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Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn's-like inflammatory bowel disease.

Kontoyiannis D, Boulougouris G, Manoloukos M, Armaka M, Apostolaki M, Pizarro T, Kotlyarov A, Forster I, Flavell R, Gaestel M, Tsichlis P, Cominelli F, Kollias G - J. Exp. Med. (2002)

Bottom Line: Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity.Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background.Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, Biomedical Sciences Research Center "Al. Fleming," Vari 166-72, Greece. Division of Gastroenterology and Hepatology, University of Virginia Health Sciences Center, Charlottesville, VA 22906, USA.

ABSTRACT
Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor-deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow-derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

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Myeloid- and lymphoid-specific deregulation of TNF biosynthesis. (A) Structure of the TNF/LTα locus on mouse chromosome 17 and the strategy for the Cre-specific removal of floxed neor allele. Filled boxes represent exons and gray shaded boxes represent untranslated regions. Filled arrowheads indicate the position of the loxP sequences. The positions of the TNF AU-rich element (ARE) and the floxed neo marker are indicated. The location of the 3′ 0.4-Kb BglII probe is also indicated. Restriction enzyme sites are Bg:BglII, K:KpnI, N:NotI, and S:SacI. (B) Southern blot analysis of BglII-digested DNA from tail, peritoneal cavity cells (PEC), total exudate peritoneal macrophages, and T and B lymphocytes from LysM-Cre or lck-Cre TnfΔAREneomice. Detected with the 3′ probe are the neo+ (1.6 Kb), neo- (0.5 Kb), and wild-type (0.4 Kb) fragments. (C) TNF protein production as determined by ELISA in culture supernatants from peritoneal macrophages (left) or splenic T cells (right) isolated from individual LysM-Cre (LysC) or lck-Cre (lckC) TnfΔAREneo(Δn) mice and stimulated with LPS or anti-CD3, respectively. Data shown as mean ng/ml (± SD) per group. P < 0.02 relative to (*) TnfΔAREneo/+ or (*) TnfΔAREneo/ΔAREneovalues with n = 3 mice/group. Histologic examination of ileal sections from 5-mo-old (D) TnfΔAREneo/ΔAREneo, (E) LysM-Cre TnfΔAREneo/+, (F) LysM-Cre TnfΔAREneo/ΔAREneo, and 7-mo-old (G) lck-Cre TnfΔAREneo/ΔAREneomutant mice. Paraffin sections stained with hematoxylin and eosin. ×100.
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fig3: Myeloid- and lymphoid-specific deregulation of TNF biosynthesis. (A) Structure of the TNF/LTα locus on mouse chromosome 17 and the strategy for the Cre-specific removal of floxed neor allele. Filled boxes represent exons and gray shaded boxes represent untranslated regions. Filled arrowheads indicate the position of the loxP sequences. The positions of the TNF AU-rich element (ARE) and the floxed neo marker are indicated. The location of the 3′ 0.4-Kb BglII probe is also indicated. Restriction enzyme sites are Bg:BglII, K:KpnI, N:NotI, and S:SacI. (B) Southern blot analysis of BglII-digested DNA from tail, peritoneal cavity cells (PEC), total exudate peritoneal macrophages, and T and B lymphocytes from LysM-Cre or lck-Cre TnfΔAREneomice. Detected with the 3′ probe are the neo+ (1.6 Kb), neo- (0.5 Kb), and wild-type (0.4 Kb) fragments. (C) TNF protein production as determined by ELISA in culture supernatants from peritoneal macrophages (left) or splenic T cells (right) isolated from individual LysM-Cre (LysC) or lck-Cre (lckC) TnfΔAREneo(Δn) mice and stimulated with LPS or anti-CD3, respectively. Data shown as mean ng/ml (± SD) per group. P < 0.02 relative to (*) TnfΔAREneo/+ or (*) TnfΔAREneo/ΔAREneovalues with n = 3 mice/group. Histologic examination of ileal sections from 5-mo-old (D) TnfΔAREneo/ΔAREneo, (E) LysM-Cre TnfΔAREneo/+, (F) LysM-Cre TnfΔAREneo/ΔAREneo, and 7-mo-old (G) lck-Cre TnfΔAREneo/ΔAREneomutant mice. Paraffin sections stained with hematoxylin and eosin. ×100.

Mentions: To identify the cellular source(s) of pathogenic TNF in our model, we set up a system of tissue-specific activation of the TnfΔAREallele. To this end, we used a targeted mutant mouse strain, which contains a “hypomorphic” TnfΔAREneoallele generated by the introduction of a neomycin acetyltransferase gene (neor gene) flanked by loxP sequences next to the ΔARE mutation (Fig. 3 A; reference 5). Mice of the TnfΔAREneogenotype show a low to normal range of myeloid- and lymphoid-specific TNF production compared with Tnf+mice and do not develop any signs of intestinal pathology even at homozygosity (Fig. 3, C and D). After germ line or tissue-specific expression of Cre recombinase, the loxP sequences allow for the complete or tissue-specific removal of the “floxed” neorgene and the respective activation of the TnfΔAREallele (Fig. 3, A–C).


Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn's-like inflammatory bowel disease.

Kontoyiannis D, Boulougouris G, Manoloukos M, Armaka M, Apostolaki M, Pizarro T, Kotlyarov A, Forster I, Flavell R, Gaestel M, Tsichlis P, Cominelli F, Kollias G - J. Exp. Med. (2002)

Myeloid- and lymphoid-specific deregulation of TNF biosynthesis. (A) Structure of the TNF/LTα locus on mouse chromosome 17 and the strategy for the Cre-specific removal of floxed neor allele. Filled boxes represent exons and gray shaded boxes represent untranslated regions. Filled arrowheads indicate the position of the loxP sequences. The positions of the TNF AU-rich element (ARE) and the floxed neo marker are indicated. The location of the 3′ 0.4-Kb BglII probe is also indicated. Restriction enzyme sites are Bg:BglII, K:KpnI, N:NotI, and S:SacI. (B) Southern blot analysis of BglII-digested DNA from tail, peritoneal cavity cells (PEC), total exudate peritoneal macrophages, and T and B lymphocytes from LysM-Cre or lck-Cre TnfΔAREneomice. Detected with the 3′ probe are the neo+ (1.6 Kb), neo- (0.5 Kb), and wild-type (0.4 Kb) fragments. (C) TNF protein production as determined by ELISA in culture supernatants from peritoneal macrophages (left) or splenic T cells (right) isolated from individual LysM-Cre (LysC) or lck-Cre (lckC) TnfΔAREneo(Δn) mice and stimulated with LPS or anti-CD3, respectively. Data shown as mean ng/ml (± SD) per group. P < 0.02 relative to (*) TnfΔAREneo/+ or (*) TnfΔAREneo/ΔAREneovalues with n = 3 mice/group. Histologic examination of ileal sections from 5-mo-old (D) TnfΔAREneo/ΔAREneo, (E) LysM-Cre TnfΔAREneo/+, (F) LysM-Cre TnfΔAREneo/ΔAREneo, and 7-mo-old (G) lck-Cre TnfΔAREneo/ΔAREneomutant mice. Paraffin sections stained with hematoxylin and eosin. ×100.
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Related In: Results  -  Collection

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fig3: Myeloid- and lymphoid-specific deregulation of TNF biosynthesis. (A) Structure of the TNF/LTα locus on mouse chromosome 17 and the strategy for the Cre-specific removal of floxed neor allele. Filled boxes represent exons and gray shaded boxes represent untranslated regions. Filled arrowheads indicate the position of the loxP sequences. The positions of the TNF AU-rich element (ARE) and the floxed neo marker are indicated. The location of the 3′ 0.4-Kb BglII probe is also indicated. Restriction enzyme sites are Bg:BglII, K:KpnI, N:NotI, and S:SacI. (B) Southern blot analysis of BglII-digested DNA from tail, peritoneal cavity cells (PEC), total exudate peritoneal macrophages, and T and B lymphocytes from LysM-Cre or lck-Cre TnfΔAREneomice. Detected with the 3′ probe are the neo+ (1.6 Kb), neo- (0.5 Kb), and wild-type (0.4 Kb) fragments. (C) TNF protein production as determined by ELISA in culture supernatants from peritoneal macrophages (left) or splenic T cells (right) isolated from individual LysM-Cre (LysC) or lck-Cre (lckC) TnfΔAREneo(Δn) mice and stimulated with LPS or anti-CD3, respectively. Data shown as mean ng/ml (± SD) per group. P < 0.02 relative to (*) TnfΔAREneo/+ or (*) TnfΔAREneo/ΔAREneovalues with n = 3 mice/group. Histologic examination of ileal sections from 5-mo-old (D) TnfΔAREneo/ΔAREneo, (E) LysM-Cre TnfΔAREneo/+, (F) LysM-Cre TnfΔAREneo/ΔAREneo, and 7-mo-old (G) lck-Cre TnfΔAREneo/ΔAREneomutant mice. Paraffin sections stained with hematoxylin and eosin. ×100.
Mentions: To identify the cellular source(s) of pathogenic TNF in our model, we set up a system of tissue-specific activation of the TnfΔAREallele. To this end, we used a targeted mutant mouse strain, which contains a “hypomorphic” TnfΔAREneoallele generated by the introduction of a neomycin acetyltransferase gene (neor gene) flanked by loxP sequences next to the ΔARE mutation (Fig. 3 A; reference 5). Mice of the TnfΔAREneogenotype show a low to normal range of myeloid- and lymphoid-specific TNF production compared with Tnf+mice and do not develop any signs of intestinal pathology even at homozygosity (Fig. 3, C and D). After germ line or tissue-specific expression of Cre recombinase, the loxP sequences allow for the complete or tissue-specific removal of the “floxed” neorgene and the respective activation of the TnfΔAREallele (Fig. 3, A–C).

Bottom Line: Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity.Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background.Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, Biomedical Sciences Research Center "Al. Fleming," Vari 166-72, Greece. Division of Gastroenterology and Hepatology, University of Virginia Health Sciences Center, Charlottesville, VA 22906, USA.

ABSTRACT
Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor-deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow-derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

Show MeSH
Related in: MedlinePlus