Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC-SIGN-TLR2 Crosstalk

February Porphyromonas gingivalis Evasion of Autophagy and Intracellular Killing by Human Myeloid Dendritic Cells Involves DC- SIGN-TLR2 Crosstalk Ahmed R. El-Awady 0 1 2 Brodie Miles 0 1 2 Elizabeth Scisci 0 1 2 Zoya B. Kurago 0 1 2 Chithra D. Palani 0 1 2 Roger M. Arce 0 1 2 Jennifer L. Waller 0 1 2 Caroline A. Genco 0 1 2 Connie Slocum 0 1 2 Matthew Manning 0 1 2 Patricia V. Schoenlein 0 1 2 Christopher W. Cutler 0 1 2 0 1 Department of Periodontics, Georgia Regents University , Augusta , Georgia , United States of America, 2 Department of Medicine, University of Colorado, Aurora, Colorado, United States of America, 3 School of Dental Medicine, Stony Brook University , Stony Brook , New York, United States of America, 4 Department of Oral Health and Diagnostic Sciences, Georgia Regents University , Augusta , Georgia , United States of America, 5 Department of Biostatistics and Epidemiology, Georgia Regents University , Augusta , Georgia , United States of America, 6 Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, Massachusetts, United States of America, 7 Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts, United States of America, 8 Department of Cellular Biology and Anatomy, Georgia Regents University , Augusta, Georgia , United States of America 1 Funding: This work was supported by U.S. Public Health Service Grants from the National Institutes of Health/National Institute of Dental and Craniofacial Research R01 DE014328 and R21 DE020916-01 to CWC and R56 DE024350 to CAG and NIH/National Institute of Allergy and Infectious Diseases T32 AI007447-22 to BM, and T32 AI089673 and PO1 AI078894 to CAG and CS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Editor: Dana J. Philpott, University of Toronto , CANADA Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autop- - hagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular Competing Interests: The authors have declared that no competing interests exist. persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs. Among the most successful of human microbes are intracellular pathogens. By entering the intracellular milieu, these pathogens are protected from harsh environmental factors in the host, including the humoral and cellular immune responses. Porphyromonas gingivalis is an opportunistic pathogen that colonizes the oral mucosa and accesses the bloodstream and distant sites such as the blood vessel walls, brain, placenta and other organs. Still unclear is how P. gingivalis traverses from oral mucosa to these distant sites. Dendritic cells are highly migratory antigen presenting cells that patrol the blood, skin, mucosa and all the major organ systems. Capture of microbes by dendritic cells activates a tightly regulated series of events, including directed migration towards the secondary lymphoid organs, where processed antigens are ostensibly presented to T cells. Autophagy is now recognized as an integral component of microbial clearance, antigen processing and presentation by dendritic cells. We report here that P. gingivalis is able to subvert autophagic destruction within dendritic cells. This occurs through its glycoprotein fimbriae, called Mfa-1, which targets the C-type lectin DC-SIGN on dendritic cells. The other major fimbriae on P. gingivalis, FimA, targets TLR2, which promotes autophagic destruction of P. gingivalis. We conclude that DC-SIGN-TLR2 crosstalk determines the intracellular fate of this pathogen within dendritic cells, and may have profound implications for the treatment of many chronic diseases involving low-grade infections. Antimicrobial autophagy or xenophagy plays an important role in controlling bacterial infection and promoting innate immunity. Recent evidence has revealed critical roles for autophagy in the ability of immune cells to recognize and selectively target microbes for elimination. [14][1]. Dendritic cells (DCs) are innate immune cells that serve as a bridge to the adaptive immune response. DCs capture a wide variety of microbes in the peripheral tissues for which they are equipped with broad spectrum of pattern recognition receptors (PRRs). The major classes of PRRs expressed by DCs include Toll-like receptors (TLRs), NOD-like family receptors, CARD helicases and C-type lectin receptors [5,6]. Many of the PRRs come equipped with unique phagocytic machinery evolved for efficient antigen processing and presentation [79]. Phagocytosis relies on a network of endocytic vesicles such as early endosomes and/or autophagosomes, which fuse with lysosomes for degradation [10]. Intracellular vesicle maturation does not necessarily proceed through similar steps in different phagocytic cells [11]. Moreover, different phagosomal maturation pathways have been reported in the same cell type [11,12]. These different pathways are dictated primarily by the initial recognition step by PRRs and by the cargo contained in the vesicle [13]. Hence the immune cell type, the PRRs engaged and the properties of the microbe seem to be crucial for microbial clearance by autophagy. DC-SIGN (DC specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor involved in pathogen uptake, signaling and antigen presentation in DCs [1416]. For uptake DC-SIGN cont (...truncated)