deacetylase (HDAC) activity (38), and that the ability of p50 to recruit HDAC activity prevents the acetylation and transcriptional activation of several NF-B-dependent genes (37). Thus, it is possible that decreased recruitment of HDAC activity to the IL-12 p40 promoter in 3X BMDM leads to increased acetylation-dependent promoter remodeling and over exuberant activation in response to stimulation with H. hepaticus. Alternatively, it is possible that inhibition of IL-12 p40 expression by NF-B subunits is not mediated by a direct affect of NF-B on the IL-12 p40 promoter, but rather is mediated in an indirect fashion. IL-10 is a potent inhibitor of IL-12 p40, but differences in IL-10 secretion between cultures of WT and 3X BMDM were small and, we believe, unlikely to explain the large differences in IL-12 p40 expression observed. Type I IFNs have also been reported to inhibit IL-12 p40 expression (39), although we have not examined H. hepaticus-induced expression of type I IFNs in this study.

Finally, we believe that the results we reported strongly support the hypothesis that within cells of the innate immune system NF-B subunits can have critical inhibitory function, essential for maintaining bowel homeostasis after exposure to inflammatory microflora. These inhibitory functions of NF-B must be integrated with the known proinflammatory functions of NF-B to gain a broader understanding of the significance of NF-B in control of IBD. Demonstration that the p50 and p65 subunits can have critical inhibitory roles within the innate immune system, and that these inhibitory functions closely correlate with limiting expression of IL-12 p40, may have implications for future therapeutic strategies.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grants AI50952 and T32 RR07036 (to J.G.F.), a research grant from the Crohn’s and Colitis Foundation of America (to B.H.H.), and a grant from the Eli and Edythe L. Broad Foundation (to B.H.H.).

² Address correspondence and reprint requests to Dr. Bruce H. Horwitz, Department of Pathology, Brigham and Women’s Hospital, LMRC-511, 221 Longwood Avenue, Boston, MA 02115. E-mail address: bhorwitz{at}rics.bwh.harvard.edu

³ Abbreviations used in this paper: IBD, inflammatory bowel disease; WT, wild-type; 3X, p50^-/-p65^+/-; Rag, recombination-activating gene; RAG, Rag-2^-/-; HDAC, histone deacetylase; BMDM, bone marrow-derived macrophages.

Received for publication March 12, 2003. Accepted for publication May 21, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Scribano, M. L., C. Prantera. 2002. Medical treatment of active Crohn’s disease. Aliment Pharmacol. Ther. 16:(Suppl 4):35.
2. Podolsky, D. K.. 2002. Inflammatory bowel disease. N. Engl. J. Med. 347:417.[Free Full Text]
3. Kuhn, R., J. Lohler, D. Rennick, K. Rajewsky, W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263.[Medline]
4. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75:253.[Medline]
5. Schultz, M., S. L. Tonkonogy, R. K. Sellon, C. Veltkamp, V. L. Godfrey, J. Kwon, W. B. Grenther, E. Balish, I. Horak, R. B. Sartor. 1999. IL-2-deficient mice raised under germfree conditions develop delayed mild focal intestinal inflammation. Am. J. Physiol. 276:G1461.
6. Sellon, R. K., S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, E. Balish, D. M. Rennick, R. B. Sartor. 1998. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice. Infect. Immun. 66:5224.[Abstract/Free Full Text]
7. Dianda, L., A. M. Hanby, N. A. Wright, A. Sebesteny, A. C. Hayday, M. J. Owen. 1997. T cell receptor--deficient mice fail to develop colitis in the absence of a microbial environment. Am. J. Pathol. 150:91.[Abstract]
8. Fox, J. G., L. Yan, B. Shames, J. Campbell, J. C. Murphy, X. Li. 1996. Persistent hepatitis and enterocolitis in germfree mice infected with Helicobacter hepaticus. Infect. Immun. 64:3673.[Abstract]
9. Cahill, R. J., C. J. Foltz, J. G. Fox, C. A. Dangler, F. Powrie, D. B. Schauer. 1997. Inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with Helicobacter hepaticus. Infect. Immun. 65:3126.[Abstract]
10. Kullberg, M. C., J. M. Ward, P. L. Gorelick, P. Caspar, S. Hieny, A. Cheever, D. Jankovic, A. Sher. 1998. Helicobacter hepaticus triggers colitis in specific-pathogen-free interleukin-10 (IL-10)-deficient mice through an IL-12- and interferon-dependent mechanism. Infect. Immun. 66:5157.[Abstract/Free Full Text]
11. Fox, J. G., F. E. Dewhirst, J. G. Tully, B. J. Paster, L. Yan, N. S. Taylor, M. J. Collins, Jr, P. L. Gorelick, J. M. Ward. 1994. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J. Clin. Microbiol. 32:1238.[Abstract/Free Full Text]
12. Erdman, S. E., T. Poutahidis, M. Tomczak, A. B. Rogers, K. Cormier, B. Plank, B. H. Horwitz, J. G. Fox. 2003. CD4⁺CD45RB^lo CD25⁺ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice. Am. J. Pathol. 162:691.[Abstract/Free Full Text]
13. Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, F. Powrie. 2003. CD4⁺CD25⁺ TR cells suppress innate immune pathology through cytokine-dependent mechanisms. J. Exp. Med. 197:111.[Abstract/Free Full Text]
14. Grilli, M., J. J. Chiu, M. J. Lenardo. 1993. NF-B and Rel: participants in a multiform transcriptional regulatory system. Int. Rev. Cytol. 143:1.[Medline]
15. DiDonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, M. Karin. 1997. A cytokine-responsive IB kinase that activates the transcription factor NF-B. Nature 388:548.[Medline]
16. Murphy, T. L., M. G. Cleveland, P. Kulesza, J. Magram, K. M. Murphy. 1995. Regulation of interleukin 12 p40 expression through an NF-B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
17. Plevy, S. E., J. H. Gemberling, S. Hsu, A. J. Dorner, S. T. Smale. 1997. Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins. Mol. Cell. Biol. 17:4572.[Abstract]
18. Kullberg, M. C., A. G. Rothfuchs, D. Jankovic, P. Caspar, T. A. Wynn, P. L. Gorelick, A. W. Cheever, A. Sher. 2001. Helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation. Infect. Immun. 69:4232.[Abstract/Free Full Text]
19. Schmitz, M. L., P. A. Baeuerle. 1991. The p65 subunit is responsible for the strong transcription activating potential of NF-B. EMBO J. 10:3805.[Medline]
20. Kang, S. M., A. C. Tran, M. Grilli, M. J. Lenardo. 1992. NF-B subunit regulation in nontransformed CD4⁺ T lymphocytes. Science 256:1452.[Abstract/Free Full Text]
21. Kastenbauer, S., H. W. Ziegler-Heitbrock. 1999. NF-B1 (p50) is upregulated in lipopolysaccharide tolerance and can block tumor necrosis factor gene expression. Infect. Immun. 67:1553.[Abstract/Free Full Text]
22. Bohuslav, J., V. V. Kravchenko, G. C. Parry, J. H. Erlich, S. Gerondakis, N. Mackman, R. J. Ulevitch. 1998. Regulation of an essential innate immune response by the p50 subunit of NF-B. J. Clin. Invest. 102:1645.[Medline]
23. Lawrence, T., D. W. Gilroy, P. R. Colville-Nash, D. A. Willoughby. 2001. Possible new role for NF-B in the resolution of inflammation. Nat. Med. 7:1291.[Medline]
24. Caamano, J. H., C. A. Rizzo, S. K. Durham, D. S. Barton, C. Raventos-Suarez, C. M. Snapper, R. Bravo. 1998. Nuclear factor (NF)-B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med. 187:185.[Abstract/Free Full Text]
25. Speirs, K., J. Caamano, M. H. Goldschmidt, C. A. Hunter, P. Scott. 2002. NF-B2 is required for optimal CD40-induced IL-12 production but dispensable for Th1 cell differentiation. J. Immunol. 168:4406.[Abstract/Free Full Text]
26. Erdman, S., J. G. Fox, C. A. Dangler, D. Feldman, B. H. Horwitz. 2001. Typhlocolitis in NF-B-deficient mice. J. Immunol. 166:1443.[Abstract/Free Full Text]
27. Berg, D. J., N. Davidson, R. Kuhn, W. Muller, S. Menon, G. Holland, L. Thompson-Snipes, M. W. Leach, D. Rennick. 1996. Enterocolitis and colon cancer in interleukin-10-deficient mice are associated with aberrant cytokine production and CD4⁺ Th1-like responses. J. Clin. Invest. 98:1010.[Medline]
28. Kanai, T., M. Watanabe, A. Okazawa, T. Sato, M. Yamazaki, S. Okamoto, H. Ishii, T. Totsuka, R. Iiyama, R. Okamoto, et al 2001. Macrophage-derived IL-18-mediated intestinal inflammation in the murine model of Crohn’s disease. Gastroenterology 121:875.[Medline]
29. Sha, W. C., H. C. Liou, E. I. Tuomanen, D. Baltimore. 1995. Targeted disruption of the p50 subunit of NF-B leads to multifocal defects in immune responses. Cell 80:321.[Medline]
30. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715.[Medline]
31. Wysocka, M., M. Kubin, L. Q. Vieira, L. Ozmen, G. Garotta, P. Scott, G. Trinchieri. 1995. Interleukin-12 is required for interferon- production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
32. Davidson, N. J., S. A. Hudak, R. E. Lesley, S. Menon, M. W. Leach, D. M. Rennick. 1998. IL-12, but not IFN-, plays a major role in sustaining the chronic phase of colitis in IL-10-deficient mice. J. Immunol. 161:3143.[Abstract/Free Full Text]
33. Kullberg, M. C., D. Jankovic, P. L. Gorelick, P. Caspar, J. J. Letterio, A. W. Cheever, A. Sher. 2002. Bacteria-triggered CD4⁺ T regulatory cells suppress Helicobacter hepaticus-induced colitis. J. Exp. Med. 196:505.[Abstract/Free Full Text]
34. von Freeden-Jeffry, U., N. Davidson, R. Wiler, M. Fort, S. Burdach, R. Murray. 1998. IL-7 deficiency prevents development of a non-T cell non-B cell-mediated colitis. J. Immunol. 161:5673.[Abstract/Free Full Text]
35. Sanjabi, S., A. Hoffmann, H. C. Liou, D. Baltimore, S. T. Smale. 2000. Selective requirement for c-Rel during IL-12 p40 gene induction in macrophages. Proc. Natl. Acad. Sci. USA 97:12705.[Abstract/Free Full Text]
36. Weinmann, A. S., D. M. Mitchell, S. Sanjabi, M. N. Bradley, A. Hoffmann, H. C. Liou, S. T. Smale. 2001. Nucleosome remodeling at the IL-12 p40 promoter is a TLR-dependent, Rel-independent event. Nat. Immun. 2:51.[Medline]
37. Zhong, H., M. J. May, E. Jimi, S. Ghosh. 2002. The phosphorylation status of nuclear NF-B determines its association with CBP/p300 or HDAC-1. Mol. Cell 9:625.[Medline]
38. Ashburner, B. P., S. D. Westerheide, A. S. Baldwin, Jr. 2001. The p65 (RelA) subunit of NF-B interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol. Cell. Biol. 21:7065.[Abstract/Free Full Text]
39. Cousens, L. P., J. S. Orange, H. C. Su, C. A. Biron. 1997. Interferon-/ inhibition of interleukin 12 and interferon- production in vitro and endogenously during viral infection. Proc. Natl. Acad. Sci. USA 94:634.[Abstract/Free Full Text]

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