The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity

This study analyzed IL-23p19 expression in inflamed mucosa of IBD and the role in the induction of IEL and NK cell activation as well as Th17 cell differentiation. Expression of IL-23p19 was performed by immunohistochemistry and quantitative real-time PCR. Expression of IL-23R was assessed by flow cytometry. Cytolytic activities of IEL and NK cells by IL-23 were determined by a standard (51)Cr-release assay. Cytokine levels were analyzed by ELISA and quantitative real-time PCR. Expression of IL-23p19 was increased significantly in inflamed mucosa of CD compared with that in UC and healthy controls. Double-staining confirmed that IL-23p19(+) cells were mainly CD68(+) macrophages/DCs. IL-23R(+) cells were increased significantly in PB- and LP-CD4(+) and -CD8(+) T and NK cells. IL-23 markedly promoted IBD IEL and NK cell activation and cytotoxicity and triggered IBD PB- and LP-T cells to secrete significantly higher levels of IFN-γ, TNF, IL-2, and IL-17A compared with controls. Importantly, IL-23 promoted IBD PB- or LP-CD4(+) T cells to differentiate into Th17 cells, characterized by increased expression of IL-17A and RORC. Anti-TNF treatment could markedly reduce IL-23 expression and Th17 cell infiltration in inflamed mucosa of CD patients. These data indicate that IL-23 is highly expressed in inflamed mucosa of IBD and plays an important role in the induction of IEL, NK, and T cell activation, proinflammatory cytokine secretion, and Th17 cell differentiation. Targeted therapy directed against IL-23p19 may have a therapeutic role in treatment of IBD.     

Impaired development of HIV-1 gp160-specific CD8(+) cytotoxic T cells by a delayed switch from Th1 to Th2 cytokine phenotype in mice with Helicobacter pylori infection

Th1 and Th2 cells play a central role in immunoregulation during infection. We show that Helicobacter pylori induces Th1 cytokine responses early (2 weeks) but predominantly Th2 responses later (6 weeks) in infection. The switch is principally mediated by urease-specific CD4(+) T cells, and correlates with a loss of urease-specific high-avidity JNK(+) Th1 and gain of low-avidity JNK(-) (possibly Th2) cells at the later stage of infection, concomitant with a 100-fold higher colonization level of H. pylori at 6 weeks than at 2 weeks that might tolerize high-avidity Th1 cells. Furthermore, differentiation of HIV gp160-specific CD4(+) Th and CD8(+) cytotoxic T lymphocytes (CTL) into effector cells is impaired in 6-week H. pylori-infected mice immunized with vaccinia expressing gp160, and serum IL-12 stimulated by vaccinia infection is barely detectable. Adoptive transfer of urease-specific Th2 cells to mice infected only with gp160-expressing vaccinia abrogates Th1 polarization of the gp120 response, down-modulates virus-specific CTL responses, and delays virus clearance. Therefore, the H. pylori urease-mediated immunoregulation in the switch from JNK(+) Th1 to JNK(-) Th2 phenotype, and the preceding low IL-12 response, are likely critical steps in the impairment of antiviral immunity.     

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.     

Helicobacter pylori-specific CD4+ T cells home to and accumulate in the human Helicobacter pylori-infected gastric mucosa

Helicobacter pylori infects the stomach and duodenal mucosa. T cells are important components of the H. pylori-induced immune response, but little is currently known about how these cells are recruited to the infected mucosa. Here, we have characterized stomach and duodenal T cells isolated from H. pylori-infected and noninfected subjects with regard to subtype, expression of homing and chemokine receptors, and in vitro reactivity to H. pylori antigens. Higher numbers of CD4(+) but similar numbers of CD8(+) lamina propria T cells were isolated from stomach biopsies from H. pylori-positive compared to H. pylori-negative individuals. CD4(+) T cells from infected stomach expressed increased levels of the homing receptor L-selectin and the chemokine receptor CCR4 compared to CD4(+) T cells from uninfected stomach. Infected stomach mucosa also contained increased levels of the CCR4 chemokine ligand MDC/CCL22. In contrast, comparable numbers of CD4(+) T cells with similar receptor expression were isolated from the duodenum of H. pylori-positive and H. pylori-negative individuals. In vitro proliferation of mucosal T cells was strongly enhanced by the addition of interleukin-2 (IL-2) and IL-7 to the cell cultures. Using this approach, H. pylori-specific T-cell responses were detected in stomach CD4(+) T cells from H. pylori-positive but not H. pylori-negative individuals. Duodenal T cells from only a few individuals responded to H. pylori stimulation, and the responsiveness was not restricted to H. pylori-positive individuals, suggesting limited H. pylori specificity in the duodenum and possible cross-reactivity with antigens from other bacteria in this compartment. In conclusion, these results suggest that H. pylori-specific CD4(+) T cells preferentially home to and accumulate in the infected stomach and that L-selectin and CCR4/MDC are important for this recruitment.     

Comparative analysis of the interaction of Helicobacter pylori with human dendritic cells, macrophages, and monocytes

Helicobacter pylori may cause chronic gastritis, gastric cancer, or lymphoma. Myeloid antigen-presenting cells (APCs) are most likely involved in the induction and expression of the underlying inflammatory responses. To study the interaction of human APC subsets with H. pylori, we infected monocytes, monocyte-derived dendritic cells (DCs), and monocyte-derived (classically activated; M1) macrophages with H. pylori and analyzed phenotypic alterations, cytokine secretion, phagocytosis, and immunostimulation. Since we detected CD163(+) (alternatively activated; M2) macrophages in gastric biopsy specimens from H. pylori-positive patients, we also included monocyte-derived M2 macrophages in the study. Upon H. pylori infection, monocytes secreted interleukin-1β (IL-1β), IL-6, IL-10, and IL-12p40 (partially secreted as IL-23) but not IL-12p70. Infected DCs became activated, as shown by the enhanced expression of CD25, CD80, CD83, PDL-1, and CCR7, and secreted IL-1β, IL-6, IL-10, IL-12p40, IL-12p70, and IL-23. However, infection led to significantly downregulated CD209 and suppressed the constitutive secretion of macrophage migration inhibitory factor (MIF). H. pylori-infected M1 macrophages upregulated CD14 and CD32, downregulated CD11b and HLA-DR, and secreted mainly IL-1β, IL-6, IL-10, IL-12p40, and IL-23. Activation of DCs and M1 macrophages correlated with increased capacity to induce T-cell proliferation and decreased phagocytosis of dextran. M2 macrophages upregulated CD14 and CD206 and secreted IL-10 but produced less of the proinflammatory cytokines than M1 macrophages. Thus, H. pylori affects the functions of human APC subsets differently, which may influence the course and the outcome of H. pylori infection. The suppression of MIF in DCs constitutes a novel immune evasion mechanism exploited by H. pylori.     

A mechanism for the action of the compound DA-6034 on NF-κB pathway activation in Helicobacter pylori-infected gastric epithelial cells

DA-6034 is a synthetic derivative of eupatilin, a flavonoid with anti-inflammatory effects. The aim of this study was to investigate the effects of DA-6034 on the interactions between IκB kinase (IKK) and heat shock protein 90 (Hsp90), and activation of the nuclear factor-kappaB (NF-κB) signalling pathway in human gastric epithelial cells infected with Helicobacter pylori. MKN-45 gastric epithelial cell line was treated with DA-6034 and H. pylori. DA-6034 significantly inhibited NF-κB activation and upregulated the expressions of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 in MKN-45 cells infected with H. pylori. However, DA-6034 did not influence activator protein-1 DNA binding activity in H. pylori-infected gastric epithelial cells. Pretreatment with DA-6034 attenuated the H. pylori-induced increase in IKK activity, and Hsp90 was associated with IKK-α and IKK-γ in MKN-45 cells. Treatment with DA-6034 dissociated the Hsp90 and IKK-γ complex in H. pylori-infected cells, leading to the inhibition of IL-8 expression. These results suggest that the eupatilin derivative 7-carboxymethyloxy-3',4',5-trimethoxy flavone has anti-inflammatory activity in gastric epithelial cells infected with H. pylori through the promotion of the dissociation of the IKK-γ-Hsp90 complex and suppression of NF-κB signalling.     

Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.     

Human memory Th17 cells express a functional histamine H4 receptor

The histamine H4 receptor is functionally expressed on CD4(+) T cells and in particular on human CD4(+) Th2-polarized T cells. Interleukin (IL)-17-producing T cells (Th17 cells) represent a newly defined major CD4(+) T-cell subset, having been identified in psoriatic plaques and in acute skin lesions of atopic dermatitis where histamine is also present in high concentrations. To elucidate the role of the histamine H4 receptor (H4R) on these effector T cells, we polarized human memory T cells into Th17 cells. Further, we investigated H4R expression and assessed its function by real-time PCR, by a cytokine secretion assay of IL-17, and by electrophoretic mobility shift assay of activating protein-1 (AP-1). We show that Th17 cells polarized by IL-1β together with IL-23 express the H4R on mRNA and protein level. Additionally, we identified IL-17-positive cells in psoriatic skin lesions. The IL-17-positive lymphocytes were all positive also for functional H4R. Stimulation with histamine or a H4R agonist increased the production of IL-17 and induced activating protein-1 in Th17 cells. In inflammatory skin diseases with enhanced histamine release, such as psoriasis and atopic dermatitis, histamine might foster the immunomodulatory potency of skin-infiltrating Th17 cells.     

Chicken IL-26 regulates immune responses through the JAK/STAT and NF-κB signaling pathways

Chicken interleukin 26 (ChIL-26), a member of the IL-10 family, is expressed in T cells and can induce expression of proinflammatory cytokines. We examined the response of signal transduction pathways to ChIL-26 stimulation in the chicken T (CU91), macrophage (HD11), and fibroblast (OU2) cell lines. ChIL-26 activated JAK2 and TYK2 phosphorylation, as well as activation of STAT1, STAT3, and SHP2 via tyrosine/serine residues. We also showed that ChIL-26 activates the phosphorylation of NF-κB1, TAK1, and MyD88 kinase, which are key regulators of NF-κB signaling pathways. Moreover, ChIL-26 stimulation upregulated mRNA expression of chemokines (CCL4, CCL20, and CXCL14), Th1 (IFN-α, IFN-β, IFN-γ, IL-1β, and IL-6), Th2 (IL-4 and IL-10), and Th17 (IL-12p40, IL-17A, and IL-17F), and the Treg cytokines (TGF-β4); additionally, it increased Th1 and Th17 protein levels and nitric oxide production but did not affect cell proliferation. Together, these results suggest that ChIL-26-induced activation of chemokines, Th1, Th2, and, Th17, and the Treg cytokines is mediated through JAK/STAT and NF-κB signaling pathways in chicken T, macrophage, and fibroblast cell lines. These results indicate a key role for ChIL-26-induced polarization of the immune response and could reveal new therapeutic approaches for use in combination with molecules that activate T and macrophage cells via activation JAK/STAT and NF-κB signaling pathways.     

Priming microenvironments dictate cytokine requirements for T helper 17 cell lineage commitment

Activation of pattern recognition receptors on dendritic cells (DCs) and macrophages leads to secretion of cytokines that control differentiation of CD4(+) T cells. The current understanding is that interleukin-6 (IL-6) in combination with transforming growth factor-β (TGF-β) leads to generation of T helper 17 (Th17) lineage cells. Here, we have discovered that the cytokine requirements for Th17 cell polarization depend on the site of priming. Although IL-6 played a critical role in Th17 cell lineage priming in the skin and mucosal tissues, it was not required for Th17 cell priming in the spleen. In contrast, IL-1 played an irreplaceable role for priming of Th17 lineage cells in all tissues. Importantly, we have demonstrated that IL-6-independent and -dependent pathways of Th17 cell differentiation are guided by DCs residing in various tissues. These results reveal fundamental differences by which the systemic, mucosal, and cutaneous immune systems guide Th17 cell lineage commitment.     

SLAMF6-driven co-stimulation of human peripheral T cells is defective in SLE T cells

The CD28 co-stimulatory pathway is well established for T cell activation. However, there is evidence suggesting the existence of additional co-stimulatory pathways. Here we report that a member of the SLAM superfamily, SLAMF6, or CD352 plays an important role in T cell co-stimulation. Cross-linking of SLAMF6 with anti-CD3 primes human T cell to secrete Th1 cytokines. Among the T cell subsets, CD8(+) and CD3(+)CD4(-)CD8(-) cells display the highest Th1 production responses. Engagement of SLAMF6 mobilizes the modulation of the same set of NF-κB-associated genes. Although the expression of SLAMF6 on the surface of T cells from patients with systemic lupus erythematosus (SLE) T cells is comparable to that on the normal T cells, engagement of SLAMF6 results in severely reduced Th1 and IL-2 cytokine production. Our results suggest the existence of an additional co-stimulatory pathway in human T cells, which is defective in SLE T cells.     

Wild-type and interleukin-10-deficient regulatory T cells reduce effector T-cell-mediated gastroduodenitis in Rag2-/- mice, but only wild-type regulatory T cells suppress Helicobacter pylori gastritis

CD4(+) CD45RB(hi) CD25(-) effector T cells (T(E)) promote Helicobacter pylori gastritis in mice, and CD4(+) CD45RB(lo) CD25(+) regulatory T cells (T(R)) are anti-inflammatory. Using adoptive transfer into H. pylori-infected Rag2(-/-) mice, we evaluated effects of wild-type (wt) C57BL/6 or congenic interleukin-10-deficient (IL-10(-/-)) T(R) cells on gastritis, gastric cytokines, and H. pylori colonization. Infected Rag2(-/-) mice colonized in the corpus and antrum with 10(5) to 10(6) H. pylori CFU/gram without associated gastritis. T(E) cell transfer caused morbidity and an H. pylori-independent pangastritis and duodenitis (gastroduodenitis) associated with increased expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha. T(E) cell transfer to H. pylori-infected mice led to additive corpus gastritis associated with inflammatory cytokine expression and reduced colonization. wt T(R) cells reduced morbidity, H. pylori corpus gastritis, gastroduodenitis, and inflammatory cytokine expression and reversed the decline in H. pylori colonization attributable to T(E) cells. Although less effective than wt T(R) cells, IL-10(-/-) T(R) cells also reduced morbidity and gastroduodenitis but did not reduce H. pylori corpus gastritis or impact T(E) cell inhibition of colonization. Gastric tissues from mice receiving wt T(R) cells expressed higher levels of Foxp3 compared to recipients of IL-10(-/-) T(R) cells, consistent with lower regulatory activity of IL-10(-/-) T(R) cells. These results demonstrate that wt T(R) cells suppressed T(E)-cell-mediated H. pylori-independent gastroduodenitis and H. pylori-dependent corpus gastritis more effectively than IL-10(-/-) T(R) cells. Compartmental differences in T(E)-cell- and H. pylori-mediated inflammation and in regulatory effects between wt T(R) and IL-10(-/-) T(R) cells suggest that IL-10 expression by wt T(R) cells is important to regulatory suppression of gastric inflammation.     

NKG2D receptor activation of NF-κB enhances inflammatory cytokine production in murine effector CD8 T cells

To induce strong immune responses, naïve CD8⁺ T cells require stimulation through the TCR and costimulatory receptors. However, the biological effect of activating costimulatory receptors on effector T cells is still unclear. One costimulatory receptor that is likely to be engaged at the target site is NKG2D. This activating receptor is expressed on human and murine CD8⁺ T cells with its ligands expressed on the majority of tumor cells and during some infections. In order to determine how activation of costimulatory receptors alters effector CD8⁺ T cell functions, this study compared the activation of the NF-κB signaling pathway by two costimulatory receptors, CD28 and NKG2D. Compared to CD28 costimulation, activation of murine effector CD8⁺ T cells through CD3 and NKG2D receptors enhanced activation of NF-κB as shown by increased phosphorylation of IKKα, IκBα, and NF-κB and IκBα degradation. NKG2D costimulation also increased activation, nuclear translocation, and DNA binding of NF-κB p65/p50 dimers. Activation of the NF-κB pathway also lead to increased gene expression and secretion of pro-inflammatory cytokines, including IFNα and IFNγ, and decreased gene expression and secretion of anti-inflammatory cytokines, including IL-10 and CCL2. Altered NF-κB activation also increased expression of the effector molecules TNFα, lymphotoxins α and β, and Fas ligand, and increased tumor cell killing through FasL. These data show that compared to CD28 costimulation, activation through the NKG2D receptor leads to the differential activation of the NF-κB signaling pathway and potentially enhances the anti-tumor and anti-viral functions of effector CD8⁺ T cells.     

The inducible costimulator (ICOS) is critical for the development of human T(H)17 cells

Human T helper 17 (T(H)17) cells regulate host defense, autoimmunity, and tumor immunity. Although cytokines that control human T(H)17 cell development have been identified, the costimulatory molecules important for T(H)17 cell generation are unknown. Here, we found that the inducible costimulator (ICOS) was critical for the differentiation and expansion of human T(H)17 cells. Human cord blood contained a subset of CD161(+)CD4(+) T cells that were recent emigrants from the thymus, expressed ICOS constitutively, and were imprinted as T(H)17 cells through ICOS signaling. ICOS stimulation induced c-MAF, RORC2, and T-bet expression in these cells, leading to increased secretion of interleukin-21 (IL-21), IL-17, and interferon-γ (IFN-γ) compared with cells stimulated with CD28. Conversely, CD28 ligation abrogated ICOS costimulation, dampening RORC2 expression while promoting the expression of the aryl hydrocarbon receptor, which led to reduced secretion of IL-17 and enhanced production of IL-22 compared with cells stimulated with ICOS. Moreover, ICOS promoted the robust expansion of IL-17(+)IFN-γ(+) human T cells, and the antitumor activity of these cells after adoptive transfer into mice bearing large human tumors was superior to that of cells expanded with CD28. The therapeutic effectiveness of ICOS-expanded cells was associated with enhanced functionality and engraftment in vivo. These findings reveal a vital role for ICOS signaling in the generation and maintenance of human T(H)17 cells and suggest that components of this pathway could be therapeutically targeted to treat cancer or chronic infection and, conversely, that interruption of this pathway may have utility in multiple sclerosis and other autoimmune syndromes. These findings have provided the rationale for designing new clinical trials for tumor immunotherapy.