A multifactorial role for P. falciparum malaria in endemic Burkitt\u27s lymphoma pathogenesis

Endemic Burkitt\u27s lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria

Germinal Center B Cells Latently Infected with Epstein-Barr Virus Proliferate Extensively but Do Not Increase in Number▿

In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo

How <i>P</i>. <i>falciparum</i> increases the risk of endemic Burkitt’s lymphoma.

<p>Essentially all adults are persistently infected with EBV (A). As a consequence, newly infected B cells are continually being produced that transit the GC on their way to becoming latently infected memory B cells (the site of viral persistence) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref014" target="_blank">14</a>]. Malaria is immunosuppressive (B) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref016" target="_blank">16</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005331#ppat.1005331.ref017" target="_blank">17</a>], and Torgbor et al. have shown that this results in a highly elevated throughput of EBV-infected cells in the GC (C). Torgbor et al. also showed that <i>P</i>. <i>falciparum</i> induces deregulated expression of the DNA-mutating and -cutting enzyme AID in GC cells (D). Robbiani et al. subsequently showed in a mouse model that this deregulated expression led to DNA damage, translocations, and, ultimately, lymphoma (E). Thus, infection with <i>P</i>. <i>falciparum</i> has been shown to have two effects on the GC, where eBL originates. Together, these increase the risk that a GC cell will undergo a c-myc translocation and that this cell will also be EBV-infected and, therefore, able to tolerate the translocation, synergistically increasing the likelihood that eBL will arise.</p

A Multifactorial Role for <i>P. falciparum</i> Malaria in Endemic Burkitt's Lymphoma Pathogenesis

<div><p>Endemic Burkitt's lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of <i>P. falciparum</i> malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that <i>P. falciparum</i> targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria.</p></div

c-myc is expressed in GC B cells.

<p>A. Flow cytometric analysis demonstrating c-myc expression in tonsil GC cells (CD10+). The arrow indicate the CD10+, c-myc positive GC population. B. Western blot analysis confirming c-myc expression in 3 independent tonsil GC B cell preparation (GC1-3). Raji and Rael are EBV positive BL cell lines. The molecular weight in KD is shown to the left. C. ImageStream analysis of c-myc positive tonsil GC B cells. Staining for a known nuclear protein bcl-6 is shown for comparison. N.B. For this study only Boston control tonsils were used.</p

<i>P. falciparum</i> extracts stimulate expression of AID protein in normal tonsil B cells.

<p>The same protocol was followed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004170#ppat-1004170-g001" target="_blank">Figure 1</a>, except the cells were analyzed by staining for AID protein expression and FACS analysis after a 5 day incubation period. - Flow cytometry histograms of cell populations demonstrate equivalent levels of AID expression in B cells activated by CpG or <i>P. falciparum</i> extracts. MFI  =  Mean fluorescence intensity. A. and C. Percentage of all B cells expressing AID (B) and percent of B cells expressing high levels of AID (C) suggest equal levels of AID expression in B cells activated by CpG or <i>P. falciparum</i> extract. We observed two overlapping populations of cells staining positive for AID. An arbitrary gate imposed on the brighter population defined high level expressers. The same gate was applied to all samples.</p

Hemozoin is taken up by B cells and activates AID expression.

<p>A. Hemozoin (sHz) (red) and CpG (green) were incubated with two different B cell lines. Note presence of red granules inside both cell types. BL2 is an EBV negative BL line and IM171 a spontaneous EBV positive lymphoblastoid line. B. Hemozoin stimulates expression of AID mRNA to an equivalent level to that obtained with surface Ig cross-linking and CpG. The stimulation was independent of hemozoin being complexed with parasite DNA. The same protocol was followed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004170#ppat-1004170-g001" target="_blank">Figure 1</a>. The cells were analyzed after a 5 day incubation period.</p

Higher levels of EBV infected cells in tonsil GCs from individuals infected with <i>P. falciparum</i> malaria compared to controls.

<p>A. The percentage of B cells (CD19+) is unchanged. (ns p = 0.18). B. The percentage of GC B cells (CD10+) is significantly elevated in the malaria tonsils (*** p<0.001). C. The frequency of GC B cells latently infected with EBV is dramatically increased in the malaria tonsils (*** p = 0.001). For details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004170#ppat-1004170-t001" target="_blank">Table 1</a>. D. The level of EBV infected GC B cells and AID expression do not directly correlate.</p