Daniele Fina

IL-21 Counteracts the Regulatory T Cell-Mediated Suppression of Human CD4+ T Lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4+CD25+ regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4+CD25+ T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4+CD25− T cells and counteracts the suppressive activities of CD4+CD25+ T cells on CD4+CD25− T cells without affecting the percentage of Foxp3+ cells or survival of Treg. Additionally, CD4+CD25+ T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8+CD25− T cells but does not revert the CD4+CD25+ T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4+ T cells resistant to suppression rather than inhibiting CD4+CD25+ T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4+CD25+ T cells. Data indicate that IL-21 renders human CD4+CD25− T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.

Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21

BACKGROUND & AIMS Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation. METHODS Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay. RESULTS High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes. CONCLUSIONS These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

IL-21 regulates experimental colitis by modulating the balance between Treg and Th17 cells

Regulatory T (Treg) cells play a key role in the maintenance of the immune system homeostasis. Treg cells can be generated in the periphery under control of TGF‐β, a cytokine involved in the negative control of the immune system. However, TGF‐β cooperates with IL‐6 in the generation of Th17 cells, a novel class of effector cells involved in numerous inflammatory diseases, including colitis. Therefore, TGF‐β emerges as a mediator of both anti‐inflammatory and pro‐inflammatory processes, depending on the local cytokine milieu. Here we demonstrate that IL‐21, a type‐1 cytokine produced by T cells and involved in the pathogenesis of immune‐mediated diseases, prevents the TGF‐β‐dependent expression of FoxP3, the master regulator of Treg cell commitment, and the induction of suppressive capacity in naive CD4+ T cells, while promoting the differentiation of Th17 cells. In vivo, CD4+ naive T cells activated in the presence of TGF‐β and IL‐21 failed to suppress colitis while inducing an inflammatory response characterized by high levels of IL‐17 and RORγt, the transcription factor expressed by Th17 cells. Therefore, IL‐21 emerges as a key modulator of TGF‐β signaling, leading to the reciprocal differentiation of Treg and Th17 cells.

Control of matrix metalloproteinase production in human intestinal fibroblasts by interleukin 21

Background: T cell-mediated immunity plays a central part in the pathogenesis of tissue damage in inflammatory bowel disease (IBD). The mechanism by which T cells mediate tissue damage during IBD remains unclear, but evidence indicates that T cell-derived cytokines stimulate fibroblasts to synthesise matrix metalloproteinases (MMPs), which then mediate mucosal degradation. We have previously shown that, in IBD, there is high production of interleukin (IL) 21, a T cell-derived cytokine, which enhances Th1 activity. Aim: To investigate whether IL21 controls MMP production by intestinal fibroblasts. Methods: IL21 receptor (IL21R) was evaluated in intestinal fibroblasts by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Fibroblasts were stimulated with IL21 and MMPs were evaluated by RT-PCR and western blotting. The effect of a neutralising IL21R fusion protein (IL21R/Fc) on the induction of MMPs in fibroblasts stimulated with IBD lamina propria mononuclear cell (LPMC) supernatants was also evaluated. Results: Intestinal fibroblasts constitutively express both IL21R and the common γ chain receptor, which are necessary for IL21-driven signalling. IL21 enhances fibroblast production of MMP-1, MMP-2, MMP-3 and MMP-9, but not tissue inhibitors of MMP-1 and MMP-2. Moreover, IL21 synergises with tumour necrosis factor α to increase synthesis of MMP synthesis. IL21 enhances MMP secretion without affecting gene transcription and protein synthesis. IBD LPMC supernatants stimulate MMP secretion by intestinal fibroblasts, and this effect is partly inhibited by IL21R/Fc. Conclusions: These results suggest that fibroblasts are a potential target of IL21 in the gut and that IL21 controls MMP secretion by fibroblasts.

Interleukin 21 contributes to the mucosal T helper cell type 1 response in coeliac disease

Background: In coeliac disease (CD), the upper bowel lesion is associated with a marked infiltration of the mucosa with Th1 cells secreting interferon γ (IFNγ) and expressing the Th1-associated transcription factor, T-bet. However, the molecular mechanisms which regulate T-bet and promote the Th1 cell response are unknown. Objective: To examine whether interleukin 21 (IL21), a cytokine that regulates T cell activation, has a role in CD. Methods: Duodenal mucosal samples were taken from CD patients and normal controls. IL21 and T-bet were examined by real-time PCR and western blotting, and IFNγ was assessed by real-time PCR and ELISA. The effect of blockade of endogenous IL21 on the expression of T-bet was examined in an ex vivo culture of biopsies taken from untreated CD patients. Finally, the role of IL21 in controlling T-bet and IFNγ was also evaluated in cultures of biopsies taken from treated CD patients and cultured with a peptic–tryptic digest of gliadin (PT) in the presence or absence of a neutralising IL21 antibody. Results: Enhanced IL21 RNA and protein expression was seen in duodenal samples from untreated CD patients. Blockade of IL21 activity in biopsies of untreated CD patients reduced T-bet and IFNγ secretion. Stimulation of treated CD biopsies with PT enhanced IL21 expression, and neutralisation of IL21 largely prevented PT-driven T-bet and IFNγ induction. Conclusions: IL21 is overproduced in the mucosa of CD patients, where it helps sustain T-bet expression and IFNγ production.

Smad7 Controls Resistance of Colitogenic T Cells to Regulatory T Cell-Mediated Suppression

BACKGROUND & AIMS Foxp3-expressing regulatory T cells (Tregs) play a key role in the maintenance of the gut immune homeostasis, and an intact transforming growth factor (TGF)-beta signaling is required for their function. In inflammatory bowel disease (IBD), the TGF-beta signaling is impaired because of high expression of the inhibitory molecule Smad7. Although no intrinsic defects in Tregs function have been shown in IBD, it is still unknown whether colitogenic T cells are susceptible to Treg-mediated suppression. In this study, we have investigated whether IBD mucosal CD4+ T cells are resistant to Tregs and whether Smad7 is involved in this process. METHODS IBD lamina propria mononuclear cells (LPMC) were cultured with or without Tregs, and proliferation was assessed by flow cytometry. Proliferation of IBD LPMC was also evaluated after Smad7 antisense oligonuclotide treatment. Treg-mediated suppression of T-cell proliferation and proinflammatory cytokine expression was investigated in murine Smad7 transgenic cells. In vivo, the Smad7-dependent resistance of colitogenic naïve T cells to Tregs was studied in the adoptive transfer model of colitis. RESULTS IBD LPMC were resistant to Treg-mediated suppression, and this phenomenon was reverted by Smad7 antisense treatment. Consistently, CD4+ T cells isolated from Smad7 transgenic mice showed high proliferation, produced considerable amount of inflammatory cytokines following activation, and induced a severe colitis when transferred in immunodeficient RAG1 knockout mice even in the presence of wild-type Tregs. CONCLUSIONS Smad7 makes CD4+ T cells resistant to Tregs-mediated suppression thus fine-tuning their proinflammatory potential.

IL‐23‐mediated regulation of IL‐17 production in Helicobacter pylori‐infected gastric mucosa

Helicobacter pylori (Hp) infection is associated with a marked infiltration of the gastric mucosa by inflammatory cells. The molecular pathways that control Hp‐associated inflammatory reaction are complex, but locally induced cytokines seem to contribute to maintaining the ongoing inflammation. We have previously shown that IL‐17 is over‐produced in Hp‐infected gastric mucosa, and that IL‐17 stimulates the synthesis of IL‐8, the major neutrophil chemoattractant. Factors/mechanisms that regulate IL‐17 expression remain, however, unknown. In this study, we initially expanded our previous data, showing that CD4+ and CD8+ T cells are a source of IL‐17 in Hp‐infected samples. Since IL‐23 enhances T cell‐derived IL‐17 during bacterial infections, we then assessed the role of IL‐23 in controlling IL‐17 expression in Hp‐colonized stomach. Using real‐time PCR and ELISA, IL‐23 was detected in all gastric biopsies, but its expression was more pronounced in Hp‐infected samples in comparison to controls. Treatment of normal gastric lamina propria mononuclear cells (LPMC) with IL‐23 enhanced Stat3 activation and IL‐17 secretion, and pharmacological inhibition of Stat3 prevented IL‐23‐driven IL‐17 synthesis. Consistently, blockade of IL‐23 in cultures of LPMC from Hp‐infected patients reduced Stat3 activation and IL‐17 production. Data show that IL‐23 is overexpressed in Hp‐infected gastric mucosa where it could contribute to sustaining IL‐17 production.

Autocrine Regulation of IL-21 Production in Human T Lymphocytes

IL-21 has pathologic function in immune-inflammatory diseases. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21R, and the common γ-chain. IL-21 is mostly produced by CD4+ T cells, but molecular mechanisms that regulate IL-21 synthesis are not fully understood. The fact that CD4+ T cells express high levels of IL-21R and are capable of functionally responding to IL-21 raises the possibility that IL-21 may regulate its own production. We here show that IL-21 enhances IL-21 RNA and protein expression in human peripheral blood CD3+ T cells in a dose- and time-dependent fashion. Additionally, both IL-7 and IL-15, but not IL-4, induce IL-21, thus suggesting that common γ-chain signals are not sufficient to promote IL-21 synthesis. Analysis of molecular mechanisms underlying IL-21 induction reveals that IL-21 activates Stat3 and enhances its recruitment to IL-21 gene promoter. Pharmacologic inhibition and knockdown of Stat3 by small interference RNA largely prevent IL-21 induction in IL-21-treated cells. Consistently, IL-21 is inducible in T cells by IL-6, another cytokine that activates Stat3. Finally, we show that IL-21 positively regulates its own expression in human intestinal CD3+ lamina propria lymphocytes, and blockade of endogenous IL-21 in cultures of CD3+ lamina propria lymphocytes isolated from patients with Crohn’s disease, a chronic inflammatory bowel disease characterized by high IL-21, down-regulates Stat3 activation and IL-21 expression. These data suggest the existence of a positive autocrine loop that could help to amplify and stabilize IL-21-driven, T cell-mediated responses.

Interleukin-25 Inhibits Interleukin-12 Production and Th1 Cell-Driven Inflammation in the Gut

BACKGROUND & AIMS During the pathogenesis of Crohns disease (CD), interleukin (IL)-12, a cytokine produced by mucosal CD14+ monocyte-like cells, promotes tissue-damaging T helper cell (Th) 1-mediated inflammation through mechanisms that are not fully understood. IL-25 promotes Th2 cell responses by activating major histocompatibility complex class II-positive non-T and non-B cells. Because Th1 and Th2 cells, and the cytokines they release, are often mutually antagonistic, we examined whether IL-25 affects IL-12 production or Th1 cell-mediated inflammation in the gut. METHODS Studies were performed using colonic samples from patients and mice with peptidoglycan (PGN)-, 2,4,6-trinitrobenzenesulphonic acid (TNBS)-, or oxazolone-induced colitis. IL-25 receptor (IL-25R) levels were evaluated in intestinal lamina propria mononuclear cells by flow cytometry, and IL-25 levels were measured by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. Mucosal CD14+ cells from patients with CD were incubated with IL-25 and/or lipopolysaccharide or PGN. Mice were injected with IL-25, and some mice first received injections of an IL-13 blocking antibody. Cytokines were quantified by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS CD14+ cells from the mucosa of CD patients expressed IL-25R and responded to IL-25 by decreasing the synthesis of IL-12 and IL-23. IL-25 prevented PGN-induced colitis in mice. IL-25 induced IL-13 production in the colon, but IL-13 was not required for suppression of PGN colitis. IL-25 ameliorated TNBS- and oxazolone-colitis. Patients with CD or ulcerative colitis produced significantly less IL-25 compared with controls. CONCLUSIONS IL-25 inhibits CD14+ cell-derived cytokines and experimental colitis. IL-25 could be a useful treatment of CD and ulcerative colitis.

Characterization of IL-17A-Producing Cells in Celiac Disease Mucosa

Celiac disease (CD) is a gluten-sensitive enteropathy associated with a marked infiltration of the mucosa with IFN-γ–secreting Th1 cells. Recent studies have shown that a novel subset of T cells characterized by expression of high levels of IL-17A, termed Th17 cells, may be responsible for pathogenic effects previously attributed to Th1 cells. In this study, we characterized the expression of IL-17A–producing cells in CD. By real-time PCR and ELISA, it was shown that expression of IL-17A RNA and protein is more pronounced in active CD biopsy specimens in comparison with inactive CD and normal mucosal biopsy specimens. Flow cytometry confirmed that IL-17A is overproduced in CD mucosa and that CD4+ and CD4+CD8+ cells were major sources. The majority of IL-17A–producing CD4+ and CD4+CD8+ cells coexpressed IFN-γ but not CD161. The addition of a peptic‑tryptic digest of gliadin to ex vivo organ cultures of duodenal biopsy specimens taken from inactive CD patients enhanced IL-17A production by both CD4+ and CD4+CD8+ cells. Because we previously showed that IL-21, a T cell-derived cytokine involved in the control of Th17 cell responses, is overproduced in CD, we next assessed whether IL-17A expression is regulated by IL-21. Blockade of IL-21 activity by a neutralizing IL-21 Ab reduced IL-17A expression in cultures of active CD and peptic–tryptic digest of gliadin-treated CD biopsy specimens. In conclusion, our data show that IL-17A is increased in CD and is produced by cells that also make IFN-γ.