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Plasmodium chabaudi infection induces AID expression in transitional and marginal zone B cells

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Endemic Burkitt's lymphoma (eBL) is associated with Epstein–Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c‐myc oncogene to the control of the immunoglobulin promoter. Activation‐induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown.

Methods: We either stimulated purified B cells from AID‐green fluorescence protein (GFP) reporter mice or infected AID‐GFP mice with Plasmodium chabaudi, AID fluorescence was monitored in B cell subsets by flow cytometry.

Results: In vitro analysis of B cells from these mice revealed that CpG (a Toll‐like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID‐GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi‐infected mouse.

Conclusions: These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

No MeSH data available.


Related in: MedlinePlus

The levels of immature B cells decrease dramatically at 17 days post Plasmodium chabaudi infection express AID. AID‐GFP mice were infected with P. chabaudi or given a control injection of uninfected RBC and sacrificed at 17 dpi. Spleens were removed and analyzed by flow cytometry. Representative flow cytometry plots demonstrating the gating strategy for each of the B cell subsets are shown. The left panel is differentiating the mature (CD19+, CD93−) and immature (CD19+, CD93+), and CD93high B cells based on expression of CD19 and CD93. Transitional B cells subsets are gated into T1 (IgM+, CD23−), T2 (IgM+, CD23+), and T3 (IgM−, CD23+) from the immature B cell subset. Germinal center B cells (PNAhigh, CD38−) and the rest of the mature B cells are further analyzed to determine the follicular (PNA−, CD38+, CD23+, CD21+) and marginal zone (PNA−, CD38+, CD23int, CD21high) B cells. The bottom panel contains representative flow cytometry histograms demonstrating the expression of AID in each B cell subset (P. chabaudi infected in black, RBC control in gray histograms) (n = 3 in two independent experiments).
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iid3134-fig-0002: The levels of immature B cells decrease dramatically at 17 days post Plasmodium chabaudi infection express AID. AID‐GFP mice were infected with P. chabaudi or given a control injection of uninfected RBC and sacrificed at 17 dpi. Spleens were removed and analyzed by flow cytometry. Representative flow cytometry plots demonstrating the gating strategy for each of the B cell subsets are shown. The left panel is differentiating the mature (CD19+, CD93−) and immature (CD19+, CD93+), and CD93high B cells based on expression of CD19 and CD93. Transitional B cells subsets are gated into T1 (IgM+, CD23−), T2 (IgM+, CD23+), and T3 (IgM−, CD23+) from the immature B cell subset. Germinal center B cells (PNAhigh, CD38−) and the rest of the mature B cells are further analyzed to determine the follicular (PNA−, CD38+, CD23+, CD21+) and marginal zone (PNA−, CD38+, CD23int, CD21high) B cells. The bottom panel contains representative flow cytometry histograms demonstrating the expression of AID in each B cell subset (P. chabaudi infected in black, RBC control in gray histograms) (n = 3 in two independent experiments).

Mentions: Previous studies performed with AID‐GFP mice demonstrated that AID expression was restricted to germinal center B cells following injection with NP‐CGG and alum, a model antigen and adjuvant commonly used to elicit a humoral immune response 19. P. chabaudi is a productive infection with a higher antigen load and inflammatory response than a model antigen injection such as NP‐CGG. Therefore, we used AID‐GFP mice in the P. chabaudi model to determine whether AID can be expressed in B cells outside of the germinal center. We performed RT‐qPCR on sections of whole spleen at 6, 12, 17, 24, and 30 days post‐infection (dpi) with P. chabaudi and found that the peak of AID expression occurred at 17 dpi (data not shown). The 17‐day time point was then used to determine the percentage of AID positive cells within the T1, T2, T3, marginal zone, follicular, and germinal center B cell subsets by multivariate flow cytometry. At 17 dpi, we found dramatic differences in splenic B cell subset distribution, including a significant decrease in immature and marginal zone B cells, and a large expansion of the germinal center B cell population (Fig. 2), which were consistent with previously reported studies 20. We determined the percentage of AID expressing cells from the immature T1 (IgM+CD23−), T2 (IgM+CD23+), and T3 (IgM‐CD23+) B cell subsets in mice 17 dpi or control mice injected with uninfected RBC. All three immature B cell populations had detectable AID expression 17 days after P. chabaudi infection, and the control mice had little or no AID expressed (Fig. 2). In addition to the CD93+ population of immature B cells, the P. chabaudi‐infected mice had an additional CD93high population of cells that were AID− (Fig. 2). Germinal center B cells (CD38‐PNAhigh) were greatly expanded during P. chabaudi infection and >80% expressed AID (Fig. 2). The control germinal center B cells also expressed AID, but at a lower frequency (Fig. 2). During acute P. chabaudi, the percentage of marginal zone B cells decreased dramatically 19; our study confirmed this finding and demonstrated the marginal zone B cell population that remained included cells that expressed AID (Fig. 2). Follicular B cells in both the P. chabaudi‐infected and control mice did not express AID (Fig. 2). The expansion of CD23− CD21− B cells is consistent with reports of age associated B cells (ABCs) that expand during infection 21, 22. Interestingly, the ABCs had low expression of AID despite their induction during infection. These data demonstrate that during P. chabaudi infection, the expression of AID is not restricted to the germinal center B cells, but is also expressed in transitional and marginal zone B cells.


Plasmodium chabaudi infection induces AID expression in transitional and marginal zone B cells
The levels of immature B cells decrease dramatically at 17 days post Plasmodium chabaudi infection express AID. AID‐GFP mice were infected with P. chabaudi or given a control injection of uninfected RBC and sacrificed at 17 dpi. Spleens were removed and analyzed by flow cytometry. Representative flow cytometry plots demonstrating the gating strategy for each of the B cell subsets are shown. The left panel is differentiating the mature (CD19+, CD93−) and immature (CD19+, CD93+), and CD93high B cells based on expression of CD19 and CD93. Transitional B cells subsets are gated into T1 (IgM+, CD23−), T2 (IgM+, CD23+), and T3 (IgM−, CD23+) from the immature B cell subset. Germinal center B cells (PNAhigh, CD38−) and the rest of the mature B cells are further analyzed to determine the follicular (PNA−, CD38+, CD23+, CD21+) and marginal zone (PNA−, CD38+, CD23int, CD21high) B cells. The bottom panel contains representative flow cytometry histograms demonstrating the expression of AID in each B cell subset (P. chabaudi infected in black, RBC control in gray histograms) (n = 3 in two independent experiments).
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iid3134-fig-0002: The levels of immature B cells decrease dramatically at 17 days post Plasmodium chabaudi infection express AID. AID‐GFP mice were infected with P. chabaudi or given a control injection of uninfected RBC and sacrificed at 17 dpi. Spleens were removed and analyzed by flow cytometry. Representative flow cytometry plots demonstrating the gating strategy for each of the B cell subsets are shown. The left panel is differentiating the mature (CD19+, CD93−) and immature (CD19+, CD93+), and CD93high B cells based on expression of CD19 and CD93. Transitional B cells subsets are gated into T1 (IgM+, CD23−), T2 (IgM+, CD23+), and T3 (IgM−, CD23+) from the immature B cell subset. Germinal center B cells (PNAhigh, CD38−) and the rest of the mature B cells are further analyzed to determine the follicular (PNA−, CD38+, CD23+, CD21+) and marginal zone (PNA−, CD38+, CD23int, CD21high) B cells. The bottom panel contains representative flow cytometry histograms demonstrating the expression of AID in each B cell subset (P. chabaudi infected in black, RBC control in gray histograms) (n = 3 in two independent experiments).
Mentions: Previous studies performed with AID‐GFP mice demonstrated that AID expression was restricted to germinal center B cells following injection with NP‐CGG and alum, a model antigen and adjuvant commonly used to elicit a humoral immune response 19. P. chabaudi is a productive infection with a higher antigen load and inflammatory response than a model antigen injection such as NP‐CGG. Therefore, we used AID‐GFP mice in the P. chabaudi model to determine whether AID can be expressed in B cells outside of the germinal center. We performed RT‐qPCR on sections of whole spleen at 6, 12, 17, 24, and 30 days post‐infection (dpi) with P. chabaudi and found that the peak of AID expression occurred at 17 dpi (data not shown). The 17‐day time point was then used to determine the percentage of AID positive cells within the T1, T2, T3, marginal zone, follicular, and germinal center B cell subsets by multivariate flow cytometry. At 17 dpi, we found dramatic differences in splenic B cell subset distribution, including a significant decrease in immature and marginal zone B cells, and a large expansion of the germinal center B cell population (Fig. 2), which were consistent with previously reported studies 20. We determined the percentage of AID expressing cells from the immature T1 (IgM+CD23−), T2 (IgM+CD23+), and T3 (IgM‐CD23+) B cell subsets in mice 17 dpi or control mice injected with uninfected RBC. All three immature B cell populations had detectable AID expression 17 days after P. chabaudi infection, and the control mice had little or no AID expressed (Fig. 2). In addition to the CD93+ population of immature B cells, the P. chabaudi‐infected mice had an additional CD93high population of cells that were AID− (Fig. 2). Germinal center B cells (CD38‐PNAhigh) were greatly expanded during P. chabaudi infection and >80% expressed AID (Fig. 2). The control germinal center B cells also expressed AID, but at a lower frequency (Fig. 2). During acute P. chabaudi, the percentage of marginal zone B cells decreased dramatically 19; our study confirmed this finding and demonstrated the marginal zone B cell population that remained included cells that expressed AID (Fig. 2). Follicular B cells in both the P. chabaudi‐infected and control mice did not express AID (Fig. 2). The expansion of CD23− CD21− B cells is consistent with reports of age associated B cells (ABCs) that expand during infection 21, 22. Interestingly, the ABCs had low expression of AID despite their induction during infection. These data demonstrate that during P. chabaudi infection, the expression of AID is not restricted to the germinal center B cells, but is also expressed in transitional and marginal zone B cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Endemic Burkitt's lymphoma (eBL) is associated with Epstein–Barr virus and repeated malaria infections. A defining feature of eBL is the translocation of the c‐myc oncogene to the control of the immunoglobulin promoter. Activation‐induced cytidine deaminase (AID) has been shown to be critical for this translocation. Malaria infection induces AID in germinal center B cells, but whether malaria infection more broadly affects AID activation in extrafollicular B cells is unknown.

Methods: We either stimulated purified B cells from AID‐green fluorescence protein (GFP) reporter mice or infected AID‐GFP mice with Plasmodium chabaudi, AID fluorescence was monitored in B cell subsets by flow cytometry.

Results: In vitro analysis of B cells from these mice revealed that CpG (a Toll‐like receptor 9 ligand) was a potent inducer of AID in both mature and immature B cell subsets. Infection of AID‐GFP mice with Plasmodium chabaudi demonstrated that AID expression occurs in transitional and marginal zone B cells during acute malaria infection. Transitional B cells were also capable of differentiating into antibody secreting cells when stimulated in vitro with CpG when isolated from a P. chabaudi‐infected mouse.

Conclusions: These data suggest that P. chabaudi is capable of inducing AID expression in B cell subsets that do not participate in the germinal center reaction, suggesting an alternative role for malaria in the etiology of eBL.

No MeSH data available.


Related in: MedlinePlus