Suppression of Experimental Cerebral Malaria by Heme Oxygenase-1 and Carbon Monoxide

Expression of the heme-degrading enzyme heme oxygenase-1 (HO-1) in response to malaria infection prevents the incidence of experimental cerebral malaria (ECM) in mice, a neurological syndrome that resembles human CM, the main cause of death from malaria. Carbon monoxide (CO), an end-product of heme catabolism by HO-1 mimics this protective effect and can be used therapeutically to prevent the lethal outcome of ECM.

Ana Pamplona1,2, Cristina D. Rodrigues1,2, ngelo Chora1, Sabrina Epiphanio1,2, Margarida CunhaRodrigues1,2, Miguel P. Soares1* and Maria M. Mota1,2*

Instituto Gulbenkian de Cincia, 2780-156 Oeiras, Portugal Unidade de Malria, Instituto de Medicina Molecular, Universidade de Lisboa, 1649-028 Lisboa,

Portugal.

*Both authors contributed equally to this work

To whom correspondence should be addressed. Email: mmota@fm.ul.pt.

Malaria causes more then one million deaths every year, mainly due to the occurrence of cerebral malaria (CM). We demonstrate hereby that expression of the heme-degrading enzyme heme oxygenase-1 (HO-1) in the host suppresses the development of experimental CM (ECM) that occurs in Plasmodium berghei ANKA infected mice, an experimental model of human CM. Carbon monoxide (CO), an end-product of heme catabolism by HO-1, can be used therapeutically (250 parts per million) to suppress ECM. While not affecting parasitemia per se, CO suppresses the major pathologic events leading to CM including blood brain barrier leakage, micro-vascular congestion and expression of the pro-inflammatory cytokines lymphotoxin-, tumor necrosis factor- and interferon-.

Erythrocyte infection by Plasmodium, the causative agent of malaria, leads to extensive haemolysis of infected as well as non-infected erythrocytes with up to 60-80% of the haemoglobin contained within each infected erythrocytes being degraded (1). This process generates free heme, a prooxidant to the parasite (2) as well as to the host (3). The parasite has developed strategies to cope with heme, polymerizing it into hemozoin (1), while host cells up-regulate the expression of heme oxygenase-1 (HO-1), which catabolizes heme into Fe++, carbon monoxide (CO) and biliverdin (4). CO acts biologically to limit the deleterious effects of a variety of inflammatory conditions (reviewed in (5)). We hypothesized that CO might counter the pathogenesis of cerebral malaria (CM), a syndrome that can develop in the course of malaria infection and lead to neurological disturbances revealed by abnormal behaviour, impairment of consciousness, seizures and coma eventually leading to death (6). We tested this hypothesis in P. berghei ANKA infected mice, a well-established experimental model of malaria infection. When infected with P. berghei ANKA some mouse strains, e.g. C57BL/6, develop neurological disturbances revealed by hemi- or paraplegia, deviation of the head, tendency to roll over on stimulation, ataxia and convulsions, dying between days 6-9 post-infection (reviewed in (7)). Due to the many similarities with human

CM this neurological syndrome is referred as experimental cerebral malaria (ECM) (reviewed in (7)). There are mouse strains, e.g. BALB/c, that do not develop ECM when infected with P. berghei ANKA, dying from hyperprasitemia about 20 days post-infection (reviewed in (7, 8)) HO-1 expression was significantly up regulated in the brain of P. berghei ANKA infected BALB/c mice, as assessed at mRNA (n=3; p<0.01; days 6 versus 0) (Fig. 1A) and protein level (Fig.1B). HO-1 mRNA expression reached maximal levels at day 6 post-infection, decreasing thereafter until day 12, the last day analyzed (Fig. 1A). HO-1 expression increased steadily in the liver and lungs until day 12 post-infection, the last day analyzed (Fig. 1C). HO-1 expression explains why BALB/c mice did not develop ECM (00%, n=14) (Fig. 2A)(9). Contrasting with the lack of ECM incidence in wild type (ho-1+/+) or heterozygous (ho-1-/+) BALB/c mice (n=14) (Fig. 2A), HO-1 deficient (ho-1-/-) BALB/c mice infected with P. berghei died from ECM (66.70% incidence; n=6, p<0.001 versus wild-type; p<0.001 versus heterozygote) (Fig. 2A). Despite increased ECM incidence, ho-1-/-mice did not display higher levels of parasitemia, i.e. percentage of infected erythrocytes, before ECM incidence, as compared to ho-1+/+ or ho-1+/- mice (n=14) (Fig. 2B). This suggests that the protective effect of HO-1 does not rely on modulation of parasitemia. In two out of six ho-1-/- mice that did not succumb to ECM there was an apparent decrease in parasitemia between days 10 and 12 post-infection (the last day analyzed), as compared to ho-1/+/+ or ho-1+/- mice (Fig. 2B). However, due to the small number of ho-1-/- mice that survived infection (33%; 2/6) this decrease is not statically significant and its biological meaningfulness is therefore difficult to ascertain at this point. Consonant with this data, inhibition of HO-1 enzymatic activity by zinc protoporphyrin (ZnPPIX) also lead to death by ECM in 77.52.5% (n=9) of ho-1+/+ BALB/c mice (Fig. 2C). Control infected mice treated with cobalt protoporphyrin (CoPPIX), a protoporphyrin that induces HO-1 expression and activity, did not develop ECM (0% incidence, n=8) (Fig. 2C). Inhibition of HO activity by ZnPPIX did not influence parasitemia (Fig. 2D), suggesting again that the 3

protective effect of HO-1 does not rely on inhibition of parasitemia. CoPPIX delayed parasitemia significantly (p<0.05 versus PBS controls) between days 9-11 post-infection, an effect lost shortly after its administration was discontinued, i.e. day 9 post-infection (Fig. 2D). It is likely that as for other non-Fe porphyrins (10), CoPPIX might interfere with parasitemia by inhibiting -haematin polymerization in the parasite. Altogether, these results demonstrate that it is the ability to upregulate HO-1 expression following malaria infection that suppresses the development of ECM in BALB/c mice. In contrast with BALB/c, C57BL/6 mice develop ECM when infected with P. berghei ANKA, dying between days 6 and 9 after infection (9). ECM was associated with significant lower levels of HO-1 mRNA expression in C57BL/6 mice (n=3; p<0.01)(Fig. 3A) in the brain, as compared to BALB/c mice. We hypothesized that the lower HO-1 expression in C57BL/6 mice was responsible for an increase of ECM incidence. In support of this notion induction of HO-1 expression by CoPPIX (11) reduced ECM incidence in C57BL/6 mice to 1010% (n=10), as compared to control C57BL/6 mice treated with PBS (n=10) or ZnPPIX (n=10) in which ECM incidence remained at 1000% (Fig. 3B). Here too, CoPPIX led to a significant delay in parasitemia (p<0.05 versus PBS treated controls) (Fig. 3C), which as argued above may result from interference with parasite heme clearance, independently of HO-1 expression (10). As parasitemia was not drug-treated, all P. berghei ANKA infected mice protected from ECM succumbed from hyperparasitemia (>60%) later on infection. Congruous with the previous observations that CO can mimic the protective effects of HO1 in other experimental conditions (12), exposure of C57BL/6 mice, i.e. expressing low levels of HO-1 in the brain following P. berghei ANKA infection (Fig. 3A), to inhaled CO (250 parts per million, ppm) prevented the development of ECM (Fig. 3D-F). None of the C57BL/6 mice (n=10) exposed to CO, 3 days post-infection (250 ppm, 24-72h), developed ECM (Fig. 3D-F). The protective effect of CO was still observed (80% protection, n=10) when applied 4 (Fig. 3F) but not 4

5 days (data not shown) post-infection (250 ppm, 24h), i.e. 2 and 1 day before expected ECM incidence, respectively. No significant changes in parasitemia were noticed upon CO exposure (Fig. 3G), suggesting that, as with HO-1, the protective effect of CO does not rely on inhibition of parasitemia. This data suggests that at a dosage as low as 250 ppm, CO can be used therapeutically, i.e. after infection, to prevent death from ECM, even when applied as late as two days before the expected time of ECM incidence (Fig. 3E,F). CO had several salutary effects that could account for its protective effects. When infected by P. berghei ANKA, C57BL/6 mice developed blood brain barrier (BBB) leakage (Fig. 4A)(13, 14). CO (250 ppm; 24h, 3 days post-infection) suppressed BBB leakage (Fig. 4A), an effect that should contribute significantly to suppress the pathogenesis of ECM (13, 14). Development of ECM in C57BL/6 mice was associated with moderate to severe vascular occlusion in 58.413.6% of the brain micro-vasculature i.e. arterioles, capillaries and/or post capillary venules. Up to 79.210.3% of congested micro-vessels presented intra-luminal accumulation of infected erythrocyte and leukocyte (n=91 vessels analyzed; 4 mice) (Fig. 4B). CO (250 ppm; 24h; 3 days post-infection) reduced significantly the percentage of occluded microvessels, i.e. 248.3% (p<0.001 versus air control). Only 42.610.8% of these micro-vessels contained infected erythrocytes and/or leukocytes (n=250 vessels analyzed; 10 mice) (Fig. 4B). Non-infected mice had 10.812.5% of brain micro-vessels with low level of intra-vascular erythrocyte congestion and only 5.56.8% of those containing leukocytes (n=56 vessels analyzed; 4 mice) (Fig. 4B). This suggests that CO prevents brain micro-vascular congestion, a key pathologic feature of ECM (reviewed in (15)) (9). We tested whether CO would interfere with endothelial cell activation in a manner that could account for decreased micro-vascular congestion (Fig. 4B). Expression of intracellular adhesion molecule-1 (ICAM-1/CD54)(13) and presumably that of vascular cell adhesion molecule 1 (VCAM-1/CD106) promote the pathogenesis of ECM. As expected (13), ICAM-1 and VCAM-1 5

mRNA expression were significantly increased in the brains of C57BL/6 mice undergoing ECM (n=3, p<0.001 infected versus non-infected mice) (fig. S1A-D). ICAM-1 protein was detected by immunostaining on both endothelial cells and brain infiltrating leukocytes, while VCAM-1 expression was confined to the vascular endothelium (fig. S1A and C). CO (250 ppm; 24h; 3 days post-infection) did not inhibit the expression of these adhesion molecules (fig. S1B and D) (16). Expression of P-selectin, associated with vascular congestion and/or platelet aggregation, contributes to the pathogenesis of ECM (17). P-selectin expression in C57BL76 mice undergoing ECM was associated with brain vascular congestion (fig. S1E) (17). Expression of HO-1 can inhibit platelet aggregation (18), an effect mimicked by CO (12, 19). However, exposure to CO (250 ppm; 24h; 3 days post-infection) failed to inhibit P-selectin expression (fig. S1E). As expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-), lymphotoxin-alpha (LT-) and interferon-gamma (IFN-) is required for the pathogenesis of ECM (20-22)(reviewed in (15), we asked whether CO modulated their expression. TNF-, LT- and IFN- mRNA expression were significantly up-regulated in the brain of P. berghei ANKA infected C57BL/6 mice (n=3, TNF- p<0.001, IFN- p<0.001 and LT- p<0.05; day 6 post-infection versus non-infected mice) (Fig. 4C, D, E). Exposure to CO (250 ppm; 24h; day 3 post-infection) reduced significantly TNF- (539 %; n=3; p<0.005), LT- (4719%; n=3; p<0.005) and IFN- (329%; n=3; p<0.05) mRNA expression, as compared to air controls (Fig. 4C, D, E). Taking into account their critical role in the pathogenesis of ECM it is likely that the protective effect of CO is mediated at least in part via its ability to inhibit the expression of these pro-inflammatory cytokines. Understanding the pathogenesis of malaria is crucial for the development of efficient clinical intervention strategies. Adhesion of infected erythrocytes and leukocytes to the brain vascular endothelium (a process referred to as sequestration) has been suggested to be a major cause of pathology (23). As suggested by others, the host inflammatory response triggered by 6

malaria infection is an important pathologic event leading to CM as well (6). Our present data demonstrates that expression of HO-1 by the infected host suppresses vascular congestion and inflammation as well as the incidence of ECM (Fig. 2). The relative contribution of each of these factors to the suppression of ECM remains to be established. However, the observation that CO, an end-product of HO-1 activity, inhibited BBB leakage (Fig. 4A) and decreased the expression of key pro-inflammatory mediators implicated in the pathogenesis of ECM (20-22)(reviewed in (15)), i.e. TNF-, LT- and IFN-, suggests that it is the anti-inflammatory (Fig. 4)(24) and presumably the cytoprotective (3) effects of HO-1 that prevail in the protection against ECM. The inability of HO-1 (Fig. 2) or CO (Fig. 3) to inhibit parasitemia is also in keeping with the anti-inflammatory and/or cytoprotective effects of HO-1 and/or CO underlying the suppression of ECM. These observations may have important implications for the understanding of the pathogenesis of human CM. Expression of HO-1 has been shown to occur during human malaria infections (25, 26). Although ECM may not reflect fully the pathophysiology of human CM, it certainly allows to identify genes that control its pathogenesis, such as proven for the involvement of TNF- (27-30) or ICAM-1/CD54 (31, 32) in both ECM and human CM (reviewed in (33)). Thus, while our present data does not allow concluding that HO-1 is a susceptible gene for human CM, it is likely that variations in the level of HO-1 expression, known to occur in human populations (34), could dictate susceptibility to human CM. Taken together, and with the necessary caution when extrapolating from rodent models to human malaria infection, the data obtained hereby reveals that the pathogenesis of CM can be controlled via expression of a so called protective gene (34) in the host. While we have shown this unequivocally for HO-1 in the context of ECM we do not exclude that other protective genes that control the extent of inflammatory reactions (35) might act in a similar manner not only to this syndrome but other forms of severe malaria.

In conclusion, our findings provide a novel insight into the pathogenesis of ECM on the basis of which new therapeutic approaches based on modulation of HO-1 expression or the administration of end products of its enzymatic activity may be used therapeutically to overcome this disease.

Figure Legends: Fig. 1. HO-1 expression is up regulated during the course of P. berghei ANKA infection in BALB/c mice. (A) HO-1 mRNA was quantified by qRT-PCR in the brain of infected mice. Shown is mean number of HO-1 per HPRT mRNA molecules (x103)standard deviation (n=3 mice per plot). (B) Representative Western Blot analysis of HO-1 and -tubulin protein expression in the brain, 6 days post-infection (6) versus non-infected mice (0). (C) Quantification of HO-1 mRNA by qRT-PCR in the liver () and lungs () following infection. Shown is the mean number of HO-1 per HPRT mRNA molecules (x103)standard deviation (n=3 mice per plot).

Fig. 2 HO-1 activity is required to prevent death by ECM in BALB/c mice infected with P. berghei ANKA. (A, B) Survival (A) and parasitemia (B) of ho-1-/- (), ho-1+/- () and ho-1+/+ () infected mice. Meanstandard deviation (n=14 mice per plot for ho-1+/- and ho-1+/+, and n=6 mice per plot for ho-1-/-) is shown for parasitemias. (C, D) Effect of HO inhibition by ZnPPIX () or HO induction by CoPPIX () on survival (C) and parasitemia (D) infected mice. PBS () treated mice were used as controls. Meanstandard deviation (n=5 mice per plot) is shown for parasitemias.

Fig. 3 Induction of HO-1 expression/activity or exogenous CO protects C57BL/6 mice from death by ECM following P. berghei ANKA infection. (A) HO-1 mRNA was quantified by qRT-PCR in the brain of infected BALB/c and C57BL/6 mice, 6 days after infection (6) and compared to uninfected strain matched controls (0). Shown is the mean number of HO-1 per HPRT mRNA molecules (x103)standard deviation (n=3 mice per histogram). (B, C) Effect of activation of HO activity by CoPPIX () on survival (B) and parasitemia (C) of P. berghei ANKA infected C57BL/6 mice. Infected mice treated with ZnPPIX () or PBS () were used as controls. (D-G) Effect of CO (250 ppm, inhalation) on survival (D-F) and parasitemia (G) of P. berghei ANKA 9

infected C57BL/6 mice. Normal atmosphere (), CO between days 3 and 6 (), CO between days 3 and 4 () or CO between days 4 and 5 (). Gray shadow (D-F) represents period of CO exposure. Meanstandard deviation (n=5 mice per plot) is shown for parasitemias.

Fig. 4 CO (250 ppm) prevents BBB leakage and decreases brain vascular congestion as well as TNF-, LT- and IFN- mRNA expression in the brain of P. berghei ANKA infected C57BL/6 mice. (A) Vascular permeability in the brain of non-infected (a), infected/air (b) and infected/CO treated (c) mice, 6 days post-infection. Images are representative of at least 3 mice per group. (B) Hematoxylin-Eosin stained brain tissue sections (5 m), in non-infected (a), infected/air (b) and infected/CO treated (c) mice, 6 days post-infection. Bar represents 100 m. (C, D, E) TNF- (C), IFN- (D) and LT- (E) mRNA qRT-PCR quantification in the brain of non infected (NI), infected/air (I) and infected/CO (I+CO) mice, 6 days post-infection. Shown is mean number of TNF-, IFN- or LT- per HPRT mRNA molecules (x103)standard deviation (n=3 mice per histogram).

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We thank Ana Rodriguez (NYU, Medical Center), Fritz H. Bach (Harvard Medical School), Teresa Pais and Christophe Gregoire (Instituto Gulbenkian de Cincia) for critically reviewing the manuscript, Sofia Rebelo (Instituto Gulbenkian de Cincia) for mouse breeding and genotyping as well as Departamento de Anatomia Patolgica (FML, Universidade de Lisboa) for histopathology studies. This work was partially supported by Fundao para a Cincia e Tecnologia (Grants POCTI/SAU-IMI/57946/2004 to MMM and POCTI/SAU-MNO/56066/2004 to MPS) and European Science Foundation (Grant EURYI 2004 to MMM). AP, CDR, AC, SE and MCR were supported by Fundao para a Cincia e Tecnologia fellowships SFRH: BPD/10510/2002, BD/14232/2003; BD/3106/2000; BPD/12188/2003 and BD/8435/2002, respectively. MMM is a fellow of the EMBO YIP and is a Howard Hughes Medical Institute International Research Scholar.

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Pamplona et al., Fig. 1

A
HO-1 x 103/HPRT 400 300 200 100 0 0 3 6 9 12 Days post-infection

B
Days 0 -Tubulin 51.1 kDa 36.6 kDa HO-1 6

C
400 300 200 100 0 0 3 6 9 Days post- infection 12

HO-1 x 103/HPRT

Pamplona et al., Fig. 2 A

100 Survival (%) 80 60 40 20 0 0 3 6 9 12 Days post- infection

60 50 40 30 20 10 0 0 3 6 9 12 Days post- infection

C
100 Survival (%) 80 60 40 20 0 0 3 6 9 12 Days post- infection

D
Parasitemia (%) 40 30 20 10 0 0 3 6 9 12 Days post- infection

Parasitemia (%)

Pamplona et al., Fig. 3 A

HO-1 x 103/HPRT

700 600 500 400 300 200 100 0

0 6 C57BL/6

0 6 BALB/c

B
Survival (%)

80 60 40 20 0 0 6 12 18 24 30 Days post- infection

Parasitemia (%)

100

70 60 50 40 30 20 10 0 0 6 12 18 24 Days post- infection

D
Survival (%)

100 80 60 40 20 0 0 6 12 18 24 30 Days post- infection

E
Survival (%)

100 80 60 40 20 0 0 6 12 18 24 30 Days post- infection

F
100 Survival (%) 80 60 40 20 0 0 6 12 18 24 30 Days post- infection

G
Parasitemia (%)

60 50 40 30 20 10 0 0 6 12 18 24 Days post- infection

Pamplona et al., Fig. 4

A a b c

B a b c

C
100 TNF- x 103/HPRT 80 60 40 20 0
NI I I+CO

D
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
NI I I+CO

E
0.12 0.10 0.08 0.06 0.04 0.02 0.00
NI I I+CO

IFN- x 103/HPRT

LT- x 103/HPRT