Furthermore, the caspase-1 activation in 18-month old thymi was significantly attenuated in Nlrp3 deficient mice (Figure 2B and Supplementary Figure 3A). demonstrate that decreasing inflammasome-dependent caspase-1 activation raises thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the reestablishment of a varied T cell repertoire in middle-aged or elderly individuals undergoing HSCT. and demonstrated as relative manifestation (Ct). Data are offered as means SD mean, 0.01, (D) The free cholesterol levels in the thymi derived from 1month BMS-345541 HCl and 12 month old (n = 6/group) C57/B6 mice were measured using gas liquid chromatography. (E) The thymic cryosections from 24month aged C57/B6 mice (n = 3) were imunostained with CD11b and filipin that binds to free cholesterol. The nuclei were counterstained with propidium iodide (reddish). The confocal immofluorescence analysis shows co-localization of macrophages with free cholesterol in medullary part of thymus. (F) The LPS primed BMDMs were loaded with methyl -cyclodextrin-cholesterol (cholesterol concentration: 80mg/ml), which raises free cholesterol levels in BMDM to 60g/mg protein (Supplementary Number 1E). The cells were exposed to LPS for 6h and FC for 1h. The immunoblot analysis shows triggered p20 form of caspase-1 in FC loaded LPS primed cells. (G) The western blot analysis of p20 subunit of caspase-1 in BMDMs from WT and 0.05. We next investigated the age-related DAMPs that cause caspase-1 activation. Interestingly, in contrast to young animals (one month aged), middle-aged mice (12 month aged) had a significant increase in thymic concentration of potentially cytotoxic free cholesterol (FC) (Number 1D). Immunostaining of thymic cryosections exposed that the majority of FC staining in the ageing thymus was localized to CD11b+ thymic macrophages in the medullary region (Number 1E and Supplementary Number 1D). Remarkably, the thymic cholesterol ester content material was reduced to undetectable levels with age (Supplementary Number 1C). Interestingly, in the presence of elevated intracellular FC, LPS-primed bone marrow derived macrophages (BMDMs) displayed autocatalytic control of caspase-1 (Number 1F). The FC induced caspase-1 cleavage and IL-1 secretion in BMDMs was abolished in the absence of Nlrp3 (Number 1G, H). Of notice, this was not due to variations in FC loading or cholesterol ester content in the BMDMs of WT and causes caspase-1 activation via the Nlrp3 inflammasome dependent mechanism. Nlrp3 inflammasome activation BMS-345541 HCl promotes age-related thymic involution We next investigated whether the Nlrp3 inflammasome regulates the age-related increase in caspase-1 activation. Our data exposed that caspase-1 processing in thymi derived from 9-month aged mice is dependent on Nlrp3 inflammasome activation (Number 2A). Furthermore, the caspase-1 activation in 18-month aged thymi was significantly attenuated in Nlrp3 deficient mice (Number 2B and Supplementary Number 3A). Consistent with these data, compared to 18-month aged WT mice, IL-1 activation in thymi Mouse monoclonal to CSF1 from Nlrp3 deficient mice was also substantially reduced (Number 2C, Supplementary Number 3B). IL-1 levels in the plasma and sera of aged WT mice were below the detection limit of the assay (data not shown) suggesting that intra-thymic IL-1 and caspase-1 activation effects the thymic involution process. Open in a separate window Number 2 The ablation of Nlrp3 inflammasome protects against age-related thymic involutionThe western blot analysis of caspase-1 activation in thymus of WT and and Asc1 (UEA-1). Nuclei were counterstained with BMS-345541 HCl DAPI. In aged WT mice, mTEC staining was localized within the periphery of medullary areas while in axis of the scenery denotes change from WT specific CDR3 size or size ( 0.05 (n = 6C8/group). We next investigated whether the donor or sponsor derived cells contribute towards changes in thymic reconstitution in 2- and 9 month aged mice. There was no switch in overall quantity of thymocyte subsets derived from sponsor (CD45.2) or donor (CD45.1) source HSC in 2-month aged irradiated WT and with a normal chow diet consisting of 4.5% fat (5002; LabDiet) and aged in specific-pathogen free animal facility in ventilated cage racks that delivers HEPA.