Animal studies find effects of CO produced as byproduct of HO-1 expression ultimately lead to tolerance to parasite.

Scientists have thrown new light on the mechanism by which the sickle cell trait protects against Plasmodium falciparum malaria. The international team claims the protective effects of sickle hemoglobin are related to the production of carbon monoxide (CO) as a result of heme catabolism catalyzed by the stress-responsive enzyme heme oxygenase-1 (HO-1), which is induced by sickle Hb. Effectively, the CO prevents further accumulation of circulating free heme after Plasmodium infection and confers tolerance to the parasite in the host, rather than directly impacting on its life cycle.

The researchers, led by Ana Ferreira, Ph.D., and Miguel P. Soares, Ph.D., at the Instituto Gulbenkian de Ciência in Portugal, claim the HO-1-related mechanism could also be associated with other clinically silent genetic red blood cell defects and might provide a general protective mechanism against Plasmodium infection in human populations. They report their findings in Cell in a paper titled “Sickle Hemoglobin Confers Tolerance to Plasmodium Infection.”

HO-1 was previously found to be protective against a wide variety of immune-mediated inflammatory diseases including experimental cerebral malaria (ECM), which develops in experimental mice infected with P. berghei ANKA infection, the researchers write. This protective effect is mediated by CO, generated as a byproduct of HO-1 activity, which binds cell-free Hb and inhibits its oxidation, thus preventing heme release from oxidized Hb and hence pathogenesis of ECM.

Given that expression of HO-1 and production of CO are induced by the sickle hemoglobin mutation, the researchers investigated whether the same mechanism is responsible for protection against malaria observed in heterozygous carriers of the sickle hemoglobin gene.

Their research in mouse models confirmed that either blocking HO-1 gene expression or chemically inhibiting the activity of the HO-1 protein increased the animals susceptibility to P. berghei ANKA infection. Importantly, the protective effects of HO-1 were independent of parasitic load or spread of the parasite to different organs, and didn’t impact on the parasite’s life cycle. Induction of HO-1 by sickle Hb also blocked the production of chemokines involved in the pathogenesis of ECM.

The team further confirmed the pivotal protective role of CO generated as a result of HO-1 activity. Administering the gas by inhalation to wild type mice significantly suppress the pathogenesis of ECM via a mechanism that hinged on the inhibition of heme release from Hb. The protective effects of CO were also evident in HO-1 gene knockout mice, or animals in which HO-1 activity was chemically blocked. Again, the results were not related to any reduction in parasitemia.

It had previously been shown that the transcription factor Nrf2 plays a central role in the transcriptional regulation of HO-1 expression, so the researchers further assessed whether induction of HO-1 expression in whole blood leukocytes of their mouse model also involved Nrf2. They confirmed that deleting just one copy of the Nrf2 gene was sufficient to reduce expression of the HO-1 mRNA and was associated with an increased incidence of ECM when the animals were infected with P. berghei ANKA.

Interestingly, the researchers found that sickle Hb appears to block the production of pathogenic CD8+ T cells involved in triggering disease onset, although the mechanism by which this occurs probably doesn’t involve HO-1, and has yet to be defined, they admit.

Interestingly, knocking out one of the Nrf2 genes was not associated with a regain of CD8+ T-cell activation and/or expansion in the spleen. This suggests the immunoregulatory activity of sickle hemoglobin probably doesn’t involve Nrf2, “which is consistent with the observation that this effect also does not seem to involve HO-1,” the team notes.

“While the immunoregulatory effect of sickle Hb appears to be driven by free heme, its molecular mechanism acts via a signal transduction pathway that remains to be established and that might target antigen presenting cells, e.g. dendritic cells, and/or CD8+ T cells.”

The researchers conclude that release of Hb from sickle red blood cells leads to chronic accumulation of cell-free Hb and release of its heme prosthetic groups. The free heme induces HO-1 expression in bone marrow and white blood cells, via a mechanism involving Nrf2. Heme catabolism by HO-1 then produces CO that prevents further heme release from the cell-free Hb after Plasmodium infection, suppressing the pathogenesis of ECM.

“We propose that modulation of the Nrf2/HO-1 system might be used therapeutically to treat severe forms of malaria and in particular cerebral malaria,” they write.

“Due to its protective effects against malaria, the sickle mutation may have been naturally selected in sub-Saharan Africa, where malaria is endemic and one of the major causes of death,” Dr. Soares suggests. “Similarly, other clinically silent mutations may have been selected throughout evolution, for their ability to provide survival advantage against Plasmodium infection.”

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