Strong Type 1, but Impaired Type 2, Immune Responses Contribute to Orientia tsutsugamushi-Induced Pathology in Mice Lynn Soong 1,2 * . , Hui Wang 1. , Thomas R. Shelite 1,2. , Yuejin Liang 1 , Nicole L. Mendell 2 , Jiaren Sun 1 , Bin Gong 2 , Gustavo A. Valbuena 2 , Donald H. Bouyer 2 , David H. Walker 2 1 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas, United States of America, 2 Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, Center for Tropical Diseases, Sealy Center for Vaccine Development, Institute of Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas, United States of America Abstract Scrub typhus is a neglected, but important, tropical disease, which puts one-third of the world’s population at risk. The disease is caused by Orientia tsutsugamushi, an obligately intracellular Gram-negative bacterium. Dysregulation in immune responses is known to contribute to disease pathogenesis; however, the nature and molecular basis of immune alterations are poorly defined. This study made use of a newly developed murine model of severe scrub typhus and focused on innate regulators and vascular growth factors in O. tsutsugamushi-infected liver, lungs and spleen. We found no activation or even reduction in base-line expression for multiple molecules (IL-7, IL-4, IL-13, GATA3, ROR-ct, and CXCL12) at 2, 6 and 10 days post-infection. This selective impairment in type 2-related immune responses correlated with a significant activation of the genes for IL-1b, IL-6, IL-10, TNF-a, IFN-c, as well as CXCR3- and CXCR1-related chemokines in inflamed tissues. The elevated angiopoietin (Ang)-2 expression and Ang-2/Ang-1 ratios suggested excessive inflammation and the loss of endothelial integrity. These alterations, together with extensive recruitment of myeloperoxidase (MPO)-expressing neutrophils and the influx of CD3 + T cells, contributed to acute tissue damage and animal death. This is the first report of selective alterations in a panel of immune regulators during early O. tsutsugamushi infection in intravenously inoculated C57BL/6 mice. Our findings shed new light on the pathogenic mechanisms associated with severe scrub typhus and suggest potential targets for therapeutic investigation. Citation: Soong L, Wang H, Shelite TR, Liang Y, Mendell NL, et al. (2014) Strong Type 1, but Impaired Type 2, Immune Responses Contribute to Orientia tsutsugamushi-Induced Pathology in Mice. PLoS Negl Trop Dis 8(9): e3191. doi:10.1371/journal.pntd.0003191 Editor: Benjamin L. Makepeace, University of Liverpool, United Kingdom Received May 19, 2014; Accepted August 14, 2014; Published September 25, 2014 Copyright: ß 2014 Soong 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. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by a pilot grant from the University of Texas Medical Branch Center for Biodefense and Emerging Infectious Diseases and start-up funds (to LS), the Carmage and Martha Walls Distinguished University Chair in Tropical Diseases (to DHW), as well as the McLaughlin post-doctoral fellowship (to TRS). 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 declared that no competing interests exist. * Email: lysoong@utmb.edu . These authors contributed equally to this work. Introduction Scrub typhus is an acute, febrile and often fatal disease, caused by infection with O. tsutsugamushi (formerly known as Rickettsia orientalis or R. tsutsugamushi) [1]. Every year, approximately one million people are infected globally, especially in the Asia-Pacific region. Humans are infected through the bites of the larva of trombiculid mites, and the Karp- and Gilliam-like genotypes account for 50% and 25% of all human infections, respectively [2,3]. Scrub typhus is responsible for a large proportion of severe undifferentiated fevers, as well as up to 23% of all febrile episodes in rural endemic areas, and has relatively high mortality rates [4]. Some individuals can progress to persistent infection even after antibiotic treatment [5]. There are no effective vaccines for scrub typhus [3,6]. Adaptive immunity following O. tsutsugamushi infection in humans appears short-lived [4,7,8], but the underlying mechanisms of protective immunity and its disappearance are unclear. O. tsutsugamushi predominantly replicates in disseminated endothelial cells (EC), and degree in macrophages and dendritic cells in the inoculation eschar [9,10,11]. The bacteria invade cells by induced phagocytosis, escape from the phagosome, and replicate in the cytoplasm. The histopathological features include perivascular infiltration of monocytes/macrophages and T cells and generalized vasculitis involving tissues from several organ systems [12,13]. Unlike other Gram-negative bacteria, O. tsutsugamushi lacks lipopolysaccharides (LPS) and peptidoglycan in its cell wall. Yet, both live and heat-killed O. tsutsugamushi are highly competent in stimulating proinflammatory cytokines [14,15], presumably by stimulating the cytosolic NOD-like receptor family proteins and NF-kB-mediated pathways. Endo- thelial ICAM-1 expression and Th1 cell activation are often seen in patients [16,17]. Pro-inflammatory responses are often crucial in bacterial clearance; however, excessive inflammation, apoptosis and TNF-a, IL-12, IL-10, CCL2 and CCL3 production can also PLOS Neglected Tropical Diseases | www.plosntds.org 1 September 2014 | Volume 8 | Issue 9 | e3191