Neil E. McCarthy

Randomised, double-blind, placebo-controlled trial of fructo-oligosaccharides in active Crohn's disease

Introduction The commensal intestinal microbiota drive the inflammation associated with Crohns disease. However, bacteria such as bifidobacteria and Faecalibacterium prausnitzii appear to be immunoregulatory. In healthy subjects the intestinal microbiota are influenced by prebiotic carbohydrates such as fructo-oligosaccharides (FOS). Preliminary data suggest that FOS increase faecal bifidobacteria, induce immunoregulatory dendritic cell (DC) responses and reduce disease activity in patients with Crohns disease. Aims and methods To assess the impact of FOS in patients with active Crohns disease using an adequately powered randomised double-blind placebo-controlled trial with predefined clinical, microbiological and immunological end points. Patients with active Crohns disease were randomised to 15 g/day FOS or non-prebiotic placebo for 4 weeks. The primary end point was clinical response at week 4 (fall in Crohns Disease Activity Index of ≥70 points) in the intention-to-treat (ITT) population. Results 103 patients were randomised to receive FOS (n=54) or placebo (n=49). More patients receiving FOS (14 (26%) vs 4 (8%); p=0.018) withdrew before the 4-week end point. There was no significant difference in the number of patients achieving a clinical response between the FOS and placebo groups in the ITT analysis (12 (22%) vs 19 (39%), p=0.067). Patients receiving FOS had reduced proportions of interleukin (IL)-6-positive lamina propria DC and increased DC staining of IL-10 (p<0.05) but no change in IL-12p40 production. There were no significant differences in the faecal concentration of bifidobacteria and F prausnitzii between the groups at baseline or after the 4-week intervention. Conclusion An adequately powered placebo-controlled trial of FOS showed no clinical benefit in patients with active Crohns disease, despite impacting on DC function. ISRCTN50422530.

Smokers with active Crohn's disease have a clinically relevant dysbiosis of the gastrointestinal microbiota

Background: Patients with Crohns disease (CD) have an intestinal dysbiosis with components of the microbiota exerting differential immune effects. Smoking is associated with an increased incidence of CD, more frequent relapse, and greater burden of surgery. This study aimed to investigate the association between smoking and the intestinal microbiota in patients with active CD. Methods: Patients with active CD (n = 103) and healthy controls (n = 66) were recruited and demographic and clinical data recorded including current smoking behavior. Fecal samples were collected and analyzed by fluorescent in situ hybridization using probes targeting 16S rRNA of bacteria previously shown to be altered in active CD (bifidobacteria, bacteroides, Clostridium coccoides‐Eubacterium rectale, Escherichia coli, and Faecalibacterium prausnitzii). Results: In total, 29/101 (29%) patients with CD and 8/58 (14%) controls were current smokers (P = 0.032). Following multivariate analysis, smoking was found to have a significant and independent effect on the microbiota of patients with CD, with higher Bacteroides‐Prevotella in smokers (38.4%) compared with nonsmokers (28.1%) (F(1,93) = 12.6, P = 0.001). Healthy controls who smoked also had higher Bacteroides‐Prevotella (34.8%) than nonsmokers (24.1%) (F(1,55) = 4.5, P = 0.038). In the pooled multivariate analysis, patients with CD had higher bifidobacteria (F(1,156) = 30.5, P < 0.001), higher Bacteroides‐Prevotella (F(1,156) = 6.5, P = 0.012), and lower F. prausnitzii (F(1,156) = 3.8, P = 0.052) compared with healthy controls. Conclusions: Smokers have luminal microbiota that consist of significantly higher bacteroides. Investigation of whether this is one mechanism through which the negative effects of smoking on CD are mediated is warranted. (Inflamm Bowel Dis 2012;)

Relationship Between Human Intestinal Dendritic Cells, Gut Microbiota, and Disease Activity in Crohn's Disease

Background: Altered intestinal dendritic cell (DC) function underlies dysregulated T‐cell responses to bacteria in Crohns disease (CD) but it is unclear whether composition of the intestinal microbiota impacts local DC function. We assessed the relationship between DC function with disease activity and intestinal microbiota in patients with CD. Methods: Surface expression of Toll‐like receptor (TLR)‐2, TLR‐4, and spontaneous intracellular interleukin (IL)‐10, IL‐12p40, IL‐6 production by freshly isolated DC were analyzed by multicolor flow cytometry of cells extracted from rectal tissue of 10 controls and 28 CD patients. Myeloid DC were identified as CD11c+HLA‐DR+lin‐/dim cells (lin = anti‐CD3, CD14, CD16, CD19, CD34). Intestinal microbiota were analyzed by fluorescent in situ hybridization of fecal samples with oligonucleotide probes targeting 16S rRNA of bifidobacteria, bacteroides‐prevotella, C. coccoides‐E. rectale, and Faecalibacterium prausnitzii. Results: DC from CD produced higher amounts of IL‐12p40 and IL‐6 than control DC. IL‐6+ DC were associated with the CD Activity Index (r = 0.425; P = 0.024) and serum C‐reactive protein (CRP) (r = 0.643; P = 0.004). DC expression of TLR‐4 correlated with disease activity. IL‐12p40+ DC correlated with ratio of bacteroides: bifidobacteria (r = 0.535, P = 0.003). IL‐10+ DC correlated with bifidobacteria, and IL‐6+ DC correlated negatively with F. prausnitzii (r = −0.50; P = 0.008). The amount of TLR‐4 on DC correlated negatively with the concentration of F. prausnitzii. Conclusions: IL‐6 production by intestinal DC is increased in CD and correlates with disease activity and CRP. Bacterially driven local IL‐6 production by intestinal DC may overcome regulatory activity, resulting in unopposed effector function and tissue damage. Intestinal DC function may be influenced by the composition of the commensal microbiota. (Inflamm Bowel Dis 2011;)

Altered intestinal microbiota and blood T cell phenotype are shared by patients with Crohn's disease and their unaffected siblings

Objective Crohns disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups. Design Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subjects genotype relative risk was determined by Illumina Immuno BeadChip. Results Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell β7 integrin expression (p=0.01) compared with controls. FC was elevated (>50 μg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ2=80.4, df=20, p<0.001). Siblings were separated from controls by immunological and microbiological variables. Conclusions Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis.

Increased production of retinoic acid by intestinal macrophages contributes to their inflammatory phenotype in patients with Crohn's disease.

BACKGROUND & AIMS Reduced generation of all-trans retinoic acid (RA) by CD103(+) intestinal dendritic cells (DCs) is linked to intestinal inflammation in mice. However, the role of RA in intestinal inflammation in humans is unclear. We investigated which antigen-presenting cells (APCs) produce RA in the human intestine and whether generation of RA is reduced in patients with Crohns disease (CD). METHODS Ileal and colonic tissues were collected from patients with CD during endoscopy or surgery, and healthy tissues were collected from subjects who were undergoing follow-up because of rectal bleeding, altered bowel habits, or cancer (controls). Cells were isolated from the tissue samples, and APCs were isolated by flow cytometry. Retinaldehyde dehydrogenase (RALDH) activity was assessed by Aldefluor assay, and ALDH1A expression was measured by quantitative real-time polymerase chain reaction. Macrophages were derived by incubation of human blood monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF). RESULTS CD103(+) and CD103(-) DCs and CD14(+) macrophages from healthy human intestine had RALDH activity. Although ALDH1A1 was not expressed by DCs, it was the predominant RALDH enzyme isoform expressed by intestinal CD14(+) macrophages and their putative precursors, CD14(+) monocytes. RALDH activity was up-regulated in all 3 populations of APCs from patients with CD; in CD14(+) macrophages, it was associated with local induction of ALDH1A1 expression. Blocking of RA receptor signaling during GM-CSF-mediated differentiation of monocytes into macrophages down-regulated CD14 and HLA-DR expression and reduced the development of tumor necrosis factor α-producing inflammatory macrophages. CONCLUSIONS RA receptor signaling promotes differentiation of human tumor necrosis factor α-producing inflammatory macrophages in vitro. In vivo, more CD14(+) macrophages from the intestinal mucosa of patients with CD than from controls are capable of generating RA, which might increase the inflammatory phenotype of these cells. Strategies to reduce the generation of RA by CD14(+) macrophages could provide new therapeutic options for patients with CD.

Inhaled allergen-driven CD1c up-regulation and enhanced antigen uptake by activated human respiratory-tract dendritic cells in atopic asthma

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory‐tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD1 antigen presentation by hRTDC may contribute to asthma pathogenesis.

Human gut‐specific homeostatic dendritic cells are generated from blood precursors by the gut microenvironment

Background: Dendritic cells (DC) dictate not only the type of T‐cell immunity, but also homing patterns of T cells in mice. In humans, we characterized normal human gut DC and tested whether gut‐specific homeostatic DC could be generated from blood precursors by factors in the gut microenvironment. Methods: We characterized the phenotype and function of healthy human gut DC compared with blood and skin DC, and studied whether conditioning of blood DC in the presence of colonic biopsy supernatants (Bx‐SN) induced gut‐like phenotype and functions. Results: Blood DC mostly expressed both gut and skin homing markers, indicating potential to migrate to both major immune surface organs, and induced multi‐homing T cells. However, DC within gut or skin did not demonstrate this multi‐homing phenotype, were tissue‐specific, and induced tissue‐specific T cells. Human gut DC were less stimulatory for allogeneic T cells than their dermal and blood counterparts. Human blood DC cultured in vitro lost homing marker expression. Conditioning of human enriched blood DC with colonic Bx‐SN from healthy controls induced a gut‐homing phenotype and a homeostatic profile. Moreover, Bx‐SN‐conditioned DC demonstrated a restricted T‐cell stimulatory capacity and preferentially induced gut‐specific T cells. Retinoic acid and transforming growth factor beta (TGF‐&bgr;) mediated the acquisition of the gut‐homing and homeostatic properties, respectively, induced by colonic Bx‐SN on blood enriched DC. Conclusions: Tissue‐specific factors manipulate immunity via modulating characteristics of DC and may provide tools to generate tissue‐specific immunotherapy. (Inflamm Bowel Dis 2011;)

Proinflammatory Vδ2+ T Cells Populate the Human Intestinal Mucosa and Enhance IFN-γ Production by Colonic αβ T Cells

In nonhuman primates, Vγ9Vδ2+ (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4β7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4β7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103+ and CD103− subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103− population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4+ T cells. Instead, phosphoantigen-activated CD103− Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αβ T cells via an IFN-γ–dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103+ and CD103− subsets that can promote inflammation by colonic αβ T cells.

Dendritic cells from control but not atopic donors respond to contact and respiratory sensitizer treatment in vitro with differential cytokine production and altered stimulatory capacity

Background Chemical haptens induce both contact and allergic respiratory disease with dendritic cells (DCs) controlling and directing immune responses in vivo. Contact and respiratory haptens may promote differential cytokine production yet distinguishing these effects in vitro remains difficult due to human donor variability.

Skin‐ and gut‐homing molecules on human circulating γδ T cells and their dysregulation in inflammatory bowel disease

Changes in phenotype and function of γδ T cells have been reported in inflammatory bowel disease (IBD), including Crohns disease (CD) and ulcerative colitis (UC). Dysregulation of lymphocyte migration plays a key role in IBD pathogenesis; however, data on migratory properties of γδ T cells are scarce. Human circulating γδ T cells from healthy controls (n = 27), patients with active CD (n = 15), active UC (n = 14) or cutaneous manifestations of IBD (n = 2) were characterized by flow cytometry. Circulating γδ T cells in healthy controls were CD3hi and expressed CD45RO. They expressed gut‐homing molecule β7 but not gut‐homing molecule corresponding chemokine receptors (CCR)9, or skin‐homing molecules cutaneous lymphocyte‐associated antigen (CLA) and CCR4, despite conventional T cells containing populations expressing these molecules. CCR9 expression was increased on γδ T cells in CD and UC, while skin‐homing CLA was expressed aberrantly on γδ T cells in patients with cutaneous manifestations of IBD. Lower levels of CD3 expression were found on γδ T cells in CD but not in UC, and a lower proportion of γδ T cells expressed CD45RO in CD and UC. Enhanced expression of gut‐homing molecules on circulating γδ T cells in IBD and skin‐homing molecules in cutaneous manifestations of IBD may be of clinical relevance.