Organisms were quantitated by fluorescence microscopy after mixing 10 l aliquots of culture material with 10 l of an acridine orange solution to concentrations

Organisms were quantitated by fluorescence microscopy after mixing 10 l aliquots of culture material with 10 l of an acridine orange solution to concentrations. cause different clinical symptoms in Europe. sensu stricto is the main cause of Lyme arthritis, most often induces neurologic manifestations, while is mainly responsible for skin disorders [4;5]. Cytokines play an important role in the pathogenesis of Lyme disease by regulating the immune responses against [6]. It has been reported that is able to induce a pro- inflammatory cytokine response, characterized especially by production of IL-1 [7]. In patients diagnosed with a typical skin disorder near the location of the tick bite, called an erythema migrans (EM), high amounts of both IL-1 and IFN- were found [8]. Furthermore, the recently described IL-17-producing T-cells, called Th17 cells, are capable of producing high amounts of IL-17 after exposure to infection, severe destructive arthritis could be induced in IFN- knock-out mice after challenge with spirochetes. When mice were given antibodies against IL-17, the development of AZ876 Lyme arthritis was strongly reduced, with the diminished severity of joint swelling [10]. Caspase-1 AZ876 is an enzyme involved in processing of the cytokines IL-1, IL-18, and is activated by a protein platform called the inflammasome [11;12]. Host defense against several pathogens have been linked to the proper activation of the inflammasome, including [13][14], [15], and [16]. Interestingly, IL-1 Rabbit polyclonal to AHSA1 has been implicated in Th17 development [17C20], while IL-18 that was first called IGIF (IFN- inducing factor) is associated with the induction of Th1 cells [21]. In the present study we investigated the role of caspase-1 in the host defense against Caspase-1 deficient cells were unable to induce a Th1 or Th17 response upon challenge with Importantly, IL-1 was responsible for the induction of the IL-17 pathway induced by while IL-18 was crucial for the induction of IFN-. In contrast, IL-18 has an inhibitory effect on IL-17 production, providing further evidence for counter-regulatory regulation between Th1 and Th17 responses. Results induces inflammasome activation and bioactive IL-1 It has been previously reported that caspase-1 is activated by several different microorganisms [14C16]. Here we demonstrate for the first time that caspase-1 is also activated by in bone marrow derived macrophages (BMDM) from wild-type C57BL/6 mice. After stimulation for 4 hours with 1106 /ml heat-killed spirochetes, with the last 30 minutes in the presence of ATP, cleaved caspase-1 was clearly induced (Figure 1A). As a control for caspase-1 activation, BMDMs were stimulated with LPS plus ATP, which also resulted in cleaved caspase-1 (Figure 1A). Since we found strong caspase-1 activation, we next examined whether IL-1 production by murine macrophages could be induced by Borrelia burgdorferi. Peritoneal macrophages from wild-type mice stimulated for 24 hours with 1106/ml heat-killed spirochetes. exposure induced IL-1 production in peritoneal macrophages (Figure 1B). In addition, IL-6 was strongly produced in peritoneal macrophages (Figure 1B). Open in a separate window Figure 1 induces caspase-1-dependent IL-1 production(A) 1 106 Bone marrow derived macrophages per ml from 5 WT C57/BL6 mice were incubated for 24 hours with or without 1 105 spirochetes/ml heat-killed or LPS (10 g/ml) with or without ATP (3 mM) for 30 AZ876 minutes. Cleaved caspase-1 was detected by Western blot using antibodies to detect the inactive caspase-1 (p45) or cleaved or active caspase-1 (p20). (B, C) Peritoneal macrophages (1 105/well) from 5 WT C57BL/6 mice and 5 caspase-1 knock-out mice were incubated for 24 hours with (B) 3 106 spirochetes/ml heat-killed or (C) 1106/ml or or medium alone. Supernatant cytokine.