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Mitochondrial function provides instructive signals for activation-induced B-cell fates.

Jang KJ, Mano H, Aoki K, Hayashi T, Muto A, Nambu Y, Takahashi K, Itoh K, Taketani S, Nutt SL, Igarashi K, Shimizu A, Sugai M - Nat Commun (2015)

Bottom Line: During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD).In this study, we show a strong association between individual B-cell fates and mitochondrial functions.In PCD-committed cells, Blimp1 reduces mitochondrial mass, thereby reducing mROS levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD). In this study, we show a strong association between individual B-cell fates and mitochondrial functions. CSR occurs specifically in activated B cells with increased mitochondrial mass and membrane potential, which augment mitochondrial reactive oxygen species (mROS), whereas PCD occurs in cells with decreased mitochondrial mass and potential. These events are consequences of initial slight changes in mROS in mitochondria(high) B-cell populations. In CSR-committed cells, mROS attenuates haeme synthesis by inhibiting ferrous ion addition to protoporphyrin IX, thereby maintaining Bach2 function. Reduced mROS then promotes PCD by increasing haeme synthesis. In PCD-committed cells, Blimp1 reduces mitochondrial mass, thereby reducing mROS levels. Identifying mROS as a haeme synthesis regulator increases the understanding of mechanisms regulating haeme homeostasis and cell fate determination after B-cell activation.

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Differential haeme synthesis in P1 and P2 cell populations.(a) Haeme levels in sorted P1 and P2 cells from in vitro-activated B cells (left) or GC B cells in an immunized spleen (right). (b) Haeme levels in sorted P1 cells treated with indicated antioxidants. (c) Haeme levels in in vitro-activated B cells treated with indicated antioxidants. (d) Flow cytometric analysis of the differentiation status monitored by CD138 and IgG1 expression after 4 days of culture with LPS+IL-4 in the presence or absence of MitoTEMPO. (e) Flow cytometric analysis of PpIX fluorescence in P1 and P2 cells. In vitro-activated B cells treated with indicated reagents were assessed. (f) Mean fluorescence intensities (MFI) of PpIX as shown in (e) are plotted. (g) Increased MFI of PpIX after treatment with indicated reagents is plotted. Data shown are representative of three independent experiments. Data are shown as mean±s.e.m. **P<0.05, ***P<0.005. (two-tailed Student's t-test).
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f4: Differential haeme synthesis in P1 and P2 cell populations.(a) Haeme levels in sorted P1 and P2 cells from in vitro-activated B cells (left) or GC B cells in an immunized spleen (right). (b) Haeme levels in sorted P1 cells treated with indicated antioxidants. (c) Haeme levels in in vitro-activated B cells treated with indicated antioxidants. (d) Flow cytometric analysis of the differentiation status monitored by CD138 and IgG1 expression after 4 days of culture with LPS+IL-4 in the presence or absence of MitoTEMPO. (e) Flow cytometric analysis of PpIX fluorescence in P1 and P2 cells. In vitro-activated B cells treated with indicated reagents were assessed. (f) Mean fluorescence intensities (MFI) of PpIX as shown in (e) are plotted. (g) Increased MFI of PpIX after treatment with indicated reagents is plotted. Data shown are representative of three independent experiments. Data are shown as mean±s.e.m. **P<0.05, ***P<0.005. (two-tailed Student's t-test).

Mentions: Because Bach2 promotes CSR and inhibits PCD via the repression of Blimp1 expression2141, the reduced level of Bach2 correlates strongly with reciprocal Blimp1 expression in the normal course of B-cell activation. However, we found significant expression levels of both Blimp1 and Bach2 in P2 cells (Fig. 3e). Expression levels of Bach2 and Bcl6 were similar between P1 and P2 cells. We first determined the haeme levels that inhibit Bach2 function36. Haeme levels were higher in P2 cells than in P1 cells (Fig. 4a). In vivo-generated P2 cells also showed higher haeme levels than P1 cells (Fig. 4a). These results indicated a direct relationship between haeme levels and mitochondrial function. We then investigated the possible role of ROS in haeme synthesis. Haeme levels (Fig. 4b,c) were increased and PCD (Figs 2 and 4d and Supplementary Fig. 9) was promoted after treatment of P1 cells or activated B cells with ascorbic acid or MitoTEMPO, a mitochondria-targeted antioxidant. In MitoTEMPO treated cells, cellular ROS level was little reduced by day 4 (Supplementary Fig. 9c). We accordingly performed a time-course analysis of cellular ROS and found an antioxidant effect of MitoTEMPO on day 2 cells (24 h after MitoTEMPO treatment, Supplementary Fig. 9c). These data indicated that ROS at early time points was important in B-cell fate determination.


Mitochondrial function provides instructive signals for activation-induced B-cell fates.

Jang KJ, Mano H, Aoki K, Hayashi T, Muto A, Nambu Y, Takahashi K, Itoh K, Taketani S, Nutt SL, Igarashi K, Shimizu A, Sugai M - Nat Commun (2015)

Differential haeme synthesis in P1 and P2 cell populations.(a) Haeme levels in sorted P1 and P2 cells from in vitro-activated B cells (left) or GC B cells in an immunized spleen (right). (b) Haeme levels in sorted P1 cells treated with indicated antioxidants. (c) Haeme levels in in vitro-activated B cells treated with indicated antioxidants. (d) Flow cytometric analysis of the differentiation status monitored by CD138 and IgG1 expression after 4 days of culture with LPS+IL-4 in the presence or absence of MitoTEMPO. (e) Flow cytometric analysis of PpIX fluorescence in P1 and P2 cells. In vitro-activated B cells treated with indicated reagents were assessed. (f) Mean fluorescence intensities (MFI) of PpIX as shown in (e) are plotted. (g) Increased MFI of PpIX after treatment with indicated reagents is plotted. Data shown are representative of three independent experiments. Data are shown as mean±s.e.m. **P<0.05, ***P<0.005. (two-tailed Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403446&req=5

f4: Differential haeme synthesis in P1 and P2 cell populations.(a) Haeme levels in sorted P1 and P2 cells from in vitro-activated B cells (left) or GC B cells in an immunized spleen (right). (b) Haeme levels in sorted P1 cells treated with indicated antioxidants. (c) Haeme levels in in vitro-activated B cells treated with indicated antioxidants. (d) Flow cytometric analysis of the differentiation status monitored by CD138 and IgG1 expression after 4 days of culture with LPS+IL-4 in the presence or absence of MitoTEMPO. (e) Flow cytometric analysis of PpIX fluorescence in P1 and P2 cells. In vitro-activated B cells treated with indicated reagents were assessed. (f) Mean fluorescence intensities (MFI) of PpIX as shown in (e) are plotted. (g) Increased MFI of PpIX after treatment with indicated reagents is plotted. Data shown are representative of three independent experiments. Data are shown as mean±s.e.m. **P<0.05, ***P<0.005. (two-tailed Student's t-test).
Mentions: Because Bach2 promotes CSR and inhibits PCD via the repression of Blimp1 expression2141, the reduced level of Bach2 correlates strongly with reciprocal Blimp1 expression in the normal course of B-cell activation. However, we found significant expression levels of both Blimp1 and Bach2 in P2 cells (Fig. 3e). Expression levels of Bach2 and Bcl6 were similar between P1 and P2 cells. We first determined the haeme levels that inhibit Bach2 function36. Haeme levels were higher in P2 cells than in P1 cells (Fig. 4a). In vivo-generated P2 cells also showed higher haeme levels than P1 cells (Fig. 4a). These results indicated a direct relationship between haeme levels and mitochondrial function. We then investigated the possible role of ROS in haeme synthesis. Haeme levels (Fig. 4b,c) were increased and PCD (Figs 2 and 4d and Supplementary Fig. 9) was promoted after treatment of P1 cells or activated B cells with ascorbic acid or MitoTEMPO, a mitochondria-targeted antioxidant. In MitoTEMPO treated cells, cellular ROS level was little reduced by day 4 (Supplementary Fig. 9c). We accordingly performed a time-course analysis of cellular ROS and found an antioxidant effect of MitoTEMPO on day 2 cells (24 h after MitoTEMPO treatment, Supplementary Fig. 9c). These data indicated that ROS at early time points was important in B-cell fate determination.

Bottom Line: During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD).In this study, we show a strong association between individual B-cell fates and mitochondrial functions.In PCD-committed cells, Blimp1 reduces mitochondrial mass, thereby reducing mROS levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.

ABSTRACT
During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD). In this study, we show a strong association between individual B-cell fates and mitochondrial functions. CSR occurs specifically in activated B cells with increased mitochondrial mass and membrane potential, which augment mitochondrial reactive oxygen species (mROS), whereas PCD occurs in cells with decreased mitochondrial mass and potential. These events are consequences of initial slight changes in mROS in mitochondria(high) B-cell populations. In CSR-committed cells, mROS attenuates haeme synthesis by inhibiting ferrous ion addition to protoporphyrin IX, thereby maintaining Bach2 function. Reduced mROS then promotes PCD by increasing haeme synthesis. In PCD-committed cells, Blimp1 reduces mitochondrial mass, thereby reducing mROS levels. Identifying mROS as a haeme synthesis regulator increases the understanding of mechanisms regulating haeme homeostasis and cell fate determination after B-cell activation.

Show MeSH
Related in: MedlinePlus