Introduction Mucosal E coli, increased in Crohn's 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 Crohn's.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 Crohn's.
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 Crohn's 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 Crohn's.
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 Crohn's 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.