Siew C. Ng

Mechanisms of Action of Probiotics : Recent Advances

&NA; The intestinal microbiota plays a fundamental role in maintaining immune homeostasis. In controlled clinical trials probiotic bacteria have demonstrated a benefit in treating gastrointestinal diseases, including infectious diarrhea in children, recurrent Clostridium difficile‐induced infection, and some inflammatory bowel diseases. This evidence has led to the proof of principle that probiotic bacteria can be used as a therapeutic strategy to ameliorate human diseases. The precise mechanisms influencing the crosstalk between the microbe and the host remain unclear but there is growing evidence to suggest that the functioning of the immune system at both a systemic and a mucosal level can be modulated by bacteria in the gut. Recent compelling evidence has demonstrated that manipulating the microbiota can influence the host. Several new mechanisms by which probiotics exert their beneficial effects have been identified and it is now clear that significant differences exist between different probiotic bacterial species and strains; organisms need to be selected in a more rational manner to treat disease. Mechanisms contributing to altered immune function in vivo induced by probiotic bacteria may include modulation of the microbiota itself, improved barrier function with consequent reduction in immune exposure to microbiota, and direct effects of bacteria on different epithelial and immune cell types. These effects are discussed with an emphasis on those organisms that have been used to treat human inflammatory bowel diseases in controlled clinical trials.

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.

Prospective Evaluation of Anti-Tumor Necrosis Factor Therapy Guided by Magnetic Resonance Imaging for Crohn ' s Perineal Fistulas

OBJECTIVES:Anti-tumor necrosis factor (TNF) therapy heals Crohns fistulas clinically, but the rate, extent, and duration to achieve fistula track healing are unknown.METHODS:We sought to monitor deep healing, as indicated by magnetic resonance imaging (MRI), and to use this to determine treatment duration. Clinical and MRI fistula healing (at 6, 12, and 18 months), Crohns Disease Activity Index (CDAI), Perianal Crohns Disease Activity Index (PDAI), and the Inflammatory Bowel Disease Questionnaire were prospectively assessed.RESULTS:Thirty-four consecutive patients with perineal fistulas were treated with infliximab (19), adalimumab (7; all infliximab failures) and thalidomide (8). Median follow-up was 110 weeks (range, 74–161). Baseline MRI: 38% ⩾2 tracks, 21% anolabial/rectovaginal. At latest follow-up, clinical fistula ‘response’ and ‘closure’ were seen in 50 and 46% of antibody-treated patients, respectively. All patients stopped thalidomide early due to side effects. Of 26 antibody-treated patients, at 6 (n=25), 12 (n=25), and 18 (n=20) months, respectively, MRI showed complete healing (20, 28, and 30%, respectively), improvement (68, 72, and 65%), no change (12, 0, and 0%) or worsening (0, 0, and 5%). MRI healing at 6 months (n=5) persisted at 12 and 18 months, including in two patients who stopped treatment at 6 months. Fistula history length and complexity did not influence the outcome. The only surgical intervention was seton insertion in one patient. The PDAI and CDAI scores decreased, and quality of life improved significantly at last follow-up.CONCLUSIONS:MRI fistula resolution was variable and slower than clinical healing. Prolonged treatment is often required for internal track resolution. Preliminary data suggest once MRI healing has occurred fistulas remain healed, while remaining on, or stopping anti-TNFα therapy. The use of a second antibody is clinically valuable.

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;)

Immunosuppressive effects via human intestinal dendritic cells of probiotic bacteria and steroids in the treatment of acute ulcerative colitis

Background: In ulcerative colitis (UC) gut bacteria drive inflammation. Bacterial recognition and T‐cell responses are shaped by intestinal dendritic cells (DCs); therapeutic effects of probiotic bacteria may relate to modulation of intestinal DC. The probiotic mixture, VSL#3, increases interleukin (IL)‐10 and downregulates IL‐12p40 production by DC in vitro. We evaluated in vivo effects of oral VSL#3 and steroids on colonic DC in patients with acute UC. Methods: Rectal biopsies were obtained from patients with active UC before and after treatment with VSL#3, corticosteroids, or placebo, and from healthy controls. Myeloid colonic DC were studied from freshly isolated lamina propria cells using multicolor flow cytometry. Surface expression of activation markers, CD40, CD86, pattern recognition receptors, Toll‐like receptor (TLR)‐2 and TLR‐4 were assessed. Changed function was measured from ongoing intracellular IL‐10, IL‐12p40, IL‐6, and IL‐13 production. Results: Acute UC colonic myeloid DC were producing more IL‐10 and IL‐12p40 than control DC (P = 0.01). In VSL#3‐treated patients DC TLR‐2 expression decreased (P < 0.05), IL‐10 production increased and IL‐12p40 production decreased (P < 0.005); 10/14 patients on VSL#3 showed a clinical response. Corticosteroids also resulted in increased IL‐10 and reduced IL‐12p40 production by DC. Conversely, in patients on placebo, TLR‐2 expression and intensity of staining for IL‐12p40 and IL‐6 increased (all P < 0.05); 5/14 patients on placebo showed a clinical response (P = NS). Conclusions: Despite small numbers of human colonic DC available, we showed that treatment of UC patients with probiotic VSL#3 and corticosteroids induced “favorable” intestinal DC function in vivo, increasing regulatory cytokines and lowering proinflammatory cytokines and TLR expression. These effects may contribute to therapeutic benefit. (Inflamm Bowel Dis 2010)

Homing of immune cells: role in homeostasis and intestinal inflammation.

Abstract: Rather like a satellite navigation system directing a vehicle to a particular destination defined by post‐code, immune cells have homing molecules or “immune post‐codes” enabling them to be recruited to specific organs, such as the intestine or skin. An efficient system would be designed such that the site of entry of an antigen influences the homing of effector T cells back to the appropriate organ. For example, to mount an immune response against an intestinal pathogen, T cells with a propensity to home to the gut to clear the infection would be induced. In health, there is such a sophisticated and finely tuned system in operation, enabling an appropriate balance of immune activity in different anatomical compartments. In disease states such as inflammatory bowel disease (IBD), which is characterized by intestinal inflammation and often an inflammatory process involving other organs such as skin, joints, liver, and eye, there is accumulating evidence that there is malfunction of this immune cell trafficking system. The clinical importance of dysregulated immune cell trafficking in IBD is reflected in recently proven efficacious therapies that target trafficking pathways such as natalizumab, an &agr;4 integrin antibody, and Traficet‐EN, a chemokine receptor‐9 (CCR9) antagonist. Here we review the mechanisms involved in the homing of immune cells to different tissues, in particular the intestine, and focus on alterations in immune cell homing pathways in IBD. Unraveling the mechanisms underlying the immune post‐code system would assist in achieving the goal of tissue‐specific immunotherapy. (Inflamm Bowel Dis 2010)

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;)

Intestinal dendritic cells: Their role in bacterial recognition, lymphocyte homing, and intestinal inflammation

&NA; Dendritic cells (DCs) play a key role in discriminating between commensal microorganisms and potentially harmful pathogens and in maintaining the balance between tolerance and active immunity. The regulatory role of DC is of particular importance in the gut where the immune system lies in intimate contact with the highly antigenic external environment. Intestinal DC constantly survey the luminal microenvironment. They act as sentinels, acquiring antigens in peripheral tissues before migrating to secondary lymphoid organs to activate naive T cells. They are also sensors, responding to a spectrum of environmental cues by extensive differentiation or maturation. Recent studies have begun to elucidate mechanisms for functional specializations of DC in the intestine that may include the involvement of retinoic acid and transforming growth factor‐&bgr;. Specialized CD103+ intestinal DC can promote the differentiation of Foxp3+ regulatory T cells via a retinoic acid‐dependent process. Different DC outcomes are, in part, influenced by their exposure to microbial stimuli. Evidence is also emerging of the close interaction between bacteria, epithelial cells, and DC in the maintenance of intestinal immune homeostasis. Here we review recent advances of functionally specialized intestinal DC and their mechanisms of antigen uptake and recognition. We also discuss the interaction of DC with intestinal microbiota and their ability to orchestrate protective immunity and immune tolerance in the host. Lastly, we describe how DC functions are altered in intestinal inflammation and their emerging potential as a therapeutic target in inflammatory bowel disease. (Inflamm Bowel Dis 2010)

Prospective assessment of the effect on quality of life of anti-tumour necrosis factor therapy for perineal Crohn's fistulas.

Background  Anti‐tumour necrosis factor (TNF) therapy effectively treats Crohn’s perineal fistulas (CPF); the effect on health‐related quality of life (HRQoL) remains unknown.

A novel population of human CD56+ human leucocyte antigen D-related (HLA-DR+) colonic lamina propria cells is associated with inflammation in ulcerative colitis

Ulcerative colitis (UC) involves inappropriate mucosal immune responses to intestinal microbiota. Gut dendritic cells (DC) are central immunoregulators of the response to commensal bacteria, and the subset of CD11c+ cells within the human leucocyte antigen D‐related (HLA‐DR+) lineage (lin)–/dim population are activated in inflammatory bowel disease. We hypothesized that CD11c‐ cells within this population may also be involved in intestinal inflammation. HLA‐DR+ lin–/dim cells were identified in freshly isolated lamina propria mononuclear cells by multi‐colour flow cytometry in 54 UC patients and 22 controls. Proportion and number of CD11c+ and CD11c‐ cells, and surface expression of activation markers CD40, CD86, Toll‐like receptor (TLR)‐2, TLR‐4, and CD56+[natural killer (NK) marker], were determined. Cytokine production was assessed by intracellular staining. Lamina propria colonic CD11c‐ HLA‐DR+ lin–/dim cells were increased significantly in inflamed and ‘non‐inflamed’ UC tissue, compared with control tissue. CD11c+ HLA‐DR+ lin–/dim cells were unchanged. Fewer CD11c‐ cells expressed activation markers and produced intracellular cytokines than their CD11c+ counterparts, and they were weakly stimulatory in mixed leucocyte reactions. Few CD11c‐ cells expressed blood plasmacytoid DC markers, but a major subset expressed high levels of CD56. CD11c‐ cells decreased after inflammation resolved. Intestinal inflammation in UC is associated with the presence of cells that share phenotypic features of both DC and NK cells. This novel population of human colonic CD56+ HLA‐DR+ cells may play a role in immune regulation or tissue repair. Their increase in quiescent UC may be a marker of subclinical inflammation.