J Neuropathol Exp Neurol Vol. 65, No. 6 Copyright Ó 2006 by the American Association of Neuropathologists, Inc. June 2006 pp. 540 Y 548 ORIGINAL ARTICLE Borrelia burgdorferi Induces TLR1 and TLR2 in Human Microglia and Peripheral Blood Monocytes but Differentially Regulates HLA-Class II Expression Riccardo Cassiani-Ingoni, MS, Erik S. Cabral, MS, Jan D. Lu¨nemann, MD, Zoila Garza, MS, Tim Magnus, MD, PhD, Harald Gelderblom, MD, Peter J. Munson, PhD, Downloaded from https://academic.oup.com/jnen/article/65/6/540/2645260 by guest on 23 September 2021 Adriana Marques, MD, and Roland Martin, MD Key Words: Glia, HLA class II, Lyme disease, Microglia, Abstract Monocyte, Outer surface protein A, Toll-like receptor. The spirochete Borrelia burgdorferi is the agent of Lyme disease, which causes central nervous system manifestations in up to 20% of patients. We investigated the response of human brain microglial cells, glial progenitors, neurons, astrocytes, as well as INTRODUCTION peripheral blood monocytes to stimulation with B. burgdorferi.We Lyme disease is the most frequently occurring vector- used oligoarrays to detect changes in the expression of genes borne infection in the United States and is also endemic in important for shaping adaptive and innate immune responses. We Europe and parts of Asia (1). Transmitted by Ixodes ticks, its found that stimulation with B. burgdorferi lysate increased the causative agent Borrelia burgdorferi initially propagates expression of Toll-like receptors (TLRs) 1 and 2 in all cell types locally in the skin before it disseminates hematogenously to except neurons. However, despite similarities in global gene other organ systems. Neurologic involvement occurs in up to profiles of monocytes and microglia, only microglial cells 20% of patients and presents as cranial neuritis, meningor- responded to the stimulation with a robust increase in HLA-DR, adiculitis, or encephalitis. Tissue tropism of B. burgdorferi HLA-DQ, and also coexpressed CD11-c, a dendritic cell marker. In for the nervous system and the inflammatory mechanisms contrast, a large number of HLA-related molecules were repressed that are involved in the local immune responses are at at both the RNA and the protein levels in stimulated monocytes, present only partly understood. The immune privilege of the whereas secretion of IL-10 and TNF-> was strongly induced. central nervous system (CNS) and the functional character- These results show that signaling through TLR1/2 in response to istics of resident CNS cells likely play important roles. B. burgdorferi can elicit opposite immunoregulatory effects in In animal models of Lyme disease, CNS invasion blood and in brain immune cells, which could play a role in the leads to local tissue damage as a consequence of potent different susceptibility of these compartments to infection. induction of proinflammatory mediators by microglial cells (2, 3). Microglia are resident immune cells within the CNS with a slow rate of turnover from circulating monocytes. When activated, microglial cells are capable of initiating a strong and prompt immune reaction against invading From the Cellular Immunology Section (RCI, ESC, JDL, HG, RM), Neuroimmunology Branch, National Institute of Neurological Disorders pathogens and orchestrate peripheral leukocyte infiltration and Stroke, Bethesda, Maryland; Mathematical and Statistical Comput- (4). These responses are probably under tight spatial and ing Laboratory (ZG, PJM), Analytical Biostatistics Section, Center for temporal regulation, depending on the inducing stimulus/ Information Technology, Bethesda, Maryland; Stem Cell Biology Unit inflammatory context, to prevent immune-mediated brain (TM), Laboratory of Neuroscience, National Institute on Aging, damage. If activated in an uncontrolled fashion, microglial Bethesda, Maryland; Laboratory of Clinical Infectious Diseases (AM), National Institute of Allergy and Infectious Diseases, National Institutes cells can amplify deleterious chronic immune responses within of Health, Bethesda, Maryland; and Department of Human Physiology the CNS as suggested for cerebral malaria or multiple sclerosis and Pharmacology (RCI), Center of Excellence in Biology and (4, 5). Besides microglial cells, B. burgdorferi-mediated Molecular Medicine, University of Rome BLa Sapienza,[ Rome, Italy. inflammation affects other glial and neuronal cells as well, Send correspondence and reprint requests to: Riccardo Cassiani-Ingoni, MS, Bldg.10, Room 5B06, 10 Center Drive, Bethesda, MD 20892; E-mail: which may occur directly through engagement of pattern [email protected] recognition receptors expressed on these cells or indirectly as This research was supported in part by the Intramural Research Program of a result of mediator release from activated microglia. Pattern the National Institutes of Health, NINDS, NIAID, and NIMH. RCI is recognition receptors, which comprise the Toll-like receptors supported by the Integrative Neural Immune Program (INIP) of NIMH (TLR), can act as pathogen Bsensors^ by recognizing specific and is a doctoral candidate in the Research in Neurophysiology program at the University of Rome BLa Sapienza.^ molecular structures that are conserved among members of Supplementary data is available online at http://www.jneuropath.com. each class of infectious agents. In this way, they confer 540 J Neuropathol Exp Neurol Volume 65, Number 6, June 2006 Copyright @ 2006 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited. J Neuropathol Exp Neurol Volume 65, Number 6, June 2006 Cellular Response to Borrelia crucial information for shaping the specific immune response, least 48 hours before stimulation with bacterial antigens to for example, antibacterial as opposed to antiviral (6). TLR2 allow complete removal of the antibodies used for sorting as mediates immune responses to a broad range of microbial shown previously (13). products and is critical for the recognition of bacterial lipopeptides or live infectious organisms (7); when it Stimulation with Borrelia burgdorferi Antigens functions in combination with another member, TLR1, it K can bind triacylated lipopeptides like mycobacterial lip- Stimulation with bacterial antigens was done using 1 g/ mL low-passage sonicate of B. burgdorferi sensu stricto strain oprotein and the outer surface protein A of B. burgdorferi K (8, 9). B31 (Biodesign, Saco, ME) or with 0.1 g/mL recombinant In vitro studies in murine microglia have shown that Lipidated-outer surface protein A (L-OspA) of B. burgdorferi B. burgdorferi is capable of inducing the expression of (SmithKline Beecham Biologicals, Rixensart, Belgium). pattern recognition receptors like TLR2 and CD14 in these Antigen stimulation was performed in 2 mL fresh medium cells (10), a critical step in immune activation in the CNS. for 48 hours. No bacterial molecules were added in the Downloaded from https://academic.oup.com/jnen/article/65/6/540/2645260 by guest on 23 September 2021 Based on the potential impact of B. burgdorferi-induced control cultures. The final endotoxin concentration in medium for all antigens tested was G0.05 EU/mL (Endosafe-PTS) or cellular activation patterns for the human disease, it was G important to investigate the cellular response of human 0.1 EU/mL (Cambrex Bioscience, East Rutherford, NJ). brain and blood cells to these molecules. We stimulated This time point and antigen concentration were chosen microglia, astrocytes, neurons, and glial progenitors with according to pilot time course and dose-titration experiments B. burgdorferi in vitro. Using oligoarrays, we compared (data not shown) as well as published data using different the individual cellular responses with that of peripheral cells (14, 15). blood monocytes in the same conditions. This analysis highlighted overlapping but characteristic expression pro- Oligoarray Hybridization files of monocytes and microglial cells as well as pre- Total RNA was isolated with RNeasy (Qiagen, Valencia, viously unrecognized differences in their response after CA) from parallel cultures of stimulated and unstimulated cells TLR engagement. from individual donors. We independently hybridized RNA samples from a number of cell preparations of microglia (4 challenged, 3 mock), of monocytes (4 challenged, 3 MATERIALS AND METHODS control), neurons (1 challenged, 3 control), glial progenitors (2 challenged, 2 control), and astrocytes (1 challenged, 1 Isolation and Characterization of Brain Cells K and Blood Monocytes control). A minimum of 5 g total RNA per cell preparation/ condition was reverse-transcribed with T7-polyA primers We used primary microglial cells, neuronal and glial (Invitrogen Life Technologies, Carlsbad, CA). In vitro progenitor cells isolated from human fetal tissue (ScienCell, transcription of cDNA was performed in the presence of San Diego, CA). Fluorescence-activated cell sorting (FACS) biotinylated ribonucleotides (ENZO Diagnostics, New York, was used to further purify individual cell populations based NY). Hybridization of 15 Kg of cRNA to each Human on the expression of lineage-specific surface markers. We Genome 133A GeneChip (Affymetrix, Santa Clara, CA) was used antibodies against CD11-b (BD Biosciences, San Jose, done overnight. Secondary detection and scanning followed CA) to sort microglial cells, A2B5 staining (Roche, Indian- the manufacturer`s protocols. apolis, IN) for progenitors, and a mixture of tetanus toxin fragment C (TnTx) (Roche) together with a mouse mono- clonal anti-TnTx