Heligmosomoides polygyrus bakeri Infection Decreases Smad7 Expression in Intestinal CD4+ T Cells, Which Allows TGF-ß to Induce IL-10-Producing Regulatory T Cells That Block Colitis.

Helminthic infections modulate host immunity and may protect their hosts from developing immunological diseases like inflammatory bowel disease. Induction of regulatory T cells (Tregs) may be an important part of this protective process. Heligmosomoides polygyrus bakeri infection also promotes the production of the regulatory cytokines TGF-ß and IL-10 in the gut. In the intestines, TGF-ß helps induce regulatory T cells. This study used Foxp3/IL-10 double reporter mice to investigate the effect of TGF-ß on the differentiation of colon and mesenteric lymph node-derived murine Foxp3- IL-10- CD4+ T cells into their regulatory phenotypes. Foxp3- IL-10- CD4+ T cells from H. polygyrus bakeri-infected mice, as opposed to T cells from uninfected animals, cultured in vitro with TGF-ß and anti-CD3/CD28 mAb differentiated into Foxp3+ and/or IL-10+ T cells. The IL-10-producing T cells nearly all displayed CD25. Smad7 is a natural inhibitor of TGF-ß signaling. In contrast to gut T cells from uninfected mice, Foxp3- IL10- CD4+ T cells from H. polygyrus bakeri-infected mice displayed reduced Smad7 expression and responded to TGF-ß with Smad2/3 phosphorylation. The TGF-ß-induced Tregs that express IL-10 blocked colitis when transferred into the Rag/CD25- CD4+ T cell transfer model of inflammatory bowel disease. TGF-ß had a greatly diminished capacity to induce Tregs in H. polygyrus bakeri-infected transgenic mice with constitutively high T cell-specific Smad7 expression. Thus, infection with H. polygyrus bakeri causes down-modulation in Smad7 expression in intestinal CD4+ T cells, which allows the TGF-ß produced in response to the infection to induce the Tregs that prevent colitis.

Generation of Rat Monoclonal Antibodies Against a Deubiquitinase, Ovarian Tumor Domain-Containing Protein 1.

Ovarian tumor domain-containing protein 1 (OTUD1), an OTU-family deubiquitinating enzyme, has been reported to be involved in cancer progression through the regulation of p53 and SMAD7. However, the precise pathophysiological functions of OTUD1 remain elusive. Here, we report the establishment of OTUD1-specific monoclonal antibodies (mAbs), using the rat medial iliac lymph node method. The generated antibodies recognize the N-terminal portion (aa. 1-290) of human and mouse OTUD1 proteins. In addition, immunofluorescent staining and subcellular fractionation analyses using these antibodies indicated that OTUD1 is predominantly localized in the cytosol. Thus, these mAbs can be further used to elucidate cellular functions of OTUD1 and its involvement in processes such as cancer progression.

Lessons Learned From Trials Targeting Cytokine Pathways in Patients With Inflammatory Bowel Diseases.

Insights into the pathogenesis of inflammatory bowel diseases (IBDs) have provided important information for the development of therapeutics. Levels of interleukin 23 (IL23) and T-helper (Th) 17 cell pathway molecules are increased in inflamed intestinal tissues of patients with IBD. Loss-of-function variants of the IL23-receptor gene (IL23R) protect against IBD, and, in animals, blocking IL23 reduces the severity of colitis. These findings indicated that the IL23 and Th17 cell pathways might be promising targets for the treatment of IBD. Clinical trials have investigated the effects of agents designed to target distinct levels of the IL23 and Th17 cell pathways, and the results are providing insights into IBD pathogenesis and additional strategies for modulating these pathways. Strategies to reduce levels of proinflammatory cytokines more broadly and increase anti-inflammatory mechanisms also are emerging for the treatment of IBD. The results from trials targeting these immune system pathways have provided important lessons for future trials. Findings indicate the importance of improving approaches to integrate patient features and biomarkers of response with selection of therapeutics.

Effect of compound Maqin decoction on TGF-ß1/Smad proteins and IL-10 and IL-17 content in lung tissue of asthmatic rats.

In this research, compound Maqin decoction (CMD) has been shown to positively affect in airway inflammation of asthma models. We evaluated the effects of CMD on the expression of transforming growth factor (TGF)-ß1/Smad proteins, interleukin (IL)-17, and IL-10 in lung tissue of asthmatic rats. Asthma was induced in a rat model using ovalbumin. After a 4-week treatment with CMD, rats were killed to evaluate the expression of TGF-ß1 and Smad proteins in lung tissue. IL-10 and IL-17 levels in lung tissue homogenates were determined by ELISA. The expression of TGF-ß1 and Smad3 protein increased, whereas expression of Smad7 protein decreased upon high-dose or low-dose treatment with CMD or by intervention with dexamethasone, compared to the control. There was a significant difference between treatment with a high dose CMD and the control treatment, but no significant difference was found between high-dose CMD treatment and dexamethasone intervention. The expression of TGF-ß1 and Smad7 protein increased, whereas the expression of Smad3 protein decreased in the model group compared to other groups. In the CMD high-dose group, low-dose group, and dexamethasone intervention group, the IL-17 concentrations in lung tissue homogenates were decreased, while IL-10 levels were increased. Again, there was a significant difference between CMD high-dose and control treatment, but not between CMD high-dose treatment and dexamethasone intervention. Thus, positive effects of CMD against asthmatic airway remodeling may be due to its regulatory effect on TGF-ß1, Smad3, and Smad7 protein levels and on cytokines such as IL-10 and IL-17.

Exchange protein directly activated by cAMP modulates regulatory T-cell-mediated immunosuppression.

The cAMP signalling pathway plays an essential role in immune functions. In the present study we examined the role of the cAMP/EPAC1 (exchange protein directly activated by cAMP) axis in regulatory T-cell (Treg)-mediated immunosuppression using genetic and pharmacological approaches. Genetic deletion of EPAC1 in Tregs and effector T-cells (Teffs) synergistically attenuated Treg-mediated suppression of Teffs. Mechanistically, EPAC1 inhibition enhanced activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) and up-regulated SMAD7 expression while down-regulating expression of SMAD4. Consequently, CD4+ T-cells were desensitized to transforming growth factor (TGF) ß1, a cytokine employed by Tregs to exert a broad inhibitory function within the immune system. Furthermore, deletion of EPAC1 led to production of significant levels of ovalbumin IgG antibodies in a low-dose, oral-tolerance mouse model. These in vivo observations are consistent with the finding that EPAC1 plays an important role in Treg-mediated suppression. More importantly, pharmacological inhibition of EPAC1 using an EPAC-specific inhibitor recapitulates the EPAC1 deletion phenotype both in vivo and in vitro. The results of the present study show that EPAC1 boosts Treg-mediated suppression, and identifies EPAC1 as a target with broad therapeutic potential because Tregs are involved in numerous pathologies, including autoimmunity, infections and a wide range of cancers.

Relationship of TGF-ß1 and Smad7 expression with decreased dendritic cell infiltration in liver gastrointestinal cancer metastasis.

Immune responses and their modulation within the liver are critical to the outcome of liver malignancies. In late-stage tumors, secreted TGF-ß promotes oncogenic functions and can confer tolerogenicity to some immune cells like DCs. The TGF-ß signaling pathway is involved in the control of several biological processes, including immunosurveillance. The aim of the present study was to assess CD1a(+) and CD83(+) DCs and to evaluate the impact of TGF-ß pathway on DCs maturation and distribution in the liver metastases from gastric and colorectal tumors. The percentage of CD83(+) DCs in the liver tissue, surrounding metastasis and in the metastasis-free liver was measured by flow cytometry, and TGF-ß levels were assessed in the tissue supernatant from the peritumoral liver after mononuclear cell isolation and in the sera of the same patients. CD1a(+) and CD83(+) DCs were observed in the tumor stroma and border. Out of 73 patients, there was cytoplasmic reactivity: of TGF-ß1 in 37 (50.7%); of Smad4 in 62 (84.9%); of Smad7 in 46 (63%), and of TGFßRII in 39 (53.4%) of the metastases. The TGF-ß1 expression in tumor cell cytoplasm correlated with low CD1a(+) and low CD83(+) DCs infiltration. The tissue levels of TGF-ß1, measured by ELISA in the supernatant were significantly increased in metastases than in normal liver. Using a two-color FACS analysis, we found that the percentage of HLA-DR(+) CD83(+) DCs in metastases was significantly decreased as compared with metastasis-free liver tissue. In conclusion, the positive and negative correlations between the mediators from the TGF-ß pathway implied the existence of imbalance and suppression of this cytokine activity. The presence of increased TGF-ß expression by immunohistochemistry in tumor cells was confirmed by detection of increased TGF-ß tissue level in the supernatant from the tissue homogenate. The observation of low numbers of CD1a(+) and CD83(+) DCs in tumor stroma correlated with TGF-ß overexpression in tumor cells, a fact that well documents the immunosuppressive role of TGF-ß in metastasis development. The increased percentage of CD83(+) DCs in the peritumoral tissue supposes that there could be active recruitment or local differentiation of DCs in the metastasis border, but inside the tumor the immune cells recruitment and activity are suppressed by TGF-ß and by other cytokines.

Attenuated TGF-ß1 responsiveness of dendritic cells and their precursors in atopic dermatitis.

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF-ß1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF-ß signaling in monocytes and monocyte-derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF-ß receptors, phospho-Smad2/3 and Smad7 were evaluated. Furthermore, TNF-α expression and synergistic effects of TNF-α upon TGF-ß signaling and DC generation were evaluated. We found LC-like DC differentiation of monocytes from AD patients in response to TGF-ß1 was remarkably reduced and TGF-ß1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF-ß1 responsiveness mirrored by lower phospho-Smad2/3 expression after TGF-ß1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC-like DCs of AD patients. Lower TNF-α expression of monocytes from AD patients might further contribute to attenuated TGF-ß signaling in the disease since TNF-α had synergistic effects on TGF-ß1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF-ß1 signaling together with the amount of soluble co-factors might direct the nature of differentiating DCs.

Smad7 antisense oligonucleotide-based therapy for inflammatory bowel diseases.

The aetiology of both Crohn's disease and ulcerative colitis, the major forms of inflammatory bowel diseases in human beings, remains unknown. However, compelling evidence suggests that the associated pathological process in inflammatory bowel disease is driven by an excessive immune response directed against normal components of the bacterial microflora and marked by defects in counter-regulatory mechanisms, such as those involving transforming growth factor-ß1. Indeed, a diminished activity of transforming growth factor-ß1, as indicated by a reduced phosphorylation of Smad3, a signalling molecule associated with the activated transforming growth factor-ß receptor, is evident in the inflamed gut of inflammatory bowel disease patients and this alteration is due to high Smad7, an intracellular inhibitor of Smad3 phosphorylation. Consistently, silencing of Smad7 with a specific antisense oligonucleotide restores transforming growth factor-ß1/Smad3 signalling, thereby leading to inhibition of inflammatory cytokine production and attenuation of experimental colitis in mice. These findings together with the demonstration that Smad7 antisense oligonucleotide is safe and well-tolerated in patients with Crohn's disease indicate that Smad7 antisense oligonucleotide-based pharmaceutical compounds could enter the therapeutic armamentarium of these disorders. In this article we review the available data supporting the pathogenic role of Smad7 in the gut and discuss why Smad7 antisense therapy could help dampen the mucosal inflammation in inflammatory bowel disease.

Early administration of probiotic Lactobacillus acidophilus and/or prebiotic inulin attenuates pathogen-mediated intestinal inflammation and Smad 7 cell signaling.

Immaturity of gut-associated immunity may contribute to pediatric mortality associated with enteric infections. A murine model to parallel infantile enteric disease was used to determine the effects of probiotic, Lactobacillus acidophilus (La), prebiotic, inulin, or both (synbiotic, syn) on pathogen-induced inflammatory responses, NF-κB, and Smad 7 signaling. Newborn mice were inoculated bi-weekly for 4 weeks with La, inulin, or syn and challenged with Citrobacter rodentium (Cr) at 5 weeks. Mouse intestinal epithelial cells (CMT93) were exposed to Cr to determine temporal alterations in NF-Kappa B and Smad 7 levels. Mice with pretreatment of La, inulin, and syn show reduced intestinal inflammation following Cr infection compared with controls, which is associated with significantly reduced bacterial colonization in La, inulin, and syn animals. Our results further show that host defense against Cr infection correlated with enhanced colonic IL-10 and transforming growth factor-ß expression and inhibition of NF-κB in syn-treated mice, whereas mice pretreated with syn, La, or inulin had attenuation of Cr-induced Smad 7 expression. There was a temporal Smad 7 and NF-κB intracellular accumulation post-Cr infection and post-tumor necrosis factor stimulation in CMT93 cells. These results, therefore, suggest that probiotic, La, prebiotic inulin, or synbiotic may promote host-protective immunity and attenuate Cr-induced intestinal inflammation through mechanisms affecting NF-κB and Smad 7 signaling.

TGF-ß signaling is altered in the peripheral blood of subjects with multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system inflammatory disorder with evidence of peripheral immune dysregulation. Abnormalities of the immune suppressive cytokine TGF-ß have been reported, but not fully defined, in MS. Through a pathway-focused expression profiling of the peripheral blood, we found abnormalities of TGF-ßRII, SMAD4 and SMAD7 expression in subjects with MS, and reduction in the levels of TGF-ß regulated genes, indicating an overall reduction in TGF-ß signaling in MS. The response to exogenous TGF-ß was intact, however, indicating an extrinsic defect of TGF-ß signaling in MS. These results indicate that TGF-ß control is diminished in MS.

Simvastatin induces Foxp3+ T regulatory cells by modulation of transforming growth factor-beta signal transduction.

Statins are widely used drugs for the treatment of hypercholesterolaemia. A number of recent studies have suggested that statins also have pleiotropic effects on immune responses and statins have proven to be effective in the treatment of autoimmune diseases in animal models. Foxp3(+) T regulatory cells are a unique subset of CD4(+) T cells that mediate immunosuppression. Foxp3(+) T cells develop in the thymus, but can also be induced in peripheral sites in the presence of transforming growth factor-beta (TGF-beta). We demonstrate here that simvastatin blockade of the mevalonate pathway can mediate induction of mouse Foxp3(+) T cells and that simvastatin can synergize with low levels of TGF-beta to induce Foxp3(+) T cells. The effects of simvastatin are secondary to a blockade of protein geranylgeranylation, are mediated at late time-points after T-cell activation, and are associated with demethylation of the Foxp3 promoter. One major effect of simvastatin was inhibition of the induction of Smad6 and Smad7, inhibitory Smads that inhibit TGF-beta signalling. Our results suggest that one mechanism responsible for the immunosuppressive effects of statins is the ability to promote the generation of Foxp3(+) T regulatory cells.

Impaired cutaneous wound healing with excess granulation tissue formation in TNFalpha-null mice.

We examined the effects of lacking tumor necrosis factor alpha (TNFalpha) on the healing process of a cutaneous wound in mice using TNFalpha-deficient mice. A full-thickness circular cutaneous wound 5.0 mm in diameter was produced in the dorsal skin of wild-type (WT) or TNFalpha-null (KO) mice. After specific intervals of healing, the healing pattern was evaluated by macroscopic observation, histology, immunohistochemistry, or real-time reverse transcription-polymerase chain reaction. Effect of Smad7 gene transfer on the healing phenotype of KO mice was also examined. The results showed that loss of TNFalpha promotes granulation tissue formation and retards reepithelialization in a circular wound in mouse dorsal skin. Immunohistochemistry showed that distribution of macrophages and myofibroblasts in newly generated granulation tissue seemed similar between WT and KO mice. However, lacking TNFalpha enhanced mRNA expression of TGFbeta1 and collagen Ialpha2 in such tissue. Smad7 gene transfer counteracted excess granulation tissue formation in KO mice. In conclusion, lacking TNFalpha potentiates Smad-mediated fibrogenic reaction in healing dermis and retards reepithelialization in a healing mouse cutaneous wound.

TGF-beta1 and Smad7 in the regulation of IBD.

Inflammatory bowel diseases (IBDs) are caused by an aberrant and excessive local immune response to components of the bacterial microflora that are: poorly controlled by endogenous counter regulatory mechanisms such as the immunosuppressive cytokine transforming growth factor-beta1 (TGF-beta1). Studies in human IBD tissues have documented a disruption of TGF-beta1 signaling marked by a block in the phosphorylation of the activated TGF-beta receptor-associated signaling molecule, Smad3, caused by the upregulation of the intracellular inhibitor of Smad signaling, Smad7. Inhibition of Smad7 with a specific antisense oligonucleotide restores TGF-beta1/Smad3 signaling, resulting in a marked suppression of inflammatory cytokine production. The functional relevance of Smad7 in gut inflammation was confirmed by studies in murine models of IBD. In inflamed tissues of mice with colitis induced by either the trinitrobenzene sulfonic acid or oxazolone, p-Smad3 was low despite active TGF-beta1 being produced in excess. In vivo administration of Smad7 antisense oligonucleotides to mice with colitis restored TGF-beta1 signaling and decreased the synthesis of inflammatory molecules and the extent of gut damage. These data support a role for Smad7 in maintaining intestinal inflammation, and suggest that blocking Smad7 could be a promising way to dampen the ongoing inflammation in IBD.

What lurks beneath: IL-11, via Stat3, promotes inflammation-associated gastric tumorigenesis.

Chronic inflammation in the stomach induces cellular transformation and gastric cancer primarily in the distal stomach or antrum. In this issue of the JCI, a study in mice by Ernst et al. provides new insight into the role of IL-11 and its glycoprotein 130 (gp130) receptor in inflammation-associated gastric epithelial cell oncogenic transformation, which they show is mediated by and dependent on increased activation of Stat3 and, to a lesser extent, Stat1 (see the related article beginning on page 1727). Prior studies from this group have shown that Stat3 hyperactivity stimulates the TGF-beta inhibitor Smad7. Collectively, the studies suggest that an important pathway of oncogenic transformation in the stomach is through suppression of growth inhibitory signals, such as members of the TGF-beta family, that originate from the stroma.

Cutting edge: trans-signaling via the soluble IL-6R abrogates the induction of FoxP3 in naive CD4+CD25 T cells.

Chronic inflammatory diseases may develop when regulatory T cells (Tregs) fail to control the balance between tolerance and immunity. Alternatively, activated immune cells might prevent the induction or activation of Tregs in such diseases. In this study, we demonstrate that trans-signaling into T cells via the soluble IL-6 receptor completely abrogates the de novo induction of adaptive Tregs. Mechanistically, IL-6 trans-signaling augmented the expression of the TGF-beta signaling inhibitor SMAD7. Consequently, SMAD7 overexpression in T cells using newly created transgenic mice rendered CD4(+)CD25(-) T cells resistant to the induction of FoxP3. Finally, IL-6 trans-signaling inhibited Treg-mediated suppression in a murine model of colitis. In summary, IL-6 trans-signaling into T cells emerges as a key pathway for blockade of the development of adaptive Tregs and thus may play a pivotal role in shifting the balance between effector and regulatory T cell numbers in chronic inflammatory and autoimmune diseases.

Deletion of exon I of SMAD7 in mice results in altered B cell responses.

The members of the TGF-beta superfamily, i.e., TGF-beta isoforms, activins, and bone morphogenetic proteins, regulate growth, differentiation, and apoptosis, both during embryonic development and during postnatal life. Smad7 is induced by the TGF-beta superfamily members and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. In addition, Smad7 is induced by other stimuli. Thus, it can fine-tune and integrate TGF-beta signaling with other signaling pathways. To investigate the functional role(s) of Smad7 in vivo, we generated mice deficient in exon I of Smad7, leading to a partial loss of Smad7 function. Mutant animals are viable, but significantly smaller on the outbred CD-1 mouse strain background. Mutant B cells showed an overactive TGF-beta signaling measured as increase of phosphorylated Smad2-positive B cells compared with B cells from wild-type mice. In agreement with this expected increase in TGF-beta signaling, several changes in B cell responses were observed. Mutant B cells exhibited increased Ig class switch recombination to IgA, significantly enhanced spontaneous apoptosis in B cells, and a markedly reduced proliferative response to LPS stimulation. Interestingly, LPS treatment reverted the apoptotic phenotype in the mutant cells. Taken together, the observed phenotype highlights a prominent role for Smad7 in development and in regulating the immune system's response to TGF-beta.