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Inhibition of p38 MAP kinase- and RICK/NF-B-signaling suppresses inflammatory bowel disease

EIKE HOLLENBACH^*,,1, MANFRED NEUMANN^*,1, MICHAEL VIETH, ALBERT ROESSNER, PETER MALFERTHEINER^,2 and MICHAEL NAUMANN^*,2,3

^* Institute of Experimental Internal Medicine;
Department of Gastroenterology, Hepatology and Infectiology; and
Institute of Pathology Otto-von-Guericke-University, Magdeburg, Germany

³ Correspondence: Institute of Experimental Internal Medicine, Otto-von-Guericke-University, Leipziger Strasse 44, Magdeburg D-39120, Germany. E-mail: naumann{at}medizin.uni-magdeburg.de

SPECIFIC AIMS

Inhibition of p38-MAPK is therapeutically beneficial for treatment of inflammatory bowel disease (IBD) indicating the importance of p38 for IBD pathogenesis. Using DSS-induced ulcerative colitis as an established mouse model, we pursued the following specific aims: 1) analysis of the effect of SB203580, an inhibitor supposedly specific for p38, on the macroscopic disease activity index (DAI), histological alterations, and the cytokine profile of inflamed tissue; 2) studying the effects of SB203580 on key signaling factors known to be critical for inflammation such as p38 and NF-B; and 3) identification of new target molecules which may be useful for future therapies of IBD.

PRINCIPAL FINDINGS

1. SB203580 reduces the disease activity index and the histological disease score of DSS-induced colitis in mice
The DAI provides a well-characterized scoring system to quantify disease severity that correlates with histological healing and includes parameters such as weight loss, bleeding, and stool consistency. As reflected by the DAI, SB203580-treated animals showed a marked reduction in the progression of colitis as well as an enhanced improvement of clinical parameters after termination of DSS administration. In parallel, a differential white blood cell count revealed that the colitis-induced increase of monocytes and granulocytes in inflamed tissue is substantially reduced in SB203580-treated mice compared with untreated animals. SB203580 treatment clearly reduced impairment of the glandular architecture and infiltration of macrophages, lymphocytes, occasional eosinophils, and neutrophils between days 3 and 7 as shown by microscopic analysis of hematoxilin/eosin (H&E) -stained bowel tissue (Fig. 1 A). Scoring was performed semiquantitatively grading inflammation, extension, regeneration, and crypt damage (Fig. 1B ).

View larger version (65K): [in this window] [in a new window]   Figure 1. Influence of SB203580 on histological alterations in DSS-induced colitis. A) Bowel tissue from Balb/c mice after DSS-induced colitis without (upper panel) and with SB203580 administration (lower panel) were fixed in 3% formaldehyde and stained with H&E. DSS-induced damages of mucosa in mice not treated with SB203580 were seen starting at day 3 and showing extensive ulcerations at day 7. DSS administration was discontinued after day 7 resulting in a resolution of inflammatory infiltrates mainly associated with a diffuse infiltration of mononuclear cells, plasma cells, and granulocytes. Treatment with SB203508 reduced mucosal injury, edema, and infiltration of inflammatory cells in the colonic bowel. Histology of the distal large intestine in the control group without and with daily i.p. injections of SB 203508 were normal. H&E staining, magnification x20. B) Histological scoring was performed semiquantitatively in H&E-stained sections. Values are means ± SD of 7 animals/group/day. Results for DSS-administered mice with SB203508 treatment were compared with mice without SB 203580 treatment (*P0.05)<.

2. SB203580 inhibits proinflammatory cytokines
The cytokine profile is the decisive parameter of any inflammatory process. Alterations of this profile during development of colitis and after SB203580 treatment were assessed at the transcriptional level by real-time quantitative PCR. With the exception of IL-2, all proinflammatory cytokines including TNF-, which is supposed to be of special importance for IBD pathogenesis, were up-regulated during colitis induction and this up-regulation was blocked by SB203580 treatment of the mice. In contrast, the mRNA level of anti-inflammatory cytokine IL-10 was induced by SB203580. Both findings indicate that anti-inflammatory responses are induced by this inhibitor.

3. SB203580 inhibits p38 activity in mononuclear and plasma cells of the lamina propria
Since p38 activation is a marker of inflammation in IBD, we analyzed the expression of p38 MAPK as well as upstream kinases MKK3/6 in inflamed bowel tissue by immunofluorescence microscopy using FITC-coupled, phospho-specific antibodies against these two kinases. p38 and MKK 3/6 were only activated in mononuclear and plasma cells of the lamina propria. In general, severity of inflammation detected by immunohistological analysis correlated with the clinical score in SB203580-treated and untreated animals. Having identified cell types mediating the inflammatory process, we next wanted to assess the activity of p38 and the impact of SB20380 on this activation by performing in vitro kinase assays with immunopurified p38 using ATF-2 as a substrate. We found that p38 was drastically activated after day 3 following colitis induction. This activation was almost abolished by SB203580 treatment.

4. SB203580 inhibits the "classical" and "alternative" NF-B pathways
Transcription factor NF-B is a key regulator of the inflammatory response. The classical IB-dependent pathway mainly mediates signal-induced activation of NF-B. We examined activation of this pathway during development of experimentally induced colitis as well as the impact of SB203580 on NF-B activation. After preparation of cytoplasmic and nuclear protein extracts from cells of murine bowel tissue, we performed immunoblot analysis assessing degradation of cytoplasmic IB (Fig. 2 A) and nuclear translocation of p65/RelA (Fig. 2B ). We analyzed phosphorylation-induced activation of IKKs by immunoblotting with phospho-specific antibodies (Fig. 2A ). All three parameters are markers for NF-B activity. NF-B is strongly activated during experimentally induced colitis. This NF-B activation is markedly inhibited in cells from bowel tissue derived from SB203580-treated mice. In addition to the classical NF-B activation pathway, the novel alternative pathway based on NIK/IKK-dependent processing of the p100 precursor to active p52/NF-B2 is strongly up-regulated during colitis induction and markedly inhibited by SB203580 as shown by immunoblotting using an antibody recognizing p100 as well as p52/NF-B2 (Fig. 2C ).

View larger version (55K): [in this window] [in a new window]   Figure 2. SB203580 inhibits NF-B and RICK in DSS-induced colitis. A) Protein extracts from the bowel of DSS-treated Balb/c mice were analyzed by immunoblotting using phospho-specific IKK and IB antibodies. Mice (n=7 mice/group/day) were either left untreated or administered SB203580. To control equal loading, blots were reprobed with ERK 1/2 antibody. Cytoplasmic (c) and nuclear (n) protein extracts were prepared from bowel tissue and immunoblotted with p65 (B) or p100/p52 specific antibodies (C). Protein concentration was measured for subcellular protein extracts. The filters were reprobed with an ERK1/2 antibody as a loading control. D) RICK was immunoprecipitated with RICK-specific antibody from whole cellular extracts of bowel tissue from DSS-fed Balb/c mice which were either treated or untreated with SB203580 (n=7 mice/group/day). Immunocomplexes were subjected to an in vitro kinase assay using MBP as substrate for RICK-dependent phosphorylation followed by immunoblotting with anti-phospho-MBP antibody. To control equal loading, blots were reprobed with RICK antibody. All results are representative of three independent experiments.

5. RICK is activated in IBD and blocked by SB203580
NF-B activation reflects the severity of inflammation in IBD pathogenesis. Bacterial products seem to trigger intestinal inflammation. NOD proteins are assumed to serve as an intracellular receptor for bacterial peptidoglycan. One critical downstream kinase of NOD2 is RICK [receptor interacting protein-like interacting CLARP (caspase-like apoptosis regulatory protein) kinase], which can activate IKK finally leading to activation of NF-B. RICK has been shown to be blocked by SB203580. To explore whether RICK might also be important for SB203580-mediated inhibition of NF-B in the course of IBD, we performed in vitro kinase assays using MBP as substrate. RICK was immunopurified from protein extracts derived from bowel tissue of untreated or SB203580-treated mice. These experiments clearly demonstrated that RICK is dramatically activated during development of experimentally induced ulcerative colitis and that this activation can be almost completely inhibited by SB203580 (Fig. 2D ).

CONCLUSIONS AND SIGNIFICANCE

p38 MAPK inhibitors are newly introduced agents in the treatment of IBD. Here we confirm the beneficial effects in a murine model of chronic intestinal inflammation. Both the clinical and histological score were significantly improved by SB203580 treatment. Proinflammatory cytokines were dramatically suppressed, whereas anti-inflammatory IL-10 was up-regulated by SB203580 treatment. At the molecular level, our findings demonstrate that p38 MAPK is not the only target of inhibitor SB203580 in IBD (Fig. 3 ). NF-B, a crucial regulator of the immune system and the inflammatory response, is strongly activated during experimentally induced colitis and this activation is drastically inhibited by SB203580. Surprisingly, this is not only true for the well-characterized IB-dependent mechanism of NF-B activation, but also for the novel pathway based on the signal-induced, NIK/IKK-dependent processing of the p100 precursor to active NF-B factor p52/NF-B2. This processing is up-regulated during colitis induction and blocked by SB203580 treatment of the mice. p52/RelB heterodimers are preferentially generated by p100 processing. These factors regulate a distinct set of target genes which may be functionally important for the pathogenesis of IBD. We analyzed the activity of RICK protein kinase in the course of colitis development. RICK is a key component of the NOD-dependent pathway which is triggered by intestinal bacteria and finally leads to NF-B activation. Our observations that RICK is strongly activated during colitis induction and that this activation is strongly suppressed by SB203580 (Fig. 3) indirectly confirm the pivotal role played by the NOD/RICK/NF-B signaling sequence in the pathogenesis of IBD. Further, it identifies RICK as a potential target for novel therapeutic agents in the treatment of IBD.

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic model of inflammatory pathways inhibited by SB203580. Oral administration of DSS induces ulcerative colitis in mice. This triggers p38 MAPK activity via upstream kinases MKK3/6 and NF-B activation via the classical IB-dependent and alternative pathway based on processing of p100. Activation of both pathways is mediated by IKK complex. RICK is a kinase upstream of IKK, which is able to activate IKK complex. RICK activity is also induced upon colitis induction. SB203580 inhibits p38 MAPK as well as RICK activity. Suppression of RICK leads to down-regulation of NF-B. Solid arrows indicate direct activation of downstream targets and dotted arrows indicate indirect or not yet defined activation processes.

FOOTNOTES

¹ These authors contributed equally to this work.

² These authors share senior authorship.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1642fje;

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