LYME DISEASE: RECENT ADVANCES AND PERSPECTIVES EDITED BY : Tanja Petnicki-Ocwieja and Catherine A. Brissette PUBLISHED IN : Frontiers in Cellular and Infection Microbiology 1 August 2015 | Lyme Disease: Recent Advances and Perspectives Frontiers in Cellular and Infection Microbiology Frontiers Copyright Statement © Copyright 2007-2015 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA (“Frontiers”) or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers. The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. 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Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: researchtopics@frontiersin.org 2 August 2015 | Lyme Disease: Recent Advances and Perspectives Frontiers in Cellular and Infection Microbiology The interplay between host and pathogen is a complex co-evolutionary battle of surveillance and evasion. The pathogen continuously develops mechanisms to subvert the immune response in order to establish infection while the immune system responds with novel mechanisms of detection. Because the majority of Lyme disease pathology is due to an over- exuberant immune response, much research in Borrelia burgdorferi pathogenesis has been devoted to understanding the mammalian host response to the bacterium. Immunological studies continue to be an active area of research employing emerging techniques, such as intra-vital imaging. These studies have furthered our understanding of inflammatory processes during long-term infection and provided some surprising insights, such as the continued presence of bacterial products after clearance. The field of Lyme disease has long debated the etiology of long- term inflammation and recent studies in the murine host have shed light on relevant cell types and inflammatory mediators that participate in the pathology of Lyme arthritis. Live imaging and bioluminescent studies have allowed for a novel view of the bacterial life cycle, including the tick mid-gut, tick-to-mammal transmission and dissemination throughout a mouse. A number of tick and bacterial proteins have been shown to participate in the completion of the enzootic cycle. Novel mechanisms of gene regulation are continuously being identified. However, B. burgdorferi lacks many traditional virulence factors, such as toxins or specialized secretion systems. Many genes in the B. burgdorferi genome have no known homolog in other bacteria. Therefore, studies focusing on host-pathogen interactions have therefore been limited by an incomplete understanding of the repertoire of bacterial virulence factors. Questions such as how the pathogen causes disease, colonizes the tick LYME DISEASE: RECENT ADVANCES AND PERSPECTIVES Topic Editors: Tanja Petnicki-Ocwieja, Tufts Medical Center, Boston, USA Catherine A. Brissette, University of North Dakota, USA Borrelia burgdorferi interacting with mammalian cells. Image by: Tanja Petnicki-Ocwieja 3 August 2015 | Lyme Disease: Recent Advances and Perspectives Frontiers in Cellular and Infection Microbiology and evades host immune-surveillance have been difficult to address. Genetic studies involving single gene deletions have identified a number of important bacterial proteins, but a large-scale genomics approach to identify virulence factors has not been attempted until recently. The generation of a site-directed mutagenesis library is an important step towards a detailed analysis of the B. burgdorferi genome and pathogenome. Using this library, high-throughput genomic studies, utilizing techniques such as massively parallel sequencing have been promising and could be used to identify novel virulence determinants of disease in the mammalian host or persistence in the tick vector. Continued research on this unique pathogen and its specific interaction with host and vector may have far reaching consequences and provide insights for diverse disciplines including ecology, infectious disease, and immunology. Here, several reviews will discuss the most recent advances and future studies to be undertaken in the field of B. burgdorferi biology. Citation: Petnicki-Ocwieja, T., Brissette, C. A., eds.(2015). Lyme Disease: Recent Advances and Perspectives. Lausanne: Frontiers Media. doi: 10.3389/978-2-88919-557-2 4 August 2015 | Lyme Disease: Recent Advances and Perspectives Frontiers in Cellular and Infection Microbiology Table of Contents 05 Lyme disease: recent advances and perspectives Tanja Petnicki-Ocwieja and Catherine A. Brissette 08 Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling Tanja Petnicki-Ocwieja and Aurelie Kern 15 Blocking pathogen transmission at the source: reservoir targeted OspA-based vaccines against Borrelia burgdorferi Maria Gomes-Solecki 22 Immunity-related genes in Ixodes scapularis —perspectives from genome information Alexis A. Smith and Utpal Pal 34 Dual role for Fc γ receptors in host defense and disease in Borrelia burgdorferi- infected mice Alexia A. Belperron, Nengyin Liu, Carmen J. Booth and Linda K. Bockenstedt 45 Forward genetic approaches for elucidation of novel regulators of Lyme arthritis severity Kenneth K. C. Bramwell, Cory Teuscher and Janis J. Weis 54 Emergence of Ixodes scapularis and Borrelia burgdorferi , the Lyme disease vector and agent, in Ohio Peng Wang, Meaghan N. Glowacki, Armando E. Hoet, Glen R. Needham, Kathleen A. Smith, Richard E. Gary and Xin Li 63 The role of eicosanoids in experimental Lyme arthritis Carmela L. Pratt and Charles R. Brown 69 Transposon mutagenesis as an approach to improved understanding of Borrelia pathogenesis and biology Tao Lin, Erin B. Troy, Linden T. Hu, Lihui Gao and Steven J. Norris 79 Phagosomal TLR signaling upon Borrelia burgdorferi infection Jorge L. Cervantes, Kelly L. Hawley, Sarah J. Benjamin, Bennett Weinerman, Stephanie M. Luu and Juan C. Salazar 91 The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi Elizabeth A. Novak, Syed Z. Sultan and Md. A. Motaleb 102 That’s my story, and I’m sticking to it—an update on B. burgdorferi adhesins Catherine A. Brissette and Robert A. Gaultney EDITORIAL published: 01 April 2015 doi: 10.3389/fcimb.2015.00027 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org April 2015 | Volume 5 | Article 27 | Edited and reviewed by: Yousef Abu Kwaik, University of Louisville School of Medicine, USA *Correspondence: Tanja Petnicki-Ocwieja, tpetnicki.ocwieja@gmail.com; Catherine A. Brissette, catherine.brissette@med.und.edu Received: 09 February 2015 Accepted: 09 March 2015 Published: 01 April 2015 Citation: Petnicki-Ocwieja T and Brissette CA (2015) Lyme disease: recent advances and perspectives. Front. Cell. Infect. Microbiol. 5:27. doi: 10.3389/fcimb.2015.00027 Lyme disease: recent advances and perspectives Tanja Petnicki-Ocwieja 1 * and Catherine A. Brissette 2 * 1 Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA, USA, 2 Department of Basic Sciences, University of North Dakota, Grand Forks, ND, USA Keywords: Lyme Disease, Borrelia burgdorferi , Ixodes scapularis , innate immunity, Lyme arthritis, adhesins, c-di-GMP, Tnseq Lyme Disease, caused by the spirochete Borrelia burgdorferi and transmitted by Ixodes scapularis (deer tick or blacklegged tick), has been gaining in incidence over the past decade. Without treat- ment, it is a long-term infection characterized by inflammation of the joints, heart and nervous system. The Centers for Disease Control and Prevention (CDC) classifies it as an Emerging Infec- tious Disease with an expanding geographical area of occurrence and have recently revised their incidence cases in the United States by 10 fold. In this Lyme Disease Research Topic we have gathered reviews and original research in the fields of microbiology and immunology of B. burgdorferi infection. Included in this Topic are the abstracts from the 13th International Congress on Lyme Borreliosis and we thank the organizers Dr. Linda Bockenstedt and Dr. Linden Hu for publishing them in this issue at the following link http://www.frontiersin.org/books/13th_International_Conference_on_Lyme_Borreliosis_and_oth er_tick_Borne_Diseases_/357. Increased Geographical Distribution As an emerging infectious disease the incidence and geographical distribution of Lyme disease cases are being monitored by a number of US states. Based on the CDC, Ohio is considered a non-endemic area for Lyme Disease, largely due to the low incidence of the arthropod vector I. scapularis . A tick surveillance program established by the Ohio Department of Public Health indicated a sharp increase in the prevalence of this tick in the state. Here, Wang et al. provide data that suggest an establishment of the enzootic cycle in Ohio (Wang et al., 2014). Vaccine Development—a Different Approach The development of a Lyme disease vaccine has been a hot topic for researchers and the pub- lic. Currently, the Food and Drug Administration has not given its approval for a human Lyme Disease vaccine. Therefore, researchers have sought alternate approaches to address control of B. burgdorferi infection in humans through pest management intervention. These methods have mainly consisted of vector-targeted or reservoir-targeted vaccines aiming to reduce tick density or control different aspects of the enzootic cycle. Gomes-Solecki writes a detailed review of the current status of those studies (Gomes-Solecki, 2014). Host Immunity—Novel Pathways and Genetic Approaches Because the majority of Lyme disease pathology is due to an over-exuberant immune response, much research in B. burgdorferi pathogenesis has been devoted to understand- ing the mammalian host response to the bacterium. A significant focus of immune 5 Petnicki-Ocwieja and Brissette Lyme disease: recent advances and perspectives studies has been the innate immune response as an initiator of inflammation. Recent studies have elucidated novel components of the innate immune response and intracellular pathways that participate in B. burgdorferi induced inflammation. Here, Cer- vantes et al. and Petnicki-Ocwieja and Kern review the most recent studies dissecting the numerous innate immune response pathways involved in B. burgdorferi recognition (Cervantes et al., 2014; Petnicki-Ocwieja and Kern, 2014). Although much progress has been made in identifying immune pathways that participate in the B. burgdorferi induced response, it is unclear how these pathways lead to arthritis resis- tance or susceptibility. The field of Lyme disease has long debated the etiology of long-term inflammation and recent studies in the murine host have shed light on relevant cell types and inflamma- tory mediators that participate in the pathology of Lyme arthri- tis. Pratt and Brown review the role of eicosanoids as important mediators of arthritis (Pratt and Brown, 2014). Also, Bramwell et al. review the challenges of genome wide association studies for studying complex genetic traits in humans and the power of for- ward genetic approaches in model animals leading to the identifi- cation of genetic loci responsible for arthritis severity phenotypes (Bramwell et al., 2014). Finally, in a research paper, Belperron et al. present data that implicate the FcRY receptor as an impor- tant contributor to acute phase Lyme arthritis (Belperron et al., 2014). Lessons from the Tick Much of the research in Lyme Disease immunology has focused on understanding the immune response in the mammalian host. However, the importance of the enzootic cycle and the mode of survival of the bacterium in its arthropod host cannot be understated. The I. scapularis tick maintains the bacterium in its mid-gut, but precisely how tick immunity functions and influ- ences persistence of pathogens remains unknown. Here, Smith and Pal discuss exciting insights that could be gained from min- ing the sequenced Ixodes genome and highlight future areas of investigation (Smith and Pal, 2014). What about the Bug? In nature, B. burgdorferi cycles between the vastly different environments of the Ixodes tick vector and mammalian host. B. burgdorferi must be able to detect changes in its environ- ment, and rapidly respond to these changes. c-di-GMP, a sec- ond messenger unique to bacteria, is a global regulator that facilitates adaption to changing environmental circumstances. Novak et al. provide up-to-date information on how c-di- GMP signaling is instrumental in orchestrating the adapta- tion of B. burgdorferi to the tick environment (Novak et al., 2014). Adhesion is the initial event in the establishment of any infec- tion. B. burgdorferi modulates adhesion to host tissues in order to colonize, disseminate, and persist in its mammalian host. Brissette and Gaultney update current knowledge on the struc- ture, function, and role of borrelial adhesins in Lyme disease pathogenesis (Brissette and Gaultney, 2014). The Next Generation: Massively Parallel Sequencing Tnseq B. burgdorferi lacks many traditional virulence factors, such as toxins or specialized secretion systems. Therefore, studies focusing on host-pathogen interactions have been limited by an incomplete understanding of the repertoire of bacterial virulence factors. Questions such as how the pathogen causes disease, col- onizes the tick and evades host immune-surveillance have been difficult to address. Genetic studies involving single gene deletions have identified a number of important bacterial proteins, but a large-scale genomics approach to identify virulence fac- tors has not been attempted until recently. Lin et al. review the generation of a site-directed mutagenesis library as an important step toward a detailed analysis of the B. burgdor- feri genome and pathogenome (Lin et al., 2014). Using this library, high-throughput genomic studies, utilizing techniques such as massively parallel sequencing or Tnseq have shown to be a powerful tool in understanding the pathogen. Lyme disease is endemic through much of the Northern hemi- sphere, including North America, Europe, and Asia, and will con- tinue to be a public health concern for the foreseeable future. This Topic highlights the important work that is currently being done to understand the pathogen, the vector, and the disease. We hope that you find this Topic as enlightening and thought-provoking as we did. References Belperron, A. A., Liu, N., Booth, C. J., and Bockenstedt, L. K. (2014). Dual role for Fcgamma receptors in host defense and disease in Borrelia burgdorferi -infected mice. Front. Cell. Infect. Microbiol. 4:75. doi: 10.3389/fcimb.2014.00075 Bramwell, K. K., Teuscher, C., and Weis, J. J. (2014). Forward genetic approaches for elucidation of novel regulators of Lyme arthritis severity. Front. Cell. Infect. Microbiol. 4:76. doi: 10.3389/fcimb.2014.00076 Brissette, C. A., and Gaultney, R. A. (2014). That’s my story, and I’m sticking to it–an update on B. burgdorferi adhesins . Front. Cell. Infect. Microbiol. 4:41. doi: 10.3389/fcimb.2014.00041 Cervantes, J. L., Hawley, K. L., Benjamin, S. J., Weinerman, B., Luu, S. M., and Salazar, J. C. (2014). Phagosomal TLR signaling upon Borrelia burgdor- feri infection. Front. Cell. Infect. Microbiol. 4:55. doi: 10.3389/fcimb.2014. 00055 Gomes-Solecki, M. (2014). Blocking pathogen transmission at the source: reservoir targeted OspA-based vaccines against Borrelia burgdorferi Front. Cell. Infect. Microbiol. 4:136. doi: 10.3389/fcimb.2014.00136 Lin, T., Troy, E. B., Hu, L. T., Gao, L., and Norris, S. J. (2014). Transposon mutagenesis as an approach to improved understanding of Borrelia pathogen- esis and biology. Front. Cell. Infect. Microbiol. 4:63. doi: 10.3389/fcimb.2014. 00063 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org April 2015 | Volume 5 | Article 27 | 6 Petnicki-Ocwieja and Brissette Lyme disease: recent advances and perspectives Novak, E. A., Sultan, S. Z., and Motaleb, M. A. (2014). The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi Front. Cell. Infect. Microbiol. 4:56. doi: 10.3389/fcimb.2014.00056 Petnicki-Ocwieja, T., and Kern, A. (2014). Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling. Front. Cell. Infect. Microbiol. 4:175. doi: 10.3389/fcimb.2014.00175 Pratt, C. L., and Brown, C. R. (2014). The role of eicosanoids in experimental Lyme arthritis. Front. Cell. Infect. Microbiol. 4:69. doi: 10.3389/fcimb.2014.00069 Smith, A. A., and Pal, U. (2014). Immunity-related genes in Ixodes scapularis – perspectives from genome information. Front. Cell. Infect. Microbiol. 4:116. doi: 10.3389/fcimb.2014.00116 Wang, P., Glowacki, M. N., Hoet, A. E., Needham, G. R., Smith, K. A., Gary, R. E., et al. (2014). Emergence of Ixodes scapularis and Borrelia burgdorferi , the Lyme disease vector and agent, in Ohio. Front. Cell. Infect. Microbiol. 4:70. doi: 10.3389/fcimb.2014.00070 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2015 Petnicki-Ocwieja and Brissette. This is an open-access article dis- tributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org April 2015 | Volume 5 | Article 27 | 7 REVIEW ARTICLE published: 15 December 2014 doi: 10.3389/fcimb.2014.00175 Mechanisms of Borrelia burgdorferi internalization and intracellular innate immune signaling Tanja Petnicki-Ocwieja* and Aurelie Kern Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA, USA Edited by: Chad J. Roy, Tulane University, USA Reviewed by: Janakiram Seshu, The University of Texas at San Antonio, USA Eric Ghigo, Centre National de la Recherche Scientifique, France *Correspondence: Tanja Petnicki-Ocwieja, Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, 800 Washington Street, Box 41, Boston, MA 02111, USA e-mail: tpetnickiocwieja@ tuftsmedicalcenter.org Lyme disease is a long-term infection whose most severe pathology is characterized by inflammatory arthritis of the lower bearing joints, carditis, and neuropathy. The inflammatory cascades are initiated through the early recognition of invading Borrelia burgdorferi spirochetes by cells of the innate immune response, such as neutrophils and macrophage. B. burgdorferi does not have an intracellular niche and thus much research has focused on immune pathways activated by pathogen recognition molecules at the cell surface, such as the Toll-like receptors (TLRs). However, in recent years, studies have shown that internalization of the bacterium by host cells is an important component of the defense machinery in response to B. burgdorferi . Upon internalization, B. burgdorferi is trafficked through an endo/lysosomal pathway resulting in the activation of a number of intracellular pathogen recognition receptors including TLRs and Nod-like receptors (NLRs). Here we will review the innate immune molecules that participate in both cell surface and intracellular immune activation by B. burgdorferi Keywords: Borrelia burgdorferi , Lyme disease, Toll-like receptor signaling, Nod-like receptor signaling, phagocytosis, endosomal signaling INTRODUCTION Lyme disease is caused by the spirochete Borrelia burgdorferi transmitted through a tick bite. The disease manifests as early localized skin inflammation (erythema migrans) occurring at the site of the tick bite. Late stage disease is characterized by inflam- mation of the heart, the joints, the nervous system or the skin. The incidence of human infections has risen steadily over the last 15 years and Lyme disease is the most common tick-borne dis- ease in the United States and Europe. More than 30,000 cases are reported annually in the US and the number of cases is estimated around 85,000 yearly in Europe (Lindgren et al., 2006; Centers for Disease Control and Prevention, 2013). Innate immune responses are the first responders to infec- tion and the catalyst of inflammation causing much of Lyme disease pathology. Recently, studies have shown that phagocy- tosis plays a role in initiating inflammatory responses (Moore et al., 2007; Shin et al., 2008; Salazar et al., 2009). Because of the requirement for pathogen internalization, much interest has been generated in studying intracellular signaling pathways. As a result, in addition to pathogen recognition by Toll-like receptors (TLRs), intracellular receptors such as the NOD-like receptors (NLRs) have been shown to participate in B. burgdorferi signal- ing. These families of receptors do not act in isolation and there is considerable cross talk among the innate immune signaling pathways activated (Takeuchi and Akira, 2010). TLR2 has been shown to initiate a significant portion of the inflammatory output in response to B. burgdorferi . The TLR2 ligands are Borrelia cell surface lipoproteins, the best-characterized being Outer Surface Protein A (OspA) (Hirschfeld et al., 1999; Lien, 1999; Takeuchi and Akira, 2010). Cellular compartmentalization is increasingly recognized as having a significant role in the regulation of innate immune sig- naling. Although this concept has been broadly understood as distinguishing between cell surface receptors, such as TLRs 2, 4, and 5 and intracellular sensors, such as TLRs 3, 7, and 9, as well as NLRs, it has recently become apparent that intracellular traffick- ing to different sub-cellular compartments and organelles, such as mitochondria and peroxisomes, plays a more intricate role in innate immune regulation than previously thought (Eisenbarth and Flavell, 2009; Blasius and Beutler, 2010; Dixit et al., 2010; Kagan, 2012). Here we will review various intracellular innate immune pathways activated by B. burgdorferi and how they may collectively contribute to inflammatory signaling. TOLL-LIKE RECEPTORS CELL SURFACE SIGNALING Recognition of B. burgdorferi at the cell surface Due to the importance of bacterial phagocytosis into host cells, significant research in recent years has been devoted to under- stand the involvement of cell surface molecules in the inter- nalization of B. burgdorferi . Spirochete internalization involves attachment or tethering of the bacteria to the host cell followed by engulfment into the host cell. These two processes, although linked, often involve different sets of cells surface molecules. B. burgdorferi contains a number of molecules known to function as adhesins and participate in the attachment of the bacterium to the host cell. One group of receptors shown to participate in tethering of B. burgdorferi to the cell surface is the integrin family, specifically integrins α v β 3 , α 5 β 1 ,and α M β 2 (CD18/CD11b, Mac-1, CR3) (Cinco et al., 1997; Coburn et al., 1998). Integrin α v β 3 has Frontiers in Cellular and Infection Microbiology www.frontiersin.org December 2014 | Volume 4 | Article 175 | CELLULAR AND INFECTION MICROBIOLOGY 8 Petnicki-Ocwieja and Kern B. burgdorferi intracellular signaling been shown to bind to the p66 protein of B. burgdorferi, but has not been shown to play a direct role in the initiation of immune responses (Coburn et al., 1998; Coburn and Cugini, 2003). It does, however, participate in adherence and potentially internal- ization of the bacterium into the host cell. Interestingly, integrin α v β 3 has been suggested to tether TLR2 ligands to the host cell via interaction with the serum protein vitronectin (Gerold et al., 2008). This has not been explored in the context of B. burgdor- feri adhesion. Integrin α M β 2 has been shown to participate in the attachment of B. burgdorferi to the cell surface (Cinco et al., 1997) and together with the TLR2 associated GPI anchored receptor, CD14, α M β 2 mediates the internalization of the bacterium into the host cell (Hawley et al., 2012). Signal transduction from the cell surface Signaling from the plasma membrane is a necessary compo- nent of the B. burgdorferi response, the mechanisms of which remain to be completely described. Understanding how cell sur- face accessory molecules contribute to the recognition of the ligand by the TLR is an active area of research. In the model of TLR4 signaling, integrin α M β 2 participates in the recruitment of the Toll-interleukin 1 receptor (TIR) domain-containing adapter protein (TIRAP) to Phosphatidylinositol 4,5-bisphosphate (PIP2) rich membranes, where TIRAP interacts with PIP2 and initiates cells surface signaling by recruiting the MyD88 signaling adaptor (Kagan and Medzhitov, 2006). TLR2 is the only other TLR that has been shown to utilize TIRAP for signaling and thus is likely that interaction with PIP2 is also important for TLR2 signaling (Yamamoto et al., 2002). However, there is significant interest in uncovering the signaling mechanism behind the ability of α M β 2 to traffic ligands and the TLR receptor to PIP2 rich locations and recruit signaling molecules. B. burgdorferi does not contain lipopolysaccharide (LPS) and does not activate TLR4 (Takayama et al., 1987; Berende et al., 2010). However, it contains other ligands that activate different TLRs. Specifically, B. burgdorferi activates TLR2/1 heterodimers through recognition of the triacylated lipid moiety on its cell sur- face localized lipopeptides (Hirschfeld et al., 1999; Alexopoulou et al., 2002). B. burgdorferi has been shown to activate TLR5, potentially through the spirochetal flagellin (Shin et al., 2008). Recruitment of MyD88 to the plasma membrane by TIRAP for TLR2 signaling or directly to TLR5 results in a signaling cascade which proceeds through the recruitment of the IRAK kinases, the E3 ubiquitin ligase, TRAF6, and TAK1 leading to the acti- vation of MAP kinases and of NF-kB through the IKK complex (Takeda and Akira, 2004). This signaling pathway leads to the activation of pro-inflammatory cytokines such as IL-6, IL-12, TNF- α , and pro-IL1 β . Type I IFNs have not been shown to be activated from this signaling cascade initiated at the plasma membrane. INTRACELLULAR SIGNALING Internalization of B. burgdorferi into host cells Although there are a number of cell surface molecules that medi- ate phagocytosis of B. burgdorferi , the signaling events during the process of internalization are under investigation. Early stud- ies into the mechanisms behind B. burgdorferi internalization indicate that it occurs through coiling, rather than conventional, phagocytosis, in which the bacteria attach to the host cell surface and are rolled into a single fold of the plasma membrane (Rittig et al., 1992). In studies with primary human macrophages it was shown that interaction with the bacteria leads to the formation of f-actin rich structures. The actin polymerization is medi- ated by the Wiskott-Aldrich syndrome family protein (WASP) and Arp2/3 complex, particularly during integrin α M β 2 and Fc γ receptor mediated phagocytosis (Linder et al., 2001; Shin et al., 2009). The regulatory pathways influencing actin polymeriza- tion and internalization of B. burgdorferi are mediated by the small GTPases Cdc42 and Rac1 (Linder et al., 2001). In addi- tion, PI3K signaling was shown to be required for phagocytosis of B. burgdorferi by murine macrophages (Shin et al., 2009). Although some cell surface molecules may serve as tethers, other molecules seem to have a more direct role in internalization. As described, CD14 is a molecule that mediates endocytosis of B. burgdorferi (Hawley et al., 2012). However, this is in itself a con- fusing finding in that CD14 is not known to have any cytoplasmic signaling domains that could initiate a signaling cascade leading to phagocytosis. CD14 has been shown to bind integrin α M β 2 and localize it to lipid rafts and it is possible that it then interacts with other molecules that can mediate endocytosis (Hawley et al., 2013). The primary role of this integrin seems to be in the attach- ment of the B. burgdorferi , rather than its internalization, and the signaling pathways activated by the integrin to mediate phago- cytosis have not been described in the context of B. burgdorferi Interestingly, integrin α 3 β 1 has been shown to mediate B. burgdor- feri and TLR2/1 ligand signaling (Marre et al., 2010). However, unlike α M β 2 , α 3 β 1 was not shown to mediate the attachment of B. burgdorferi or other TLR2 ligands to the cell surface but rather to participate in internalization (Behera et al., 2006; Marre et al., 2010). Overall, the β 1 integrin is required for internaliza- tion of B. burgdorferi into murine fibroblasts and to utilize the Src kinase signaling pathway for internalization (Wu et al., 2011). It is unclear if α 3 β 1 directly activates the Src signaling cascade, nor if CD14 and integrin α 3 β 1 cooperate to mediate phagocytosis of B. burgdorferi The complexity of molecule involvement in the phagocytic process is increased by the added role of scavenger recep- tors, which have also been shown to participate in TLR2 and B. burgdorferi signaling. CD36, a type B scavenger receptor, has been shown to be important for the internalization of TLR2 lig- ands and numerous studies have been devoted to understanding the mechanisms behind CD36 cooperation with TLR2. However, its role in B. burgdorferi internalization has not been explored (Shamsul et al., 2010). The scavenger receptor Macrophage Receptor with Collagenous Structure (MARCO), which plays a role in the internalization of a variety of microbial ligands, does mediate B. burgdorferi phagocytosis. In our studies we showed that MARCO was significantly up-regulated upon B. burgdorferi stimulation. The up-regulation of MARCO was dependent on MyD88 and MARCO deficient macrophage showed a decrease in the phagocytosis of B. burgdorferi (Petnicki-Ocwieja et al., 2013). These findings offered one possible explanation for the partial phagocytic defect previously observed in MyD88 deficient macrophage (Shin et al., 2008; Petnicki-Ocwieja et al., 2013). It is Frontiers in Cellular and Infection Microbiology www.frontiersin.org December 2014 | Volume 4 | Article 175 | 9 Petnicki-Ocwieja and Kern B. burgdorferi intracellular signaling yet unclear if MARCO participates in B. burgdorferi attachment to the cell surface. Significant work still needs to be done to determine how these cell surface accessory molecules: integrins, scavenger receptors and the GPI anchored receptor, CD14, coop- erate to mediate signaling and internalization of B. burgdorferi into the host cell. Signal transduction from the endosomal compartment TLR signaling can be initiated from both the plasma membrane and intracellular compartments. TLR5 has not been shown to signal from intracellular compartments and thus will not be dis- cussed further in this context. Interestingly, we and others have recently shown that in addition to being localized at the plasma membrane, TLR2 mediates signaling from endosomal vesicles in response to B. burgdorferi and TLR2 synthetic ligands. Inhibition of endosomal acidification upon B. burgdorferi stimulation results in a decrease in type I IFN and pro-inflammatory cytokine activation, such as IL-6 (Marre et al., 2010; Cervantes et al., 2011). TLR2 cooperates with other endosomal TLRs to generate a B. burgdorferi specific inflammatory response. B. burgdorferi acti- vates TLR7/8 and TLR9, which are endosomally localized TLR receptors (Shin et al., 2008; Petzke et al., 2009; Cervantes et al., 2011, 2013). Adaptor molecules, such as MyD88, are recruited to the endosomal compartment to transduce signals for the acti- vation of inflammatory cytokines and type I IFN from these endosomal TLRs (for a detailed review on TLR 7/8 and 9 in B. burgdorferi signaling, please see Cervantes, Hawley and Salazar in this issue). The localization of TLR2 at two different cellular locations requires that signaling molecules are able to distinguish the cel- lular localization of TLRs and assemble cell location specific signaling complexes. The only other TLR that has been shown to signal from two different cellular locations is TLR4. In the model of TLR4 signaling, signaling pathways from the cell surface vs. the endosome are clearly distinguished. TLR4 mediates signaling from the plasma membrane via TIRAP/MyD88 for MAP kinase and NF-kB activation resulting in pro-inflammatory cytokine activation. From the endosome TLR4 utilizes an entirely different set of adaptors, TRAM and TRIF, which are the signaling plat- form used to signal for type I IFNs, although TRAM/TRIF also mediate a delayed wave of NF-kB and pro-inflammatory cytokine activation (Kawai and Akira, 2011). Investigating the TLR2 signaling complex at the plasma mem- brane in comparison to the endosome, we found that TLR2 was also able to utilize the adaptor TRIF. TRIF deficient macrophage showed a reduction in type I IFN activation and secretion of IL-6 (Petnicki-Ocwieja et al., 2013). This was an unexpected result as TRIF was previously thought not to participate in TLR2 signaling. Interestingly, as opposed to the clear separation of signaling path- ways in the TLR4 model, TLR2/TRIF signaling was dependent on MyD88, suggesting that the MyD88 and TRIF signaling path- ways were interconnected. In addition to participating in TLR7/8 and 9 signaling, MyD88 may also participate in TLR2 signal- ing at the endosome. In vivo , TRIF deficient mice did not show any deficiencies in the ability to control bacterial loads in the joints of B. burgdorferi infected mice in comparison to wild type mice (Petnicki-Ocwieja et al., 2013). However, TRIF deficient mice did have increased levels of inflammatory cytokines in the joints, suggesting that TRIF has an important role in controlling immune responses leading to inflammation but not responses leading to control of pathogen burden. This might in part be due to the fact that, unlike MyD88 deficient cells, TRIF deficient cells do not have any observable phagocytic defects. Intracellular activation of immune pathways has been exten- sively studied in the case of viral infections. The intracellular activation of type I interferons (IFNs) was for a long time consid- ered to be strictly a viral response. Recently, type I IFN activation has been shown to play an important role in a large number of bacterial infections (Katze et al., 2002; Perry et al., 2005). In B. burgdorferi infection, the type I IFN response has also been shown to be important for the development of murine Lyme arthritis (Miller et al., 2008; Petzke et al., 2009; Salazar et al., 2009; Cervantes et al., 2011). Type I IFN activation initiated by TLRs is mediated by interferon regulatory factors (IRFs). In studies with TLR2 signaling, IRF1, and IRF7, both of which have been shown to bind MyD88, participate in TLR2 signaling (Dietrich et al., 2010). Downstream of the adaptor TRIF, TRAF3 is responsible for localizing an IRF3 signaling complex to the endosome lead- ing to type I IFN activation. B. burgdorferi stimulation has also been shown to proceed through IRF7 via TLR2 and TLR7 and 9 (Petzke et al., 2009; Petnicki-Ocwieja et al., 2013). Interestingly, studies have also shown that IRF3 is required for the type I IFN response to B. burgdorferi (Miller et al., 2010). NUCLEOTIDE BINDING OLIGOMERIZATION DOMAIN RECEPTORS (NOD-LIKE RECEPTORS) TLRs sense the extracellular and the endosomal compart- ments whereas RIG-like receptors (RLRs) and Nod-like receptors (NLRs) are intracellular sensors. In addition to TLRs, NLRs also participate in B. burgdorferi mediated intracellular signal- ing. From the receptors in the NLR family, Nod1, and 2 and the inflammasome complex are the best studied. NOD1 AND NOD2