Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of Borrelia burgdorferi Sensu Stricto

et al. (2012) Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of Borrelia burgdorferi Sensu Stricto. PLoS ONE 7(6): e40046. doi:10.1371/journal.pone.0040046 Microarray Analyses of Inflammation Response of Human Dermal Fibroblasts to Different Strains of Borrelia burgdorferi Sensu Stricto Fre de ric Schramm 0 Aure lie Kern 0 Cathy Barthel 0 Sophie Nadaud 0 Nicolas Meyer 0 Benot Jaulhac 0 Nathalie Boulanger 0 Roman Ganta, Kansas State University, United States of America 0 1 EA 4438, Physiopathologie et Me decine Translationnelle, Faculte s de Me decine et de Pharmacie, Universite de Strasbourg, Strasbourg, France, 2 INSERM UMR-S 956, UPMC Universite Paris 06, Paris, France, 3 Laboratoire de Biostatistique et Informatique Me dicale, Faculte de Me decine, Universite de Strasbourg , Strasbourg , France In Lyme borreliosis, the skin is the key site of bacterial inoculation by the infected tick, and of cutaneous manifestations, erythema migrans and acrodermatitis chronica atrophicans. We explored the role of fibroblasts, the resident cells of the dermis, in the development of the disease. Using microarray experiments, we compared the inflammation of fibroblasts induced by three strains of Borrelia burgdorferi sensu stricto isolated from different environments and stages of Lyme disease: N40 (tick), Pbre (erythema migrans) and 1408 (acrodermatitis chronica atrophicans). The three strains exhibited a similar profile of inflammation with strong induction of chemokines (CXCL1 and IL-8) and IL-6 cytokine mainly involved in the chemoattraction of immune cells. Molecules such as TNF-alpha and NF-kB factors, metalloproteinases (MMP-1, -3 and -12) and superoxide dismutase (SOD2), also described in inflammatory and cellular events, were up-regulated. In addition, we showed that tick salivary gland extracts induce a cytotoxic effect on fibroblasts and that OspC, essential in the transmission of Borrelia to the vertebrate host, was not responsible for the secretion of inflammatory molecules by fibroblasts. Tick saliva components could facilitate the early transmission of the disease to the site of injury creating a feeding pit. Later in the development of the disease, Borrelia would intensively multiply in the skin and further disseminate to distant organs. - Funding: Aurelie Kern was supported by grant 2009.60.053 from the Conseil Regional dAlsace and Direction Generale de lArmement. Part of the research project was supported by the Pasteur Institute, PTR 309 (Programme Transversal de Recherche, Paris, France). 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. . These authors contributed equally to this work. Lyme borreliosis (LB) caused by spirochetes of the B. burgdorferi sl group is the most common vector-borne disease in the Northern Hemisphere. These bacteria are transmitted by the tick Ixodes spp. [1]. LB is a multisystemic infection that starts generally with an erythema migrans (EM) lesion at the site of the tick bite. Untreated, the infection can progress and disseminate, with inflammatory complications commonly affecting distant skin sites, joints, heart, and nervous system [2]. LB differs in clinical features based upon its geographic distribution and in relation to its pathogenic potential and/or tissue tropism [3]. The skin represents a key interface in LB since it is the target of the spirochetes at the early stage of the disease, the EM and at later stages of the disease, the borrelial lymphocytoma and a typical manifestation of late european LB, the acrodermatitis chronica atrophicans (ACA) [4,5]. The skin constitutes a complex physical barrier [6]. The external multilayered part, the epidermis, mainly composed of keratinocytes (KCs) and Langerhans cells, is tightly connected to the dermis, in which fibroblasts are the main resident cells [7]. Dermal fibroblasts not only play an active role in synthesizing and remodeling the extracellular matrix (ECM), but also communicate with other cell types such as dermal dendritic cells, mast cells, macrophages and KCs. They also participate in tissue homeostasis, leukocyte recruitment and inflammation regulation [8]. Due to their broad and highly specialized roles in conditioning the cellular and cytokine/chemokine environment, resident sentinel fibroblasts function as part of the immune system [9]. To date, most studies of the cutaneous phase of LB have focused on the interaction of Borrelia with dendritic cells [10,11], mast cells [12], and KCs [13,14]. A few studies have investigated fibroblast responses to this disease. A recent study indicated that the interaction of B. burgdorferi ss with dermal fibroblasts induced the proinflammatory chemokine IL-8, along with the antimicrobial peptides defensin and cathelicidin [15]. Borrelia has also been shown to internalize and survive within fibroblasts [16]. Although KCs are the first cells to be injured by the tick mouthparts, biting pieces penetrate deeply into the skin [17]. Spirochetes are inoculated into the dermis, interacting with additional immune cells (dermal dendritic cells, mast cells) and the fibroblasts. We found it therefore particularly relevant to assess how Borrelia infection impacts dermal fibroblasts. In this study we investigated the role of dermal fibroblasts in skin inflammation in response to Borrelia. Since the inflammation could be related to the specific environments from which the strains were isolated, we tested one strain isolated from a tick and two strains isolated from different stages of the disease, potentially providing a link between spirochetal-related factors and LB outcome. Toward this end, we used specific skin cDNA microarrays to compare the global transcriptional response elicited in human dermal fibroblasts by three different strains of B. burgdorferi ss, isolated from an infected tick (N40) and from patients affected by EM (Pbre) or ACA (1408). Then, we investigated more precisely whether one of the major lipoproteins of Borrelia, OspC, which is necessary for the transmission of Borrelia to the vertebrate host [18,19], could be responsible for the induction of inflammatory molecules secreted by fibroblasts. Finally, we tested the effect of tick salivary gland extracts (SGE) on Borrelia-induced fibroblast response. Fibroblasts Stimulated by B. burgdorferi ss N40, Pbre and 1408 Strains Secrete Inflammatory Genes B. burgdorferi ss N40 has been shown to induce a proinflammatory response when coincubated with human primary fibroblasts. In this response, IL-8 was induced in a dose-dependent manner [15]. To check whether B. burgdorferi ss N40, Pbre and 1408 behave similarly when co-incubated in vitro with fibroblasts, we measured IL-8 synthesis. The chemokine was secreted in a dose- and timedependent manner, with peak secretion a (...truncated)