Hiroshi Chinen

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.

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.

Dysbiosis of Salivary Microbiota in Inflammatory Bowel Disease and Its Association With Oral Immunological Biomarkers

Analysis of microbiota in various biological and environmental samples under a variety of conditions has recently become more practical due to remarkable advances in next-generation sequencing. Changes leading to specific biological states including some of the more complex diseases can now be characterized with relative ease. It is known that gut microbiota is involved in the pathogenesis of inflammatory bowel disease (IBD), mainly Crohns disease and ulcerative colitis, exhibiting symptoms in the gastrointestinal tract. Recent studies also showed increased frequency of oral manifestations among IBD patients, indicating aberrations in the oral microbiota. Based on these observations, we analyzed the composition of salivary microbiota of 35 IBD patients by 454 pyrosequencing of the bacterial 16S rRNA gene and compared it with that of 24 healthy controls (HCs). The results showed that Bacteroidetes was significantly increased with a concurrent decrease in Proteobacteria in the salivary microbiota of IBD patients. The dominant genera, Streptococcus, Prevotella, Neisseria, Haemophilus, Veillonella, and Gemella, were found to largely contribute to dysbiosis (dysbacteriosis) observed in the salivary microbiota of IBD patients. Analysis of immunological biomarkers in the saliva of IBD patients showed elevated levels of many inflammatory cytokines and immunoglobulin A, and a lower lysozyme level. A strong correlation was shown between lysozyme and IL-1β levels and the relative abundance of Streptococcus, Prevotella, Haemophilus and Veillonella. Our data demonstrate that dysbiosis of salivary microbiota is associated with inflammatory responses in IBD patients, suggesting that it is possibly linked to dysbiosis of their gut microbiota.

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.

Dietary Histidine Ameliorates Murine Colitis by Inhibition of Proinflammatory Cytokine Production From Macrophages

BACKGROUND & AIMS Elemental diet (ED) is effective for human Crohns disease (CD). Although some of this effectiveness may be due to its low antigenic load and low fat content, the mechanisms remain unclear. We sought to assess the role of histidine, one of the constituent amino acids of ED, in controlling colitis. METHODS The interleukin (IL)-10-deficient (IL-10(-/-)) cell transfer model of colitis was used. SCID mice with colitis induced by transfer of IL-10(-/-) cells were maintained on experimented diets containing either single amino acids or a mixture. The severity of colitis was assessed by wet colon weight. Colonic tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) expression was detected by quantitative reverse-transcription polymerase chain reaction. Mouse peritoneal macrophages were stimulated by lipopolysaccharides (LPS), with or without amino acids. The concentration of cytokines in the supernatant was determined by enzyme-linked immunosorbent assay. Inhibitor of nuclear factor (NF)-kappaB-alpha and nuclear p65 were confirmed by immunoblotting. RESULTS In the IL-10(-/-) transfer model, dietary histidine, but not alanine, reduced histologic damage and colon weight and TNF-alpha mRNA expression. Histidine inhibited LPS-induced TNF-alpha and IL-6 production by mouse macrophages in a concentration-dependent manner, whereas alanine or histidine-related metabolites had no such effect. Histidine inhibited LPS-induced NF-kappaB in macrophages. CONCLUSIONS These results showed that histidine could be a novel therapeutic agent for CD by inhibition of NF-kappaB activation, following down-regulation of proinflammatory cytokine production by macrophages. Thus, our studies provided new insights into the roles of amino acid metabolism in the pathophysiology of CD and for therapeutic strategies.

Nonpathogenic Escherichia coli strain Nissle1917 prevents murine acute and chronic colitis

Background: Nonpathogenic Escherichia coli strain Nissle1917 has been used as a probiotics in human inflammatory bowel disease; however, there are few reports examining its therapeutic effect on animal colitis models, and its therapeutic mechanisms remain unknown. The aim of this study was to elucidate the therapeutic effect and mechanism of Nissle1917 using murine acute and chronic colitis models. Methods: Two models were used. (1) Acute model: colitis was induced by administration of 1.3% dextran sodium sulfate for 7 days. Nissle1917 or phosphate‐buffered saline were orally administered for 10 days. Mice were killed at day 10, and the colonic lesions were assessed macro‐ and microscopically. (2) Chronic model: IL‐10−/− mice were treated with Nissle1917 or phosphate‐buffered saline for 8 weeks. After 8 weeks of treatment, mice were killed to assess the colonic lesions macro‐ and microscopically. In the acute dextran sodium sulfate colitis model, viable, heat‐killed, or genomic DNA of Nissle1917 was orally administered for 10 days, and the therapeutic effect was assessed. Results: In the acute model, Nissle1917 ameliorated body weight loss, disease activity index, and macro‐ and microscopic damage. In the chronic model, it also suppressed the mucosal inflammatory findings and histologic damages. Moreover, heat‐killed Nissle1917 or its genomic DNA alone also ameliorated the acute DSS colitis and viable bacteria macro‐ and microscopically. Conclusions: Nonpathogenic E. coli strain Nissle1917 prevents both acute and chronic colitis, and its anti‐inflammatory effect is exhibited not only by viable bacteria but also by heat‐killed bacteria or its DNA.

Exclusive increase of CX3CR1+CD28-CD4+ T cells in inflammatory bowel disease and their recruitment as intraepithelial lymphocytes

Background: CX3CL1/Fractalkine (FKN) has been reported to play important roles in various inflammatory diseases. We examined the role of FKN and its receptor CX3CR1 in T‐cell migration in the inflammatory bowel diseases (IBDs), ulcerative colitis (UC) and Crohns disease (CD). Methods: CX3CR1 expression on peripheral CD4+ cells from normal controls (NL n = 24) and IBD patients (UC n = 28, CD n = 26) was examined using flow cytometry. CX3CR1+CD4+ T cells were further characterized for surface antigens, cytokine production, and cytotoxic granule release by flow cytometry and ELISA. FKN expression in 53 colonic biopsy specimens (UC n = 20, CD n = 23, NL n = 10) was analyzed by quantitative PCR and immunohistochemistry. Isolated lamina propria and intraepithelial lymphocytes were also analyzed by flow cytometry (UC n = 10, CD n = 10, NL n = 6). Results: CX3CR1+CD4+ cells were increased in IBD while they were virtually absent in controls. Upregulation of CX3CR1 on CD4+ T cells was positively correlated with disease activity. These unique T cells expressed markers for both effector memory and cytotoxic cells. Interestingly, CX3CR1 was expressed on CD4+ T cells lacking CD28. CX3CR1+CD28−CD4+ cells produced more IFN‐&ggr; and TNF‐&agr; than CX3CR1− counterparts and released cytotoxic granules. FKN mRNA was upregulated in inflamed colonic tissues and robust expression of FKN was immunohistochemically observed on epithelial cells. Although CX3CR1+ CD4+ cells could not be detected in the gut, CD28−CD4+ cells were found in IBD mainly as intraepithelial lymphocytes. Conclusions: FKN/CX3CR1 may contribute to the pathogenesis of IBD through the emergence of unique CX3CR1+CD28−CD4+ T cells that can act both as proinflammatory and cytotoxic cells. (Inflamm Bowel Dis 2007)