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Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus.

Fan H, Liu F, Dong G, Ren D, Xu Y, Dou J, Wang T, Sun L, Hou Y - Cell Death Dis (2014)

Bottom Line: We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days.Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors.These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing, China.

ABSTRACT
B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19(+)CD27(-) and CD19(+)IgM(+) B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19(+) B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients.

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Related in: MedlinePlus

Co-activation of TLR7 and BCR pathways promotes the formation of necrotic ultrastructure, mitochondrial dysfunction and hypoxia of B cells. (a) The ultrastructure of mouse B cells treated with anti-IgM/CD40 or joint stimulation (R848+anti-IgM/CD40) for 4 days by transmission electron microscopy. Black arrowheads denote cell membrane integrity in the anti-IgM/CD40 treated cells. White arrowheads denote the swelling of cellular organelles and membrane breakdown in cells treated with joint stimulation. Among 200 cells counted, 92 showed necrotic morphology as presented in (a) in joint stimulated sample, whereas only 16 such cells were seen in anti-IgM/CD40-treated sample. (b) The mouse B cells stained with DCFH-DA for 4 days were detected by flow cytometry, and ROS production was assessed according to changes in the fluorescence intensity of DCF, the oxidation product of DCFH-DA. (c) The mouse B cells stained with Flou3-AM for 4 days were detected by flow cytometry, and Ca2+ influx was assessed according to changes in the fluorescence intensity of Flou3 and Ca2+combination. (d) The intracellular ATP levels were determined by the luciferase method and normalized by protein content. (e) Mitochondrial membrane potential (ΔΨm) of mouse B cells in each treatment group was analyzed by JC-1 staining for 5 days. Loss of ΔΨm was demonstrated by the percentage of JC-1 FITC. (f) Mitochondrial DNA quantification of mouse B cells using nuclear DNA (nDNA) as a reference was carried out by real-time PCR, and the results are presented as mtDNA/nDNA ratio. (g) The relative mRNA expression of target genes GAPDH, HIF-1α and BNIP3 in mouse B cells with different stimulations were detected by real-time PCR on day 4, and β-tubulin was used as a reference. Mouse B cells without treatment as the control group were collected immediately after isolation from the spleen that were used to compare the target gene expression level
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fig6: Co-activation of TLR7 and BCR pathways promotes the formation of necrotic ultrastructure, mitochondrial dysfunction and hypoxia of B cells. (a) The ultrastructure of mouse B cells treated with anti-IgM/CD40 or joint stimulation (R848+anti-IgM/CD40) for 4 days by transmission electron microscopy. Black arrowheads denote cell membrane integrity in the anti-IgM/CD40 treated cells. White arrowheads denote the swelling of cellular organelles and membrane breakdown in cells treated with joint stimulation. Among 200 cells counted, 92 showed necrotic morphology as presented in (a) in joint stimulated sample, whereas only 16 such cells were seen in anti-IgM/CD40-treated sample. (b) The mouse B cells stained with DCFH-DA for 4 days were detected by flow cytometry, and ROS production was assessed according to changes in the fluorescence intensity of DCF, the oxidation product of DCFH-DA. (c) The mouse B cells stained with Flou3-AM for 4 days were detected by flow cytometry, and Ca2+ influx was assessed according to changes in the fluorescence intensity of Flou3 and Ca2+combination. (d) The intracellular ATP levels were determined by the luciferase method and normalized by protein content. (e) Mitochondrial membrane potential (ΔΨm) of mouse B cells in each treatment group was analyzed by JC-1 staining for 5 days. Loss of ΔΨm was demonstrated by the percentage of JC-1 FITC. (f) Mitochondrial DNA quantification of mouse B cells using nuclear DNA (nDNA) as a reference was carried out by real-time PCR, and the results are presented as mtDNA/nDNA ratio. (g) The relative mRNA expression of target genes GAPDH, HIF-1α and BNIP3 in mouse B cells with different stimulations were detected by real-time PCR on day 4, and β-tubulin was used as a reference. Mouse B cells without treatment as the control group were collected immediately after isolation from the spleen that were used to compare the target gene expression level

Mentions: Next, we observed that activated B cells were significantly distinguished as two subgroups, named as FSC subset and SSC subset according to their size and particle density (Figure 5d). Meanwhile, B cells showed more percentage of SSC subset and less percentage of FSC subset with joint stimulation (Figure 5b). Interestingly, the FSC subset was almost PI− cells (living or early apoptosis cells) (Figure 5e). At the same time, the morphology of B cells treated with anti-IgM/CD40 or R848+anti-IgM/CD40 was observed by transmission electron microscopy (TEM). The results showed that most of anti-IgM/CD40-treated B cells possessed normal cellular morphology including intact cytoplasmic membranes or apoptotic cellular morphology such as fragmented nuclei with condensed chromatin and formation of apoptotic bodies (Figure 6a), whereas most of R848+anti-IgM/CD40-treated B cells displayed necrotic cellular morphology, including electron-lucent cytoplasm, mitochondrial swelling and loss of plasma membrane integrity without severe damage to nuclei (Figure 6a). These data adequately indicated that co-activation of TLR7 and BCR pathways promoted activation-induced cell necroptosis (AICN) of B cells.


Activation-induced necroptosis contributes to B-cell lymphopenia in active systemic lupus erythematosus.

Fan H, Liu F, Dong G, Ren D, Xu Y, Dou J, Wang T, Sun L, Hou Y - Cell Death Dis (2014)

Co-activation of TLR7 and BCR pathways promotes the formation of necrotic ultrastructure, mitochondrial dysfunction and hypoxia of B cells. (a) The ultrastructure of mouse B cells treated with anti-IgM/CD40 or joint stimulation (R848+anti-IgM/CD40) for 4 days by transmission electron microscopy. Black arrowheads denote cell membrane integrity in the anti-IgM/CD40 treated cells. White arrowheads denote the swelling of cellular organelles and membrane breakdown in cells treated with joint stimulation. Among 200 cells counted, 92 showed necrotic morphology as presented in (a) in joint stimulated sample, whereas only 16 such cells were seen in anti-IgM/CD40-treated sample. (b) The mouse B cells stained with DCFH-DA for 4 days were detected by flow cytometry, and ROS production was assessed according to changes in the fluorescence intensity of DCF, the oxidation product of DCFH-DA. (c) The mouse B cells stained with Flou3-AM for 4 days were detected by flow cytometry, and Ca2+ influx was assessed according to changes in the fluorescence intensity of Flou3 and Ca2+combination. (d) The intracellular ATP levels were determined by the luciferase method and normalized by protein content. (e) Mitochondrial membrane potential (ΔΨm) of mouse B cells in each treatment group was analyzed by JC-1 staining for 5 days. Loss of ΔΨm was demonstrated by the percentage of JC-1 FITC. (f) Mitochondrial DNA quantification of mouse B cells using nuclear DNA (nDNA) as a reference was carried out by real-time PCR, and the results are presented as mtDNA/nDNA ratio. (g) The relative mRNA expression of target genes GAPDH, HIF-1α and BNIP3 in mouse B cells with different stimulations were detected by real-time PCR on day 4, and β-tubulin was used as a reference. Mouse B cells without treatment as the control group were collected immediately after isolation from the spleen that were used to compare the target gene expression level
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4225223&req=5

fig6: Co-activation of TLR7 and BCR pathways promotes the formation of necrotic ultrastructure, mitochondrial dysfunction and hypoxia of B cells. (a) The ultrastructure of mouse B cells treated with anti-IgM/CD40 or joint stimulation (R848+anti-IgM/CD40) for 4 days by transmission electron microscopy. Black arrowheads denote cell membrane integrity in the anti-IgM/CD40 treated cells. White arrowheads denote the swelling of cellular organelles and membrane breakdown in cells treated with joint stimulation. Among 200 cells counted, 92 showed necrotic morphology as presented in (a) in joint stimulated sample, whereas only 16 such cells were seen in anti-IgM/CD40-treated sample. (b) The mouse B cells stained with DCFH-DA for 4 days were detected by flow cytometry, and ROS production was assessed according to changes in the fluorescence intensity of DCF, the oxidation product of DCFH-DA. (c) The mouse B cells stained with Flou3-AM for 4 days were detected by flow cytometry, and Ca2+ influx was assessed according to changes in the fluorescence intensity of Flou3 and Ca2+combination. (d) The intracellular ATP levels were determined by the luciferase method and normalized by protein content. (e) Mitochondrial membrane potential (ΔΨm) of mouse B cells in each treatment group was analyzed by JC-1 staining for 5 days. Loss of ΔΨm was demonstrated by the percentage of JC-1 FITC. (f) Mitochondrial DNA quantification of mouse B cells using nuclear DNA (nDNA) as a reference was carried out by real-time PCR, and the results are presented as mtDNA/nDNA ratio. (g) The relative mRNA expression of target genes GAPDH, HIF-1α and BNIP3 in mouse B cells with different stimulations were detected by real-time PCR on day 4, and β-tubulin was used as a reference. Mouse B cells without treatment as the control group were collected immediately after isolation from the spleen that were used to compare the target gene expression level
Mentions: Next, we observed that activated B cells were significantly distinguished as two subgroups, named as FSC subset and SSC subset according to their size and particle density (Figure 5d). Meanwhile, B cells showed more percentage of SSC subset and less percentage of FSC subset with joint stimulation (Figure 5b). Interestingly, the FSC subset was almost PI− cells (living or early apoptosis cells) (Figure 5e). At the same time, the morphology of B cells treated with anti-IgM/CD40 or R848+anti-IgM/CD40 was observed by transmission electron microscopy (TEM). The results showed that most of anti-IgM/CD40-treated B cells possessed normal cellular morphology including intact cytoplasmic membranes or apoptotic cellular morphology such as fragmented nuclei with condensed chromatin and formation of apoptotic bodies (Figure 6a), whereas most of R848+anti-IgM/CD40-treated B cells displayed necrotic cellular morphology, including electron-lucent cytoplasm, mitochondrial swelling and loss of plasma membrane integrity without severe damage to nuclei (Figure 6a). These data adequately indicated that co-activation of TLR7 and BCR pathways promoted activation-induced cell necroptosis (AICN) of B cells.

Bottom Line: We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days.Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors.These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing, China.

ABSTRACT
B-cell abnormality including excessive activation and lymphopenia is a central feature of systemic lupus erythematosus (SLE). Although activation threshold, auto-reaction and death of B cells can be affected by intrinsical and/or external signaling, the underlying mechanisms are unclear. Herein, we demonstrate that co-activation of Toll-like receptor 7 (TLR7) and B-cell receptor (BCR) pathways is a core event for the survival/dead states of B cells in SLE. We found that the mortalities of CD19(+)CD27(-) and CD19(+)IgM(+) B-cell subsets were increased in the peripheral blood mononuclear cells (PBMCs) of SLE patients. The gene microarray analysis of CD19(+) B cells from active SLE patients showed that the differentially expressed genes were closely correlated to TLR7, BCR, apoptosis, necroptosis and immune pathways. We also found that co-activation of TLR7 and BCR could trigger normal B cells to take on SLE-like B-cell characters including the elevated viability, activation and proliferation in the first 3 days and necroptosis in the later days. Moreover, the necroptotic B cells exhibited mitochondrial dysfunction and hypoxia, along with the elevated expression of necroptosis-related genes, consistent with that in both SLE B-cell microarray and real-time PCR verification. Expectedly, pretreatment with the receptor-interacting protein kinase 1 (RIPK1) inhibitor Necrostatin-1, and not the apoptosis inhibitor zVAD, suppressed B-cell death. Importantly, B cells from additional SLE patients also significantly displayed high expression levels of necroptosis-related genes compared with those from healthy donors. These data indicate that co-activation of TLR7 and BCR pathways can promote B cells to hyperactivation and ultimately necroptosis. Our finding provides a new explanation on B-cell lymphopenia in active SLE patients. These data suggest that extrinsic factors may increase the intrinsical abnormality of B cells in SLE patients.

Show MeSH
Related in: MedlinePlus