Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells Matthew A. Pettengill1,2, Verissa W. Lam1, Ikechukwu Ollawa 1 , Camila Marques-da-Silva1,3, David M. Ojcius1* 1 Molecular Cell Biology, Health Sciences Research Institute, University of California Merced, Merced, California, United States of America, 2 Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, United States of America, 3 Biophysics Institute Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil Abstract Ivermectin is currently approved for treatment of both clinical and veterinary infections by nematodes, including Onchocerca cervicalis in horses and Onchocerca volvulus in humans. However, ivermectin has never been shown to be effective against bacterial pathogens. Here we show that ivermectin also inhibits infection of epithelial cells by the bacterial pathogen, Chlamydia trachomatis, at doses that could be envisioned clinically for sexually-transmitted or ocular infections by Chlamydia. Citation: Pettengill MA, Lam VW, Ollawa I, Marques-da-Silva C, Ojcius DM (2012) Ivermectin Inhibits Growth of Chlamydia trachomatis in Epithelial Cells. PLoS ONE 7(10): e48456. doi:10.1371/journal.pone.0048456 Editor: Jean Kanellopoulos, University Paris Sud, France Received February 21, 2012; Accepted October 1, 2012; Published October 30, 2012 Copyright: 2012 Pettengill et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work in the authors’ laboratory is supported by National Institutes of Health grant R01 DE019444 and a University of California Presidential Chair. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts: Co-author DMO is a PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: dojcius@ucmerced.edu Introduction Avermectins are macrocyclic lactone derivatives which are produced during fermentation of Streptomyces avermectinius, a species of actinomycete isolated from soil samples in Japan. Eight avermectins are produced by S. avermectinius (A1a, A1b, A2a, A2b, B1a, B1b, B2a, and B2b). The A series molecules contain a 59- methoxyl group, while the B series contain a 59-hydroxyl group and have more potent anti-parasitic activity [1,2,3]. Improved efficacy as a broad-spectrum anti-parasitic was achieved for the B1 compounds by selective hydrogenation utilizing Wilkinson’s catalyst (RhCl(PPh3)3), with the resultant product (22,23-dihy- droavermectin B1) being given the name ivermectin [4]. Following considerable success as an anti-helminthic agent in veterinary practice, including use for treatment of Onchocerca cervicalis in horses, ivermectin entered clinical trials for use in humans against Onchocerca volvulus, a nematode which causes onchocerciasis (‘‘river blindness’’) [5]. Efficacious use in humans has extended to lymphatic filariasis [6], head-lice infestation [7], and scabies [8]. However, avermectins have not been previously described to have anti-bacterial activity [5]. Here we demonstrate that ivermectin inhibits growth of Chlamydia trachomatis during infection of human cervical epithelial cells, suggesting that avermectins may have previ- ously undescribed anti-bacterial activity against pathogenic obligate intracellular bacteria, potentially via indirect effects on the host-cell. Chlamydiae are obligate intracellular bacteria which mature through a unique biphasic developmental cycle, infecting as metabolically inert elementary bodies (EBs) and maturing into metabolically active but non-infectious reticulate bodies (RBs), which proliferate before condensing into infectious EBs to complete the cycle [9,10,11]. C. trachomatis strains, which are the leading cause of bacterial sexually transmitted disease and first cause of preventable blindness [12], primarily infect mucosal epithelial cells. Results and Discussion Five mM ivermectin added one hour post infection (hpi) significantly inhibited the production of infectious EB (Figure 1A) and chlamydial 16s rRNA accumulation (Figure 1B), as evaluated by harvesting infected HeLa cultures and infecting fresh HeLa monolayers followed by fluorescent microscopy to determine infectious units per mL (IFU/ml, Figure 1A), or by RNA extraction of infected HeLa cultures followed by cDNA synthesis and qPCR for C. trachomatis 16s rRNA (Figure 1B). Treatment with 1 mM ivermectin at 1 hpi modestly decreased the size of inclusions, while inclusions in cells treated with 5 mM ivermectin had significantly reduced dimensions (Figure 2). Treatment with 10 mM ivermectin completely inhibited inclusion development. Of note, 5 mM ivermectin did not reduce the number of cells in which inclusions were apparent, suggesting that the effect of ivermectin was not related to uptake of bacteria into host cells. To exclude the possibility that ivermectin may be inhibiting infection through an indirect cytotoxic effect on the host cells, we verified that treatment of HeLa cells with up to 10 mM ivermectin for 4 to 24 hours did not cause increased cell death (Figure 3A) or lactate dehydrogenase (LDH) release (Figure 3B). For use in humans as an anti-helminthic, ivermectin is typically received orally at a dose of 150 mg/kg body weight, and peak PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e48456