Mina T. Kitazume

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.

Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease

BACKGROUND & AIMS Dendritic cells (DCs) possess the most potent ability to induce acquired immunity. However, their involvement in the pathogenesis of Crohns disease (CD) has not yet been determined. We aimed to establish the immune status of mesenteric lymph nodes, the major gut-associated lymphoid tissue, and isolated DCs and determine their involvement in the pathogenesis of CD. METHODS CD4(+) T cells and DCs were isolated from mesenteric lymph nodes of CD, ulcerative colitis, and normal control. The immune status of CD4(+) T cells was analyzed by cytokine production and transcriptional profile. Surface phenotype of DCs was analyzed by flow cytometry. Cytokine production by myeloid DCs was analyzed by real-time polymerase chain reaction and exogenous bacterial stimulation. Immune stimulating activity of DCs was determined by mixed lymphocyte reaction. RESULTS In CD, mesenteric lymph node CD4(+) T cells produced higher amounts of interferon-gamma and interleukin (IL)-17 compared with ulcerative colitis and normal control, and this was dictated by increased T-bet and retinoic acid-related orphan receptor-gamma expression. Three subtypes of DCs, myeloid DC, plasmacytoid DC, and mature DC, were identified in all groups. When stimulated with exogenous bacterial derivative, myeloid DCs from CD produced a higher amount of IL-23 and a lower amount of IL-10. Myeloid DCs from CD induced stronger T helper cell (Th)1 immune response in mixed lymphocyte reaction compared with those from ulcerative colitis and normal control. CONCLUSIONS Our findings revealed that mesenteric lymph node is the key pathogenic location of CD elicited by the unique cytokine milieu produced by DCs leading to a dysregulated Th1/Th17 immune response.

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.

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.

Monocyte Chemoattractant Protein-1 Contributes to Gut Homeostasis and Intestinal Inflammation by Composition of IL-10-Producing Regulatory Macrophage Subset

Lamina propria macrophages (LPMϕs) spontaneously produce large amounts of anti-inflammatory IL-10 and play a central role in regulation of immune responses against commensal bacteria. MCP-1 is a chemokine that plays an important role in recruitment of monocytes and macrophages to inflamed tissues. We demonstrated that, in addition to IL-10, LPMϕs produced large amounts of MCP-1, even in a steady state. MCP-1 deficiency caused impaired IL-10 production by LPMϕs and led to exacerbation of dextran sulfate sodium-induced acute colitis. As an explanation of this impaired IL-10 production by LPMϕs, we found that LPMϕs could be separated into two subsets with distinct side-scattered properties, namely LPMϕ1 (CD11b+F4/80+CD11c–SSChi) and LPMϕ2 (CD11b+F4/80+CD11c–SSClo). Unlike LPMϕ1, the LPMϕ2 subset migrated in response to MCP-1 and produced a larger amount of IL-10 in response to commensal bacteria. LPMϕs isolated from MCP-1–deficient mice produced less IL-10 as a consequence of the lack of the MCP-1–dependent LPMϕ2 population. This imbalanced composition in LPMϕ population may be involved in the susceptibility to DSS-induced colitis in MCP-1–deficient mice. Our results suggest that endogenous MCP-1 contributes to the composition of resident LPMϕ subsets in the intestine. Moreover, MCP-1–dependent LPMϕ2 subset may play an important role in maintenance of gut homeostasis in the steady state, and in the termination of excess inflammatory responses in the intestine, by producing IL-10.

Novel, Objective, Multivariate Biomarkers Composed of Plasma Amino Acid Profiles for the Diagnosis and Assessment of Inflammatory Bowel Disease

Background Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram). Methodology and Principal Findings We measured fasting plasma aminograms in 387 IBD patients (Crohns disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898–0.983) and 0.894 (95%CI: 0.853–0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853–0.935) and 0.849 (95%CI: 0.770–0.928), and correlated with clinical disease activity indexes for CD (rs = 0.592, 95%CI: 0.385–0.742, p<0.001) or UC (rs = 0.598, 95%CI: 0.452–0.713, p<0.001), respectively. Conclusions and Significance In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the 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.

Reduced numbers and proapoptotic features of mucosal-associated invariant T cells as a characteristic finding in patients with inflammatory bowel disease

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. Methods:Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohns disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. Results:Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. Conclusions:The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.