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The survival of macrophage-adapted chlamydiae correlates with the multiplicity of infection (MOI), and optimal chlamydial growth occurs in macrophages infected at an MOI of \u22641. In this study, we examined the replicative capacity of<jats:named-content content-type=\"genus-species\">Chlamydia muridarum<\/jats:named-content>in the RAW 264.7 murine macrophage cell line at different MOIs.<jats:named-content content-type=\"genus-species\">C. muridarum<\/jats:named-content>productively infected these macrophages at low MOIs but yielded few viable elementary bodies (EBs) when macrophages were infected at a moderate (10) or high (100) MOI. While high MOIs caused cytotoxicity and irreversible host cell death, macrophages infected at a moderate MOI did not show signs of cytotoxicity until late in the infectious cycle. Inhibition of host protein synthesis rescued<jats:named-content content-type=\"genus-species\">C. muridarum<\/jats:named-content>in macrophages infected at a moderate MOI, implying that chlamydial growth was blocked by activated defense mechanisms. Conditioned medium from these macrophages was antichlamydial and contained elevated levels of interleukin 1\u03b2 (IL-1\u03b2), IL-6, IL-10, and beta interferon (IFN-\u03b2). Macrophage activation depended on Toll-like receptor 2 (TLR2) signaling, and cytokine production required live, transcriptionally active chlamydiae. A hydroxyl radical scavenger and inhibitors of inducible nitric oxide synthase (iNOS) and cathepsin B also reversed chlamydial killing. High levels of reactive oxygen species (ROS) led to an increase in cathepsin B activity, and pharmacological inhibition of ROS and cathepsin B reduced iNOS expression. Our data demonstrate that MOI-dependent TLR2 activation of macrophages results in iNOS induction via a novel ROS- and cathepsin-dependent mechanism to facilitate<jats:named-content content-type=\"genus-species\">C. muridarum<\/jats:named-content>clearance.<\/jats:p>","DOI":"10.1128\/iai.00382-15","type":"journal-article","created":{"date-parts":[[2015,5,27]],"date-time":"2015-05-27T15:24:51Z","timestamp":1432740291000},"page":"3164-3175","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":37,"title":["Chlamydia muridarum Infection of Macrophages Elicits Bactericidal Nitric Oxide Production via Reactive Oxygen Species and Cathepsin B"],"prefix":"10.1128","volume":"83","author":[{"given":"Krithika","family":"Rajaram","sequence":"first","affiliation":[{"name":"Department of Biology, Indiana University, Bloomington, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David E.","family":"Nelson","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-695X.2010.00772.x"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1086\/652397"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jir333"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.181.3.2092"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.4049\/jimmunol.172.4.2407"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1189\/jlb.0711336"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.immuni.2013.12.013"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1128\/iai.20.2.439-445.1978"},{"key":"e_1_3_3_10_2","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.4049\/jimmunol.139.4.1297","article-title":"Differential antimicrobial activity of human mononuclear phagocytes against the human biovars of Chlamydia trachomatis","volume":"139","author":"Yong EC","year":"1987","unstructured":"Yong EC, Chi EY, Kuo CC. 1987. 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