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B1
* 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;
Department of Medicine and Microbiology, Columbia University Medical Center, New York, NY 10032; and
Division of Emergency Medicine, Children’s Hospital, Boston, MA 02115
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Abstract |
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B-deficient (p50–/–; p65+/–) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-B-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2–/– (RAG) and p50–/–; p65+/–; RAG-2–/– (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50–/–; RAG-2–/– (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-B within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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NF-B is a family of transcription factors consisting of homo- and heterodimers of p50, p52, p65, rel-B, and c-Rel subunits, which play a central role in regulating inflammatory gene expression, including expression of IL-12 (16, 17), as well as a number of other proinflammatory chemokines and cytokines postulated to play a role in the pathogenesis of colitis. Consistent with proinflammatory functions of NF-B, general inhibition of NF-B activity with antisense or decoy oligonucleotides has been demonstrated to ameliorate the severity of colitis (18, 19). However, we have demonstrated previously that individual NF-B subunits can also function as inhibitors of colitis (3, 13, 20). Mice that lack the p50 subunit of NF-B are susceptible to Hh-induced colitis, and mice that lack p50 and are also heterozygous for p65 (p50–/–;p65+/–, referred to in this work as 3X mice) develop more severe colitis after Hh infection than mice that lack p50 alone, although mice that are only heterozygous for p65 are not susceptible (3) (mice that are homozygously deficient for p65 are not viable (21)). Colitis in these NF-B-deficient mice is characterized by the elevated expression of a number of proinflammatory genes within the lower bowel, including IL-12 p40, and depletion of IL-12 p40 ameliorates disease (3, 13). Susceptibility to disease in NF-B-deficient mice appears to be a property of the innate immune system, as we have shown recently that although both wild-type (WT) and 3X splenocytes inhibit Hh-induced colitis after adoptive transfer into RAG mice, WT splenocytes fail to inhibit colitis after adoptive transfer into 3X mice that also lack the gene for RAG-2 (3X/RAG) (13).
Although these studies clearly demonstrate a defect that sensitizes 3X/RAG mice to Hh-induced colitis exists in the innate immune compartment, the nature of this defect has not yet been delineated. Although it is possible that the presence of NF-B subunits is necessary for the recruitment and/or activation of regulatory T cells necessary to prevent colitis, an alternative possibility is that the innate inflammatory response in 3X/RAG mice is less sensitive to suppression by regulatory T cells. This possibility is supported by our observations that Hh challenge induces higher levels of IL-12 p40 expression in 3X macrophages than in WT macrophages despite similar levels of IL-10 production (13). Because it has been demonstrated previously that IL-10 is necessary and sufficient for regulatory T cells to suppress Hh-induced colitis in RAG mice (14, 15), we have addressed the latter possibility by evaluating the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit colitis in RAG-deficient mice lacking NF-B subunits. We have found that the ability of IL-10-Ig to inhibit Hh-induced colitis in RAG mice depends on the presence of the NF-B subunit p50/p105 within hemopoietic cells of the innate immune system. In addition, we demonstrate that the ability of IL-10 to suppress expression of IL-12 p40 is markedly compromised in NF-B-deficient macrophages. Thus, we postulate that NF-B subunits play a critical role in facilitating the ability of IL-10 to suppress inflammation of the lower bowel by limiting the innate inflammatory response to colonic microflora.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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. RAG-2–/– (RAG), p50–/–; RAG-2–/– (p50/RAG), p50–/–; p65+/–; RAG-2–/– (3X/RAG), and IL-10–/– (IL-10) mice were described previously (13, 14, 20). p50–/–; IL-10–/– (p50/IL-10) mice were generated by crossing the p50-deficient allele onto the IL-10-deficient background. All mice used in this study were maintained on the 129S6/SvEvTac background for at least six generations. All mice were maintained under conditions free of known Helicobacter species before targeted infection.
Generation of IL-10-Ig fusion protein
Using a PCR-based cloning strategy, murine IL-10 was fused to the IgG2a CH2-CH3 regions mutated at the Fc receptor binding site. The chimeric gene was cloned into an adenoviral vector to obtain infectious virus (Ad-IL-10-Ig), as previously described (22). Higher titer stocks necessary for these studies were generated by growing Ad-IL-10-Ig in 293 cells, followed by ultracentrifuge purification through two cesium chloride gradients. To obtain fusion protein (IL-10-Ig), RAG mice were infected i.v. with 1011 particles (5 x 109 PFU) of virus, and serum was harvested 10 days after the infection. The concentration of fusion protein in the serum, quantified by an IgG2a-specific ELISA, was 
2 mg/ml. Serum containing IL-10-Ig was used as the source of IL-10-Ig. The t1/2 of the protein in the serum was 12 days (data not shown). Using an in vitro assay, we have determined that 150 ng/ml IL-10-Ig was comparable to 1 ng/ml rIL-10 in its ability to inhibit the production of IL-12 p40 and IFN-
-inducible protein 10 (IP-10) in LPS-stimulated IL-10-deficient macrophages (data not shown).
Hh culture
Hh (strain 3B1; American Type Culture Collection, 51449) was grown on blood agar plates under microaerobic conditions at 37°C (6). Cultures were examined by Gram stain and phase microscopy for bacteria quality and purity. Bacteria were resuspended in Brucella broth with 30% glycerol. Quantifications of Hh were assessed by spectrophotometry, as described previously (23).
Experimental design
In colitis inhibition studies, mice received IL-10-Ig fusion protein or an equivalent dose of IgG2a (Sigma-Aldrich) in 200 µl of PBS i.p. twice per week for a total of 6 wk. On the day after the first dose, mice were infected with Hh. Mice received 2 x 107 bacteria by gastric gavage every other day for total of three doses, as described previously (3). In colitis treatment studies, mice were infected with Hh, and then 5 wk later they received three i.p. doses of either IgG2a or IL-10-Ig every other day. On the day following the last treatment, mice were euthanized by CO2 inhalation.
In selected experiments in which mice received 0.1 µg of IL-10-Ig twice per week, serum was collected at the time of necropsy, and sandwich ELISA was performed using a combination of anti-IgG2a (capture) and anti-IL-10 (detecting) Abs to determine the serum concentration of IL-10-Ig. We determined that the IL-10-Ig concentration ranged between 75 and 150 ng/ml without significant differences between groups (data not shown). Given our estimate that 150 ng/ml IL-10-Ig had similar effects to 1 ng/ml rIL-10, these results suggests that in vivo concentrations of IL-10-Ig were within a physiologically inhibitory range.
Radiation chimeras
RAG and p50/RAG hosts were irradiated with doses of 800 and 400 rad, separated by 3 h, using a 137Cs source. Irradiated mice received 2 x 106 bone marrow cells harvested from either RAG or p50/RAG mice by retro-orbital injection after lysis of RBC. In these experiments, RAG bone marrow cells were derived from RAG mice carrying a GFP transgene (GFP-RAG), such that 80% of the Mac1+ cells in RAG donors were GFP positive. Flow cytometry analysis of bone marrow isolated from GFP-RAG
RAG and GFP-RAGp50/RAG mice at necropsy 6 wk after infection demonstrated that close to 80% of Mac1+ cells were GFP positive, indicating successful and nearly complete reconstitution with donor marrow cells (data not shown). Host mice were maintained on water containing trimethoprim-sulfamethoxazole (Septra) for 1 mo. Six weeks after transplantation, mice were dosed with 0.1 µg of IL-10-Ig twice per week, and infected with Hh, as described above.
Histopathologic evaluation
At necropsy, the entire large intestine, including the ileocecocolic junction and the colon, was harvested, fixed in formalin, embedded in paraffin, sectioned at 5 µm, and stained with H&E. Inflammation and hyperplasia in the ileocecocolic junction and colon were scored on an ascending scale from 0 to 4 by a board-certified veterinary pathologist. The scale was based on the degree of severity: 0 (absent), 1 (mild), 2 (moderate), 3 (marked), and 4 (severe). Colitis score was calculated as a sum of the inflammation and hyperplasia scores (scale 0–8).
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), and RNA was isolated, according to the manufacturer’s instruction. RNase protection analyses (RPA) were performed on 10–20 µg of total RNA using RiboQuant Multi Probe Template Sets (BD Pharmingen). Intensities of the protected fragments were quantified by phosphor imager analysis, and expressed as a ratio, relative to GAPDH, which was included as an internal control for each sample.
Hh quantification in cecum using real-time PCR
At the time of necropsy, distal ceca were collected and total DNA was isolated using High Pure PCR kit (Roche), according to manufacturer’s instructions. The number of bacterial genomes per µg of host genomic DNA was quantified using a real-time PCR method based on cdtB gene using Prism Sequence Detection System 7700 (Applied Biosystems), as described previously (24).
In vitro stimulation of bone marrow-derived macrophages (BMDM)
BMDM were grown, as previously described (13), and replated in 6-well dishes at concentrations of 3 x 106/well. BMDM were challenged with LPS from Escherichia coli 0127:B8 (Sigma-Aldrich) at the final concentration of 1 ng/ml. To evaluate the ability of IL-10 to suppress LPS-induced gene expression, rIL-10 (BD Biosciences) was added to macrophage cultures simultaneously with the challenging stimulus, at final concentration of 0.3 ng/ml.
Analysis of gene expression in BMDM
BMDM were lysed on the plates with 1 ml of TriReagent, and RNA was isolated, according to the manufacturer’s instructions. RPA were performed on 5 µg of total RNA using RiboQuant Multi Probe Template sets, as described above. Intensities of the protected fragments were quantified by phosphor imager analysis, and the relative expression of the analyzed gene to GAPDH was calculated.
Statistical evaluation
Colitis scores between experimental groups were compared using the Mann-Whitney t test for nonparametric data. Unpaired two-tailed t tests were used to compare inflammatory gene expression and ELISA data. Differences were considered statistically significant when p < 0.05.
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Results |
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To determine whether IL-10-Ig was able to inhibit the development of Hh-induced colitis in RAG mice and whether this function was compromised in 3X/RAG mice, we evaluated the severity of Hh-induced colitis in RAG and 3X/RAG mice treated with control IgG2a or increasing doses of IL-10-Ig initiated before Hh infection. As expected, although Hh infection induced colitis in RAG mice treated with control IgG2a, treatment of RAG mice with 0.1 µg of IL-10-Ig twice per week strongly suppressed the development of colitis (Fig. 1, A and C). Furthermore, treatment of RAG mice with either 0.1 or 1.0 µg of IL-10-Ig markedly decreased both the median colitis score (p < 0.01) and the expression of the inflammatory cytokines IL-12 p40, TNF-
, and IP-10 within the cecum (p < 0.05), compared with RAG mice treated with IgG2a (Fig. 2, A and B, compare lanes C, E, and G). These results demonstrate that either 0.1 or 1.0 µg of IL-10-Ig prevents the development of colitis in Hh-infected RAG mice.
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, and IP-10 mRNA in the cecum than RAG mice treated with 0.1 µg of IL-10-Ig (Fig. 2B, compare lanes E and F; p < 0.01). Whereas treatment with 1 µg of IL-10-Ig twice per week was able to reduce colitis scores and inflammatory gene expression in Hh-infected 3X/RAG mice compared with 3X/RAG mice treated with control IgG2a (p < 0.05) (Fig. 2, A and B, compare lanes D and H), even under these conditions we observed higher colitis scores (1.5 vs 0.0; p < 0.05) and expression of proinflammatory cytokines (p < 0.01 for comparisons of TNF- and IP-10) in 3X/RAG mice than in RAG mice (Fig. 2, A and B, compare lane G with H). These results indicate that there is a defect within the innate immune system of 3X/RAG mice that compromises the ability of IL-10-Ig to prevent Hh-induced inflammation. The ability of IL-10-Ig to treat established colitis is compromised in 3X/RAG mice
The experiments described above examined differences in the ability of IL-10-Ig to prevent the development of colitis in RAG and 3X/RAG mice. To determine whether IL-10-Ig could also treat established colitis, and whether this function was compromised in 3X/RAG mice, we compared the severity of colitis in RAG and 3X/RAG mice treated for 1 wk with control IgG2a or IL-10-Ig, initiated 5 wk after infection with Hh. As expected, untreated RAG and 3X/RAG mice showed signs of moderate colitis (median colitis scores of 3.0 and 4.0, respectively; p < 0.01) (Fig. 3). In contrast, whereas 0.1 and 1 µg of IL-10-Ig both significantly lowered the median colitis score in RAG mice (p < 0.01), the median colitis score in 3X/RAG mice was only significantly lowered by treatment with the higher dose of IL-10-Ig (p < 0.01) (Fig. 3). Interestingly, colitis scores were significantly higher in 3X/RAG mice treated with 0.1 or 1.0 µg of IL-10-Ig than in RAG mice treated with these doses (p < 0.01) (Fig. 3). These experiments suggest that the ability of IL-10-Ig to treat Hh-induced innate inflammation is compromised in 3X/RAG mice.
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To determine whether the absence of the p50/p105 subunit alone resulted in a defect in the ability of IL-10 to prevent inflammation, we compared the severity of Hh-induced colitis in RAG, p50/RAG, and 3X/RAG mice treated with either IgG2a Ab or 0.1 µg/dose of IL-10-Ig starting before Hh infection. Six weeks after infection, there was little difference in the colitis scores between groups of RAG, p50/RAG, and 3X/RAG mice treated with IgG2a (Fig. 4A, lanes A–C). Furthermore, mice from these groups expressed similar levels of proinflammatory genes within the colon (Fig. 4B, lanes A–C). As expected, although the treatment of RAG mice with IL-10-Ig significantly suppressed colitis scores and inflammatory gene expression compared with RAG mice treated with IgG2a, colitis scores and inflammatory gene expression in p50/RAG mice treated with IL-10-Ig could not be distinguished from IL-10-Ig-treated 3X/RAG mice (Fig. 4, A and B, lanes D–F). Thus, the absence of p50/p105 is sufficient to interfere with the ability of IL-10-Ig to prevent Hh-induced innate inflammation within the lower bowel.
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It has been demonstrated previously that although hemopoietic cells are most likely the primary target of IL-10’s suppressive function, certain nonhemopoietic cells are able to respond to IL-10 as well (25). To determine whether p50/p105 is required within hemopoietic or nonhemopoietic cells of the innate immune system to facilitate prevention of colitis by IL-10-Ig, we produced radiation chimeras in which RAG bone marrow cells were engrafted into either irradiated RAG (RAGRAG) or p50/RAG hosts (RAGp50/RAG), or p50/RAG bone marrow cells were engrafted into RAG (p50/RAGRAG) or p50/RAG (p50/RAG p50/RAG) hosts. Cells were allowed to engraft for 6 wk, and then mice were treated with 0.1 µg of IL-10-Ig twice per week for 6 wk. Mice were infected with Hh on the day after the first dose of IL-10-Ig.
As expected, colitis scores and inflammatory gene expression were significantly higher in p50/RAGp50/RAG mice than in RAGRAG mice (Fig. 5). Remarkably, whereas colitis scores and inflammatory gene expression in RAGRAG mice and RAGp50/RAG mice were not statistically different (Fig. 5), colitis scores and inflammatory gene expression were significantly higher in p50/RAGRAG mice than in either RAGRAG or RAGp50/RAG mice, and little different from the levels observed in p50/RAGp50/RAG (Fig. 5). Thus, it appears that the ability of IL-10-Ig to prevent Hh-induced innate inflammation in the lower bowel is facilitated by the presence of the p50/p105 subunit of NF-B within hemopoietic cells of the innate immune system.
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It has been demonstrated previously that adoptive transfer of CD4+CD25+ regulatory T cells into RAG mice does not influence Hh colonization levels (15). However, it is conceivable that a defect within the innate immune system of NF-B-deficient mice leads to higher colonization levels, which secondarily exacerbates inflammation. To address this issue, we compared Hh colonization levels within the lower bowel of RAG and 3X/RAG mice in the presence or absence of IL-10-Ig. Consistent with previous results using regulatory T cells, treatment with IL-10-Ig had little effect on colonization levels (Fig. 6). Furthermore, there was little difference in colonization levels in RAG or 3X/RAG mice in the presence or absence of IL-10-Ig. Thus, a defect in controlling Hh colonization levels is unlikely to explain the observation that inflammation is more severe in IL-10-Ig-treated 3X/RAG mice than in IL-10-Ig-treated RAG mice.
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The observations reported in this study are consistent with the possibility that the absence of p50/p105 leads to a defect in the ability of IL-10 to control critical target genes in hemopoietically derived APC populations. One well-recognized target gene of IL-10 that is highly relevant to the development of colitis is IL-12 p40. In fact, we have shown previously that Hh induces higher expression of IL-12 p40 in p50-deficient BMDM than in WT. However, because macrophages secrete a considerable amount of endogenous IL-10 after TLR stimulation, it is difficult to definitively evaluate the response of p50-deficient BMDM to exogenous IL-10. To circumvent this potential problem, we have produced mice that lack both p50 and IL-10 (p50/IL-10) and compared LPS-induced IL-12 p40 expression in BMDM derived from these mice and those lacking IL-10 alone, in the presence and absence of exogenous IL-10. In the absence of IL-10, LPS induced expression of IL-12 p40 in both p50/IL-10 BMDM and IL-10-deficient BMDM (Fig. 7, left). However, whereas exogenous IL-10 was able to inhibit LPS-induced IL-12 p40 expression to some degree in both IL-10-deficient and p50/IL-10 BMDM (Fig. 7, left), inhibition was considerably less efficient in p50/IL-10 BMDM than in IL-10-deficient BMDM. Similar results were obtained for another IL-10-dependent target gene, IP-10 (Fig. 7, right). These results demonstrate that suppression of IL-10 target genes is markedly compromised in the absence of p50/p105.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B, as the ability of IL-10-Ig to inhibit Hh-induced colitis was as compromised in p50/RAG mice as in 3X/RAG mice. Furthermore, studies with chimeric mice revealed that the ability of IL-10-Ig to efficiently inhibit colitis required the presence of the p50/p105 subunit within hemopoietic cells of the innate immune system. The requirement for p50/p105 within hemopoietically derived APCs for adequate suppression of proinflammatory gene expression was verified by demonstrating aberrant regulation of IL-12 p40 expression in p50/IL-10 BMDM. These results indicate that the p50/p105 subunit of NF-B plays a critical role in facilitating the ability of IL-10 to inhibit Hh-induced innate inflammatory responses in the lower bowel, and suggest that this defect may play a central role in sensitizing NF-B-deficient mice to Hh-induced colitis.
Our previous results have shown that whereas WT lymphocyte populations were able to inhibit Hh-induced colitis after adoptive transfer into RAG mice, they were unable to inhibit colitis upon adoptive transfer into 3X/RAG mice (13). We and others (3, 11, 15) have demonstrated that IL-10 is necessary and sufficient for inhibitory lymphocyte populations to prevent Hh-induced colitis in RAG mice. Thus, the observation that the ability of IL-10-Ig to inhibit Hh-induced colitis is compromised in p50/RAG and 3X/RAG mice confirms that there is a defect within the innate immune system of these animals, and suggests that this defect interferes with the ability of lymphocyte-derived IL-10 to adequately inhibit Hh-induced inflammatory gene expression. The defect in the ability of IL-10 to inhibit inflammatory gene expression may facilitate clinical inflammation in several ways. Elevated levels of proinflammatory chemokines and cytokines such as MIP-2 and TNF- within the colon may directly induce leukocyte migration and activation in situ, ultimately leading to inflammation-mediated tissue damage (26). Alternatively, in mice with intact lymphocyte compartments (such as p50 and 3X), it is possible that elevated levels of IL-12 may lead to exaggerated Th1-like responses and/or interfere with the suppressive function of regulatory T cell populations (5, 27). Thus, we suggest that failure of IL-10 to adequately control Hh-induced inflammatory gene expression in intact 3X and p50 mice most likely exacerbates mucosal inflammation at multiple levels.
Although these studies indicate that the ability of IL-10-Ig to inhibit Hh-induced inflammation is compromised in the absence of the p50/p105 subunit of NF-B, several possibilities exist for this observation. One possibility is that inhibition of inflammatory gene expression by IL-10 requires the presence of p50/p105. This is supported by the observations that there are relatively small differences between colitis indexes and inflammatory gene expression in RAG, p50/RAG, and 3X/RAG mice in the absence of exogenous IL-10, despite the fact that inflammation is significantly more severe in p50/RAG and 3X/RAG mice in the presence of 0.1 µg of IL-10-Ig. However, it is clear that p50/p105 is not absolutely required for suppression of Hh-induced inflammation, as higher doses of IL-10-Ig are able to suppress inflammation in 3X/RAG mice, albeit not quite to the levels observed in RAG mice treated with similar doses.
An alternative explanation for the reduced efficiency of suppression by IL-10-Ig in the absence of p50/p105 is that baseline Hh-induced inflammation may be more severe in p50/RAG and 3X/RAG mice than in RAG mice. Although differences in colitis indexes and levels of inflammatory gene expression between genotypes in the absence of IL-10-Ig are modest, there is a general trend that inflammation is more severe in p50/RAG and 3X/RAG mice than in RAG mice. Furthermore, it seems possible that our ability to detect differences in the inflammatory response to Hh is not linear, and that differences in the severity of inflammation that are difficult to detect when inflammation is moderate or severe are more apparent when inflammation is absent or mild. If this is correct, it would suggest that differences in the severity of inflammation observed in the presence of IL-10-Ig are the result of underlying differences in the innate inflammatory response to Hh in the absence of p50/p105, and are mechanistically unrelated to a defect in responding to IL-10.
Studies in BMDM lacking either IL-10 alone, or both IL-10 and p50/p105, support the concept that the absence of p50/p105 strongly affects the expression of critical IL-10 target genes. Interestingly, preliminary gene expression profiling experiments from our laboratory confirm that p50/p105 inhibits the ability of LPS to induce a wide range of IL-10 target genes while having relatively modest effects on LPS-induced genes that are not regulated by IL-10 (M. Tomczak and B. Horwitz, manuscript in preparation). These data strongly support the concept that p50/p105 facilitates the ability of IL-10 to adequately suppress critical proinflammatory target genes. Interestingly, a previous study had found that IL-10 was able to inhibit LPS-induced TNF production by peritoneal macrophages isolated from p50/p105-deficient mice. However, LPS-induced TNF expression in these experiments was relatively weak, and the levels of endogenous IL-10 were not evaluated. Thus, it is not clear how these previously reported results relate to those reported in this study (28).
Links between inhibition by IL-10 and p50/p105 have been previously suggested. There are strong data indicating that STAT-3 activation mediates many of the suppressive effects of IL-10 (29, 30, 31). IL-10-mediated STAT-3 activation induces expression of Bcl-3 (32, 33), and overexpression of Bcl-3 inhibits inflammatory gene expression in the absence of IL-10. Furthermore, Bcl-3-deficient macrophages are resistant to IL-10-mediated inhibition of TNF expression (32). Interestingly, it has been demonstrated that Bcl-3 physically interacts with p50, and it has been shown that there are lower levels of Bcl-3 in a p50-deficient macrophage-derived cell line than in a comparable WT cell line (34, 35). Thus, it is possible that IL-10 collaborates with p50 to facilitate the inhibitory potential of Bcl-3. Clearly, there are multiple other pathways in which IL-10 and p50 may potentially converge. Elucidating the biochemical links between the inhibitory pathways mediated by p50 and those mediated by IL-10 may lead to important new insights into the pathogenesis of IBD.
The present study demonstrates that defects manifest within the innate immune system interfere with the ability of IL-10 to suppress colitis. This indicates that a defect within the innate immune system could potentially play a critical role in development of colitis even in the presence of physiological levels of IL-10. Interestingly, it has been demonstrated that patients with IBD express normal or elevated levels of IL-10 (36, 37) and that treatment of patients with IL-10 has limited efficacy (38, 39). Although one explanation for these findings is that suppression of lower bowel inflammation by IL-10 does not play the central role in humans that it does in mice, an alternative possibility is that some patients with IBD have defects with the innate immune system that interfere with the ability of appropriate levels of IL-10 to adequately suppress inflammation. Recent studies demonstrated a robust association between certain mutation in the NOD2 gene and Crohn’s disease (40, 41). Because NOD2 is expressed predominately within cells of the innate immune system and is involved in NF-B activation (42, 43, 44), it is interesting to speculate whether mutations in NOD2 might also interfere with suppression by IL-10. If correct, this might supply further evidence to the hypothesis that a defect in response to IL-10, rather than in its production, could play a central role in disease development. Hopefully, unraveling these regulatory networks within the innate immune system will lead to development of novel therapeutic strategies for human IBDs.
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant AI52267 (to B.H.H. and S.E.E.), Crohn’s and Colitis Foundation of America and the William and Shelby Modell Family Foundation Senior Research Grant (to B.H.H.), National Institutes of Health Grant CA67529 (to J.G.F.), and a Crohn’s and Colitis Foundation of America Research Fellowship Grant (to M.F.T.). 
2 Address correspondence and reprint requests to Bruce H. Horwitz, Department of Pathology, Brigham and Women’s Hospital, Harvard New Research Building 630E, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: bhorwitz{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: IBD, inflammatory bowel disease; BMDM, bone marrow-derived macrophage; Hh, Helicobacter hepaticus; IP-10, IFN--inducible protein 10; RPA, RNase protection analysis; WT, wild type.
Received for publication July 14, 2006. Accepted for publication August 25, 2006.
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References |
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  Y. Wang, B. H. Rickman, T. Poutahidis, K. Schlieper, E. A. Jackson, S. E. Erdman, J. G. Fox, and B. H. Horwitz c-Rel Is Essential for the Development of Innate and T Cell-Induced Colitis J. Immunol., June 15, 2008; 180(12): 8118 - 8125. [Abstract] [Full Text] [PDF]
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 S. Frantz, J. Tillmanns, P. J. Kuhlencordt, I. Schmidt, A. Adamek, C. Dienesch, T. Thum, S. Gerondakis, G. Ertl, and J. Bauersachs Tissue-Specific Effects of the Nuclear Factor {kappa}B Subunit p50 on Myocardial Ischemia-Reperfusion Injury Am. J. Pathol., August 1, 2007; 171(2): 507 - 512. [Abstract] [Full Text] [PDF]
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) and 3X/RAG mice (•) were infected with Hh, and after 5 wk were treated for 1 wk with 0.1 µg of IL-10-Ig, 1.0 µg of IL-10-Ig, or left untreated. Colitis scores in the ileocecocolic junction were assessed based on histopathology. Horizontal bar represents the median value of the group. Asterisks denote significant differences (p < 0.05) between indicated groups.
), and 3X/RAG (•) mice that received IgG2a (lanes A–C) or 0.1 µg of IL-10-Ig twice per week (lanes D–F). Horizontal bar represents the median value of the group. Asterisks denote significant differences (p < 0.05) between the groups. B, Inflammatory gene expression within the colon as measured by RPA in groups of RAG (
), p50/RAG (
), and 3X/RAG (
) mice that received control IgG2a (lanes A–C) or IL-10-Ig (lanes D–F). Relative expression compared with GAPDH is represented on yaxis. SEM is shown. Asterisks denote significant differences.
