Dysregulation of the haem-haemopexin axis is associated with severe malaria in a case–control study of Ugandan children

SM is a complex, multi-organ syndrome that is poorly understood. This study provides
evidence that increased generation of plasma haem, combined with lower levels of haemopexin
that normally binds and clears haem, are associated with both SMA and CM in Ugandan
children. Furthermore, decreased or depleted levels of haemopexin, and increased plasma
haem, are associated with a worse outcome in a pre-clinical model of CM. These observations
support the hypothesis that haem-induced pathology, exacerbated by decreased levels
of haemopexin, may be a pathway of injury contributing to disease severity and death
in malaria infections.

The observed median plasma levels of haemin in children with CM and SMA were ten-fold
or higher than that observed in UM 12], and equivalent to those reported in haemolytic disorders such as sickle cell disease
4], where haem-induced vasculopathy is a frequent cause of organ dysfunction and mortality
8]. Although there were no differences in cell-free haemoglobin levels between groups,
given the 11–15-fold higher levels of plasma haemin observed in children with SMA
and CM and that cell-free haemoglobin generates haem, it is likely that there were
also differences in levels of plasma haemoglobin during the course of infection, but
the timing of sample collection did not capture these events.

Previously it has been shown that plasma levels of haem are higher on day 6 post infection
in the ECM-susceptible C57BL/6 mice compared to ECM-resistant BALB/c mice 13]. Injections of haemin into resistant BALB/c mice infected with P. berghei ANKA resulted in increased fatalities compared to controls (100 vs 0 %) 13], 14], suggesting that haem may play an important role in mediating, or enhancing, disease
severity. While a role for haem has previously been implicated in ECM, it had yet
to be confirmed in informative human populations. Recently, Dalko et al. showed that
adults with SM in India had elevated levels of plasma haem compared to those with
mild malaria, which were both higher than levels measured in endemic controls 15]. These findings were extended in this study to show an association between increased
haem and disease severity in African children with SM compared to those with UM.

It is hypothesized that haem is mediating adverse outcomes during malaria infection
by inducing endothelial dysfunction and microvascular injury. Haem is implicated,
directly or indirectly, as a key mediator of endothelial dysfunction by a variety
of pathways. Plasma haem is a major source of reactive oxygen species (ROS) and subsequent
pro-oxidant stress on endothelium 8]. Haem also induces activation of the endothelium by: inducing Weibel-Palade body
exocytosis and the release of their contents, including the release of von Willebrand
factor (vWF), p-selectin 4], 6], and Ang-2, and increasing expression of endothelial adhesion markers that bind both
inflammatory cells and parasitized erythrocytes 6]. Haem is also a pro-inflammatory molecule that can stimulate complement activation
4] and the release of pro-inflammatory cytokines from monocytes/macrophages 16]. Haem-induced endothelial injury may be further exacerbated by decreases in bioavailable
nitric oxide. Cell-free haemoglobin in addition to generating plasma haem, is a potent
scavenger of nitric oxide, further amplifying endothelial dysfunction 8], 17]. Many of these pathways induced by haem are also implicated in the pathogenesis of
SM 3], 17]–23]. Further prospective studies are required to define which of the above pathways are
induced by haem in the context of SM.

The toxic effects of cell-free haemoglobin and haem are normally mitigated by high
affinity-binding to haptoglobin and haemopexin, respectively, followed by transport
and degradation. Haem is degraded by haem oxygenase-1 resulting in the generation
of biliverdin and carbon monoxide which have anti-inflammatory and endothelial stabilizing
effects 8]. Therefore, the excess generation of haemin observed in children with CM and SMA
appears to be further complicated by a relative deficiency in circulating haemopexin.
Previous studies have primarily focused on levels of haptoglobin 12], 24]–26] or haemopexin 24]–26] in UM. However, it was recently shown that adults with SM in India had decreased
levels of haemopexin compared to those with mild malaria 15]. Similarly, in this study it was observed that children with SMA had significantly
lower levels of both haemopexin and haptoglobin, in agreement with a previous report
25]. This study extends these previous findings and reports that African children with
CM also have lower levels of both haemopexin and haptoglobin, suggesting a common
pathway of endothelial injury in both CM and SMA, and in both adults and children
with severe disease. Despite the markedly low levels of these protective proteins
in children with CM or SMA, there were no observed differences in either haemopexin
or haptoglobin levels between children who survived or died of the infection. These
data suggest that these proteins are already depleted during severe infection. Whether
these relative deficiencies are due to consumption or due to a decreased capacity
to synthesize these proteins in response to haemolysis is unknown. Nonetheless, these
low levels would be expected to further compromise clearance and degradation of haem
and haemoglobin, and contribute to worse clinical outcomes, as was illustrated by
the observed higher plasma levels of haemin and cell-free haemoglobin in children
who subsequently died of malaria.

Due to the associations observed in children with CM or SMA, a potential mechanistic
role for haemopexin was investigated using a pre-clinical model of ECM. As with all
animal models, there are inherent limitations. However, there is evidence that this
model shares several features, but not all, with human malaria 27]–32]. Pre-clinical models enable investigation of mechanistic questions that are difficult
to safely or ethically address in human populations and can provide direct genetic
evidence that the haem axis is causally involved in severe and fatal infection.

Similar to this patient population, mice that were more susceptible to ECM had significantly
lower haemopexin levels compared to more resistant mice, despite comparable parasite
burdens. Furthermore, mice with targeted deletion of the hpx gene, and no measurable circulating haemopexin, were more susceptible to ECM than
their wild-type littermates suggesting that haemopexin is playing a mechanistic role
during malaria infection. The increased susceptibility in the Hpx KO animals was associated
with increased levels of plasma haemin prior to the onset of ECM, further implicating
a role for plasma haem.

A dose response to ECM was predicted due to the gene dosing effect observed in these
mice; however, both the haemopexin-null and heterozygous mice responded similarly
to ECM. The haemopexin-null mice appear to compensate for their lack of haemopexin
by increasing the production of haptoglobin. The haemopexin-null mice have higher
baseline levels of haptoglobin compared to both the wild type and heterozygous mice.
When infected with P. berghei ANKA, haemopexin-deficient mice further upregulate circulating haptoglobin to at
least 1300 times the levels seen in either the wild type or heterozygous mice. This
compensation in a protein upstream of haemopexin in the haemolytic pathway is hypothesized
to mitigate the severity of the observed phenotype. The haemopexin heterozygous mice
may more accurately reflect the impact of haemopexin deficiency in ECM due to their
inability to compensate with increased levels of haptoglobin to the levels observed
in haemopexin-null mice. It would be of interest to further explore the phenotype
of a double haemopexin, haptoglobin knockout mouse in the ECM model.

Despite the marked increase in haptoglobin, haemopexin-null mice are still more susceptible
than their wild-type counterparts. This is likely attributable to malaria-induced
generation of plasma haemoglobin and haem that overwhelms both haptoglobin and/or
haemopexin pathways, as demonstrated by significantly higher levels of plasma haem
prior to the onset of ECM in the KO animals. Alternatively, haemopexin may provide
additional protection independent of its ability to clear haem from the circulation.
Haemopexin has been shown to exert haem-independent, anti-inflammatory effects by
decreasing the release of pro-inflammatory cytokines from lipopolysaccharide (LPS)
stimulated macrophages 33], and decreased differentiation of TH17 cells in a model of experimental autoimmune
encephalitis 34].

Collectively, the above observations suggest that malaria-induced haem, and corresponding
haemopexin deficiency may represent a component of disease pathogenesis in which haem
amplifies complement activation, pro-coagulant activity and endothelial injury, culminating
in microangiopathy, multi-organ dysfunction and adverse clinical outcomes. The recent
observation that both cases of CM and SMA are associated with parasitized erythrocytes
that bind to endothelial protein C receptor (EPCR) lends further support to this hypothesis
35]. Binding to EPCR may disrupt the anti-inflammatory and endothelial barrier effects
of activated protein C (APC), resulting in enhanced pro-coagulant activity, complement
activation and endothelial injury. Of note, APC mediates its endothelial cytoprotective
and barrier protective activities via the Ang-Tie2 pathway 36], which is dysregulated in SM 37]. Moreover, since parasitized erythrocyte sequestration focuses parasite burdens and
erythrocyte haemolysis directly onto the microvasculature of vital organs, this would
be expected to result in higher local haem concentrations in the cerebral microvascular
bed, than those observed in the peripheral circulation.

Similar to atypical haemolytic uremic syndrome (aHUS) associated with infection, where
haem-induced complement activation is a proposed common pathway of microvascular injury
and organ failure, severe disease does not occur in all infected individuals, but
rather only in those with genetic or acquired defects in complement regulation 4]. This suggests that similar or related susceptibility determinants, including defects
in haemopexin expression or haem metabolism, may contribute to the onset and outcome
of severe malarial syndromes.