- Email: [email protected]
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Abstract / Cytokine 70 (2014) 28–79 NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. Because of its involvement in many diseases such as atherosclerosis and Alzheimer’s Disease, a better understanding of NLRP3 regulation may help to shape therapeutic approaches. The aim of this study was to identify posttranslational modifications on NLRP3 and their impact on inflammasome activation. We applied mass spectrometry of NLRP3 to identify post-translational modifications. We identified ubiquitinated and phosphorylated residues. To analyze the function of the modifications, we mutated the residues in NLRP3 expression plasmids and used them for reconstitution of NLRP3-deficient macrophages or overexpression studies in HEK293T cells. Ubiquitinylated residues were found in the NACHT and LRR domains of NLRP3 and mutation of these residues resulted in hyperactive NLRP3. In addition, mutation of one of the phosphorylated residues to a phosphomimetic residue showed reduced IL-1beta release and caspase-1 cleavage upon NLRP3 inflammasome activation, whereas mutation to alanine had no effect. Further studies in HEK293T cells found that the recruitment of the downstream adaptor ASC as well as the self-interaction of NLRP3 were reduced with the phosphomimetic residue. Thus, NLRP3 is likely regulated by post-translational modification and needs to be deubiquitinylated and possibly dephosphorylated before activation. Knowledge about the modifications and the pathways involved may help shape novel strategies for treating diseases that involve NLRP3.
71
IAPs have emerged as attractive targets for anti-cancer therapeutics since it has been shown that overexpression of the IAP antagonist Smac sensitises tumour cells to TNF driven apoptosis as do the synthetic counterparts, Smac mimetics. We demonstrated that cell death induced by inhibition of IAPs through Smac mimetics is not strictly TNF dependent. The combined treatment of Interferon c (IFNc), a potent pro-inflammatory cytokine, and Smac mimetics shows potent synergistic killing of a wide range of tumour cell lines as well as primary and transformed cells. Further investigation showed that IFNc and Smac mimetic induced killing requires NF-jB as well as Jak/STAT signalling and displayed classic apoptotic hallmarkers. Whereas killing of several cell lines could be blocked by the inhibition of caspases, other cell lines like HT29, KATOIII, mouse dermal fibroblasts (MDFs) and keratinocytes remained sensitive. In order to elucidate the involvement of the necroptotic pathway in this apoptosis independent IFNc and Smac mimetic combined killing we performed knock down experiments and analysed primary and transformed knock out MDFs and keratinocytes lacking members of the necroptotic pathway like RIPK3 / and MLKL / . The results suggest that IFNc and Smac mimetic combined treatment can activate both apoptotic and necroptotic death pathways, involving members of the necroptotic pathway. This may provide new insights into yet unknown cell death mechanisms and extend the clinical use of Smac mimetics. http://dx.doi.org/10.1016/j.cyto.2014.07.186
http://dx.doi.org/10.1016/j.cyto.2014.07.184
178 Leucine-rich a2 glycoprotein promotes TGFb1-induced apoptosis in the lewis lung carcinoma cell lines 1,2
3
1
1
Norihiko Takemoto , Tomoshige Matsumoto , Satoshi Serada , Minoru Fujimoto , Tetsuji Naka 1, 1 Laboratory for Immune Signal, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan, 2 Osaka University, Suita, Osaka, Japan, 3 Department of Clinical Laboratory, Osaka Anti-Tuberculosis Association Osaka Hospital, Neyagawa, Osaka, Japan Aims: Leucine-rich a2 glycoprotein (LRG) is an approximately 50 kDa glycoprotein originally found in the blood. It has been reported that the expression levels of LRG were elevated in the sera of several cancers. While it has been demonstrated that LRG modulated TGFb1 signaling in endothelial cells and promoted pathogenic angiogenesis, the precise function of LRG in cancer remains unclear. In this study, we investigated the role of LRG on the cellular response by TGFb1 in Lewis lung carcinoma (LLC) cell line. Methods: Parental LLC, mLRG-overexpressing LLC or control vector transfeced LLC cells were subcutaneously transplanted into LRG Knockout(KO) or Wild-type(WT) C57BL/6 J mice and tumor growth was monitored. Cell proliferation treated with TGFb1 was evaluated by WST-8 assay. Caspase activity was measured with Casapase-GloTM assay kit, protein expression levels were analyzed by western blotting and gene expression was analyzed with Quantitative real-time PCR. LLC tumor growth were monitored in LRG KO or WT mice treated with TGFbRI inhibitor(SB431542) or vehicle. Apoptosis of LLC tumor was evaluated by TUNEL staining. Result: LLC tumor growth in LRG KO mice was significantly enhanced compared with that in WT mice. Conversely, overexpression of mLRG significantly inhibited the growth of LLC tumors in WT mice. TGFb1 inhibited the proliferation of LLC cells in vitro. Addition of TGFb1 strongly inhibited the proliferation of mLRG-overexpressing LLC cells than control vector LLC cells by induction of apoptosis. By TGFb1 stimulation, induction of apoptosis via activation of smad2 and downstream signaling pathway was significantly increased in mLRG overexpressing LLC compared with control LLC cells in vitro. TGFbRI inhibitor significantly enhanced growth and inhibited apoptosis of LLC tumor in WT mice compared with LRG KO. Conclusion: Our data showed that LRG promotes TGFb1-induced apoptosis in LLC, in vitro and in vivo. LRG might be classified as a novel protein that modulates the effects of cytokines.
http://dx.doi.org/10.1016/j.cyto.2014.07.185
179 IFNc and Smac mimetics induce synergistic killing via necroptosis and apoptosis Maria Tanzer, Nufail Khan, James Rickard, Nima Etemadi, John Silke, Walter & Eliza Hall Institute of Medical Research, Melbourne/Carlton, VIC, Australia Inhibitor of Apoptosis proteins (IAPs) are a group of E3 ligases involved in tumor necrosis factor (TNF) signalling. Upon TNF stimulation IAPs are needed for proper activation of NF-jB signalling, thereby protecting cells from cytotoxic activity of TNF.
180 Elucidation and characterization of CD4+ TCRcd+ FoxP3+ type of immune-suppressive cells with the progression of leprosy Mohd Tarique 1, Raza Ali Naqvi 1, Neena Khanna 2, D.N. Rao 1, 1 Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi, Delhi 110029, India, 2 Dermatovenerolgy, All India Institute of Medical Sciences (AIIMS), New Delhi, Delhi 110029, India Leprosy is one of the dreaded infectious diseases haunting the mankind for older ages. Owing to tremendous scientific advancements we now gradually understand the dysregulation of immunological networks in driving the pathogenesis of leprosy. As we move from borderline, tuberculoid to lepromatous patients we found a gradual increase in the proportion of Mycobacterium leprae specific CD4+ TCRcd+ cells. Interestingly, CD4+ TCRcd+ cells isolated from lepromatous patients significantly reduced the proliferation of CD4+ CD25 cells in co-culture experiments draw out attention towards the immune- suppressive nature of these cells. Furthermore, we have also evaluated TGF-b mediated SMAD3 signaling and induction of FoxP3 inside these cells. We observed comparatively increased nuclear ingress of SMAD3 in lepromatous patients as compared to SMAD7 in tuberculoid patients. Thus, the induction of FoxP3 in CD4 + TCRcd+ was found to associate with the severity of disease. To establish this molecular event further, we silenced SMAD3 in these cells in the culture. A remarkable reduction of FoxP3 and a concomitant increase in the proliferation of CD4+ CD25 cells in co-culture proved the operation of TGF-b mediated SMAD3 signaling towards the induction of FoxP3 in these cells. This study for the first time reveals the role of CD4+ TCRcd+ cells in the progression of disease.This study added a new dimension of immune suppression with the progression of leprosy. Also, for the first time we have explored the Th3 dependent TGF-b induced SMAD3 signaling inside CD4+ TCRcd+ cells with the progression of leprosy. http://dx.doi.org/10.1016/j.cyto.2014.07.187
181 Ifnar2 plays an important role in limiting inflammation and disease following influenza virus infection Michelle D. Tate, Jennifer K. Dowling, Rebecca A. Piganis, Paul J. Hertzog, MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia Aims: Excessive inflammation is associated with severe influenza virus infections in both mice and humans. The type I interferon (IFN) receptor (Ifnar) is comprised of two chains, Ifnar1 and Ifnar2. We hypothesized that Ifnar1 and Ifnar2 play differing roles during influenza virus infection. We therefore aimed to investigate and compare the involvement of Ifnar1 and Ifnar2 in host defence, using a mouse model of influenza virus infection. Methods: Wildtype, Ifnar1 / and Ifnar2 / mice were intranasally infected with 102 PFU of HKx31 (H3N2) and weighed daily. Viral loads in respiratory tact was determined by plaque assay. Cellular infiltrate in the airways was examined by flow cytometry and cytokines in bronchoalveolar lavage (BAL) fluid and sera were quantified by cytokine bead array and ELISA. Protein concentrations of BAL fluid and lung wet to dry ratios were utilised to examine vascular leakage and edema, respectively.
71
IAPs have emerged as attractive targets for anti-cancer therapeutics since it has been shown that overexpression of the IAP antagonist Smac sensitises tumour cells to TNF driven apoptosis as do the synthetic counterparts, Smac mimetics. We demonstrated that cell death induced by inhibition of IAPs through Smac mimetics is not strictly TNF dependent. The combined treatment of Interferon c (IFNc), a potent pro-inflammatory cytokine, and Smac mimetics shows potent synergistic killing of a wide range of tumour cell lines as well as primary and transformed cells. Further investigation showed that IFNc and Smac mimetic induced killing requires NF-jB as well as Jak/STAT signalling and displayed classic apoptotic hallmarkers. Whereas killing of several cell lines could be blocked by the inhibition of caspases, other cell lines like HT29, KATOIII, mouse dermal fibroblasts (MDFs) and keratinocytes remained sensitive. In order to elucidate the involvement of the necroptotic pathway in this apoptosis independent IFNc and Smac mimetic combined killing we performed knock down experiments and analysed primary and transformed knock out MDFs and keratinocytes lacking members of the necroptotic pathway like RIPK3 / and MLKL / . The results suggest that IFNc and Smac mimetic combined treatment can activate both apoptotic and necroptotic death pathways, involving members of the necroptotic pathway. This may provide new insights into yet unknown cell death mechanisms and extend the clinical use of Smac mimetics. http://dx.doi.org/10.1016/j.cyto.2014.07.186
http://dx.doi.org/10.1016/j.cyto.2014.07.184
178 Leucine-rich a2 glycoprotein promotes TGFb1-induced apoptosis in the lewis lung carcinoma cell lines 1,2
3
1
1
Norihiko Takemoto , Tomoshige Matsumoto , Satoshi Serada , Minoru Fujimoto , Tetsuji Naka 1, 1 Laboratory for Immune Signal, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan, 2 Osaka University, Suita, Osaka, Japan, 3 Department of Clinical Laboratory, Osaka Anti-Tuberculosis Association Osaka Hospital, Neyagawa, Osaka, Japan Aims: Leucine-rich a2 glycoprotein (LRG) is an approximately 50 kDa glycoprotein originally found in the blood. It has been reported that the expression levels of LRG were elevated in the sera of several cancers. While it has been demonstrated that LRG modulated TGFb1 signaling in endothelial cells and promoted pathogenic angiogenesis, the precise function of LRG in cancer remains unclear. In this study, we investigated the role of LRG on the cellular response by TGFb1 in Lewis lung carcinoma (LLC) cell line. Methods: Parental LLC, mLRG-overexpressing LLC or control vector transfeced LLC cells were subcutaneously transplanted into LRG Knockout(KO) or Wild-type(WT) C57BL/6 J mice and tumor growth was monitored. Cell proliferation treated with TGFb1 was evaluated by WST-8 assay. Caspase activity was measured with Casapase-GloTM assay kit, protein expression levels were analyzed by western blotting and gene expression was analyzed with Quantitative real-time PCR. LLC tumor growth were monitored in LRG KO or WT mice treated with TGFbRI inhibitor(SB431542) or vehicle. Apoptosis of LLC tumor was evaluated by TUNEL staining. Result: LLC tumor growth in LRG KO mice was significantly enhanced compared with that in WT mice. Conversely, overexpression of mLRG significantly inhibited the growth of LLC tumors in WT mice. TGFb1 inhibited the proliferation of LLC cells in vitro. Addition of TGFb1 strongly inhibited the proliferation of mLRG-overexpressing LLC cells than control vector LLC cells by induction of apoptosis. By TGFb1 stimulation, induction of apoptosis via activation of smad2 and downstream signaling pathway was significantly increased in mLRG overexpressing LLC compared with control LLC cells in vitro. TGFbRI inhibitor significantly enhanced growth and inhibited apoptosis of LLC tumor in WT mice compared with LRG KO. Conclusion: Our data showed that LRG promotes TGFb1-induced apoptosis in LLC, in vitro and in vivo. LRG might be classified as a novel protein that modulates the effects of cytokines.
http://dx.doi.org/10.1016/j.cyto.2014.07.185
179 IFNc and Smac mimetics induce synergistic killing via necroptosis and apoptosis Maria Tanzer, Nufail Khan, James Rickard, Nima Etemadi, John Silke, Walter & Eliza Hall Institute of Medical Research, Melbourne/Carlton, VIC, Australia Inhibitor of Apoptosis proteins (IAPs) are a group of E3 ligases involved in tumor necrosis factor (TNF) signalling. Upon TNF stimulation IAPs are needed for proper activation of NF-jB signalling, thereby protecting cells from cytotoxic activity of TNF.
180 Elucidation and characterization of CD4+ TCRcd+ FoxP3+ type of immune-suppressive cells with the progression of leprosy Mohd Tarique 1, Raza Ali Naqvi 1, Neena Khanna 2, D.N. Rao 1, 1 Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi, Delhi 110029, India, 2 Dermatovenerolgy, All India Institute of Medical Sciences (AIIMS), New Delhi, Delhi 110029, India Leprosy is one of the dreaded infectious diseases haunting the mankind for older ages. Owing to tremendous scientific advancements we now gradually understand the dysregulation of immunological networks in driving the pathogenesis of leprosy. As we move from borderline, tuberculoid to lepromatous patients we found a gradual increase in the proportion of Mycobacterium leprae specific CD4+ TCRcd+ cells. Interestingly, CD4+ TCRcd+ cells isolated from lepromatous patients significantly reduced the proliferation of CD4+ CD25 cells in co-culture experiments draw out attention towards the immune- suppressive nature of these cells. Furthermore, we have also evaluated TGF-b mediated SMAD3 signaling and induction of FoxP3 inside these cells. We observed comparatively increased nuclear ingress of SMAD3 in lepromatous patients as compared to SMAD7 in tuberculoid patients. Thus, the induction of FoxP3 in CD4 + TCRcd+ was found to associate with the severity of disease. To establish this molecular event further, we silenced SMAD3 in these cells in the culture. A remarkable reduction of FoxP3 and a concomitant increase in the proliferation of CD4+ CD25 cells in co-culture proved the operation of TGF-b mediated SMAD3 signaling towards the induction of FoxP3 in these cells. This study for the first time reveals the role of CD4+ TCRcd+ cells in the progression of disease.This study added a new dimension of immune suppression with the progression of leprosy. Also, for the first time we have explored the Th3 dependent TGF-b induced SMAD3 signaling inside CD4+ TCRcd+ cells with the progression of leprosy. http://dx.doi.org/10.1016/j.cyto.2014.07.187
181 Ifnar2 plays an important role in limiting inflammation and disease following influenza virus infection Michelle D. Tate, Jennifer K. Dowling, Rebecca A. Piganis, Paul J. Hertzog, MIMR-PHI Institute of Medical Research, Clayton, VIC, Australia Aims: Excessive inflammation is associated with severe influenza virus infections in both mice and humans. The type I interferon (IFN) receptor (Ifnar) is comprised of two chains, Ifnar1 and Ifnar2. We hypothesized that Ifnar1 and Ifnar2 play differing roles during influenza virus infection. We therefore aimed to investigate and compare the involvement of Ifnar1 and Ifnar2 in host defence, using a mouse model of influenza virus infection. Methods: Wildtype, Ifnar1 / and Ifnar2 / mice were intranasally infected with 102 PFU of HKx31 (H3N2) and weighed daily. Viral loads in respiratory tact was determined by plaque assay. Cellular infiltrate in the airways was examined by flow cytometry and cytokines in bronchoalveolar lavage (BAL) fluid and sera were quantified by cytokine bead array and ELISA. Protein concentrations of BAL fluid and lung wet to dry ratios were utilised to examine vascular leakage and edema, respectively.