Oxidative stress and intracellular infections: more iron to the fire

Abstract

The immune system’s battle against pathogens includes the “respiratory burst,” a rapid release of ROS from leukocytes, thought to play a role in destroying the invading species. In this issue of the JCI, Paiva et al. demonstrate that oxidative stress actually enhances infection with the protozoan Trypanosoma cruzi, by a mechanism that may involve facilitating parasite access to iron. Their findings suggest a novel direction for the development of drugs against intracellular parasites.

The immune system of higher vertebrates is capable of rapidly recognizing pathogens, mounting an immediate innate response. The innate response is followed by adaptive immunity, the slower-developing process that is responsible for preventing reinfection by the same organism. An important component of the rapid innate immune response is production of ROS, highly reactive and toxic molecules produced by phagocytes and other cell types. ROS have been traditionally viewed as a “necessary evil” in the battle against pathogens, and their production is coupled to antioxidant responses important for mitigating oxidative damage in cells and tissues. However, this is clearly not the whole story. In this issue of the JCI, Paiva et al. (1) demonstrate that antioxidant responses actually suppress infections with Trypanosoma cruzi, a protozoan parasite that seems to thrive in an oxidative environment. Earlier studies with viruses and bacterial pathogens suggested a similar scenario, but those observations were mostly attributed to a role of antioxidant pathways in innate immunity. Challenges to established dogma are usually slow to emerge, but this detailed T. cruzi study raises the intriguing possibility that the deleterious effects of antioxidants on intracellular pathogens may occur by a mechanism fundamentally different from classical innate or adaptive immune responses.

The role of oxidative stress

This insight came from experiments wherein mice and mouse-derived macrophages were exposed to CoPP, an activator of the transcription factor NRF2 that orchestrates antioxidant responses. NRF2 drives expression of heme oxygenase–1 (HO-1), an enzyme that shifts the cellular redox balance by degrading pro-oxidant heme (2). Surprisingly, activation of NRF2 and increased expression of HO-1 markedly reduced parasite burden in infected animals and in isolated macrophages. The process was independent of T lymphocyte–mediated immunity and did not seem to involve apoptotic clearance of infected cells or effectors known to be active against T. cruzi, such as NO, TNF, or type I IFN (1). Remarkably, NRF2 activation was still able to reduce the very high T. cruzi load that is observed in iNOS-deficient mice. NRF2 activation had the expected effect of inhibiting ROS generation in infected macrophages, but this did not affect the number of intracellular parasites found shortly after infection (1). This observation is consistent with the presence in T. cruzi and other trypanosomatid parasites of a unique and highly effective antioxidant machinery, the trypanothione-thiol system (3).

The unexpected role of high oxidative stress in promoting T. cruzi infection may have important implications for the pathology of Chagas disease, the chronic debilitating illness that affects millions of people infected by this parasite in Latin America. Although infective trypomastigote stages can invade practically every nucleated cell type, in vivo the parasites are frequently found replicating in skeletal and cardiac muscle (4). The most serious manifestation of chronic Chagas disease, cardiomyopathy, is responsible for significant mortality in infected patients and has been directly attributed to the persistence of T. cruzi within cardiomyocytes (5, 6). Interestingly, there is evidence that marked and sustained oxidative stress is established in cardiomyocytes following T. cruzi infection, due to parasite-induced disturbances in mitochondrial membrane potential and electron transport. The recurring tissue injuries that are generated in this environment were proposed to contribute to the progression of Chagas disease pathology (7). The findings of Paiva et al. now suggest that the persistent oxidative environment generated in cardiomyocytes may not only explain the heart pathology, but may also be self-perpetuating by directly promoting T. cruzi replication (1).

The challenge of finding iron inside cells

Paiva et al. found that induction of antioxidant responses reduced T. cruzi burden in macrophages, but not in other cell types — suggesting that a macrophage-specific mechanism was at play (1). This is significant because macrophages play an important role in vivo as iron stores, which are mobilized and maintained through regulated expression of a macrophage-specific iron exporter, ferroportin (8). The antioxidant response regulator NRF2 upregulates expression of ferroportin and also of ferritin, the protein responsible for cytosolic storage of iron in a redox-inert form (9, 10). Increased levels of ferroportin and ferritin are expected to reduce the levels of iron available for intracellular pathogens, suggesting that this pathway could be the basis for the surprising effect of antioxidants in inhibiting T. cruzi infection.

Iron is essential for several metabolic pathways, but its concentration inside cells has to be tightly regulated because it can catalyze the formation of dangerous free radicals. The mechanisms used by intracellular trypanosomatid protozoa (including the human parasites T. cruzi and Leishmania) to acquire this critical element in the challenging intracellular environment are only beginning to be elucidated (11). T. cruzi amastigotes take up iron-loaded transferrin when grown in vitro (12), but the physiological significance of this observation is unclear. Transferrin is restricted to the lumen of the endocytic pathway and thus is absent from the host cell cytosol, where intracellular amastigotes replicate. In Leishmania, a transferrin receptor–based mechanism for iron uptake was also initially postulated, but it was not confirmed by subsequent studies (13). Transferrin can reach the lysosome-like parasitophorous vacuoles where Leishmania resides in macrophages (14), but it appears to function mainly as a source of ferric iron (Fe³⁺) for the sequential action of two surface-associated parasite molecules: the Fe³⁺ reductase LFR1 (15) and the LIT1 transporter, which directly promotes ferrous iron (Fe²⁺) uptake (16). Intriguingly, the T. cruzi genome does not contain an obvious LIT1 ortholog, raising the possibility that this ferrous iron transporter represents a specific Leishmania adaptation to the low-iron environment of phagolysosomes. Mutations in the lysosomal iron efflux pump NRAMP1 confer susceptibility to Leishmania and other intravacuolar pathogens (17, 18), reinforcing the conclusion that Leishmania needs a high-affinity transporter such as LIT1 to compete effectively for iron within its parasitophorous vacuole (16).

Ironing out the details

So how does T. cruzi acquire iron during its replicative phase in the host cell cytosol? This question remains to be answered, but given their cytosolic location and apparent lack of a high-affinity Fe²⁺ transporter, these parasites may be particularly dependent on the intracellular labile iron pool, defined as a transitory pool of redox-active iron complexes (19). The findings of Paiva et al. support a scenario whereby parasite growth in macrophages is inhibited by depletion of the labile iron pool, as a consequence of NRF- and HO-1–mediated upregulation of ferroportin and ferritin, which cause efflux of iron and its cytosolic sequestration in a redox-inactive form. Conversely, ROS generation may help T. cruzi thrive in an environment of high oxidative stress by allowing superoxide-mediated iron release from ferritin, and/or peroxide-mediated iron extraction from heme and/or iron-sulfur cluster proteins (ref. 19 and Figure 1). Importantly, in this scenario the stimulation of T. cruzi growth by oxidative stress would not be restricted to macrophages, since a persistent oxidative environment can be generated by the parasites in other cell types, such as cardiomyocytes (7). It will be very interesting to determine whether Leishmania parasites, which are closely related to T. cruzi but replicate in the endocytic compartment of macrophages, also fare better under oxidative stress. Such studies may provide an answer to the fascinating cell biological question of whether residence in the cytosol or within a membrane-bound compartment has a differential impact on the access of pathogens to iron, on the specific iron pools utilized, and on the consequences of oxidative stress.

Figure 1

Increase in iron availability: an unexpected benefit of ROS for intracellular pathogens. Ferric iron (Fe³⁺) enters mammalian cells complexed to transferrin, and after reduction to the ferrous form (Fe²⁺), it is translocated to the cytosol by endosome transporters. Fe²⁺ is rapidly utilized for metabolic pathways such as heme and iron-sulfur cluster protein biosynthesis or oxidized to Fe³⁺ and stored as a complex with ferritin. This scenario makes access to available iron very challenging for intracellular pathogens. Paiva et al. show that oxidative stress in macrophages promotes the intracellular growth of T. cruzi, by a mechanism that may involve releasing Fe²⁺ from ferritin, or from heme and/or iron-sulfur cluster proteins. Tf-R indicate transferrin receptor.

Acknowledgments

Work in the author’s laboratory is suppoted by NIH grants R01AI067979, R01GM064625, and R37AI34867. The author thanks Bidyottam Mittra and Iqbal Hamza for critical reading of the article, and all members of her laboratory for stimulating discussions.

Address correspondence to: Norma W. Andrews, Department of Cell Biology and Molecular Genetics, 2134 Bioscience Research Building, University of Maryland, College Park, Maryland 20742-5815, USA. Phone: 301.405.8418; Fax: 301.314.9489; E-mail: andrewsn@umd.edu.

Footnotes

Conflict of interest: The author has declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(7):2352–2354. doi:10.1172/JCI64239.

See the related article at Oxidative stress fuels Trypanosoma cruzi infection in mice.

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