Kazutaka Koganei

Unique CD14+ intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-γ axis

Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-alpha, and IL-6, than typical intestinal resident macrophages (CD14-CD33+ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-alpha compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14+ macrophages contributed to IFN-gamma production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-alpha. Furthermore, the IFN-gamma produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-gamma-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.

IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn’s disease

Background: A novel T helper (Th) cell lineage, Th17, that exclusively produces the proinflammatory cytokine interleukin 17 (IL17) has been reported to play important roles in various inflammatory diseases. IL23 is also focused upon for its potential to promote Th17. Here, the roles of the IL23/IL17 axis in inflammatory bowel diseases such as ulcerative colitis (UC) and Crohn’s disease (CD) were investigated. Methods: Mucosal samples were obtained from surgically resected specimens (controls, n = 12; UC, n = 17; CD, n = 22). IL17 production by isolated peripheral blood (PB) and lamina propria (LP) CD4+ cells was examined. Quantitative PCR amplification was performed to determine the mRNA expression levels of IL17, interferon γ (IFNγ), IL23 receptor (IL23R) and retinoic acid-related orphan receptor γ (RORC) in LP CD4+ cells, and IL12 family members, such as IL12p40, IL12p35 and IL23p19, in whole mucosal specimens. The effects of exogenous IL23 on IL17 production by LP CD4+ cells were also examined. Results: IL17 production was higher in LP CD4+ cells than in PB. Significant IL17 mRNA upregulation in LP CD4+ cells was found in UC, while IFNγ was increased in CD. IL23R and RORC were upregulated in LP CD4+ cells isolated from both UC and CD. IL17 production was significantly increased by IL23 in LP CD4+ cells from UC but not CD. Upregulated IL23p19 mRNA expression was correlated with IL17 in UC and IFNγ in CD. Conclusions: IL23 may play important roles in controlling the differential Th1/Th17 balance in both UC and CD, although Th17 cells may exist in both diseases.

CD4+CD25bright T Cells in Human Intestinal Lamina Propria as Regulatory Cells

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4+CD25bright T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4+ T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4+CD25− T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4+CD25+ T cells, however, cells expressing CD25 at high levels (CD4+CD25bright) suppressed the proliferation and various cytokine productions of CD4+CD25− T cells. LP CD4+CD25bright T cells by themselves produced fewer amounts of IL-2, IFN-γ, and IL-10. Interestingly, LP CD4+CD25bright T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4+CD25bright T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.

Blockade of B7-H1 Suppresses the Development of Chronic Intestinal Inflammation

A newly identified costimulatory molecule, programmed death-1 (PD-1), provides a negative signal that is essential for immune homeostasis. However, it has been suggested that its ligands, B7-H1 (PD-L1) and B7-dendritic cells (B7-DC; PD-L2), could also costimulate T cell proliferation and cytokine secretion. Here we demonstrate the involvement of PD-1/B7-H1 and B7-DC interaction in the development of colitis. We first examined the expression profiles of PD-1 and its ligands in both human inflammatory bowel disease and a murine chronic colitis model induced by adoptive transfer of CD4+CD45RBhigh T cells to SCID mice. Second, we assessed the therapeutic potential of neutralizing anti-B7-H1 and/or B7-DC mAbs using this colitis model. We found significantly increased expression of PD-1 on T cells and of B7-H1 on T, B, and macrophage/DCs in inflamed colon from both inflammatory bowel disease patients and colitic mice. Unexpectedly, the administration of anti-B7-H1, but not anti-B7-DC, mAb after transfer of CD4+CD45RBhigh T cells suppressed wasting disease with colitis, abrogated leukocyte infiltration, and reduced the production of IFN-γ, IL-2, and TNF-α, but not IL-4 or IL-10, by lamina propria CD4+ T cells. These data suggest that the interaction of PD-1/B7-H1, but not PD-1/B7-DC, might be involved in intestinal mucosal inflammation and also show a possible role of interaction between B7-H1 and an as yet unidentified receptor for B7-H1 in inducing T cell activation.

Imbalance of NKp44+NKp46− and NKp44−NKp46+ Natural Killer Cells in the Intestinal Mucosa of Patients With Crohn's Disease

BACKGROUND & AIMS Mucosal natural killer (NK) cells that produce interleukin (IL)-22 mediate intestinal homeostasis and inflammation in mice. However, their role in the pathogenesis of human inflammatory bowel diseases (IBDs) is not known. We investigated intestinal NK cells in intestinal mucosa samples of patients with Crohns disease (CD). METHODS We isolated lamina propria NK cells from intestinal mucosal samples of patients with IBD and subjects without IBD (controls) and analyzed expression patterns of cell surface molecules and cytokine production. Interactions between lamina propria NK cells and intestinal macrophages were examined. RESULTS In intestinal mucosa samples from controls, NKp44 and NKp46 were expressed differentially on CD3(-)CD56(+) NK cells, NKp44(+)NKp46(-) (NKp44(+)) NK cells expressed CD127 and the transcription factor retinoic acid-related orphan receptor C (RORC) and produced IL-22 whereas NKp44(-)NKp46(+) (NKp46(+)) NK cells did not express CD127 or RORC and produced interferon (IFN)-gamma. NKp46(+) NK cells were predominant in intestinal mucosa of patients with CD compared with controls or patients with ulcerative colitis. Upon interaction with intestinal inflammatory macrophages NKp46(+), NK cells from patients with CD were activated via IL-23 and produced IFN-gamma; this activation required cell-to-cell contact. CONCLUSIONS The balance of NKp44(+)/NKp46(+) NK cells is disrupted in intestinal mucosa of patients with CD. NKp46(+) NK cells might mediate the pathogenesis of CD by producing IFN-gamma.

T-bet upregulation and subsequent interleukin 12 stimulation are essential for induction of Th1 mediated immunopathology in Crohn’s disease

Background and aims: Many lines of evidence suggest that T helper cell type 1 (Th1) immune responses predominate in Crohn’s disease (CD). Recently, a novel transcription factor T-box expressed in T cells (T-bet) has been reported as the master regulator of Th1 development. This study was designed to investigate the role of T-bet and proinflammatory cytokines in Th1 mediated immunopathology in CD. Materials: CD4+ lamina propria mononuclear cells (LPMCs) were isolated from surgically resected specimens (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5). Methods: (1) T-bet expression of CD4+ LPMCs was examined by quantitative real time polymerase chain reaction and western blotting. (2) T-bet expression of LPMCs stimulated by interleukin (IL)-12/IL-18 was analysed by western blotting. (3) Interferon γ (IFN-γ) production and T-bet expression of CD4+ peripheral blood mononuclear cells (PBMCs) were examined with or without stimulation by anti-CD3/CD28 monoclonal antibodies and/or IL-12. Results: (1) T-bet expression of CD4+ LPMCs was increased in CD compared with UC and NL. (2) Synergistically, augmentation of IFN-γ production by IL-12/IL-18 was independent of T-bet expression in LPMCs. (3) T-bet was induced by T cell receptor stimulation in CD4+ PBMCs. T-bet induction correlated with IFN-γ production and with augmentation of surface expressed IL-12 receptor β2. Conclusions: T-bet induction by antigenic stimulation and subsequent stimulation by macrophage derived IL-12/IL-18 are important for establishing Th1 mediated immunopathology in CD.

TL1A produced by lamina propria macrophages induces Th1 and Th17 immune responses in cooperation with IL-23 in patients with Crohn's disease

Background: Tumor necrosis factor (TNF)‐like protein 1A (TL1A) is a member of the TNF superfamily and contributes to the pathogenesis of Crohns disease (CD) by stimulating T‐helper (Th) 1 cells. In addition to Th1, recent studies have focused on the role of Th17 cells in the pathogenesis of CD. Here we tried to clarify the role of TL1A in Th1 and Th17 immunity in CD. Methods: TL1A expression was assessed by quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) in lamina propria (LP) macrophages (LP‐M&PHgr;s) from normal controls (NC) and patients with CD or ulcerative colitis (UC). Purified LP CD4+ T cells were stimulated with TL1A and/or IL‐23 and interferon gamma (IFN‐&ggr;) and interleukin (IL)‐17 levels were analyzed. We also examined the effect of TL1A on naïve CD4+ T‐cell differentiation. Results: We found that LP‐M&PHgr;s are a major producer of TL1A. TL1A expression was markedly enhanced in LP‐M&PHgr;s from CD patients compared with NC or UC patients. IL‐23, in addition to TL1A, was induced in LP‐M&PHgr;s by commensal bacteria stimulation. TL1A and IL‐23 synergistically promoted the production of IFN‐&ggr; and IL‐17 by LP T cells, while TL1A alone did not induce cytokine production. Furthermore, TL1A promoted Th17 differentiation from naïve T cells by LP‐M&PHgr;s; however, IL‐23 did not show any synergistic effects on Th17 differentiation. Conclusions: TL1A expressed in LP‐M&PHgr;s might play an important role in the pathogenesis of CD by inducing Th1 and Th17 immunity. IL‐23 differentially regulated these functions of TL1A on memory and naïve T cells. Inflamm Bowel Dis 2009

Human CD14+ Macrophages in Intestinal Lamina Propria Exhibit Potent Antigen-Presenting Ability

Intestinal APCs are considered critical in maintaining the balance between the response against harmful pathogens and the induction of tolerance to commensal bacteria and food Ags. Recently, several studies indicated the presence of gut-specific APC subsets, which possess both macrophage and dendritic cell (DC) markers. These unique APC subsets play important roles in gut immunity, especially for immune regulation against commensal bacteria. Herein, we examined a unique macrophage subset, which coexpressed the macrophage (Mφ) marker CD14 and the DC marker CD209 in human intestinal lamina propria (LP). The LP Mφ subset in both normal control subjects or Crohn’s disease (CD) patients induced proliferation of naive CD4+ T cells as well as monocyte-derived DCs, and it expressed retinoic acid synthetic enzyme retinaldehyde dehydrogenase 2 and retinol dehydrogenase 10, which induced expression of gut homing receptors on T cells in a retinoic acid-dependent manner. Moreover, the LP Mφ subset strongly evoked differentiation of Th1 cells and slightly induced Th17 cells in both normal control subjects and CD patients; the inducing potential was highest in CD patients. In CD patients, Th17, but not Th1, induction by the LP Mφ subset was enhanced in the presence of commensal bacteria Ags. This enhancement was not observed in normal control subjects. The Th17 induction by the LP Mφ subset was inhibited by neutralization of IL-6 and IL-1β, but it was enhanced by blockade of retinoic acid signaling. These observations highlight a role for LP Mφ in the enhanced Th1, and potentially in Th17 differentiation, at the inflammatory site of inflammatory bowel diseases.

Dietary fat attenuates the benefits of an elemental diet in active Crohn's disease: a randomized, controlled trial.

Objectives Although an elemental diet has been established as the primary treatment for patients with Crohns disease, the influence of dietary fat on the elemental diet remains unclear. We have designed the first randomized, controlled trial for elemental diets containing different fat percentages in patients with active Crohns disease. Methods Each patient was randomized to receive one of three dose levels of fat in an elemental diet (Elental) for 4 weeks: 10 patients received low fat (3.06 g/day), 10 patients received medium fat (16.56 g/day) and eight patients received high fat (30.06 g/day). The additional fat was composed of long-chain fatty acids. All patients were evaluated using the International Organization of Inflammatory Bowel Disease rating, plus C-reactive protein level and erythrocyte sedimentation rate, which were measured at weekly intervals. Results Although the International Organization of Inflammatory Bowel Disease rating, C-reactive protein level and erythrocyte sedimentation rate in the low-fat group decreased, the values in the medium- and high-fat groups fluctuated during the study. The remission rate after 4 weeks in each group was 80%, 40% and 25% for patients in the low-, medium- and high-fat groups, respectively. Conclusions When the fat consisted of long-chain triglycerides, a high amount of this fat in the elemental diet formula decreased its therapeutic effect against active Crohns disease.

TGR5 signalling inhibits the production of pro-inflammatory cytokines by in vitro differentiated inflammatory and intestinal macrophages in Crohn's disease

Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mϕs) and dendritic cells. Mϕs differentiated with macrophage colony‐stimulating factor and interferon‐γ (Mγ‐Mϕs), which are similar to the human intestinal lamina propria CD14+ Mϕs that contribute to Crohns disease (CD) pathogenesis by production of pro‐inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mϕ and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor‐α production in Mγ‐Mϕs stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5–cAMP pathway to induce phosphorylation of c‐Fos that regulated nuclear factor‐κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non‐inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14+ intestinal Mϕs from patients with CD expressed TGR5. In isolated intestinal CD14+ Mϕs, a TGR5 agonist could inhibit tumour necrosis factor‐α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.