Introduction
The link between Plasmodium falciparum malaria and endemic Burkitt’s lymphoma (eBL)
has been an enigma for more than 50 years, since it was first observed that the occurrence
of the two coincided [1,2]. So convincing was the association that it led to the prediction
that eBL was caused by an infectious agent that was spread by malarial mosquitoes.
The subsequent search turned up the first human oncogenic virus, Epstein-Barr virus
(EBV) [3,4]. EBV is found in nearly all cases of eBL [5] and has been shown to be
a potent transforming virus for human B cells [4]. eBL is believed to arise from germinal
center (GC) B cells [6] and is characterized by a typical translocation of the c-myc
oncogene into one of the immunoglobulin loci [7,8]. The subsequent deregulation of
c-myc expression would normally lead to rapid apoptosis of the cell, but the presence
of EBV is thought to rescue these cells and allow them to survive [9]. The enigma
remained, however, because EBV is not spread by mosquitoes. eBL therefore represents
an intriguing and unusual situation in which interactions between a protozoan parasite
(P. falciparum) and a mammalian virus (EBV) combine to cause cancer. While the mechanisms
linking EBV to lymphoma development are becoming better understood, the link between
P. falciparum malaria and eBL has remained completely unexplained until now. Finally,
we have a breakthrough in this longstanding issue from two separate studies: one through
investigation of human tissues and one in a mouse model.
A Role for Activation-Induced Cytidine Deaminase (AID)
The first breakthrough occurred with the demonstration that P. falciparum induces
the DNA-mutating and double-strand-breaking enzyme activation-induced cytidine deaminase
(AID) [10]. This is the enzyme that is normally responsible for the somatic hypermutation
and class-switch recombination of immunoglobulin genes that occur in B cells when
they enter the GC [11]. However, AID is also known to be somewhat promiscuous and
occasionally mutate off targets, including oncogenes [12]. Usually, these mutations
are repaired, but when AID expression is deregulated in mouse models, it becomes a
risk factor for lymphoma development, including the c-myc translocation characteristic
of eBL [13]. The study by Torgbor and colleagues utilized a unique sample of primary
human tissues, specifically tonsils, obtained from individuals either chronically
infected or uninfected with P. falciparum. This enabled them to isolate GC B cells
directly from individuals at risk for developing eBL. They went on to show that individuals
chronically infected with P. falciparum malaria had higher numbers of GC B cells that
expressed elevated levels of AID and, furthermore, had an extremely high level of
GC B cells latently infected with EBV [10]. To explain the mechanism behind the induction
of AID in the tonsils of individuals with malaria, the authors observed that extracts
from P. falciparum-infected red blood cells directly caused a strong activation of
AID in tonsil B cells in vitro. They additionally showed that this was due, at least
in part, to the action of hemozoin, the metabolic product of hemoglobin digestion
by P. falciparum parasites. Together, these results allowed the investigators to conclude
that P. falciparum infection increases two major risk factors for lymphoma development.
The first is induction of AID in GC B cells, putting them at increased risk for a
translocation; the second is a much higher frequency of EBV-infected cells in the
GC, increasing the chances that the translocation will occur in a cell that will tolerate
it (Fig 1). The increased combinatorial risk of these two events explains the increased
prevalence of eBL in P. falciparum-endemic areas, but many questions remain. These
include why eBL is specifically linked only with P. falciparum and not the other species
that cause malaria in humans (discussed in detail below), the origins of sporadic
BL (which is not linked with P. falciparum and is frequently EBV-negative), why eBL
is predominantly a cancer of children, and why the tumor is located in very specific
anatomical regions that tend to change with age (the jaw in children and the abdomen
in adults). Nevertheless, these experiments represent an important breakthrough in
our understanding of how P. falciparum and EBV interact to cause eBL. However, the
limitations inherent in working with human populations and the lack of an experimental
animal model of the disease has hindered a more detailed investigation.
10.1371/journal.ppat.1005331.g001
Fig 1
How P. falciparum increases the risk of endemic Burkitt’s lymphoma.
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) [14]. Malaria
is immunosuppressive (B) [16,17], 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 P. falciparum 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 P. falciparum 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.
A Mouse Model for eBL
The study described by Torgbor et al. predicted that P. falciparum infection would
lead directly to AID-dependent DNA damage and translocations in the GC, ultimately
producing lymphoma—a prediction impossible to test in an actual human infection. This
prediction has now been confirmed by employing the P. chabaudi mouse model of malaria
[14]. In this study, Robbiani et al. showed that mice repeatedly infected with this
species of malaria parasite develop prolonged expansion of GCs in which the B cells
undergo rapid expansion and express AID—exactly what was seen in the human infection
with P. falciparum. These authors went on to show that this resulted in the accumulation
of widespread DNA damage in the GC cells, including translocations, which was associated
with subsequent lymphoma development, but only when expressed on a p53-null background.
One might speculate that the p53 knockout essentially complemented the lack of EBV
infection in the mice and in the GC B cells (but see caveat below). What was lacking
in this study was evidence that the rodent parasite P. chabaudi was in any way an
accurate model for P. falciparum and, specifically, that the rapid expansion of GC
B cells and induction of AID expression are actually seen in the human disease. This
was conveniently provided by the Torgbor et al. study.
The Specificity of eBL for Infection by P. falciparum—Limitations of the Mouse Model
While the mouse studies provide a potential animal model for eBL, there remain serious
concerns, most notably with respect to specificity. Human malaria is caused by five
different species of Plasmodium; however, eBL is specifically associated with P. falciparum
and is primarily observed in regions of the world endemic for this species of parasite,
namely sub-Saharan Africa and Papua New Guinea. While P. falciparum generally causes
a more severe form of malaria, all species can cause chronic infections, and, thus,
the reason for its specific association with eBL remains unanswered. Given this specificity,
it is reasonable to question if P. chabaudi is an accurate model of P. falciparum
infection. The Torgbor et al. study implicated hemozoin as playing a role in inducing
AID expression in GC B cells through Toll-like receptor (TLR) signaling, but this
mechanism was not tested in the mouse study. Furthermore, hemozoin alone was not as
effective as complete extracts obtained from P. falciparum-infected red blood cells,
suggesting other components also play a role.
Recent studies have implicated the P. falciparum-specific protein PfEMP1 in polyclonal
B cell activation and increased survival, leading the authors to propose a possible
link to eBL [15]. PfEMP1 is an antigenic protein expressed on the surface of infected
red blood cells that is unique to P. falciparum; thus, if PfEMP1 plays a key role
in eBL, it could explain the specificity of the disease to P. falciparum. Like all
Plasmodium strains, the rodent parasites, including P. chabaudi, express antigenic
proteins on the infected red blood cell surface, but they are unrelated to PfEMP1.
If it can be shown that PfEMP1 does indeed play an important role in the development
of eBL, it would raise significant questions about the specificity of the mouse model.
A second concern with the mouse model is that it was necessary to perform the infection
on a p53-/- background. The p53-/- strain alone produces lymphoma, and infection with
P. chabaudi did not increase the incidence of lymphoma but only altered the tumors
to a more mature phenotype. This is in contrast with P. falciparum, for which compelling
epidemiological evidence has demonstrated that it dramatically increases the occurrence
of eBL. Taken together, these observations suggest the mouse model may mimic only
part of the human disease and lacks the specificity associated with P. falciparum.
Perspectives
In summary, therefore, two recent, very complementary papers finally provide the missing
link between infection with P. falciparum malaria and the development of eBL. The
first paper [10] demonstrated that the human pathogen actually induces AID expression
in the disease setting, but it could only speculate on the link between this induction
of AID and the DNA damage leading to lymphoma. The subsequent paper [14] extended
this result to show, in a mouse model, that AID induced by malaria was indeed a risk
factor for DNA damage and lymphoma, but it could only speculate that this mechanism
actually occurred in and was directly related to the human disease. Taken together,
these two independent studies demonstrate the power of combining the direct study
of human disease to validate animal models that can then be used to develop a more
detailed understanding. The first significant insights into this 50-year-old enigma
have finally come to light—P. falciparum malaria is a risk factor for eBL because
it drives a high throughput of EBV-infected cells through the GC, where it also deregulates
AID, leading to DNA damage, c-myc translocations, and lymphoma (Fig 1).