[4] It has been demonstrated that allergens in the presence of

endotoxins trigger a substantially stronger allergic inflammation, compared with that evoked in the absence of endotoxins.[5-7] After inhalation, endotoxins, such as lipopolysaccharide (LPS), encounter and activate alveolar macrophages, leading to the production and release of pro-inflammatory cytokines, chemokines, adhesion molecules and other mediators.[8] Nasal and lung lavage samples of allergic subjects show increased levels of interleukin-1β (IL-1β),[9] primarily produced by activated macrophages.[10] Production IWR-1 purchase of mature IL-1β requires distinct signals, some of which induce gene expression in the so called ‘priming step’, whereas other signals trigger the maturation of pro-IL-1β to IL-1β by a multiprotein complex called inflammasome. The NLRP3 inflammasome complex consists of NLRP3 (NOD-like receptor family pyrin domain-containing 3) sensor, caspase-1 and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) adaptor.[11, 12] NLRP3 inflammasomes play a crucial role in the detection and sensing of exogenous danger signals like pathogen-associated molecular patterns and toxins of microbes, asbestos or silica, as well as endogenous danger signals like monosodium urate and amyloid.[13, 14] Most NLRP3 activators have been shown to induce ROS Cabozantinib in vivo generation,[15]

and enough inhibitors of ROS production or ROS scavengers attenuate NLRP3 inflammasome activation[16] implying an essential role for ROS in NLRP3 function. As pollen NADPH oxidases are able to generate ROS, and ROS have been implicated in the NLRP3 inflammasome-mediated IL-1β production, we hypothesized that exposure to pollen extract may influence inflammatory responses and IL-1β production of macrophages via NLRP3 inflammasome. Here we report for the first time that ragweed

pollen extract (RWE), typically used as a model for pollen action,[3] significantly elevates LPS-induced IL-1β production of THP-1 or primary macrophages and dendritic cells in an NADPH-dependent manner. We also demonstrate that a caspase-1 inhibitor or NLRP3 silencing abolish this enhancing effect together with the original LPS-triggered inductions. We also show that RWE in the presence of NADPH enhances LPS-induced p38 and Jun N-terminal kinase (JNK) signalling pathways resulting in the activation of AP-1 transcription factors and the subsequent gene transcription/expression of pro-IL-1β and key components of the inflammasome. This effect is mediated by a ROS-dependent mechanism. The THP-1 cell line (ATCC TIB-202) was a generous gift from Professor Laszlo Nagy. THP-1 monocytes were cultured in RPMI-1640 (Gibco BRL Inc., Grand Island, NY) containing 10% heat-inactivated fetal calf serum, penicillin-streptomycin and glutamine, and maintained at 37° under 5% CO2.