Notch-1 signaling regulates intestinal epithelial barrier function, through interaction with CD4+ T cells, in mice and humans.

BACKGROUND & AIMS: Interactions between lymphocytes and intestinal epithelial cells occur in the subepithelial space of the gastrointestinal tract. Normal human lamina propria lymphocytes (LPLs) induce differentiation of intestinal epithelial cells. The absence of LPLs in mice, such as in RAG1(-/-) mice, results in defects in epithelial cell differentiation. We investigated the role of lymphoepithelial interactions in epithelial differentiation and barrier function. METHODS: We used adoptive transfer to determine if CD4(+) T cells (CD4(+)CD62L(+)CD45Rb(Hi) and/or CD4(+)CD62L(+)CD45Rb(Lo)) could overcome permeability defect (quantified in Ussing chambers). Immunofluorescence staining was performed to determine expression of cleaved Notch-1, villin, and claudin 5 in colon samples from mice and humans. Caco-2 cells were infected with a lentivirus containing a specific Notch-1 or scrambled short hairpin RNA sequence. Tight junction assembly was analyzed by immunoblot and immunofluorescence analyses, and transepithelial resistance was monitored. RESULTS: Expression of cleaved Notch-1, villin, or claudin 5 was not detected in RAG1(-/-) colonocytes; their loss correlated with increased intestinal permeability. Transfer of CD45Rb(Hi) and/or CD45Rb(Lo) cells into RAG1(-/-) mice induced expression of cleaved Notch, villin, and claudin 5 in colonocytes and significantly reduced the permeability of the distal colon. Loss of Notch-1 expression in Caco-2 cells correlated with decreased transepithelial resistance and dysregulated expression and localization of tight junction proteins. Levels of cleaved Notch-1 were increased in colonic epithelium of patients with Crohn's disease. CONCLUSIONS: LPLs promote mucosal barrier function, which is associated with activation of the Notch-1 signaling pathway. LPLs maintain intestinal homeostasis by inducing intestinal epithelial cell differentiation, polarization, and barrier function.

CCR7 deficiency in NOD mice leads to thyroiditis and primary hypothyroidism.

CCR7 is involved in the initiation of immune responses and has been recently implicated in the control of tolerance. To analyze the role of CCR7 in autoimmunity, we backcrossed CCR7(ko/ko) mice (in which ko signifies deficient) onto the autoimmune-prone NOD background. Surprisingly, NODCCR7(ko/ko) mice never developed diabetes, but showed severe inflammation in multiple tissues including thyroid, lung, stomach, intestine, uterus, and testis. NODCCR7(ko/ko) mice had a marked enlargement of the thyroid gland (goiter) that was associated with circulating autoantibodies against thyroglobulin, and development of primary hypothyroidism (decreased levels of serum thyroxin, and augmented levels of thyroid-stimulating hormone in the pituitary gland), features found in Hashimoto's thyroiditis. Cells isolated from diseased thyroids and activated splenocytes from NODCCR7(ko/ko) animals induced goiter in NOD.SCID recipients, demonstrating that autoreactive cells were generated in the absence of CCR7. Moreover, thyroid disease could be accelerated in young NODCCR7(ko/ko) mice by immunization with thyroglobulin. These results demonstrate the complexity in the generation of multiple autoimmune phenotypes in NOD mice and indicate that CCR7 is a key molecule in their development.

AP-1 activated by toll-like receptors regulates expression of IL-23 p19.

Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5'-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (-413 to +10) that contains several important elements, including NF-kappaB and AP-1. In addition to NF-kappaB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-kappaB.

SCF-mediated mast cell infiltration and activation exacerbate the inflammation and immunosuppression in tumor microenvironment.

Despite the evidence for the role of inflammation in cancer initiation, promotion, and progression, the precise mechanism by which the inflammation within tumor is orchestrated by inflammatory cells remains to be determined. Here, we report that tumor-infiltrating mast cells remodel tumor microenvironment and promote tumor growth. Mast cell infiltration and activation in tumors were mainly mediated by tumor-derived stem cell factor (SCF) and its receptor c-Kit on mast cells. Low concentrations of SCF efficiently induced the chemotactic migration of mast cells. Tumor-infiltrating mast cells, activated by higher concentrations of SCF, expressed multiple proinflammatory factors and increased IL-17 expression in tumors. The activity of NF-kappaB and AP-1 in tumor cells was intensified in the mast cell-remodeled inflammatory microenvironment. SCF-activated mast cells also exacerbated tumor immunosuppression by releasing adenosine and increasing T regulatory cells, which augmented the suppression of T cells and natural killer cells in tumors. These findings emphasize that the remodeling of the tumor microenvironment can actually be initiated by tumor cell-released SCF and suggest that mast cells are not only a participator but also a critical regulator of inflammation and immunosuppression in the tumor microenvironment.

Interleukin 10 suppresses Th17 cytokines secreted by macrophages and T cells.

IL-17 and IL-22 are typical cytokines produced by the Th17 T cell subset, but it is unclear if Th17 cytokines can be produced by other cell types. We demonstrate that IL-10-deficient and IL-10R-deficient macrophages stimulated with lipopolysaccharide produce high levels of IL-17 and IL-22. Addition of exogenous IL-10 to IL-10-deficient macrophages abolished IL-17 production. When IL-10-deficient and IL-10R-deficient splenocytes were cultured under Th17 polarizing conditions, the population of IL-17-producing cells was increased and the cultures produced significantly higher levels of IL-17 and IL-22. The addition of recombinant IL-10 to IL-10-deficient splenocytes significantly decreased the percentage of IL-17-producing CD4(+) T cells. Finally, the mRNA for the Th17 transcription factor retinoic acid-related orphan receptor (ROR)gammat was significantly elevated in IL-10-deficient spleen cells and macrophages. These data demonstrate that Th17 cytokines and RORgammat are also expressed in macrophages and that IL-10 negatively regulates the expression of Th17 cytokines and RORgammat by both macrophages and T cells.

Epstein-Barr virus-induced gene 3 negatively regulates IL-17, IL-22 and RORgamma t.

Epstein-Barr virus-induced gene 3 (EBI3) associates with p28 to form IL-27 and with IL-12p35 to form IL-35. IL-27Ralpha(-/-) mice studies indicate that IL-27 negatively regulates Th17 cell differentiation. However, no EBI3, p28 or p35-deficiency studies that directly address the role of EBI3, p28 or p35 on Th17 cells have been done. Here, we demonstrate that spleen cells derived from EBI3(-/-) mice produce significantly higher levels of IL-17 as well as IL-22 upon stimulation with OVA. In vitro derived EBI3(-/-) Th17 cells also produced significantly higher levels of IL-17 and IL-22 than WT cells. The frequency of IL-17-producing cells was also elevated when EBI3(-/-) cells were cultured under Th17 conditions. In addition, spleen cells from EBI3(-/-) mice immunized with Listeria monocytogenes produced significantly elevated levels of IL-17 and IL-22. Furthermore, the Th17 transcription factor RORgamma t was significantly enhanced in EBI3(-/-) cells. Finally, EBI3(-/-) mice exhibited a reduced bacterial load following an acute challenge with L. monocytogenes or a re-challenge of previously immunized mice, suggesting that EBI3 negatively regulates both innate and adaptive immunity. Taken together, these data provide direct evidence that EBI3 negatively regulates the expression of IL-17, IL-22 and RORgamma t as well as protective immunity against L. monocytogenes.

Listeria monocytogenes promotes tumor growth via tumor cell toll-like receptor 2 signaling.

The contribution of bacterial infection to tumorigenesis is usually ascribed to infection-associated inflammation. An alternate view is that direct interaction of bacteria with tumor cells promotes tumor progression. Here, we show that the microenvironment of large tumors favors bacterial survival, which in turn directly accelerates tumor growth by activating tumor cell Toll-like receptors (TLR). Listeria monocytogenes (Lm) survives in the microenvironment of large but not small tumors, resulting in the promotion of tumor growth. Lm did not affect the percentage of regulatory T cells or myeloid suppressor cells in the tumor. Through TLR2 signaling, Lm activated mitogen-activated protein kinases and nuclear factor-kappaB in tumor cells, resulting in the increased production of nitric oxide and interleukin-6 and increased proliferation of tumor cells. All of these effects were abrogated by silencing expression of TLR2, but not TLR4. The interaction of Helicobacter pylori with tumor cells from gastric carcinoma patients resulted in similar effects. These findings provide a new insight into infection-associated tumorigenesis and illustrate the importance of antibiotic therapy to treat tumors with bacterial infiltration.

IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways.

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.

Toll-like receptors on tumor cells facilitate evasion of immune surveillance.

The signal pathways that trigger tumor cell escape from immune surveillance are incompletely understood. Toll-like receptors (TLRs), which activate innate and adaptive immune responses, are thought to be restricted to immune cells. We show here that TLRs, including TLR4, are expressed on tumor cells from a wide variety of tissues, suggesting that TLR activation may be an important event in tumor cell immune evasion. Activation of TLR4 signaling in tumor cells by lipopolysaccharide induces the synthesis of various soluble factors and proteins including interleukin-6, inducible nitric oxide synthase, interleukin-12, B7-H1, and B7-H2, and results in resistance of tumor cells to CTL attack. In addition, lipopolysaccharide-stimulated tumor cell supernatants inhibit both T cell proliferation and natural killer cell activity. Blockade of the TLR4 pathway by either TLR4 short interfering RNA or a cell-permeable TLR4 inhibitory peptide reverses tumor-mediated suppression of T cell proliferation and natural killer cell activity in vitro, and in vivo, delays tumor growth and thus prolongs the survival of tumor-bearing mice. These findings indicate that TLR signaling results in a cascade leading to tumor evasion from immune surveillance. These novel functions of TLRs in tumor biology suggest a new class of therapeutic targets for cancer therapy.

Ubiquitin-dependent degradation of interferon regulatory factor-8 mediated by Cbl down-regulates interleukin-12 expression.

Interferon regulatory factor (IRF)-8/interferon consensus sequence-binding protein is regulated by both transcription and degradation. IRF-8 induced in peritoneal macrophages by interferon-gamma and lipopolysaccharide was degraded rapidly, and degradation of IRF-8 was blocked by MG132, the proteasome inhibitor, but inhibitors of calpain and lysosomal enzymes had no effect. The ubiquitination of IRF-8 was shown by co-immunoprecipitation from RAW264.7 macrophages retrovirally transduced with IRF-8 and hemagglutinin-ubiquitin. The dominant negative ubiquitin mutants K48R and K29R inhibited IRF-8 degradation in 293T cells, confirming the relationship between ubiquitination of IRF-8 and its degradation. IRF-8 carboxyl-terminal truncation mutants were not ubiquitinated and were consequently stable, indicating that the carboxyl-terminal domain of IRF-8 controls ubiquitination. The ubiquitin-protein isopeptide ligase (E3) that ubiquitinated IRF-8 was likely to be Cbl, which formed a complex with IRF-8, demonstrable by both immunoprecipitation and gel filtration. Furthermore, IRF-8 stability was increased by dominant negative Cbl, and IRF-8 ubiquitination was significantly attenuated in Cbl-/- cells. Reflecting increased stability and expression, the IRF-8 carboxyl-terminal deletion mutant induced interleukin (IL)-12 p40 promoter activity much more strongly than IRF-8 did. Furthermore, IRF-8-induced IL-12 p40 synthesis in RAW264.7 cells was enhanced by dominant negative Cbl, and peritoneal macrophages from Cbl-/- mice showed increased IL-12 p40 protein production. Taken together, these results suggest that the proteasomal degradation of IRF-8 mediated by the ubiquitin E3 ligase Cbl down-regulates IL-12 expression.

Interaction of TRAF6 with MAST205 regulates NF-kappaB activation and MAST205 stability.

The binding of immune complexes to macrophage Fcgamma receptor results in a subsequent inhibition of lipopolysaccharide-stimulated interleukin-12 synthesis without affecting the induction of tumor necrosis factor-alpha. RNA interference targeting MAST205, a 205-kDa serine/threonine kinase, and transfection of dominant negative MAST205 mutants also mimic this type II macrophage phenotype. Our previous epistasis experiments suggested that the position of MAST205 in the TLR4 signal pathway was proximal to the IkappaB kinase complex. We now report that MAST205 forms a complex with TRAF6, resulting in the inhibition of TRAF6 NF-kappaB activation. We have identified a peptide (residues 218-233) from the N terminus of MAST205 that, when coupled to a protein transduction domain, inhibits the lipopolysaccharide-stimulated activation of NF-kappaB, modulates the size of the MAST205.TRAF6 complex, and inhibits ubiquitination of TRAF6. A dominant negative N-terminal MAST205 deletion mutant also inhibits TRAF6 ubiquitination. The domain required for degradation of MAST205 after Fcgamma receptor activation resides within the N-terminal 261 residues, and degradation is triggered by protein kinase C isoform phosphorylation of Ser/Thr residues. These results suggest that MAST205 functions as a scaffolding protein controlling TRAF6 activity and, therefore, plays an important role in regulating inflammatory responses.

Microtubule-associated serine/threonine kinase-205 kDa and Fc gamma receptor control IL-12 p40 synthesis and NF-kappa B activation.

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-alpha, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcgammaR ligation; TNF-alpha secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-kappaB activation, but do not affect IL-1 or TNF-alpha signaling. Finally, macrophage FcgammaR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.

Complex formation of the interferon (IFN) consensus sequence-binding protein with IRF-1 is essential for murine macrophage IFN-gamma-induced iNOS gene expression.

This study describes the role of the interferon (IFN) consensus sequence-binding protein (ICSBP or IRF-8) in iNOS gene expression by murine macrophages. An ICSBP binding site in the iNOS promoter region (-923 to -913) was identified using an electrophoretic mobility shift assay and chromatin co-immunoprecipitation. Overexpression of ICSBP greatly enhanced IFN-gamma-induced iNOS promoter activation in RAW264.7 cells, and IFN-gamma-induced iNOS promoter activation was abolished in ICSBP-/- macrophages. Furthermore, transduction of retrovirus-ICSBP in ICSBP-/- macrophages rescued IFN-gamma-induced iNOS gene expression. However, transduction of retrovirus-ICSBP in the absence of IFN-gamma activation did not induce iNOS expression in either RAW264.7 cells or ICSBP-/- macrophages. Interestingly, ICSBP alone transduced into ICSBP-/- macrophages did not bind to IFN-stimulated response element site (-923 to -913) of the iNOS promoter region, although following activation with IFN-gamma, a DNA.protein complex was formed that contains ICSBP and IRF-1. Co-transduction of ICSBP with IRF-1 clearly induces nitric oxide production. In addition, interleukin-4 inhibits IFN-gamma-induced iNOS gene expression by attenuating the physical interaction of ICSBP with IRF-1. Complex formation of ICSBP with IRF-1 is essential for iNOS expression, and interleukin-4 attenuates the physical interaction of ICSBP with IRF-1 resulting in the inhibition of INOS gene expression.