Paul K Flanagan

Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer

Objective Colonic mucosa-associated Escherichia coli are increased in Crohns disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. Design A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). Results 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. Conclusions IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

Bacteria in the pathogenesis of inflammatory bowel disease

Twin studies have demonstrated the importance of environmental factors in the pathogenesis of inflammatory bowel disease, but progress has been relatively slow in identifying these, with the exception of smoking, which is positively associated with Crohns disease and negatively associated with ulcerative colitis. Genetic studies have identified risk alleles which are involved in host-bacterial interactions and the mucosal barrier, and evidence is building for a likely pathogenic role for changes in the gut microbiome, with respect to both faecal and mucosa-associated microbiota. Some of these changes may be secondary to inflammation, nevertheless promising new therapeutic targets are beginning to emerge.

Escherichia coli-host macrophage interactions in the pathogenesis of inflammatory bowel disease.

Multiple studies have demonstrated alterations in the intestinal microbial community (termed the microbiome) in Crohns disease (CD) and several lines of evidence suggest these changes may have a significant role in disease pathogenesis. In active and quiescent disease, both the faecal and mucosa-associated microbiome are discordant with matched controls with reduced biodiversity, changes in dominant organisms and increased temporal variation described. Mucosa-associated adherent, invasive Escherichia coli (E. coli) (AIEC), pro-inflammatory and resistant to killing by mucosal macrophages, appear to be particularly important. AIEC possess several virulence factors which may confer pathogenic potential in CD. Type-1 pili (FimH) allow adherence to intestinal cells via cell-surface carcinoembryonic antigen-related cell adhesion molecules and possession of long polar fimbrae promotes translocation across the intestinal mucosa via microfold (M)-cells of the follicle-associated epithelium. Resistance to stress genes (htrA, dsbA and hfq) and tolerance of an acidic pH may contribute to survival within the phagolysosomal environment. Here we review the current understanding of the role of mucosa-associated E. coli in Crohns pathogenesis, the role of the innate immune system, factors which may contribute to prolonged bacterial survival and therapeutic strategies to target intracellular E. coli.

OC-140 Hydroxychloroquine as a treatment for crohn's disease: enhancing antibiotic efficacy and macrophage killing of E coli

Introduction Mucosal E coli, increased in Crohns disease, have an adherent invasive phenotype (AIEC) and replicate within macrophages. AIEC can induce granulomas in vitro and in vivo and treatment leads to remission of colitis in animal models of Crohns.1 Hydroxychloroquine, which alters phagolysosomal pH and cellular iron mobilisation, enhances antibiotic efficacy and macrophage killing of other intra-macrophage organisms (Coxiella, Tropheryma).2 We postulate Hydroxychloroquine may be a useful treatment in Crohns. Methods We aimed to assess the effect of Hydroxychloroquine, alone or in combination with antibiotics, on intra-macrophage E coli survival. Further, we aimed to investigate the role of intracellular iron release and phagolysosomal pH as possible mechanisms of action. J774A.1 murine macrophages were infected with representative Crohns E coli isolates, HM605 (colonic) or LF82 (ileal), and the effect of Hydroxychloroquine and/or antibiotics was assessed using the gentamicin protection assay. FeNTA (pH independent ferric iron release from transferrin) and FeCitrate (pH dependent) were assessed for their ability to reverse the effect of Hydroxychloroquine. Fluorescence of macrophages co-infected with E coli and pHrodo E coli bioparticles was measured with a plate reader to determine phagolysosomal pH. Standard curves obtained by co-incubation of cells with nigericin and phosphate-citrate buffers allowed calculation of pH from fluorescence. Results Compared to untreated control, Hydroxychloroquine significantly reduced intra-macrophage E coli survival in a dose dependent manner at clinically achievable concentrations (31.4±4.6% at 2 μg/ml, p<0.001, ANOVA, N=3 where n=3). Combination with Doxycycline was significantly more effective than antibiotic treatment alone both at Cmax (34.5±4.7% vs 75.5±6.7%, p<0.001, N=6) and 10% Cmax (48.9±5.4% vs 89±5.6%, p<0.001, N=6). Similar synergy was seen with Ciprofloxacin at 10% Cmax (4.63±1.0% vs 7.9±1.3%, p<0.05, N=3) but not at Cmax where antibiotic alone markedly reduced bacterial survival (0.17±0.1%, N=3). Neither FeNTA nor FeCitrate reversed the effect of Hydroxychloroquine suggesting its effect is not mediated by changes in iron metabolism. A trend towards higher pH was seen with Hydroxychloroquine compared to control (7.22±0.016 vs 6.66±0.19) but this did not reach significance. Conclusion Hydroxychloroquine enhances antibiotic efficacy and macrophage killing of AIEC. Its mechanism of action is not via pH dependent iron metabolism but is likely due to direct phagolysosomal pH changes. Further work is required to determine its mechanism of action but it holds potential as a treatment for Crohns. Competing interests P Flanagan: None declared, B Campbell: None declared, J Rhodes consultant for: a member of advisory boards for Atlantic, Procter and Gamble and Falk, Speaker bureau with: Received speaking honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble, Schering Plough, Shire and Wyeth, Conflict with: With the University of Liverpool and Provexis UK, holds a patent for use of a soluble fibre preparation as maintenance therapy for Crohns disease plus a patent pending for its use in antibiotic-associated diarrhoea. References 1. Flanagan P, et al. Biochem Soc Trans 2011;39:1067–72. 2. Rolain JM, et al. Int J Antimicrob Agents. 2007;30:297–308.

A rare cause of weight loss

A 36-year-old man of Eastern European origin presented with a 4-week history of odynophagia, dysphagia, 10 kg weight loss and night sweats. There was no significant past medical history, he was on no regular medications but the travel history was notable with several years spent working in Africa. Clinical examination revealed loss of subcutaneous fat, muscle wasting, extensive lymphadenopathy and oral candidiasis. Initial blood tests revealed Hb 9.0 g/dL (13.0–16.7), white cell count 6.2×109/L (3.5–11), erythrocyte sedimentation rate 44 mm/h, C reactive protein 113 mg/L (<5) and albumin 25 g/L (35–50). HIV infection was suspected and confirmed by both serology …

Lessons from diversion studies and antibacterial interventions.

If bacteria cause IBD, then it should be possible to target the bacteria with therapies and cure or at least treat the disease. Discovery of a successful intervention, unless found by chance, will depend on knowing more about which bacteria are involved, where they are and how to remove them. Some evidence for the possible role of bacteria has come from in vivo studies of the effects of diverting the faecal stream away from sites of IBD. Alternative hypotheses arise from the diversion studies that could incriminate other components of the faecal stream that include bile acids and dietary components. Antibiotics will only really be adequately tested when we know what the target bacteria are and where they are, e.g. whether in the lumen or mucosa and whether intracellular or extracellular. Some encouraging responses have been observed, however, with empirical antibiotic therapy.

Original article: Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer

Objective Colonic mucosa-associated Escherichia coli are increased in Crohns disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. Design A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). Results 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. Conclusions IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

Colonic mucosa-associated diffusely adherentafaC+ Escherichia coliexpressinglpfAandpksare increased in inflammatory bowel disease and colon cancer

Objective Colonic mucosa-associated Escherichia coli are increased in Crohns disease (CD) and colorectal cancer (CRC). They variously haemagglutinate, invade epithelial cell lines, replicate within macrophages, translocate across M (microfold) cells and damage DNA. We investigated genes responsible for these effects and their co-association in colonic mucosal isolates. Design A fosmid library yielding 968 clones was prepared in E coli EPI300-T1 using DNA from a haemagglutinating CRC isolate, and resulting haemagglutinating clones were 454-pyrosequenced. PCR screening was performed on 281 colonic E coli isolates from inflammatory bowel disease (IBD) (35 patients), CRC (21) and controls (24; sporadic polyps or irritable bowel syndrome). Results 454-Pyrosequencing of fosmids from the haemagglutinating clones (n=8) identified the afimbrial adhesin afa-1 operon. Transfection of afa-1 into E coli K-12 predictably conferred diffuse adherence plus invasion of HEp-2 and I-407 epithelial cells, and upregulation of vascular endothelial growth factor. E coli expressing afaC were common in CRC (14/21, p=0.0009) and CD (9/14, p=0.005) but not ulcerative colitis (UC; 8/21) compared with controls (4/24). E coli expressing both afaC and lpfA (relevant to M-cell translocation) were common in CD (8/14, p=0.0019) and CRC (14/21, p=0.0001), but not UC (6/21) compared with controls (2/24). E coli expressing both afaC and pks (genotoxic) were common in CRC (11/21, p=0.0015) and UC (8/21, p=0.022), but not CD (4/14) compared with controls (2/24). All isolates expressed dsbA and htrA relevant to intra-macrophage replication, and 242/281 expressed fimH encoding type-1 fimbrial adhesin. Conclusions IBD and CRC commonly have colonic mucosal E coli that express genes that confer properties relevant to pathogenesis including M-cell translocation, angiogenesis and genotoxicity.

PWE-091 Crohn’s Disease Monocyte-derived Macrophages Exhibit Equivalent Responses To Intramacrophage Bacterial Infection Relative To Healthy Controls

Introduction Patients with Crohn’s disease exhibit an attenuated inflammatory response after trauma or Escherichia coli infection and have delayed clearance of subcutaneous bacteria compared to healthy controls. Adherent, invasive E. coli, increased in Crohn’s disease, replicate within macrophages and may have a primary pathogenic role. Crohn’s disease patients’ macrophages may have a primary defect in bacterial killing, allowing survival of AIEC. Methods We aimed to assess the relative ability of monocyte-derived macrophages from Crohn’s patients to kill intracellular E. coli and Staphylococcus aureus compared to healthy controls. Peripheral blood monocytes were obtained from consenting adults by centrifugation over Lymphoprep™ followed by 2h adherence to plastic Nuncò tissue culture dishes and subsequent differentiation into macrophages by 5d culture (as per Smith et al , J. Exp. Med. 2009, 206: 1883–97). Macrophages were infected with an adherent, invasive E. coli HM605, E. coli K12 or Staph. aureus Oxford strain. Intramacrophage killing was assessed using the gentamicin protection assay. Cytokine release to the culture medium was also determined by sandwich ELISA. Macrophage-mediated chemotaxis of human neutrophils, obtained from healthy controls by centrifugation over Polymorphoprep™, was quantified in Boyden chambers. Results No significant difference in the relative killing of E.coli K12 (-14 ± 11% vs. -45 ± 9%) and Staph. aureus (-52 ± 4% vs. -63 ± 5%) nor in the relative survival of AIEC HM605 (+50 ± 26% vs. +8 ± 22%) within monocyte-derived macrophages was seen (healthy controls vs. Crohn’s disease respectively; n = 10 each group, ANOVA). TNFα, IL-6 and IL-8 production were not significantly different between the two groups and macrophage mediated neutrophil chemotaxis was equivalent. Smoking status did not affect bacterial survival, with no differences observed in killing between current smokers, ex-smokers and non-smokers. Conclusion AIEC are ineffectively killed by both Crohn’s disease and healthy macrophages. Macrophages from patients with Crohn’s disease do not appear to have an inherent defect in killing and exhibit equivalent ability to induce neutrophil chemotaxis relative to controls. These data suggest circulating inhibitors of Neutrophil chemotaxis may explain the previously observed defective neutrophil chemotaxis and bacterial clearance in vivo. Disclosure of Interest P. Flanagan Grant/research support from: Awarded a Shire innovation fund for SpRs, S. Subramanian: None Declared, B. Campbell: None Declared, J. Rhodes Consultant for: A member of advisory boards for Atlantic, Procter and Gamble and Falk, Speaker bureau with: Has received speaking honoraria from Abbott, Falk, Ferring, Glaxo Smith Kline, Procter and Gamble, Schering Plough, Shire and Wyeth, Conflict with: With the University of Liverpool and Provexis UK, holds a patent for use of a soluble fibre preparation as maintenance therapy for Crohn’s disease plus a patent pending for its use in antibiotic-associated diarrhoea.