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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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Related in: MedlinePlus

Schematic representation of models of B-cell differentiation during normal course of immune reaction.On activation, B cells acquire mitochondrial mass and membrane potential (and are called P1 cells) and proliferate. Stochastic differences in mitochondrial activity within P1 cells generate diverse P1 cells having various ROS amounts (in this scheme, they are simply classified into two groups, P1 ROShigh and P1 ROSlow cells). In addition to the signalling function, excess amounts of ROS have another function of attenuating haeme synthesis. In general, newly generated haeme that cannot bind to haeme proteins is degraded immediately by HO-1. Haeme that binds to high- but not to low-affinity haeme-binding proteins (such as Bach2), are retained for a longer period. Thus, the effects of haeme on the regulation of low-affinity haeme-binding proteins are limited. Moreover, increasing amounts of free haeme induce HO-1 expression, which enhances the activity of free haeme clearance. Accordingly, the level of effective haeme concentration that regulates low-affinity haeme-binding proteins cannot be achieved by transient elevation of haeme contents. In P1 ROSlow cells, the upregulation of haeme turnover, which supports a continuous haeme supply, inhibits Bach2 function and promotes the PCD program (from P1 ROSlow to P2 cells). Theoretically, initial changes might occur at any steps described in this scheme. For example, HO-1-dependent degradation of haeme provides an antioxidant, which can cause reduction of ROS level. The important steps in the respective immune reactions await identification. The majority of naïve B cells are also P2 cells, but almost all the cells show increased mitochondrial mass, membrane potential (Fig. 1a) and Bach2 protein expression after activation (not shown). Accordingly, P1 and P2 populations observed in naïve B cells may be different from P1 and P2 populations of activated B cells.
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f7: Schematic representation of models of B-cell differentiation during normal course of immune reaction.On activation, B cells acquire mitochondrial mass and membrane potential (and are called P1 cells) and proliferate. Stochastic differences in mitochondrial activity within P1 cells generate diverse P1 cells having various ROS amounts (in this scheme, they are simply classified into two groups, P1 ROShigh and P1 ROSlow cells). In addition to the signalling function, excess amounts of ROS have another function of attenuating haeme synthesis. In general, newly generated haeme that cannot bind to haeme proteins is degraded immediately by HO-1. Haeme that binds to high- but not to low-affinity haeme-binding proteins (such as Bach2), are retained for a longer period. Thus, the effects of haeme on the regulation of low-affinity haeme-binding proteins are limited. Moreover, increasing amounts of free haeme induce HO-1 expression, which enhances the activity of free haeme clearance. Accordingly, the level of effective haeme concentration that regulates low-affinity haeme-binding proteins cannot be achieved by transient elevation of haeme contents. In P1 ROSlow cells, the upregulation of haeme turnover, which supports a continuous haeme supply, inhibits Bach2 function and promotes the PCD program (from P1 ROSlow to P2 cells). Theoretically, initial changes might occur at any steps described in this scheme. For example, HO-1-dependent degradation of haeme provides an antioxidant, which can cause reduction of ROS level. The important steps in the respective immune reactions await identification. The majority of naïve B cells are also P2 cells, but almost all the cells show increased mitochondrial mass, membrane potential (Fig. 1a) and Bach2 protein expression after activation (not shown). Accordingly, P1 and P2 populations observed in naïve B cells may be different from P1 and P2 populations of activated B cells.

Mentions: As shown in Fig. 1c, commitment of activated B cells to CSR or PCD was predicted by their mitochondrial status. P1 cells, which contained higher mitochondrial mass and membrane potential, preferentially underwent CSR, whereas P2 cells, which contained lower mitochondrial mass and membrane potential, differentiated into plasma cells. Bach2 function is required for CSR, and reduced activity of Bach2 promotes PCD. However, the expression level of Bach2 differed little between P1 and P2 cells (Fig. 3e). Thus, the differential regulation of Bach2 activity is key in these cell fate-determination processes. Bach2 function was maintained by inhibition of haeme synthesis by mROS produced in P1 cells (Fig. 4a–g). In contrast, Bach2 function was inhibited in P2 cells, allowing the expression of Bach2 target genes such as Blimp1 (Fig. 3e). P2 cells appear to arise from plasma cell-committed populations (Figs 3e and 6b). To determine whether the same scenario is applicable in the initial step of cell fate determination, we evaluated the differentiation capacity of ROShigh and ROSlow cells within P1 cells. Between ROShigh and ROSlow cells derived from the P1 cell population, we found no differences in protein levels of transcription factors essential to CSR and PCD (Fig. 6d). These results suggested that cells at this stage are not committed to either differentiation pathway. When undifferentiated ROShigh and ROSlow cells were collected and cultured for an additional 2.5 days, ROShigh cells underwent predominantly CSR, whereas ROSlow cells differentiated into plasma cells (Fig. 6c). In addition, the preferential appearance of P1 cells was observed in cultured ROShigh cells, but appearance of P2 cells was observed in cultured in ROSlow cells (Fig. 6c). Because CSR and PCD are influenced by cell proliferation, numbers of cell divisions were assessed by BRSE. As shown in Fig. 6c, there was little difference in the cell division numbers between these populations. These results indicated that change in mROS generation was the cause of the cell fate decision after B-cell activation. These findings together indicate that fate determination of activated B cells is dependent on ROS level, which negatively regulates haeme synthesis. Halflife of Bach2 protein was longer than that of Pax5 protein, because significant amount of Bach2 protein was observed in 5 days-cultured B cells (Supplementary Fig. 12). These data indicated that quantitative regulation of haeme plays an important role in promoting PCD. In other words, PCD is a reversible state before loss of Bach2 protein. Our results show the mechanism of induction of differentiation in the lineage commitment process of B cells (Fig. 7). In general, commitment to specific lineages of haematopoietic cells is an integrated interpretation of instructive and stochastic signals141516174344. Precise understanding of the mechanism of B-cell fate determination requires further identification of the mechanisms by which levels of ROS and haeme synthesis are regulated by instructive signals and by which haeme functions as a signalling molecule.


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)

Schematic representation of models of B-cell differentiation during normal course of immune reaction.On activation, B cells acquire mitochondrial mass and membrane potential (and are called P1 cells) and proliferate. Stochastic differences in mitochondrial activity within P1 cells generate diverse P1 cells having various ROS amounts (in this scheme, they are simply classified into two groups, P1 ROShigh and P1 ROSlow cells). In addition to the signalling function, excess amounts of ROS have another function of attenuating haeme synthesis. In general, newly generated haeme that cannot bind to haeme proteins is degraded immediately by HO-1. Haeme that binds to high- but not to low-affinity haeme-binding proteins (such as Bach2), are retained for a longer period. Thus, the effects of haeme on the regulation of low-affinity haeme-binding proteins are limited. Moreover, increasing amounts of free haeme induce HO-1 expression, which enhances the activity of free haeme clearance. Accordingly, the level of effective haeme concentration that regulates low-affinity haeme-binding proteins cannot be achieved by transient elevation of haeme contents. In P1 ROSlow cells, the upregulation of haeme turnover, which supports a continuous haeme supply, inhibits Bach2 function and promotes the PCD program (from P1 ROSlow to P2 cells). Theoretically, initial changes might occur at any steps described in this scheme. For example, HO-1-dependent degradation of haeme provides an antioxidant, which can cause reduction of ROS level. The important steps in the respective immune reactions await identification. The majority of naïve B cells are also P2 cells, but almost all the cells show increased mitochondrial mass, membrane potential (Fig. 1a) and Bach2 protein expression after activation (not shown). Accordingly, P1 and P2 populations observed in naïve B cells may be different from P1 and P2 populations of activated B cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403446&req=5

f7: Schematic representation of models of B-cell differentiation during normal course of immune reaction.On activation, B cells acquire mitochondrial mass and membrane potential (and are called P1 cells) and proliferate. Stochastic differences in mitochondrial activity within P1 cells generate diverse P1 cells having various ROS amounts (in this scheme, they are simply classified into two groups, P1 ROShigh and P1 ROSlow cells). In addition to the signalling function, excess amounts of ROS have another function of attenuating haeme synthesis. In general, newly generated haeme that cannot bind to haeme proteins is degraded immediately by HO-1. Haeme that binds to high- but not to low-affinity haeme-binding proteins (such as Bach2), are retained for a longer period. Thus, the effects of haeme on the regulation of low-affinity haeme-binding proteins are limited. Moreover, increasing amounts of free haeme induce HO-1 expression, which enhances the activity of free haeme clearance. Accordingly, the level of effective haeme concentration that regulates low-affinity haeme-binding proteins cannot be achieved by transient elevation of haeme contents. In P1 ROSlow cells, the upregulation of haeme turnover, which supports a continuous haeme supply, inhibits Bach2 function and promotes the PCD program (from P1 ROSlow to P2 cells). Theoretically, initial changes might occur at any steps described in this scheme. For example, HO-1-dependent degradation of haeme provides an antioxidant, which can cause reduction of ROS level. The important steps in the respective immune reactions await identification. The majority of naïve B cells are also P2 cells, but almost all the cells show increased mitochondrial mass, membrane potential (Fig. 1a) and Bach2 protein expression after activation (not shown). Accordingly, P1 and P2 populations observed in naïve B cells may be different from P1 and P2 populations of activated B cells.
Mentions: As shown in Fig. 1c, commitment of activated B cells to CSR or PCD was predicted by their mitochondrial status. P1 cells, which contained higher mitochondrial mass and membrane potential, preferentially underwent CSR, whereas P2 cells, which contained lower mitochondrial mass and membrane potential, differentiated into plasma cells. Bach2 function is required for CSR, and reduced activity of Bach2 promotes PCD. However, the expression level of Bach2 differed little between P1 and P2 cells (Fig. 3e). Thus, the differential regulation of Bach2 activity is key in these cell fate-determination processes. Bach2 function was maintained by inhibition of haeme synthesis by mROS produced in P1 cells (Fig. 4a–g). In contrast, Bach2 function was inhibited in P2 cells, allowing the expression of Bach2 target genes such as Blimp1 (Fig. 3e). P2 cells appear to arise from plasma cell-committed populations (Figs 3e and 6b). To determine whether the same scenario is applicable in the initial step of cell fate determination, we evaluated the differentiation capacity of ROShigh and ROSlow cells within P1 cells. Between ROShigh and ROSlow cells derived from the P1 cell population, we found no differences in protein levels of transcription factors essential to CSR and PCD (Fig. 6d). These results suggested that cells at this stage are not committed to either differentiation pathway. When undifferentiated ROShigh and ROSlow cells were collected and cultured for an additional 2.5 days, ROShigh cells underwent predominantly CSR, whereas ROSlow cells differentiated into plasma cells (Fig. 6c). In addition, the preferential appearance of P1 cells was observed in cultured ROShigh cells, but appearance of P2 cells was observed in cultured in ROSlow cells (Fig. 6c). Because CSR and PCD are influenced by cell proliferation, numbers of cell divisions were assessed by BRSE. As shown in Fig. 6c, there was little difference in the cell division numbers between these populations. These results indicated that change in mROS generation was the cause of the cell fate decision after B-cell activation. These findings together indicate that fate determination of activated B cells is dependent on ROS level, which negatively regulates haeme synthesis. Halflife of Bach2 protein was longer than that of Pax5 protein, because significant amount of Bach2 protein was observed in 5 days-cultured B cells (Supplementary Fig. 12). These data indicated that quantitative regulation of haeme plays an important role in promoting PCD. In other words, PCD is a reversible state before loss of Bach2 protein. Our results show the mechanism of induction of differentiation in the lineage commitment process of B cells (Fig. 7). In general, commitment to specific lineages of haematopoietic cells is an integrated interpretation of instructive and stochastic signals141516174344. Precise understanding of the mechanism of B-cell fate determination requires further identification of the mechanisms by which levels of ROS and haeme synthesis are regulated by instructive signals and by which haeme functions as a signalling molecule.

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