Oxygen and immunity to Leishmania infection

ABSTRACT

Oxygen availability plays a fundamental role in shaping immune responses to infections. Leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, manifests in a spectrum of clinical outcomes, ranging from localized cutaneous lesions to life-threatening visceral infections. Like many infections and chronic diseases, Leishmania-infected tissues are characterized by hypoxia. Despite the recognized importance of oxygen in immune regulation, our understanding of how hypoxia shapes the immune landscape in leishmaniasis remains in its early stages. Collectively, the published studies in leishmaniasis highlight the critical role of oxygen availability and hypoxia-inducible factor (HIF) in orchestrating immune responses, particularly within myeloid cells. Here, we review the literature on how oxygen availability and HIF signaling influence the immune response in leishmaniasis. By consolidating existing findings and identifying gaps in knowledge, we aim to inspire further research into the interplay between oxygen availability, immune function, disease progression, and therapeutic potential in leishmaniasis.

HYPOXIA

Oxygen is essential for the survival of most eukaryotic organisms, serving as the foundation of energy production in cells (1). Through the process of oxidative phosphorylation, eukaryotic cells efficiently generate ATP, the primary energy currency essential for cellular function. However, when the supply of oxygen fails to meet the demands of tissues or cells, a state of hypoxia develops. This physiological imbalance is not uncommon and can arise under diverse conditions. Hypoxia plays a critical role in both physiological processes, such as adaptation to high altitudes and wound healing, and pathological states, including ischemia, tumor progression, and chronic infections (2). Understanding the mechanisms and consequences of hypoxia is vital for unraveling its impact on health and disease.

The understanding of how hypoxia can alter cell metabolism began in the early 20th century with Otto Warburg’s discovery of unique metabolic adaptations in tumor cells. He observed that these cells favor aerobic glycolysis despite its lower efficiency in ATP production compared to oxidative phosphorylation, a process now known as the “Warburg Effect.” This shift is critical for cellular adaptation to low oxygen environments, offering tumor cells a survival advantage (3). Decades later, the mechanisms by which cells sense and respond to hypoxia were discovered through the pioneering work of Drs. William Kaelin, Peter Ratcliffe, and Gregg Semenza in the early 1990s. Semenza identified the transcription factor hypoxia-inducible factor (HIF) as the central regulator of cellular responses to low oxygen levels, while Ratcliffe and Kaelin delineated the molecular pathways that govern HIF stability and activity. Their collective discoveries provided a detailed understanding of oxygen-sensing machinery at the cellular level (4–8). This transformative work earned the three scientists the Nobel Prize in Physiology or Medicine in 2019, solidifying their contributions as milestones in the field of hypoxia research.

The response to hypoxia is orchestrated by three HIF isoforms: HIF-1α, HIF-2α, and HIF-3α. The stability and activity of HIF-α are tightly regulated by prolyl hydroxylase domain (PHD) enzymes (7), which require oxygen, iron, and 2-oxoglutarate as essential co-factors for their function. Additionally, HIF activity is modulated by an asparaginyl hydroxylase known as factor inhibiting HIF (FIH) (9). Under normoxic conditions, PHDs hydroxylate HIF-α, marking it for recognition by the von Hippel-Lindau (VHL) ubiquitin ligase complex (8). This modification facilitates the ubiquitination and subsequent proteasomal degradation of HIF-α, effectively suppressing HIF-mediated transcriptional activity. Concurrently, FIH hydroxylates HIF-α at a distinct asparagine residue, which inhibits its interaction with the p300 co-activator, further limiting transcriptional responses (Fig. 1a). In hypoxic conditions, however, the reduced availability of oxygen inhibits both PHD and FIH activity. This allows HIF-α to stabilize, accumulate, and translocate into the nucleus, where it dimerizes with HIF-1β (also known as the aryl hydrocarbon receptor nuclear translocator, or ARNT). The HIF-α/β dimer interacts with p300 and binds to hypoxia-response elements (HREs) in the promoter regions of target genes. The activation of these genes drives critical adaptive responses to hypoxia, including angiogenesis, erythropoiesis, cell survival, cell proliferation, and metabolic reprogramming (Fig. 1b). HIF-α transcription and translation can also be regulated independent of PHD and oxygen levels by intracellular metabolites and other pathways, for example, cytokine signaling, Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), and the mammalian target of rapamycin (mTOR) (10). Dysregulation of the HIF pathway is implicated in several pathological conditions, such as cancer progression, ischemic diseases, and chronic inflammation. Consequently, the HIF pathway presents a promising therapeutic target for addressing these conditions in clinical settings.

Fig 1.

Regulation of HIF-α under normoxic and hypoxic conditions. (a) Normoxia: In the presence of sufficient oxygen, the oxygen-dependent enzymes, prolyl hydroxylase domain proteins and factor inhibiting HIF-1 have access to their substrate to facilitate the hydroxylation of HIF-α, rendering it inactive. When PHDs hydroxylate HIF-α, it is recognized by the VHL E3 ubiquitin ligase complex, which ubiquitinates HIF-α, targeting it for proteasomal degradation. Additionally, when FIHs hydroxylate HIF-α, it inhibits the interaction with a transcriptional co-activator, p300. (b) Hypoxia: Under low oxygen conditions, PHDs and FIH are inhibited due to reduced availability of oxygen as a substrate. This allows HIF-α to become stabilized, where it will translocate to the nucleus to dimerize with HIF-1β. The HIF-α/HIF-1β heterodimer recruits co-activators, such as p300, to bind HREs in the promoter regions of target genes. This interaction drives the transcription of hypoxia-responsive genes involved in processes such as angiogenesis, metabolism, and cell survival, adapting the cellular response to low oxygen levels.

Our understanding of HIF signaling is primarily derived from studies on HIF-1α and HIF-2α, while HIF-3α is less studied (11). Despite sharing significant structural similarity and overlapping DNA binding capacities, HIF-1α and HIF-2α have distinct and nonredundant functions, largely attributed to their differential expression patterns (12, 13). HIF-1α is ubiquitously expressed across most cell types and is crucial during acute hypoxia. It shifts cellular metabolism toward anaerobic ATP production by regulating glycolytic genes, such as glucose transporter-1, and downregulating oxidative phosphorylation to reduce oxygen dependency (12, 14–17). In contrast, HIF-2α is expressed in specific cell types, including endothelial, renal, liver, cardiomyocytes, and immune cells, and plays a dominant role in chronic hypoxia, regulating genes involved in angiogenesis, erythropoiesis, and iron homeostasis (12, 18, 19). Notable HIF-2α targets include vascular endothelial growth factor (VEGF), which promotes blood vessel growth to improve oxygen delivery, and erythropoietin, which stimulates red blood cell production. Additionally, HIF-2α regulates iron homeostatic genes, such as divalent metal transporter-1 and ferroportin, to ensure adequate iron availability for erythropoiesis (19–23). These distinct functional roles highlight the complementary and time-dependent actions of HIF-1α and HIF-2α in the adaptive response to hypoxia.

Despite evolutionary adaptations to ensure adequate oxygen delivery to organs and cells, the vascularization of mammalian tissues remains highly variable. This variability results in substantial differences in oxygen availability, both between distinct tissues and within different regions of the same tissue. For example, while in vitro cell cultures are typically maintained at 21% oxygen—corresponding to atmospheric oxygen levels—the maximum oxygen concentration in the human body reaches only 14.5% in pulmonary alveoli (24). Much lower oxygen levels are found in tissues commonly infected by Leishmania parasites, such as 5% in the liver and spleen and as little as 1% in the superficial regions of the skin (25). These physiological oxygen levels, referred to as “physioxia” or tissue normoxia, vary widely depending on the organ. In contrast, hypoxia describes oxygen levels below the physiological norm for a given tissue and typically indicates insufficient oxygenation. The amount of oxygen cells are exposed to matters since optimal expression, DNA binding activity, and responses mediated by HIF-1α/β occur at oxygen levels of 1.5%–2%, reaching maximal activity at approximately 0.5% (26). In this review, we will examine studies that have used varying oxygen concentrations to assess how oxygenation impacts the ability of innate immune cells to respond to Leishmania infection in vitro. These findings are further complemented by in vitro and in vivo research utilizing animal models deficient in HIF.

Leishmaniasis

Leishmaniases are vector-borne diseases caused by protozoan parasites from the genus Leishmania, with an estimated 1 million new cases occurring each year. Parasites are transmitted to the skin by the bite of an infected phlebotomine sandfly, and over 20 species cause disease in humans (27). To date, no vaccine has been approved for human use, and the available drugs are often toxic, have limited efficacy, are difficult to administer, and face increasing parasite resistance (28). Disease manifestations can range from self-healing cutaneous ulcers to disfiguring mucosal lesions and fatal visceral disease, and in all these different organs, parasites infect and survive inside cells from the innate immune system, such as macrophages, monocytes, dendritic cells, and neutrophils (29). Therefore, in order to consider novel therapeutic strategies, we must understand how our immune system can clear or sustain Leishmania infection.

The control of Leishmania requires antigen presentation by dendritic cells in the presence of co-stimulatory molecules and interleukin-12 (IL-12), leading to the differentiation of CD4 T cells into T helper 1 (Th1) cells that produce interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) (30, 31). IFN-γ, in combination with TNF, activates infected cells to kill Leishmania by inducing the assembly of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the inducible or type 2 nitric oxide synthase (iNOS or NOS2) necessary for the production of reactive oxygen species (ROS) and nitric oxide (NO), respectively. These oxygen-dependent pathways are used as a defense mechanism against Leishmania and are required to control disease. As a successful pathogen that persists indefinitely in the mammalian host, Leishmania has developed mechanisms to adapt to the different intracellular and extracellular factors, recently named the “immunomicrotope” (32). One such microenvironmental factor is hypoxia. This review discusses the (i) development of hypoxia and HIF stabilization in Leishmania infections, followed by the effects of hypoxia and HIFs in (ii) protection and (iii) susceptibility to infection.

DEVELOPMENT OF HYPOXIA AND HIF-Α STABILIZATION IN LEISHMANIA INFECTION

Hypoxia has been observed in cutaneous leishmaniasis lesions from human and mouse skin (33–36). Correspondingly, HIF-1α expression has been detected in both cutaneous lesions (37–39) and in the spleens of mice infected with parasites causing visceral leishmaniasis (40, 41). In cutaneous infections, lesion size inversely correlates with tissue oxygenation, with oxygen levels increasing as the lesions resolve (35). This hypoxic microenvironment appears paradoxical, given that Leishmania-infected skin is highly vascularized (39). Recent studies have shed light on this apparent contradiction by analyzing mRNA sequencing from human skin lesion samples. By comparing lesions with high and low hypoxic transcriptional signatures, researchers found that hypoxic lesions contained significantly more neutrophils (36). This finding suggested that neutrophils contribute to the development of hypoxia in cutaneous lesions. Subsequent mouse studies confirmed this observation, showing that while neutrophils themselves do not become hypoxic, they are surrounded by intensely hypoxic regions (36). The mechanism underlying this phenomenon was linked to the neutrophil-mediated production of ROS, which depletes local oxygen levels (36) (Fig. 2a). It is likely that other innate immune cells and oxygen-consuming pathways, such as NO production, also contribute to the hypoxic environment.

Fig 2.

Mechanisms of HIF-α stabilization in Leishmania infection through hypoxia-dependent and -independent conditions. (a) Development of hypoxia: hypoxia can result as a by-product of immune responses to infection that require oxygen to fulfill. One major contributor to the development of hypoxia in Leishmania infection is through the production of ROS by neutrophils. (b) HIF-α stabilization independent of hypoxia: while the canonical signaling of HIF-α occurs through hypoxia, HIF-α stabilization can occur in the presence of adequate oxygen. This noncanonical signaling can be mediated through intracellular iron dysregulation facilitated by the inherent need for Leishmania to scavenge host cell iron to survive.

A significant consequence of hypoxia is the stabilization of HIF-α, and it is well-established that pathogens can stabilize HIF-α through mechanisms independent of hypoxia. For example, similar to Toxoplasma gondii (42), Leishmania infection of macrophages and dendritic cells induces HIF-α stabilization under normoxic conditions (43–45). One study suggests that this stabilization is thought to result not from oxygen depletion but from the parasite’s ability to manipulate host cell iron availability. In Leishmania donovani-infected macrophages, the depletion of intracellular iron pools inhibits PHD enzyme activity, leading to HIF-1α stabilization and conferring a survival advantage to the parasite (45) (Fig. 2b). While all Leishmania species depend on host iron for survival, evidence suggests that different species may employ distinct iron scavenging mechanisms, with some requiring higher iron supplies than others (46, 47). These variations could account for the differing effects of Leishmania infections on HIF-α stabilization. For instance, Leishmania major-infected macrophages did not exhibit normoxic HIF-1α stabilization, as reported by Schatz et al. Instead, they found that stabilization required additional inflammatory signals, such as IFN-γ and TNF, which are commonly present in L. major lesions (37). Another potential explanation for these discrepancies may result from different cells used in the experiments. While Singh et al. (45) used J774.A1 macrophage-like cell lines, studies from Schatz et al. (37) used bone-marrow-derived macrophages.

It is clear that Leishmania infection induces a hypoxic tissue microenvironment, and in L. major-infected ear pinnae, the levels of oxygen are below 0.5% (36). Furthermore, some Leishmania species induce HIF-α stabilization, even in normoxic conditions. The work described in the next two sections highlights studies attempting to understand how the amount of oxygen and HIF impact infection and disease outcomes in cutaneous and visceral diseases.

PROTECTIVE ROLES OF HYPOXIA AND HIF PATHWAYS IN HOST DEFENSE AGAINST LEISHMANIA INFECTION

The first studies examining the impact of oxygen levels on Leishmania-infected macrophages investigated the effects of 5% oxygen on peritoneal macrophages and human macrophage cell lines, comparing them to atmospheric oxygen levels (21%) (48, 49). These studies found that macrophages exposed to 5% oxygen showed enhanced control of Leishmania amazonensis infection in vitro (48, 50) despite a notable 50% reduction in NO production when activated with lipopolysaccharide (LPS) and IFN-γ (48). Similarly, human dendritic cells cultured at 5% oxygen also exhibited improved control of L. amazonensis infection (51). The greater killing of Leishmania by macrophages under these low oxygen conditions was attributed to increased ROS production. This conclusion was supported by experiments utilizing the ROS scavenger N-acetylcysteine, which significantly increased infection rates in macrophages cultured under low oxygen conditions (50). While these studies were not conducted under true hypoxic conditions (lower than 2%), they highlighted that innate immune cells cultured in physioxia (e.g., 5% oxygen) demonstrate an enhanced ability to control Leishmania parasites in vitro compared to cells maintained at ambient oxygen levels. These findings emphasize the importance of physiological oxygen levels in shaping the antimicrobial functions of innate immune cells.

Building on the role of low oxygen in modulating macrophage function, studies have explored the specific involvement of HIF-1α in the control of Leishmania infection. While pharmacological stabilization of HIF-1α alone does not appear to enhance the microbicidal activity of macrophages, its expression in L. major-infected macrophages activated by LPS and IFN-γ is crucial for controlling Leishmania in vitro (37). In vivo studies further demonstrated that myeloid-specific deletion of HIF-1α using Lysm^creHif1a^flox/flox mice leads to reduced iNOS expression in myeloid cells, resulting in increased parasite loads and larger lesions during the later stages of the disease (37). Similarly, HIF-1α-dependent induction of NADPH oxidase was shown to be necessary for adequate ROS production in L. amazonensis-infected macrophages cultured in 3% oxygen (52). Collectively, these data suggest that HIF-1α expression is important to mediate iNOS and NADPH oxidase induction in myeloid cells and control cutaneous leishmaniasis caused by L. major and L. amazonensis.

Evidence suggests that HIF-1β expression in myeloid cells is crucial for the production of VEGF-A during L. major infection. Myeloid-specific deficiency of HIF-1β results in larger lesions without affecting parasite numbers in the infected skin (53). These findings align with earlier studies demonstrating that VEGF-A deletion increases lesion size while having no impact on parasite control (39). The observed increase in lesion size in myeloid HIF-1β-deficient mice is attributed to altered vascular remodeling, which leads to an accumulation of monocytes and Th1 cells in the lesions due to reduced VEGF-A expression (53, 54). In addition to HIF-α isoforms, HIF-1β is known to dimerize with various proteins, and each dimeric complex interacts with specific DNA elements to regulate distinct gene expression pathways (55). Therefore, it is unknown which specific binding partners are responsible for the observed inflammatory effects and increased lesion size in animals with myeloid-specific deletion of HIF-1β. Identifying those specific binding partners will help clarify whether these effects are directly related to the hypoxic environment of cutaneous leishmaniasis lesions caused by L. major and/or involve other pathways.

HIF-1α also plays a protective role against L. donovani infection by regulating macrophage metabolism. In Lysm^creHif1a^flox/flox mice infected with L. donovani, the loss of HIF-1α resulted in sustained activation of the mTOR pathway, which contributed to increased fatty acid synthesis (41). This dysregulated lipid metabolism rendered mice more susceptible to L. donovani infection. Similarly, individuals carrying a single-nucleotide polymorphism that reduces HIF1A expression exhibit increased lipogenesis and are more susceptible to L. donovani infection in vitro (41). These data support a role for HIF-1α in the ability of myeloid cells to suppress fatty acid synthesis and control Leishmania species that cause visceral leishmaniasis.

Collectively, these studies highlight the importance of including low oxygen conditions when evaluating the ability of cells to control pathogens (Fig. 3). Furthermore, these studies suggest that HIF expression in myeloid cells can bolster host defenses against Leishmania infections. However, there is also substantial contrasting evidence that low oxygen tensions (5%), hypoxia (oxygen tensions below 2%), and HIF expression in immune cells play a significant role in promoting disease progression, which may be dependent or independent of the parasite burden, and which will be explored in the following section.

Fig 3.

Role of HIF-α in host defense to Leishmania infection under hypoxic, physioxic, and normoxic conditions. In vivo studies (potentially hypoxia-dependent HIF stabilization): mice with myeloid-specific HIF-1α deletion (Lysm^CreHif1a^fl/fl) infected with L. major subcutaneously (s.c.) exhibit larger lesions and increased parasite burdens due to reduced iNOS expression. Additionally, mice with myeloid-specific HIF-1β deletion (Lysm^CreHif1b^fl/fl) infected intradermally (i.d.) with L. major develop larger lesions, but the parasite burden remains unchanged. This is attributed to increased VEGF-A production, which exacerbates lesion growth through changes in vascularization but does not affect parasite replication. Physioxia: in Leishmania-infected cells cultured at physiological oxygen levels (5% oxygen), HIF-1α/HIF-1β stabilization enhances NADPH oxidase production, leading to increased ROS production and enhances iNOS expression, but this does not confer to higher NO levels as infected macrophages show diminished NO production. Normoxia: macrophages with HIF-1α deficiency (Lysm^CreHif1a^fl/fl) exhibit increased lipogenesis, which allows for a greater survival advantage to support parasite replication, suggesting that HIF-1α plays a protective role in restricting parasite growth under normal oxygen levels.

HYPOXIA AND HIF PATHWAYS IN HOST SUSCEPTIBILITY AND DISEASE PROGRESSION IN LEISHMANIA INFECTIONS

Low oxygen significantly impacts the immune response to L. major and L. amazonensis infection by altering the production of key antimicrobial molecules such as NO. While HIF-1α was shown to be important for iNOS expression (37), earlier studies by the same group showed that NO production is lower in macrophages cultured in hypoxia (0.5%–2% oxygen) (35). Experiments with L. amazonensis also showed a reduction in NO production in mouse and human macrophages cultured under 5% oxygen tension (48). Conversely, exposing L. amazonensis-infected macrophages to hyperoxic conditions in a hyperbaric chamber enhanced parasite control (56). However, it remains unclear whether this effect resulted from improved macrophage antimicrobial activity in a high-oxygen environment or a direct inhibitory impact of hyperoxia on L. amazonensis survival. Other in vivo studies provide additional clarity on the role of hypoxia in Leishmania infections. Wild-type mice exposed to systemic hypoxia exhibited reduced tissue oxygenation in cutaneous lesions, impairing parasite control (35). Interestingly, in iNOS-deficient mice, parasite loads were similar under normoxic and hypoxic conditions, demonstrating that hypoxia primarily undermines Leishmania control by depriving iNOS of the oxygen required for NO production (35). These findings reveal a critical limitation of the host’s ability to control Leishmania infections in hypoxic conditions, where insufficient oxygen availability hampers the production of NO, providing a survival advantage to the parasite.

In visceral leishmaniasis, Hammami et al. demonstrated that HIF-1α in dendritic cells supports L. donovani infection in vivo. Using CD11c^creHif1a^flox/flox mice, they showed that dendritic cells deficient in HIF-1α produced higher levels of IL-12 (40, 57), a cytokine critical for anti-parasitic immunity. Supporting this observation, human dendritic cells and macrophages infected with L. amazonensis produced significantly less IL-12 when cultured under 5% oxygen tension compared to normoxia (50, 51). Additionally, HIF-1α-deficient dendritic cells produced reduced levels of the regulatory cytokine IL-10, which is known to dampen immune responses (40, 57). This dual effect of HIF-1α—suppressing IL-12 while promoting IL-10—renders dendritic cells less effective in supporting T cell-mediated control of infection. As a consequence of the ablation of HIF-1α in dendritic cells, the authors observed enhanced development of short-lived effector CD8 T cells (40) and increased frequency of CD4 T cells producing IFN-γ (57), both of which are important for the control of visceral disease. Moreover, HIF-1α was implicated in the expansion of myeloid-derived suppressor cells in the spleens of L. donovani-infected mice, further increasing susceptibility to infection (58). Supporting these findings, pharmacological inhibition of HIF-1α in vivo significantly reduces parasite burden in the liver and spleen of infected mice (59) and suggests a detrimental role of HIF-1α in visceral disease. Taken together, these findings suggest that low oxygen levels and HIF-1α play a detrimental role in innate immunity during visceral leishmaniasis by impairing the functionality of dendritic cells and promoting immunosuppressive pathways in T cells. The discrepancy between these findings and studies showing that HIF-1α protects against L. donovani by modulating fatty acid synthesis remains unclear but may be explained by differences in the cell subsets studied (e.g., macrophages versus dendritic cells). Further investigation is needed to clarify this divergence.

While the studies in visceral leishmaniasis using CD11c^creHif1a^flox/flox mice suggest that hypoxia indirectly suppresses T cell responses by affecting dendritic cell function, hypoxia can also directly influence the fate and function of CD4 and CD8 T cells (60–77). To date, only one study has examined the direct impact of hypoxia on T cells in the context of leishmaniasis. This study demonstrated that the hypoxic environment of cutaneous leishmaniasis lesions during L. major and Leishmania braziliensis infections promotes the expression of the cytotoxic molecule granzyme B in CD8 T cells (36). Mechanistically, CD8 T cells are initially activated in draining lymph nodes, where oxygen levels are relatively higher. Once activated, these cells migrate to inflamed, hypoxic skin lesions with oxygen tensions below 0.5%. The hypoxic microenvironment reprograms CD8 T cells, driving the upregulation of granzyme B. There is ample evidence that cytotoxic CD8 T cells damage the Leishmania-infected skin, delay wound healing, and do not effectively control Leishmania infection (78–88). Thus, hypoxia skews CD8 T cell responses toward a phenotype that is detrimental to the host, highlighting the negative impact of hypoxia on adaptive immune responses in cutaneous leishmaniasis (Fig. 4).

Fig 4.

Effects of oxygen levels and HIF signaling on host susceptibility during Leishmania infection. Systemic hypoxia: wild-type (WT) mice infected subcutaneously (s.c.) with L. major exposed to systemic hypoxia exhibit a reduction in NO due to a decrease in oxygen levels needed for iNOS to turnover NO production. This dampening of NO leads to an increase in parasite burdens, as it is a critical effector molecule for Leishmania control. Hypoxia: in vitro studies demonstrate that hypoxic conditions in macrophages reduce NO production, impairing the ability to control L. major infection. Additionally, intradermal (i.d.) infection of WT mice with L. major creates highly inflamed and hypoxic regions. CD8 T cells that have newly migrated from the draining lymph node to the hypoxic skin upregulate granzyme B production. These granzyme B-positive CD8 T cells do not control parasites and only exacerbate inflammation and delay wound healing. In vivo studies (potentially hypoxia-dependent HIF stabilization): mice with dendritic cell-specific deletion of HIF-1α (CD11c^Cre Hif1a^fl/fl) infected intravenously (i.v.) with L. donovani exhibit altered cytokine profiles, including increased IL-12 and decreased IL-10 production. This cytokine shift promotes the generation of short-lived effector (Eff) CD8 T cell and CD4 Th1 cell responses, which are both necessary for the control of visceral disease. This study highlights the impact of HIF-1α signaling in dendritic cells, where it controls the production of IL-10 while dampening IL-12 production, which benefits Leishmania survival and pro-inflammatory immune evasion. Hyperoxia: in vitro studies of Leishmania-infected macrophages cultured under hyperoxic conditions show increased ROS production, leading to enhanced parasite killing. These data suggest that high oxygen levels favor parasite clearance, whereas low oxygen levels provide a greater opportunity for parasite survival.

CONCLUSIONS AND OPEN QUESTIONS

The role of low oxygen, hypoxia, and HIFs in leishmaniasis is complex and varies depending on factors such as the Leishmania species, affected cell types, mouse models used, and the degree of oxygenation (Table 1). While it is tempting to categorize microenvironmental signals and transcription factors as either beneficial or detrimental, the reality is likely more nuanced. For instance, as demonstrated by Fowler et al. (36), regions of severe hypoxia, such as those with high neutrophil density, may deprive infected macrophages of molecular oxygen necessary for NO production, impairing their ability to kill Leishmania, even if iNOS expression is elevated by HIF-1α stabilization. In contrast, macrophages in moderately hypoxic regions of the same lesion may benefit from iNOS expression induced by HIF-1α stabilization, with sufficient molecular oxygen available to sustain NO production and effectively control the infection. Similarly, immune cells closer to blood vessels may not experience severe hypoxia and thus function differently within the same microenvironment. To better understand these dynamics, future studies should focus on mapping hypoxia distribution and immune cell subsets within infected tissues. Integrating this with research conducted under varying oxygen pressures will provide deeper insights into the differential outcomes driven by hypoxia in leishmaniasis.

TABLE 1.

Summary of studies on oxygen levels, HIF deletion, and the impact on protection or susceptibility to Leishmania infection

Leishmania species	Clinical presentation	Models studied	Host response	References
				Low oxygen	HIF deletion
L. major	Cutaneous	Mouse	Protection	(37)	(37, 53, 54)
			Susceptibility	(35, 36)	None
L. braziliensis	Cutaneous	Mouse and human	Protection	None	None
			Susceptibility	(36)	None
L. amazonensis	Cutaneous	Mouse and human	Protection	(48–52)	(52)
			Susceptibility	(56)	None
L. donovani	Visceral	Mouse and human	Protection	None	(41)
			Susceptibility	None	(40, 57–59)

The existing research underscores the significant role of oxygen levels and HIFs in shaping the immune response, particularly focusing on immune cells. However, it is evident that all cells within the hypoxic microenvironment of infected tissues, not just immune cells, are influenced by oxygen deprivation. Epithelial cells, for instance, are well-known for their ability to orchestrate immune responses and react to inflammatory signals (89). While they do not express iNOS under hypoxic conditions (90), they may still be directly or indirectly impacted by low oxygen (91–93). Yet, how epithelial cells respond to hypoxia and how this response might influence disease resolution in leishmaniasis remain largely unexplored, representing an important avenue for future research.

One understudied aspect of hypoxia in Leishmania infection is its direct impact on the parasites. Leishmania is likely exposed to hypoxic conditions both within the sandfly vector and in the mammalian host. However, our understanding of how Leishmania adapts to low oxygen environments remains limited (94). Investigating the cellular and molecular mechanisms that enable the parasite to survive and thrive under hypoxia is crucial for advancing our knowledge of Leishmania biology and potentially identifying novel therapeutic targets.

Another crucial area of research is exploring how hypoxia influences the host’s ability to respond to therapy. Studies have shown a modest but significant reduction in the efficacy of anti-parasitic drugs under hypoxic conditions in vitro (95), highlighting the need to consider hypoxia as a key microenvironmental factor when developing and testing novel therapeutics. Separately, studies in mice deficient in ROS and NO production have shown impaired control of L. donovani following treatment with anti-parasitic drugs (96). Since ROS and NO production require oxygen, these mice are expected to experience reduced levels of tissue hypoxia. While the Murray et al. study (96) did not directly evaluate the hypoxic state of infected tissues, these findings highlight that factors beyond hypoxia may also critically influence drug efficacy. Collectively, these observations underscore the complexity of the tissue microenvironment and the importance of integrating multiple host factors, including oxygen availability, into the design of future drug studies to optimize treatment outcomes.

A key challenge in this field is the inconsistent use of oxygen definitions, with many studies employing 5%–6% oxygen as a model for hypoxia—despite this level aligning more closely with physioxia than true hypoxia. This variability complicates comparisons across studies and highlights the need for standardized definitions for those studying hypoxia in leishmaniasis. Despite these challenges, this review has highlighted significant progress in understanding the role of oxygen levels as a critical component of the immunomicrotope in Leishmania-induced disease. It also emphasizes how much remains unknown about this dynamic process, presenting an exciting and expansive field for future research. By addressing these gaps, we can deepen our understanding of hypoxia’s dual role in infection and therapy, paving the way for novel insights and transformative advancements in leishmaniasis research.

Biographies

Bridget Bowman earned her Bachelor of Science in Biochemistry and Molecular Biology from Otterbein University. She then worked as a Research Assistant at Nationwide Children’s Hospital and is now a Ph.D. candidate at The Ohio State University. In the Novais lab, Bridget has spent the past three years studying host-pathogen interactions, with a particular interest in how the intracellular parasite Leishmania alters macrophage function to subvert immunity.

Fernanda O. Novais, Ph.D. is an Assistant Professor at The Ohio State University, where she leads a research program focused on host-pathogen interactions, with a particular emphasis on immune regulation and tissue pathology in leishmaniasis. She received her doctoral degree from the Federal University of Bahia in Brazil, followed by postdoctoral training at the University of Pennsylvania. Dr. Novais has over 20 years of experience studying immune responses to Leishmania infections, spanning from basic immunology to translational research. Her work has contributed to the identification of disease-driving immune mechanisms and the development of host-directed therapies, including approaches currently being tested in clinical trials. Her lab is especially interested in how microenvironmental cues—such as hypoxia—influence immune function and pathogen persistence. She is passionate about mentoring and fostering collaborative science that bridges disciplines to better understand chronic infectious diseases.