Celiac Disease-Related Inflammation Is Marked by Reduction of Nkp44/Nkp46-Double Positive Natural Killer Cells.

INTRODUCTION AND AIM: Natural killer (NK) cells are a first line of defence against viruses and down-regulation of NK cell cytotoxic receptors represents one of the strategies by which viruses escape the host's immune system. Since onset of celiac disease (CD), a gluten-driven enteropathy, has been associated with viral infections, we examined whether CD-associated inflammation is characterized by abnormal distribution of NK cell receptors involved in recognition of viral-infected cells. MATERIALS AND METHODS: Intraepithelial mononuclear cells, isolated from duodenal biopsies of active and inactive CD patients and healthy controls (CTR) and jejunal specimens of obese subjects undergoing gastro-intestinal bypass, were analysed for NK cell markers by flow-cytometry. Expression of granzyme B, interleukin (IL)-22 and tumor necrosis factor (TNF)-α was as assessed in freshly isolated and toll-like receptor (TLR) ligand-stimulated cells. RESULTS: The percentages of total NK cells and NKT cells did not significantly differ between CD patients and CTR. In active CD, the fractions of NKp30+ NK cells, NKG2D+ NK cells and NKG2D+ NKT cells were significantly increased as compared to inactive CD patients and CTR. In contrast, CD-associated inflammation was marked by diminished presence of NKG2A+ NK cells and NKG2A+ NKT cells. The fractions of NK cells and NKT cells expressing either NKp44 or NKp46 did not differ between CD and controls, but in CD less NK cells and NKT cells co-expressed these receptors. NKp44/NKp46-double positive cells produced granzyme B and IL-22 but not TNF-α and responded to TLR ligands with enhanced expression of granzyme B. CONCLUSIONS: These data indicate that active phase of CD associates with reduced presence of NKp44/NKp46-double positive NK cells and NKT cells in the epithelial compartment.

Sampling of proximal and distal duodenal biopsies in the diagnosis and monitoring of celiac disease.

BACKGROUND: Since celiac disease-associated mucosal lesions are patchy, the diagnosis of the disease requires histological evaluation of multiple duodenal biopsies. AIM: To examine whether adequate biopsy sampling in either the bulb or distal duodenum is sufficient to diagnose celiac disease. METHODS: Twenty-five patients with positive celiac disease-specific serology and 17 patients with negative serology, who were on a gluten-containing diet, and 13 celiac disease patients on a gluten-free diet were consecutively and prospectively enrolled. Mucosal damage, anti-transglutaminase-2 IgA deposits, interferon-γ, interleukin-17A and interleukin-15 transcripts were evaluated in bulb and distal duodenal biopsies. RESULTS: All patients with positive celiac disease-specific serology exhibited villous atrophy in both duodenal sites. In this group, mucosal anti-transglutaminase-2 IgA deposits were found in 24/25 (96%) bulb samples and 22/25 (88%) distal duodenal samples. No villous atrophy was documented in patients with negative serology. Interferon-γ and interleukin-17A were over-expressed in both duodenal sites of patients with villous atrophy, unlike patients with normal duodenal morphology (p<0.001). Among treated celiac disease patients, 2 (15.4%) had villous atrophy exclusively in the bulb and 6 (46.2%) had minimal histological abnormalities at both sites. CONCLUSION: Sampling in the bulb and distal duodenum could be sufficient to diagnose/exclude celiac disease.

Inhibition of colitis by IL-25 associates with induction of alternatively activated macrophages.

BACKGROUND: Interleukin (IL)-25, a Th2-related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL-25, however, remains unknown. Since Th2-cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL-25-mediated anticolitic effect. METHODS: AAM-related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis treated with IL-25 and/or neutralizing IL-4, IL-13, and transforming growth factor beta 1 (TGF-ß1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL-25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL-25 on the alternative activation of peritoneal F4/80+ cells. RESULTS: IL-25 enhanced the expression of AAM-related markers in F4/80(+) cells infiltrating the peritoneum and colon of naïve and colitic mice. Peritoneal F4/80(+) cells isolated from IL-25-treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL-25 did not directly induce AAM in vitro and in vivo in mice, IL-25 administration enhanced the expression of IL-4, IL-13, and TGF-ß1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL-25-driven AAM induction. Blockade of IL-4, IL-13, and TGF-ß1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL-25 on AAM gene expression. CONCLUSIONS: The IL-25-mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties.

Smad7 expression in T cells prevents colitis-associated cancer.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colorectal cancer due to chronic inflammation. In IBD, chronic inflammation relies upon a TGFß signaling blockade, but its precise mechanistic relationship to colitis-associated colorectal cancer (CAC) remains unclear. In this study, we investigated the role of the TGFß signaling inhibitor Smad7 in CAC pathogenesis. In human colonic specimens, Smad7 was downregulated in CD4(+) T cells located in the lamina propria of patients with complicated IBD compared with uncomplicated IBD. Therefore, we assessed CAC susceptibility in a transgenic mouse model where Smad7 was overexpressed specifically in T cells. In this model, Smad7 overexpression increased colitis severity, but the mice nevertheless developed fewer tumors than nontransgenic mice. Protection was associated with increased expression of IFNγ and increased accumulation of cytotoxic CD8(+) and natural killer T cells in the tumors and peritumoral areas. Moreover, genetic deficiency in IFNγ abolished the Smad7-dependent protection against CAC. Taken together, our findings defined a novel and unexpected role for Smad7 in promoting a heightened inflammatory response that protects against CAC.

Role of IL-21 in inflammatory bowel disease.

IL-21 was first described as a critical regulator of T- and B-cell functions. More recently, it has become apparent that IL-21 controls the activity of both immune and nonimmune cells and, depending on the timing and context analyzed, it can promote either inflammatory or counter-regulatory effects. IL-21 participates in the immune responses against tumor cells and chronic viral infections, but excessive production of IL-21 has been associated with the development of immune-inflammatory diseases in various organs. In this article, we focus on data supporting the pathogenic role of IL-21 in human inflammatory bowel diseases and discuss preclinical studies that suggest that neutralization of IL-21 in vivo could be a new strategy to counteract the inflammatory bowel disease-related, tissue damaging immune response.

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-gamma-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-gamma 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-gamma.

Inhibition of colon carcinogenesis by 2-methoxy-5-amino-N-hydroxybenzamide, a novel derivative of mesalamine.

BACKGROUND & AIMS: Mesalamine has been reported to protect against inflammatory bowel disease-related colorectal cancer (CRC), but several drug-related issues have limited its use in chemopreventive programs. We evaluated the antineoplastic properties of mesalamine derivatives using in vitro and in vivo models of CRC. METHODS: CRC cell proliferation and cell-cycle progression were evaluated by flow cytometry after exposure to mesalamine or mesalamine derivatives. Cyclins, cyclin-dependent kinases, and endoplasmic reticulum stress-related molecules were examined by immunoblotting. The in vivo antineoplastic effect of 2-methoxy-5-amino-N-hydroxybenzamide (2-14) was evaluated in a syngenic, CT26-derived xenograft mouse model of CRC and in the azoxymethane/dextran sulfate sodium-induced mouse model of colitis-associated CRC. RESULTS: The mesalamine derivative 2-14 was 10-fold more potent than mesalamine in inhibiting CRC cell proliferation. After exposure to 2-14, cyclin D1 expression was reduced and G0/G1 phase cells accumulated. These events were preceded by activation of eukaryotic translation initiation factor 2-alpha kinase 3 (pancreatic endoplasmic reticulum eIF2alpha kinase), phosphorylation of eukaryotic translation initiation factor 2alpha, induction of activating transcription factor 4, and splicing of X-box binding protein 1 messenger RNA, events that define endoplasmic reticulum stress. Silencing of PERK restored cyclin D1 levels, allowing cells to overcome the cell-cycle block induced by 2-14. Mice injected with 2-14 developed fewer CRC xenograft-derived tumors. Moreover, 2-14 injection reduced the development of neoplastic lesions induced by azoxymethane and dextran sulfate sodium in mice. CONCLUSIONS: The mesalamine derivative 2-14 inhibited CRC cell proliferation in vitro and prevented CRC progression in mouse models.

Interleukin-25 inhibits interleukin-12 production and Th1 cell-driven inflammation in the gut.

BACKGROUND & AIMS: During the pathogenesis of Crohn's 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.

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.

Mesalazine negatively regulates CDC25A protein expression and promotes accumulation of colon cancer cells in S phase.

Regular consumption of mesalazine has been associated with a reduced risk of colorectal cancer (CRC) in patients with inflammatory bowel disease. The molecular mechanisms underlying the antineoplastic effect of 5-aminosalicylic acid remain, however, poorly characterized. In this study, we examined whether mesalazine affects cell cycle progression and analyzed specific checkpoint pathways in experimental models of CRC. Mesalazine inhibited the growth of HCT-116 and HT-29 cells, two CRC cell lines that express either a wild-type or mutated p53. Cell cycle analysis revealed that mesalazine induced cells to accumulate in S phase. This effect was associated with a sustained phosphorylation of the cyclin-dependent kinase (CDK)2 at threonine 14 and tyrosine 15 residues, an event that inactivates the CDK2-cyclin complex and blocks S-G(2) phase cell cycle transition. Consistently, mesalazine reduced the protein content of CDC25A, a phosphatase that regulates CDK2 phosphorylation status. Analysis of upstream kinases that negatively control CDC25A expression showed that mesalazine enhanced the activation of CHK1 and CHK2. However, silencing of CHK1 and CHK2 did not prevent the mesalazine-induced CDC25A protein downregulation. In contrast, CDC25A protein ubiquitination and degradation and accumulation of cells in S phase following mesalazine exposure were reverted by proteasome inhibitors. Notably, mesalazine also inhibited CDC25A in human CRC explants. Finally, we showed that mesalazine downregulated CDC25A in CT26, a murine CRC cell line, and prevented the formation of CT26-derived tumors in mice. Data show that mesalazine negatively regulates CDC25A protein expression, thus delaying CRC cell progression.

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-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 gamma-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 gamma-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.

Cyclooxygenase-2-dependent and -independent inhibition of proliferation of colon cancer cells by 5-aminosalicylic acid.

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway may have a pathogenic role in colorectal cancer (CRC). Recent studies suggest that 5-aminosalicylic acid (5-ASA) reduces the risk of inflammatory bowel disease-related CRC, but the mechanism by which 5-ASA interferes with CRC cell growth remains unknown. In this study, we have examined whether the negative effect of 5-ASA on CRC cells is dependent on COX-2/PGE2 axis inhibition. We show that 5-ASA down-regulates both constitutive and TNF-alpha or IL-1beta-induced COX-2 in HT-115 and HT-29 cells. Inhibition of COX-2 by 5-ASA occurs at the RNA and protein level, and is associated with a significant decrease in PGE2 synthesis, arrest of growth and enhanced death of CRC cells. However, exogenous PGE2 does not revert the 5-ASA-mediated CRC cell proliferation block. 5-ASA also inhibits the growth of DLD-1, a COX-deficient CRC cell line, thus suggesting that the anti-proliferative effect of 5-ASA on CRC cells is not strictly dependent on the inhibition of COX-2/PGE2. Taken together our data indicate that 5-ASA causes both a COX-2-dependent and -independent inhibition of CRC cell growth.

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

Regulatory T (T(reg)) cells play a key role in the maintenance of the immune system homeostasis. T(reg) cells can be generated in the periphery under control of TGF-beta, a cytokine involved in the negative control of the immune system. However, TGF-beta 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-beta 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-beta-dependent expression of FoxP3, the master regulator of T(reg) 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-beta and IL-21 failed to suppress colitis while inducing an inflammatory response characterized by high levels of IL-17 and RORgammat, the transcription factor expressed by Th17 cells. Therefore, IL-21 emerges as a key modulator of TGF-beta signaling, leading to the reciprocal differentiation of T(reg) and Th17 cells.

IL-21 is highly produced in Helicobacter pylori-infected gastric mucosa and promotes gelatinases synthesis.

Helicobacter pylori (Hp) infection is associated with gastric inflammation and ulceration. The pathways of tissue damage in Hp-infected subjects are complex, but evidence indicates that T cell-derived cytokines enhance the synthesis of matrix metalloproteinases (MMP) that contribute to mucosal ulceration and epithelial damage. In this study, we have examined the role of the T cell cytokine IL-21 in Hp-infected gastric mucosa and evaluated whether IL-21 regulates MMP production by gastric epithelial cells. We show that IL-21 is constitutively expressed in gastric mucosa and is more abundant in biopsy specimens and purified mucosal CD3(+) T cells from Hp-infected patients compared with normal patients and disease controls. We also demonstrate that IL-21R is expressed by primary gastric epithelial cells, as well as by the gastric epithelial cell lines AGS and MKN28. Consistently, AGS cells respond to IL-21 by increasing production of MMP-2 and MMP-9, but not MMP-1, MMP-3, MMP-7, or tissue inhibitors of MMP. Analysis of signaling pathways leading to MMP production reveals that IL-21 enhances NF-kappaB but not MAPK activation, and inhibition of NF-kappaB activation reduces IL-21-induced MMP-2 and MMP-9 production. Finally, we show that treatment of Hp-infected gastric explants with anti-IL-21 reduces epithelial cell-derived MMP-2 and MMP-9 production. These data indicate that IL-21 is overexpressed in Hp-infected gastric mucosa where it could contribute to increased epithelial gelatinase production.

Role of interleukin-21 in inflammation and allergy.

Interleukin-21 (IL-21) is a newly described cytokine, produced by activated CD4+ T cells. Since the discovery in 2000, IL-21 has been the object of intensive research because of its homology to IL-2, IL-4 and IL-15, and its ability to modulate both innate and adaptive immune responses. IL-21 mediates its functions through a heterodimeric receptor, composed of a specific subunit, termed IL-21 receptor (IL-21R) and the common gamma-chain, that is shared with IL-2, IL-4, IL-7, IL-9, IL-13, and IL-15 receptors. IL-21R is originally described on T, B and NK cells, which is in accordance with the cell types that mostly respond to IL-21. Indeed, IL-21 augments the proliferation of CD4+ and CD8+ T lymphocytes and regulates the profile of cytokines secreted by these cells, drives the differentiation of B cells into memory cells and terminally differentiated plasma cells, and moreover, enhances the activity of natural killer cells. More recently, IL-21R has also been documented on non-immune cells, raising the possibility that IL-21 is an important mediator in the cross-talk between immune and non-immune cells. As discussed in this review, the potential role of IL-21 in immune-mediated and allergic diseases would seem to suggest that either disrupting or enhancing IL-21 signaling may be useful in specific clinical settings.

Lactobacillus paracasei subsp. paracasei B21060 suppresses human T-cell proliferation.

Recent studies have shown that probiotics are beneficial in T-cell-mediated inflammatory diseases. The molecular mechanism by which probiotics work remains elusive, but accumulating evidence indicates that probiotics can modulate immune cell responses. Since T cells express receptors for bacterial products or components, we examined whether different strains of lactobacilli directly regulate the functions of human T cells. CD4(+) T cells were isolated from blood and intestinal lamina propria (LP) of normal individuals and patients with inflammatory bowel disease (IBD). Mononuclear cells were also isolated from Peyer's patches. Cells were activated with anti-CD3/CD2/CD28 in the presence or absence of Lactobacillus paracasei subsp. paracasei B21060, L. paracasei subsp. paracasei F19, or L. casei subsp. casei DG. Cell proliferation and death, Foxp3, intracellular pH, and cytokine production were evaluated by flow cytometry. We showed that L. paracasei subsp. paracasei B21060 but neither L. paracasei subsp. paracasei F19 nor L. casei subsp. casei DG inhibited blood CD4(+) T-cell growth. This effect was associated with no change in cell survival, expression of Foxp3, or production of gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10. L. paracasei subsp. paracasei B21060-mediated blockade of CD4(+) T-cell proliferation required a viable bacterium and was associated with decreased MCT-1 expression and low intracellular pH. L. paracasei subsp. paracasei B21060 also inhibited the growth of Peyer's patch mononuclear cells, normal lymphocytes, and IBD CD4(+) LP lymphocytes without affecting cytokine production. The data show that L. paracasei subsp. paracasei B21060 blocks T-cell growth, thus suggesting a mechanism by which these probiotics could interfere with T-cell-driven immune responses.

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.