Crosstalk between nitric oxide (NO) and carbon monoxide (CO) controls the
pathogenesis of cerebral malaria
1. Summary
The central hypothesis to be tested under this project was that the gasotransmitters nitric
oxide (NO) and carbon monoxide (CO) interact functionally to modulate the pathogenesis of
cerebral malaria. This hypothesis was strongly supported by the observation that NO can
induce the expression of heme oxygenase-1 (HO-1), an enzyme that produces CO through
heme catabolism and that suppresses the onset of cerebral malaria in mice (1, 2). Moreover,
there was also evidence that CO controls the activity of NO synthases, thus modulating NO
production. Dr. Viktória Jeney has demonstrated unequivocally that both CO and NO can
provide host tolerance against Plasmodium infection, that is, these gasotransmitters provide a
survival advantage against malaria without interfering with parasite burden. We also found
that that the protective effect of NO depends strictly on the induction of the heme
catabolyzing enzyme HO-1, via activation of the transcription factor transcription factor
nuclear factor erythroid 2-related factor (Nrf2). The same protective mechanism, involving
Nrf2 and HO-1, explains why sickle hemoglobin confers host tolerance to plasmodium
infection (2).
2. Progress of training activities
Dr. Viktória Jeney started this project with no previous experience in animal experimentation
and as such the main training objective was that Dr. Viktória Jeney acquired competence to
perform animal studies including, ethical considerations, experimental design and execution
and data interpretation. The project was designed to take advantage of well-established rodent
model of a specific inflammatory disease, i.e. cerebral malaria (1). It was planned that Dr.
Viktória Jeney learns how to use gaseous molecules (nitric oxide; NO and carbon monoxide;
CO) delivery systems in rodents. In addition we planned that she will acquire experience in
flow cytometry, immunohistochemistry, qRT-PCR based RNA/cDNA arrays, and breeding
and maintenance of mouse colonies. All these objectives were fully achieved. Briefly, Dr.
Viktória Jeney bred mice deficient for different NO synthase (Nos1-/-, Nos2-/-, Nos3-/-) and
established mouse colonies to produce double deficient (Nos1/2-/-, Nos2/3-/-, Nos1/3-/-) mice.
She has also bred mice deficient for transcription factor nuclear factor erythroid 2-related
factor (Nrf2) and for the heme catabolizing enzyme heme oxygnease-1 (HO-1). Dr. Viktória
Jeney performed over 30 animal involving approximately 400 mice. In some of the
experiments Dr. Viktória Jeney used gas delivery systems for NO and CO, flow cytometry to
monitor parasitemia and analyzed leukocytes infiltrating the central nervous system. She used
qRT-PCR to monitor the expression of several genes in the central nervous system.
Dr. Viktória Jeney completed a “Practical Course on Animal Handling and
Experimentation on Rodent and Fish“ organized by the Instituto Gulbenkian de Ciência
according to the FELASA (Federation of European Laboratory Animal Science Associations)
recommendations. She also attended a “Practical Course of Flow Cytometry” organized by
the Instituto Gulbenkian de Ciência. The fellow’s communicating skills, e.g. writing grant
proposals and scientific publications as well as project management skills, laboratory and
personnel management competencies, were improved. Dr. Viktória Jeney has attended the
course “Effective writing for Life Science Research and Grant Proposals” co-organized by
Instituto de Medicina Molecular and Instituto Gulbenkian de Ciência.
3. Experimental work progress
3.1. Introduction.
Pre-emptive induction of effector immunity, such as achieved through vaccination has been
extremely successful in conferring protection against a wide variety of infectious diseases.
1
Unexpectedly however, the same approach has failed, so far, to yield significant protection
against “ancestral” pathogens such as Plasmodium, the causative agent of malaria.
Presumably, Plasmodium co-evolved with its human host to reach an evolutionary trade-off in
which host immunity fails to clear infection without compromising its own viability.
However, if one considers that less than 1-2% of Plasmodium infected individuals, succumb
to severe forms of malaria, it is reasonable to assume that alternative host defense strategies,
presumably acting independently of protective immunity, might have evolved as to limit host
morbidity/mortality associated with Plasmodium infection (4). This is in keeping with the
notion that an infected host has, not one, but two distinct evolutionary conserved strategies to
limit disease severity. While the first relies on reducing pathogen load, i.e. resistance to
infection, the other limits tissue damage irrespectively of pathogen load, i.e. tolerance to
infection (5, 6). Mice can develop tolerance to Plasmodium infection (4), the causative agent
of malaria (7), via mechanism that we have shown to rely on the expression of heme
oxygenase-1 (HO-1)(8), a stress-responsive heme catabolizing enzyme that confers host
protection to infection irrespectively of pathogen load (9, 10).
During the blood stage of Plasmodium infection, red blood cells release into the
circulation significant amounts of intact host hemoglobin, from which their heme prosthetic
groups are released upon hemoglobin oxidation (1, 11). The protein-free heme generated in
this manner contributes in a critical manner to the development of experimental cerebral
malaria in mice (1, 11), a lethal form of severe malaria that resembles cerebral malaria, as it
occur in P. falciparum infected children (12). The protective effect of HO-1, against
experimental cerebral malaria, relies on the production of carbon monoxide (CO) via the
catabolism of heme (1, 11). There is another gasotransmitter, namely, nitric oxide (NO)
produced from L-arginine by NO synthases (NOS) -1, -2 and -3 (13), that can afford similar
protective effects against experimental cerebral malaria, via a mechanism that remains to be
established.
3.2. Introduction.
The central hypothesis to be tested under this project was that NO and CO, CO and NO
interact functionally to modulate the pathogenesis of experimental cerebral malaria. This
hypothesis was strongly supported by the observation that NO can induce the expression of
HO-1 and modulate inflammation as well as cell survival (3, 14-16). Moreover, CO also
controls the activity of NO synthases, thus modulating NO production (3).
We asked whether generation of CO by HO-1 acts as part of a so-called “therapeutic
amplification funnel” (17, 18), via which the gasotransmitter NO suppresses the pathogenesis
of cerebral malaria in mice (1, 11). To test this hypothesis we used mice infected with
Plasmodium berghei ANKA, a well-recognized model of cerebral malaria in which mice
show neurological dysfunction and succumb between day 6 and 10 post-infection (11). When
applied, starting the day before infection until day 6 post-infection, NO protected mice against
experimental cerebral malaria, i.e. 80% survival. In contrast, when applied 3 days postinfection, NO failed to protect mice from experimental cerebral malaria, i.e. 0% survival. This
suggests that the survival advantage provided by NO might rely on a “pre-sensitization“
phenomenon, presumably involving the expression of protective genes that prevent the
deleterious effects associated with Plasmodium infection.
In the presence of free radicals NO can generate peroxynitrate and hydroxyl radicals
that induce Hmox1 transcription, HO-1 protein expression and activity leading to the
production of CO via heme catabolism (19). Therefore, we tested whether the protective
effect of NO, against experimental cerebral malaria, acts in a “CO-dependent” manner, that is,
through the induction of HO-1 and the production of CO. We found that NO induces the
expression of Hmox1 mRNA in the spleen, kidney, liver, heart and circulating white blood
cells. More importantly perhaps, the survival advantage provided by NO in wild-type mice,
i.e. 80% survival, was abrogated in mice lackinh HO-1 (Hmox1-/-) mice, i.e. 0% survival. It is
well established that NO drives HO-1 expression mainly at the transcriptional level, through
activation of the transcription factor nuclear factor erythroid 2-related factor (Nrf2) (20). We
tested whether NO can rescue Nrf2 deficient (Nrf2-/-) mice from developing experimental
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cerebral malaria. We found that, in contrast to wild type mice, NO failed to provide a survival
advantage against Plasmodium infection in Nrf2-/- mice, i.e. 0% survival. We conclude that
the protective effect of NO relies on the induction of HO-1 vai Nrf2 activation, leading to the
production of CO.
We also tested whether CO arrests the pathogenesis of experimental cerebral malaria
via a NO dependent mechanism (13). We tested this hypothesis by assessing whether CO
requires the expression of different NOS isoforms to arrest the pathogenesis of experimental
cerebral malaria, using mice lacking one or two NOS isoforms, i.e. Nos1-/-, Nos2-/-, Nos3-/-,
Nos1/2-/-, Nos2/3-/-, Nos1/3-/-. We found that, in a similar manner to wild-type C57BL/6 mice,
when CO is applied between day 3 and 6 post-infection at a dose of 250 parts per million
(PPM), single and double NOS-deficient C57BL/6 mice were rescued by CO, i.e. >80%
survival after Plasmodium infection.
We have also investigated under this project the mechanism of action of a related
“pre-sensitization“ phenomenon well know to afford protection against Plasmodium
infection. Briefly, several point mutations in the -chain of human Hb, e.g. HbS (6Glu>Val)(21, 22), HbC (6Glu->Lys)(23) and HbE (26Glu->Lys)(24), can confer a survival
advantage to Plasmodium infection (25). When present in the homozygous form, some of
these mutations, e.g. HbS, become pathologic causing hemolytic anemia, leading to
accumulation of high levels of cell-free Hb and heme in plasma (26). Individuals carrying the
HbS mutation in the heterozygous form, i.e. the A/S sickle cell trait, also accumulate low
(non-pathologic) levels of heme in plasma (27) and are protected against malaria (22, 25, 28,
29). Given that expression of HO-1 (30, 31) and production of CO (32) are induced in
individuals carrying the HbS mutation we hypothesized that this might explain why
individuals carrying the A/S sickle cell trait have a survival advantage against malaria (21, 29,
33). We found that mice expressing sickle Hb do not succumb to experimental cerebral
malaria (2), irrespectively of parasite load, revealing that sickle Hb confers host tolerance to
Plasmodium infection (2). Sickle Hb induces the expression of HO-1 in hematopoietic cells,
via a mechanism involving the transcription factor Nrf2, leading to the production of CO that
suppresses the pathogenesis of experimental cerebral malaria (2).
In conclusion we have demonstrated that both CO and NO can provide host tolerance
against Plasmodium infection, that is, these gasotransmitters provide a survival advantage
against malaria without interfering with parasite burden. We also found that the protective
effect of NO depends on the induction of the heme catabolyzing enzyme, HO-1 via activation
of the transcription factor Nrf2. While this protective mechanism explains why sickle Hb
confers host tolerance to severe forms of malaria, whether the protective effect of sickle Hb
involves the production of NO remains to be established.
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