Roberta Izzo

Aryl hydrocarbon receptor-driven signals inhibit collagen synthesis in the gut

Fibrostrictures (FS) are a major complication of Crohns disease (CD). Pathogenesis of FS is not fully understood, but activation of fibroblasts and excessive collagen deposition are crucial in the development of FS. Here, we investigated the role of aryl hydrocarbon receptor (AhR) in intestinal fibrosis. AhR RNA and protein expression were evaluated in intestinal fibroblasts of CD patients and controls. CD fibroblasts were stimulated with TGF‐β1 or TNF‐α in the presence or absence of the AhR activator Ficz, an AhR antagonist CH223191, or a specific AhR‐silencing RNA. In CD fibroblasts, TGF‐β1 and TNF‐α increased Col1A1, Col3A1 and α‐SMA transcripts and collagen secretion and this effect was reduced by Ficz and upregulated by CH22319. TGF‐β1 or TNF‐α induced activation of p38 and ERK1/2 MAP kinases was decreased by Ficz and increased by CH223191. The inhibitory effect of Ficz on Map kinase activation and collagen induction was abolished by AhR silencing. To assess the role of AhR in vivo, mice with trinitrobenzene‐sulfonic‐acid induced colonic fibrosis were given Ficz or CH223191. Mice given either Ficz or CH223191 produced less or more collagen respectively as compared with control mice. Our results indicate that AhR is a negative regulator of profibrotic signals in the gut.

TNF-α Producing Innate Lymphoid Cells (ILCs) Are Increased in Active Celiac Disease and Contribute to Promote Intestinal Atrophy in Mice

Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of this study was to characterize the tissue distribution of ILCs in celiac disease (CD), a gluten-driven enteropathy, and analyze their role in gut tissue damage. ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs) isolated from duodenal biopsies of CD patients and healthy controls (CTR) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with peptidoglycan, poly I:C, or CpG, the agonists of TLR2, TLR3, or TLR9 respectively, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Although the percentage of total ILCs did not differ between CD patients and CTR, ILCs producing TNF-α and IFN-γ were more abundant in CD mucosa compared to controls. ILCs expressed TLR2, TLR3 and TLR9 but neither TLR7 nor TLR4. Stimulation of LPMC with poly I:C but not PGN or CpG increased TNF-α and IFN-γ in ILCs. RAG1-deficient mice given poly I:C exhibited increased frequency of TNF-α but not IFN-γ/IL17A-producing ILCs in the gut and depletion of ILCs prevented the poly I:C-driven intestinal damage. Our data indicate that CD-related inflammation is marked by accumulation of ILCs producing TNF-α and IFN-γ in the mucosa. Moreover, ILCs express TLR3 and are functionally able to respond to poly I:C with increased synthesis of TNF-α thus contributing to small intestinal atrophy.

IL-21 as a therapeutic target in inflammatory disorders

Introduction: IL-21, a cytokine produced by activated CD4+ cells, activated natural killer T cells and T helper cells in the germinal centers, is involved in the control of the function of both immune and parenchymal cells. Areas covered: IL-21 is overproduced in many chronic inflammatory disorders, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, type I diabetes and systemic lupus erythematosus, and studies in experimental models indicate that IL-21 plays an important role in sustaining tissue-damaging immune responses in such pathologies. However, genetic deficiency of IL-21 associates with inflammatory bowel diseases and blockade of IL-21 in the early phases exacerbates the disease progression in some models of rheumatoid arthritis and systemic lupus erythematosus, thus suggesting a dual role of IL-21 in the control of immune-mediated diseases. IL-21 can exert additional protective functions for the host as it promotes cytotoxic responses against tumors and viruses. Expert opinion: We here review the available data on the role of IL-21 in chronic inflammatory diseases and discuss the therapeutic benefit of IL-21 inhibitors in such diseases as well as the potential risks of such treatments.

Interleukin-34 Induces Cc-chemokine Ligand 20 in Gut Epithelial Cells.

BACKGROUND AND AIM Production of chemokines by intestinal epithelial cells is a key step in the amplification of the destructive immune-inflammatory response in patients with inflammatory bowel diseases [IBD]. In this study, we examined whether intestinal epithelial cells express macrophage colony-stimulating factor receptor 1 [M-CSFR-1], the functional receptor of interleukin-34 [IL-34], a cytokine that is over-produced in IBD and supposed to sustain inflammatory pathways. METHODS M-CSFR-1 expression was evaluated in intestinal samples of IBD patients, controls, and colon epithelial cell lines by real-time polymerase chain reaction [PCR], immunohistochemistry, and western blotting. DLD-1 cells were stimulated with IL-34 in the presence or absence of MAP kinase inhibitors, chemokine induction was assessed by real-time PCR and enzyme-linked immunosorbent assay [ELISA], and mitogen-activated protein (MAP) kinase activation was monitored by western blotting. The effect of a neutralising IL-34 antibody on CC chemokine ligand (CCL) 20 synthesis was tested in ex vivo organ cultures of IBD mucosal explants. RESULTS Enhanced expression of M-CSFR-1 RNA transcripts was seen in inflamed mucosa of IBD patients as compared with controls. Immunohistochemical analysis confirmed up-regulation of M-CSFR-1 in IBD and showed that both epithelial and lamina propria mononuclear cells expressed this receptor. Stimulation of DLD-1 with IL-34 increased CCL20 production through an ERK1/2-dependent mechanism. Consistently, treatment of IBD explants with anti-IL-34 reduced CCL20 production. CONCLUSIONS These data show that intestinal epithelial cells are a target of IL-34 and suggest that this cytokine contributes to mediating the cross-talk between epithelial cells and immune cells in IBD.

Smad7 knockdown activates protein kinase RNA-associated eIF2α pathway leading to colon cancer cell death

Upregulation of Smad7, an inhibitor of transforming growth factor-β1 (TGF-β1), occurs in sporadic colorectal cancer (CRC) and knockdown of Smad7 inhibits CRC cell growth, a phenomenon that associates with decreased expression of cell division cycle 25 homolog A and arrest of cells in the S phase of the cell cycle. These findings occur in CRC cells unresponsive to TGF-β1, thus suggesting the existence of a Smad7-mediated TGF-β1-independent mechanism that controls CRC cell behavior. Here we show that Smad7 inhibition with a specific Smad7 antisense oligonucleotide upregulates eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, a transcription factor involved in the regulation of cell cycle arrest and induction of cell death, and induces activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homology protein (CHOP), two downstream targets of eIF2α. Among the upstream kinases that control eIF2α phosphorylation, the serine–threonine protein kinase RNA (PKR), but not general control non-derepressible 2 (GCN2) and protein kinase RNA-like endoplasmic reticulum kinase (PERK), is activated by Smad7 knockdown. PKR silencing abolishes Smad7 antisense-induced eIF2α phosphorylation and ATF4/CHOP induction, thereby preventing Smad7 antisense-driven cell death. Smad7 inhibition diminishes interaction of PKR with protein kinase inhibitor p58 (p58IPK), a cellular inhibitor of PKR, but does not change the expression and/or activity of other factors involved in the control of PKR activation. These findings delineate a novel mechanism by which Smad7 knockdown promotes CRC cell death.

Tofacitinib for the treatment of ulcerative colitis

ABSTRACT Introduction: Management of patients with active ulcerative colitis (UC), one of the most frequent inflammatory bowel diseases in human beings, is mainly based on the use of mesalamine and corticosteroids. Since in the long-term, these two drugs may be ineffective in nearly one third of the patients, immunosuppressants and/or biologics are needed to control disease activity. Areas covered: The marked activation of JAK/STAT molecules in inflamed mucosa of UC patients and the demonstration that UC-associated mucosal injury is driven by soluble factors that signal through JAK/STAT pathways led to investigation of JAK inhibitors for the treatment of active UC. Tofacitinib, an oral inhibitor of the cytokine-driven JAK-STAT signalling cascade, has recently been proposed for the treatment of moderate-to-severe UC. Phase 2 study showed the efficacy of tofacitinib to induce clinical and endoscopic improvement/remission and the safety profile of the drug. Herein the authors review this compound. Expert opinion: The results obtained from clinical trials with tofacitinib suggest that this drug could be a new treatment option for patients with moderate to severe UC. However, further experimentation is needed to assess the efficacy of this drug in selected subgroups of patients as well as to maintain remission and to determine the long-term safety profile of the drug.

Knockdown of Smad7 With a Specific Antisense Oligonucleotide Attenuates Colitis and Colitis-Driven Colonic Fibrosis in Mice

Background In Crohns disease (CD), the pathogenic immune response is associated with high Smad7, an inhibitor of TGF-β1 signaling. Smad7 knockdown with Mongersen, a specific antisense oligonucleotide-containing compound, restores TGF-β1 activity leading to inhibition of inflammatory signals and associates with clinical benefit in CD patients. As TGF-β1 is pro-fibrogenic, it remains unclear whether Mongersen-induced Smad7 inhibition increases the risk of intestinal fibrosis. We assessed the impact of Smad7 inhibition on the course of colitis-driven intestinal fibrosis in mice. Methods BALB/c mice were rectally treated with increasing doses of trinitrobenzene sulfonic acid (TNBS) for 8 or 12 weeks. The effect of oral Smad7 antisense or control oligonucleotide, administered to mice starting from week 5 or week 8, respectively, on mucosal inflammation and colitis-associated colonic fibrosis was assessed. Mucosal samples were analyzed for Smad7 by immunoblotting and immunohistochemistry, TGF-β1 by enzyme-linked immunosorbent assay, and collagen by immunohistochemistry. Results TNBS-induced chronic colitis was associated with colonic deposition of collagen I and fibrosis, which were evident at week 8 and became more pronounced at week 12. TNBS treatment enhanced Smad7 in both colonic epithelial and lamina propria mononuclear cells. Colitic mice treated with Smad7 antisense oligonucleotide exhibited reduced signs of colitis, less collagen deposition, and diminished fibrosis. These findings were associated with diminished synthesis of TGF-β1 and reduced p-Smad3 protein expression. Conclusion Attenuation of colitis with Smad7 antisense oligonucleotide limits development of colonic fibrosis.

Sphingosine-1-phosphate receptor: a novel therapeutic target in ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory condition of the colon that affects several million people worldwide. UC is histologically characterized by mucosal infiltration with macrophages, lymphocytes, neutrophils and plasma cells, formation of crypt abscesses, and disruption of epithelial barrier. Patients with UC suffer from recurrent episodes of rectal bleeding and diarrhea and may have reduced quality of life and increased risk of developing colorectal cancer [1]. The underlying cause of UC is unknown, but there is evidence that the pathological process is triggered, in genetically predisposed individuals, by environmental insults, which promote an excessive mucosal immune response. Among environmental factors, diet seems to play a decisive role in the pathogenesis of UC [2]. Patients with mild-to-moderate UC are mainly treated with oral and rectal mesalamine and/or steroids, while patients with more severe forms need systemic steroids and/or biologics (i.e. antibodies against tumor necrosis factor [TNF] or integrins). Immunosuppressive drugs (i.e. azathioprine and 6-mercaptopurine) are also used to prevent flare-ups in steroid-dependent patients and (i.e. cyclosporine A) to control the active phases. However, not all the patients respond to these pharmacological approaches, and the use of such drugs (e.g. steroids, immunosuppressors, and biologics) can associate with enhanced risk of side effects [1]. This indicates the necessity of safer and more effective compounds.

High Smad7 sustains inflammatory cytokine response in refractory coeliac disease

Refractory coeliac disease (RCD) is a form of coeliac disease (CD) resistant to gluten‐free diet and associated with elevated risk of complications. Many effector cytokines over‐produced in the gut of patients with RCD are supposed to amplify the tissue‐destructive immune response, but it remains unclear if the RCD‐associated mucosal inflammation is sustained by defects in counter‐regulatory mechanisms. The aim of the present study was to determine whether RCD‐related inflammation is marked by high Smad7, an intracellular inhibitor of transforming growth factor‐β1 (TGF‐β1) activity. Smad7 was evaluated in duodenal biopsy samples of patients with RCD, patients with active CD, patients with inactive CD and healthy controls by Western blotting, immunohistochemistry and real‐time PCR. In the same samples, TGF‐β1 and phosphorylated (p)‐Smad2/3 were evaluated by ELISA and immunohistochemistry, respectively. Pro‐inflammatory cytokine expression was evaluated in RCD samples cultured with Smad7 sense or antisense oligonucleotide. Smad7 protein, but not RNA, expression was increased in RCD compared with active and inactive CD patients and healthy controls and this was associated with defective TGF‐β1 signalling, as marked by diminished p‐Smad2/3 expression. TGF‐β1 protein content did not differ among groups. Knockdown of Smad7 in RCD biopsy samples reduced interleukin‐6 and tumour necrosis factor‐α expression. In conclusion, in RCD, high Smad7 associates with defective TGF‐β1 signalling and sustains inflammatory cytokine production. These results indicate a novel mechanism by which the mucosal cytokine response is amplified in RCD and suggest that targeting Smad7 can be therapeutically useful in RCD.

Therapy implications for the role of IL-21 in lupus

Systemic lupus erythematosus (SLE), a chronic autoimmune disease that can affect multiple organs, is characterized by a dysregulated activation and differentiation of B cells and plasma cells with a massive production of autoreactive antibodies. Overproduction of interleukin (IL)-21, a cytokine with pleiotropic effects on a wide range of cells, occurs in SLE and is supposed to activate detrimental immune pathways in this disorder. Consistently, IL-21 blockade is beneficial in murine models of SLE. We here discuss the available data supporting the involvement of IL-21 in the pathogenesis of SLE and the rationale for the therapeutic targeting of this cytokine.