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Id Subject Object Predicate Lexical cue
T31 0-7 Sentence denotes Results
T2217 9-36 Sentence denotes Characterization of Sharpin
T32 9-36 Sentence denotes Characterization of Sharpin
T2218 38-128 Sentence denotes Orthologs of SHARPIN protein are found in various species, including human, mouse and rat.
T33 38-128 Sentence denotes Orthologs of SHARPIN protein are found in various species, including human, mouse and rat.
T2219 129-340 Sentence denotes Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain.
T34 129-340 Sentence denotes Motif prediction programming, using COILS [11] and MotifScan [12], suggests that SHARPIN contains a coiled-coil (CC) domain, a ubiquitin-like (UBL) domain, and a zinc-finger Ran-binding protein 2 (ZFRBP) domain.
T2220 341-545 Sentence denotes These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species.
T35 341-545 Sentence denotes These functional motifs constitute similar domain profiles that are present in the SHARPIN protein of all three origins (Fig. 1A), suggesting that SHARPIN exerts highly conserved functions across species.
T2221 546-782 Sentence denotes Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3].
T36 546-782 Sentence denotes Spontaneous mutations in the mouse Sharpin gene results in a complex inflammatory phenotype characterized by severe dermatitis (Fig. 1B), systemic inflammation and an enlarged spleen (Fig. 1C) caused by extramedullary hematopoiesis [3].
T2222 783-951 Sentence denotes The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS.
T37 783-951 Sentence denotes The endogenous expression of Sharpin mRNA in BMDC was determined by quantitative real time-PCR (qRT-PCR) following culture in medium only or after stimulation with LPS.
T2223 952-1074 Sentence denotes Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation.
T38 952-1074 Sentence denotes Sharpin mRNA was present in BMDC generated from WT mice (Fig. 1D) and its level was modestly decreased by LPS stimulation.
T2224 1075-1169 Sentence denotes There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice.
T39 1075-1169 Sentence denotes There was a significant reduction of Sharpin mRNA (6–7-fold) in BMDC generated from cpdm mice.
T2225 1170-1325 Sentence denotes Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).
T40 1170-1325 Sentence denotes Transfection of Flag-tagged Sharpin in fibroblasts (NIH3T3) and macrophages (RAW264.7) indicated cytoplasmic localization of the SHARPIN protein (Fig. 1E).
T41 1326-1411 Sentence denotes 10.1371/journal.pone.0031809.g001 Figure 1 In vivo and in vitro features of SHARPIN.
T12763 1370-1411 Sentence denotes In vivo and in vitro features of SHARPIN.
T12764 1412-1725 Sentence denotes (A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice.
T42 1412-1725 Sentence denotes (A) COILS and MotifScan programs were used to predict the presence of CC (coiled-coil) domain, UBL (ubiquitin-like) domain and ZFRBP (zinc-finger Ran-Binding protein 2) domain which form similar motif patterns in the SHARPIN protein of human, mouse and rat origins. (B) Eight week-old females of WT and cpdm mice.
T12765 1726-1985 Sentence denotes The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours.
T43 1726-1985 Sentence denotes The mutant mice (above) develop progressive skin inflammation starting at about four weeks. (C) Extramedullary hematopoiesis causes marked enlargement of the spleen of cpdm mice. (D) BMDC were incubated in the absence or presence of 100 ng/ml LPS for 4 hours.
T12766 1986-2108 Sentence denotes The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC.
T44 1986-2108 Sentence denotes The mRNA level of Sharpin was measured by qRT-PCR and presented relative to the mRNA expression in non-stimulated WT BMDC.
T12767 2109-2289 Sentence denotes Bars represent the mean ± s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN.
T45 2109-2289 Sentence denotes Bars represent the mean ± s.d. of 3 mice. * P<0.05; ** P<0.001. (E) Fibroblast (NIH3T3) and macrophages (RAW264.7) cells were transfected with the expression plasmid pFLAG-SHARPIN.
T12768 2290-2388 Sentence denotes After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody.
T46 2290-2388 Sentence denotes After 48 hours, cells were fixed and probed with anti-FLAG and FITC-conjugated secondary antibody.
T12769 2389-2419 Sentence denotes Nuclei were stained with DAPI.
T47 2389-2419 Sentence denotes Nuclei were stained with DAPI.
T12770 2420-2501 Sentence denotes In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.
T48 2420-2501 Sentence denotes In both transfected cell lines, FLAG-SHARPIN was found to be cytoplasm-localized.
T2872 2503-2556 Sentence denotes Phenotyping splenic DC and BMDC from WT and cpdm mice
T49 2503-2556 Sentence denotes Phenotyping splenic DC and BMDC from WT and cpdm mice
T2873 2557-2654 Sentence denotes DC are heterogeneous and can be categorized into multiple subtypes based on surface markers [13].
T50 2557-2654 Sentence denotes DC are heterogeneous and can be categorized into multiple subtypes based on surface markers [13].
T2874 2655-2890 Sentence denotes To determine if the Sharpin mutation affects DC development in lymphoid tissues, mouse spleens were examined for the distribution of conventional DC (cDC; CD11c+CD8α+ and CD11c+CD8α−) [13] and plasmacytoid DC (pDC; CD11c−PDCA-1+) [14].
T51 2655-2890 Sentence denotes To determine if the Sharpin mutation affects DC development in lymphoid tissues, mouse spleens were examined for the distribution of conventional DC (cDC; CD11c+CD8α+ and CD11c+CD8α−) [13] and plasmacytoid DC (pDC; CD11c−PDCA-1+) [14].
T2875 2891-3003 Sentence denotes The percentages for splenic cDC and pDC were both reduced in cpdm mice when compared with WT controls (Fig. 2A).
T52 2891-3003 Sentence denotes The percentages for splenic cDC and pDC were both reduced in cpdm mice when compared with WT controls (Fig. 2A).
T2876 3004-3130 Sentence denotes However, when gated on CD11c+ cDC, the percentages of CD8α+ and CD8α− cells were not affected by SHARPIN deficiency (Fig. 2A).
T53 3004-3130 Sentence denotes However, when gated on CD11c+ cDC, the percentages of CD8α+ and CD8α− cells were not affected by SHARPIN deficiency (Fig. 2A).
T2877 3131-3406 Sentence denotes Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers.
T54 3131-3406 Sentence denotes Since the spleen of cpdm mice is markedly enlarged and contains three times as many cells (Fig. 1C), the different percentages of splenic cDC and pDC between WT and mutant mice reflect the increased number of total spleen cells rather than a reduction in cDC and pDC numbers.
T2878 3407-3527 Sentence denotes These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.
T55 3407-3527 Sentence denotes These data indicate that the Sharpin mutation does not affect the distribution of the examined DC subsets in the spleen.
T56 3528-3635 Sentence denotes 10.1371/journal.pone.0031809.g002 Figure 2 Effect of Sharpin mutation on DC subpopulations and maturation.
T13265 3572-3635 Sentence denotes Effect of Sharpin mutation on DC subpopulations and maturation.
T13266 3636-3731 Sentence denotes (A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion.
T57 3636-3731 Sentence denotes (A) Spleens from WT and cpdm mice were isolated and subject to collagenase and DNase digestion.
T13267 3732-3843 Sentence denotes The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes.
T58 3732-3843 Sentence denotes The obtained splenic homogenates were centrifuged over a Percoll gradient (35% and 55% density) for 15 minutes.
T13268 3844-4191 Sentence denotes The bands at the 35%-medium and the 35–55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8α+, CD11c+CD8α− and plasmacytoid DC (CD11c−PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1− and CD11c−PDCA-1+ cells.
T59 3844-4191 Sentence denotes The bands at the 35%-medium and the 35–55% interface were pooled, washed and stained with a combination of various antibodies to stain different DC subsets, conventional CD11c+CD8α+, CD11c+CD8α− and plasmacytoid DC (CD11c−PDCA-1+).The top panels were gated on FSChiSSClo cells to show separate populations of CD11c+PDCA-1− and CD11c−PDCA-1+ cells.
T13269 4192-4332 Sentence denotes Further gating on the CD11c+PDCA-1− subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8α+ and CD11c+CD8α− cells.
T60 4192-4332 Sentence denotes Further gating on the CD11c+PDCA-1− subpopulation gave the bottom panel that showed two distinct pools of CD11c+CD8α+ and CD11c+CD8α− cells.
T13270 4333-4561 Sentence denotes Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5×105) cells were stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T61 4333-4561 Sentence denotes Percentages were calculated based on the parental population and were additionally shown as bar graphs (n = 2) (B). (C) WT and cpdm BMDC (5×105) cells were stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T13271 4562-4679 Sentence denotes The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis.
T62 4562-4679 Sentence denotes The cells were labeled with PE-labeled anti-CD40, anti-CD80, and anti-CD86, and subjected to flow cytometry analysis.
T13272 4680-4743 Sentence denotes The populations shown in histograms were gated on CD11c+ cells.
T63 4680-4743 Sentence denotes The populations shown in histograms were gated on CD11c+ cells.
T13273 4744-4788 Sentence denotes Unstained cells served as negative controls.
T64 4744-4788 Sentence denotes Unstained cells served as negative controls.
T13274 4789-4847 Sentence denotes Results are representative of two independent experiments.
T65 4789-4847 Sentence denotes Results are representative of two independent experiments.
T2879 4850-4971 Sentence denotes BMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16].
T66 4850-4971 Sentence denotes BMDC from in vitro cultures functionally resemble non-lymphoid tissue DC and monocyte-derived inflammatory DC [15], [16].
T2880 4972-5026 Sentence denotes The yields of BMDC from WT and cpdm mice were similar.
T67 4972-5026 Sentence denotes The yields of BMDC from WT and cpdm mice were similar.
T2881 5027-5125 Sentence denotes BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86.
T68 5027-5125 Sentence denotes BMDC were CD11c+ and MHC II+ with low expression of co-stimulatory molecules CD40, CD80, and CD86.
T2882 5126-5283 Sentence denotes The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18].
T69 5126-5283 Sentence denotes The TLR3 ligand poly I:C and the TLR4 ligand LPS each activate overlapping but different signaling pathways and were used to induce DC maturation [17], [18].
T2883 5284-5506 Sentence denotes Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B).
T70 5284-5506 Sentence denotes Incubation with the TLR agonists for 24 hours resulted in increased expression of CD40, CD80, and CD86 on BMDC; however, there was no difference in the expression levels of these markers between WT and cpdm BMDC (Fig. 2B).
T2884 5507-5601 Sentence denotes Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.
T71 5507-5601 Sentence denotes Thus, SHARPIN deficiency did not influence the expression of co-stimulatory molecules by BMDC.
T3823 5603-5667 Sentence denotes Production of proinflammatory mediators by cpdm BMDC is impaired
T72 5603-5667 Sentence denotes Production of proinflammatory mediators by cpdm BMDC is impaired
T3824 5668-5896 Sentence denotes Incubation with LPS or poly I:C resulted in secretion of IL6, IL12P70, and GMCSF from both WT and cpdm BMDC; however, BMDC from cpdm mice produced significantly less of all three cytokines compared with WT BMDC (Fig. 3A–C, E–F).
T73 5668-5896 Sentence denotes Incubation with LPS or poly I:C resulted in secretion of IL6, IL12P70, and GMCSF from both WT and cpdm BMDC; however, BMDC from cpdm mice produced significantly less of all three cytokines compared with WT BMDC (Fig. 3A–C, E–F).
T3825 5897-6112 Sentence denotes The amount of nitric oxide generated by mutant BMDC was also significantly reduced compared with WT cells (Fig. 3D), indicating severely disrupted production of proinflammatory mediators from Sharpin-deficient BMDC.
T74 5897-6112 Sentence denotes The amount of nitric oxide generated by mutant BMDC was also significantly reduced compared with WT cells (Fig. 3D), indicating severely disrupted production of proinflammatory mediators from Sharpin-deficient BMDC.
T3826 6113-6403 Sentence denotes In vivo complementation with a Sharpin gene-containing BAC reversed the phenotype of the mutant mice [2], and BMDC generated from these rescued mice secreted significantly more IL12P70 than BMDC from cpdm mice (Fig. 3G), supporting a necessary role of SHARPIN for the production of IL12P70.
T75 6113-6403 Sentence denotes In vivo complementation with a Sharpin gene-containing BAC reversed the phenotype of the mutant mice [2], and BMDC generated from these rescued mice secreted significantly more IL12P70 than BMDC from cpdm mice (Fig. 3G), supporting a necessary role of SHARPIN for the production of IL12P70.
T3827 6404-6619 Sentence denotes In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).
T76 6404-6619 Sentence denotes In addition, the transcript levels of the inflammatory cytokines Il6, Il12p40, Gmcsf, and Ifnb were examined, and these were all significantly reduced in stimulated cpdm BMDC when compared with WT controls (Fig. 4).
T77 6620-6741 Sentence denotes 10.1371/journal.pone.0031809.g003 Figure 3 Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.
T13806 6664-6741 Sentence denotes Defective production of pro-inflammatory mediators from stimulated cpdm BMDC.
T13807 6742-6902 Sentence denotes (A–D) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T78 6742-6902 Sentence denotes (A–D) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T13808 6903-7142 Sentence denotes Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E–F) Gradient numbers (1×104, 2×104, and 4×104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation.
T79 6903-7142 Sentence denotes Supernatants were collected for ELISA of IL12P70, IL6, and GMCSF, and for quantification of nitric oxide (NO). (E–F) Gradient numbers (1×104, 2×104, and 4×104 cells in 0.1 mL complete medium) of BMDC were used for100 ng/mL LPS stimulation.
T13809 7143-7335 Sentence denotes After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2].
T80 7143-7335 Sentence denotes After 24 hours, the amounts of IL12P70 and IL6 from the supernatant were measured. (G) The cpdm mice rescued by Sharpin-containing BAC had complete remission of the inflammatory phenotype [2].
T13810 7336-7453 Sentence denotes BMDC developed from cpdm and rescued cpdm mice were plated (1×106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS.
T81 7336-7453 Sentence denotes BMDC developed from cpdm and rescued cpdm mice were plated (1×106 cells in 0.3 mL) and stimulated with 100 ng/mL LPS.
T13811 7454-7533 Sentence denotes After 24 hours, supernatants were collected for analysis of IL12P70 production.
T82 7454-7533 Sentence denotes After 24 hours, supernatants were collected for analysis of IL12P70 production.
T13812 7534-7613 Sentence denotes Data are representative of three independent experiments. * P<0.01; ** P<0.005.
T83 7534-7613 Sentence denotes Data are representative of three independent experiments. * P<0.01; ** P<0.005.
T84 7614-7721 Sentence denotes 10.1371/journal.pone.0031809.g004 Figure 4 Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.
T14256 7658-7721 Sentence denotes Decreased mRNA levels of inflammatory cytokines from cpdm BMDC.
T14257 7722-7875 Sentence denotes Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 µg/ml poly I:C (B,D,F,H).
T85 7722-7875 Sentence denotes Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS (A,C,E,G) or 25 µg/ml poly I:C (B,D,F,H).
T14258 7876-8032 Sentence denotes At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA.
T86 7876-8032 Sentence denotes At 0, 1 and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the expression of Il12p40 (A,B), Il6 (C,D), Gmcsf (E,F), and Ifnb (G,H) mRNA.
T14259 8033-8058 Sentence denotes Bars represent mean ± SD.
T87 8033-8058 Sentence denotes Bars represent mean ± SD.
T14260 8059-8114 Sentence denotes Data are representative of two independent experiments.
T88 8059-8114 Sentence denotes Data are representative of two independent experiments.
T4475 8116-8213 Sentence denotes Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-κB signaling
T89 8116-8213 Sentence denotes Impaired cytokine production by cpdm BMDC is correlated with selective defects in NF-κB signaling
T4476 8214-8558 Sentence denotes There are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.
T90 8214-8558 Sentence denotes There are a number of possible explanations for the defective cytokine secretion in stimulated cpdm BMDC, including 1) reduced surface expression of the LPS receptor complex, 2) increased production of anti-inflammatory mediators, 3) increased expression of negative regulators of TLR pathways, and 4) impaired TLR-induced signaling activation.
T4477 8559-8726 Sentence denotes We determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19].
T91 8559-8726 Sentence denotes We determined the surface expression of the LPS receptor complex that comprises TLR4, the accessory proteins CD14 and myeloid differentiation factor 2 (MD2/LY96) [19].
T4478 8727-8854 Sentence denotes Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A).
T92 8727-8854 Sentence denotes Flow cytometric analysis shows that the expression levels of CD14 and TLR4/MD2 between WT and cpdm BMDC were similar (Fig. 5A).
T4479 8855-8988 Sentence denotes We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21].
T93 8855-8988 Sentence denotes We then quantified the secretion of the suppressive cytokines IL10 that can inhibit IL12 secretion in an autocrine manner [20], [21].
T4480 8989-9205 Sentence denotes The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC.
T94 8989-9205 Sentence denotes The supernatants from LPS-stimulated cpdm BMDC contained significantly lower levels of IL10 than stimulated WT BMDC (Fig. 5B), suggesting that IL10 was not responsible for decreased secretion of IL12P70 by cpdm BMDC.
T4481 9206-9322 Sentence denotes Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion.
T95 9206-9322 Sentence denotes Increased expression of a negative regulator of TLR signaling such as A20 [22] may also suppress cytokine secretion.
T4482 9323-9513 Sentence denotes However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.
T96 9323-9513 Sentence denotes However, the transcript level of A20 was lower in LPS-activated cpdm BMDC than WT controls (Fig. 5C), thereby ruling out overexpression of A20 as a factor in the reduced cytokine production.
T97 9514-9650 Sentence denotes 10.1371/journal.pone.0031809.g005 Figure 5 Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.
T14580 9558-9650 Sentence denotes Normal TLR4 and MD2 expression and decreased IL10 secretion and A20 expression by cpdm BMDC.
T14581 9651-9793 Sentence denotes (A) Unstimulated WT and cpdm BMDC (5×105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis.
T98 9651-9793 Sentence denotes (A) Unstimulated WT and cpdm BMDC (5×105) were labeled with PE-labeled anti-TLR4/MD2 or anti-CD14 and then subject to flow cytometry analysis.
T14582 9794-9996 Sentence denotes Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T99 9794-9996 Sentence denotes Unstained cells serve as negative controls. (B) Cultured WT and cpdm BMDC (1×105 cells in 0.1 ml complete medium) were washed and stimulated with medium, 100 ng/ml LPS or 25 µg/ml poly I:C for 24 hours.
T100 9997-10181 Sentence denotes Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 µg/ml poly I:C.
T14583 9997-10284 Sentence denotes Supernatants were collected for ELISA of IL10. (C) Cultured WT and cpdm BMDC (5×105 cells in 0.2 ml complete medium) were washed and stimulated with 100 ng/ml LPS or 25 µg/ml poly I:C. At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.
T101 10182-10284 Sentence denotes At 0, 1, and 2 hours, total RNA was extracted and subject to qRT-PCR to measure the production of A20.
T4483 10288-10458 Sentence denotes The transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-κB, TBK1/IRF3, and MAPK [23]–[27].
T102 10288-10458 Sentence denotes The transcription of TLR3/4-induced proinflammatory intermediates is tightly regulated by cellular signaling pathways, in particular NF-κB, TBK1/IRF3, and MAPK [23]–[27].
T4484 10459-10616 Sentence denotes We next determined if disrupted NF-κB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC.
T103 10459-10616 Sentence denotes We next determined if disrupted NF-κB, TBK1/IRF3, and/or MAPK signaling may underlie the impaired cytokine production from stimulated Sharpin-deficient BMDC.
T4485 10617-10867 Sentence denotes Stimulus-induced phosphorylation of the IκB kinase (IKK1/2) is an essential step in NF-κB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-κB inhibitor IκBα to release the NF-κB transcription factors into the nucleus.
T104 10617-10867 Sentence denotes Stimulus-induced phosphorylation of the IκB kinase (IKK1/2) is an essential step in NF-κB signaling, allowing phosphorylation and proteasome-mediated degradation of the NF-κB inhibitor IκBα to release the NF-κB transcription factors into the nucleus.
T4486 10868-11049 Sentence denotes The amount of phosphorylated IKK1/2 (p-IKK1/2) and IκBα (p-IκBα) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6).
T105 10868-11049 Sentence denotes The amount of phosphorylated IKK1/2 (p-IKK1/2) and IκBα (p-IκBα) following incubation with LPS or poly I:C was severely decreased in cpdm BMDC as compared with WT controls (Fig. 6).
T4487 11050-11160 Sentence denotes The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6).
T106 11050-11160 Sentence denotes The cpdm BMDC exhibited similar levels of TBK1, ERK1/2, and p38 phosphorylation to those of WT cells (Fig. 6).
T4488 11161-11314 Sentence denotes These results indicate that the absence of functional SHARPIN decreased NF-κB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.
T107 11161-11314 Sentence denotes These results indicate that the absence of functional SHARPIN decreased NF-κB activation but did not affect TBK1/IRF3, ERK1/2, and p38 signaling in BMDC.
T108 11315-11402 Sentence denotes 10.1371/journal.pone.0031809.g006 Figure 6 Inhibition of NF-κB signaling in cpdm BMDC.
T14993 11359-11402 Sentence denotes Inhibition of NF-κB signaling in cpdm BMDC.
T14994 11403-11524 Sentence denotes WT and cpdm BMDC (2×106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B).
T109 11403-11524 Sentence denotes WT and cpdm BMDC (2×106 cells in 0.5 mL complete medium) were stimulated with 100 ng/mL LPS (A) or 25 µg/mL poly I:C (B).
T14995 11525-11713 Sentence denotes At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways.
T110 11525-11713 Sentence denotes At 0, 15, 30, and 60 minutes, whole-cell lysates were obtained and subject to immunoblots with antibodies against proteins involved in NF-κB, TBK1/IRF3, ERK1/2, and p38 signaling pathways.
T14996 11714-11897 Sentence denotes Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines.
T111 11714-11897 Sentence denotes Beta-actin was used as loading control. (C) Cellular levels of p-IKK1/2 and p-IκBα in LPS- or poly I:C-stimulated BMDC were quantitated with ImageJ (NIH) and presented as trend lines.
T14997 11898-11965 Sentence denotes Results are representative of at least two independent experiments.
T112 11898-11965 Sentence denotes Results are representative of at least two independent experiments.
T5820 11967-12016 Sentence denotes Th2-biased immunogenicity of stimulated cpdm BMDC
T113 11967-12016 Sentence denotes Th2-biased immunogenicity of stimulated cpdm BMDC
T5821 12017-12230 Sentence denotes The defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells.
T114 12017-12230 Sentence denotes The defective IL12 production (Fig. 3A) and Th2-dominant cytokine profile in cpdm mice [5] suggest that the absence of SHARPIN affects the ability of cpdm BMDC to induce T cell differentiation into effector cells.
T5822 12231-12531 Sentence denotes Co-culture of allogeneic naïve CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNγ production, whereas the concentration of IFNγ in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC.
T115 12231-12531 Sentence denotes Co-culture of allogeneic naïve CD4+ T cells with WT BMDC stimulated with LPS or poly I:C elicited robust IFNγ production, whereas the concentration of IFNγ in cpdm BMDC-T cell cultures was significantly lower after LPS stimulation (Fig. 7A), indicating impaired Th1-polarizing abilities of cpdm BMDC.
T5823 12532-12668 Sentence denotes In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]–[30].
T116 12532-12668 Sentence denotes In addition to TLR3/4 agonists, the TLR2 ligand Pam3CYS was used since it has been shown to induce both Th1 and Th2 responses [28]–[30].
T5824 12669-12781 Sentence denotes Pam3CYS-matured WT BMDC induced robust IFNγ production at a significantly higher level than cpdm BMDC (Fig. 7A).
T117 12669-12781 Sentence denotes Pam3CYS-matured WT BMDC induced robust IFNγ production at a significantly higher level than cpdm BMDC (Fig. 7A).
T5825 12782-12962 Sentence denotes The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31].
T118 12782-12962 Sentence denotes The reduced Th1 differentiation following Pam3CYS stimulation is consistent with the recent report of decreased IL12 production in cpdm macrophages following TLR2 stimulation [31].
T5826 12963-13130 Sentence denotes In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC.
T119 12963-13130 Sentence denotes In contrast, more Th2-specific IL4 cytokine was produced in the cpdm BMDC co-cultures than the WT control (Fig. 7B), suggesting Th2-skewed immunogenicity of cpdm BMDC.
T5827 13131-13299 Sentence denotes The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown).
T120 13131-13299 Sentence denotes The production of Th17-specific cytokine IL17A following LPS stimulation of dendritic cells was similar between stimulated WT and cpdm BMDC cocultures (data not shown).
T5828 13300-13540 Sentence denotes Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C).
T121 13300-13540 Sentence denotes Despite the distinct Th1- and Th2-stimulating abilities of WT and cpdm BMDC, they were equally effective in IL2 production from BMDC-T cell co-cultures except for poly I:C stimulation where WT BMDC induced more IL2 than cpdm BMDC (Fig. 7C).
T5829 13541-13721 Sentence denotes The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naïve CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.
T122 13541-13721 Sentence denotes The Th2-biased stimulating capability of cpdm BMDC when co-cultured with allogeneic naïve CD4+ T cells is consistent with the Th2-dominant cytokine phenotype observed in cpdm mice.
T123 13722-13850 Sentence denotes 10.1371/journal.pone.0031809.g007 Figure 7 Stimulated cpdm BMDC induced Th2-biased cytokine production from naïve CD4+ T cells.
T15334 13766-13850 Sentence denotes Stimulated cpdm BMDC induced Th2-biased cytokine production from naïve CD4+ T cells.
T15335 13851-14009 Sentence denotes WT and cpdm BMDC (5×104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 µg/mL poly I:C or 5 µg/mL Pam3CYS.
T124 13851-14009 Sentence denotes WT and cpdm BMDC (5×104 cells in 0.1 mL complete medium; MHC haplotype: H-2b) were incubated with medium, 100 ng/mL LPS, 25 µg/mL poly I:C or 5 µg/mL Pam3CYS.
T15336 14010-14182 Sentence denotes After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naïve CD4+ T cells (2.5×105 cells in 0.1 mL complete medium; MHC haplotype: H-2d).
T125 14010-14182 Sentence denotes After 24 hours, cells were washed with PBS and incubated with freshly isolated allogeneic naïve CD4+ T cells (2.5×105 cells in 0.1 mL complete medium; MHC haplotype: H-2d).
T15337 14183-14288 Sentence denotes After 5 days, supernatants were collected and the secretion of IFNγ, IL4, and IL2 were measured by ELISA.
T126 14183-14288 Sentence denotes After 5 days, supernatants were collected and the secretion of IFNγ, IL4, and IL2 were measured by ELISA.
T15338 14289-14446 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T127 14289-14446 Sentence denotes Negative controls are 1) stimulated BMDC without co-culture with allogeneic CD4+ T cells; 2) allogeneic CD4+ T cells without co-culture with stimulated BMDC.
T15339 14447-14556 Sentence denotes Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown).
T128 14447-14556 Sentence denotes Samples from both negative controls had no detectable production of the aforementioned cytokines (not shown).
T15340 14557-14616 Sentence denotes Results are analyzed based on 2–4 mice per group. * P<0.05.
T129 14557-14616 Sentence denotes Results are analyzed based on 2–4 mice per group. * P<0.05.