Hypoxia, Hypoxia-Inducible Factor-1α, and Innate Antileishmanial Immune Responses

Low oxygen environments and accumulation of hypoxia-inducible factors (HIFs) are features of infected and inflamed tissues. Here, we summarize our current knowledge on oxygen levels found in Leishmania-infected tissues and discuss which mechanisms potentially contribute to local tissue oxygenation in leishmanial lesions. Moreover, we review the role of hypoxia and HIF-1 on innate antileishmanial immune responses.


OXYGeN level iN leiSHMANiAl leSiONS
In guinea-pigs, intravenous challenge experiments with guinea-pig adapted Leishmania enrietti demonstrate that skin tissue predisposes to the development of leishmanial lesions (22). Given that skin tissue is known to display low oxygen levels (23)(24)(25), these data suggest that low oxygen microenvironments might provide a safe haven for Leishmania parasites (22). Araújo et al. assessed lesional tissue oxygen levels with an immunohistochemical method in cutaneous L. amazonensis infection (26). For that purpose, they injected a 2-nitroimidazol derivative into L. amazonensis infected mice. Upon injection, these compounds are enriched in tissues with very low oxygen tensions and form adducts that can be visualized after staining with adduct-specific antibodies (27,28). Using this technology, they found that in L. amazonensis-induced lesions very low oxygen tensions are present (26).
To the best of our knowledge, there are no data available on tissue oxygen levels after infection with L. donovani in liver tissue. Since it is known that steep oxygen gradients exist in the liver (27), it is very likely that L. donovani-infected liver tissues display low oxygen levels as well. Using 2-nitroimidazol based techni ques to visualize hypoxic tissues, Hammami et al. revealed that in L. donovani-infected spleens, very low tissue oxygen prevail (29).
Although staining of hypoxic areas with 2-nitroimidazolderivatives allows for identification of severely hypoxic tissue, this method does not provide quantitative data and is not suitable for continuous recording of lesional tissue oxygen levels (25). Alternative methodologies to quantitatively assess tissue oxygen levels rely on polarographic oxygen sensors or imaging of tissue oxygenation by using oxygen-dependent luminescence quenching (25). For instance, non-invasive luminescence-based oxygen imaging using sensor foils can be used to quantitatively assess tissue oxygenation in a non-invasive manner over time (25). Using this technology in a mouse model of self-healing cutaneous leishmaniasis, Mahnke et al. observed that oxygen tension in leishmanial skin lesions displayed oxygen levels around ~2.8% O2 when the lesions reached their maximum size (30). Resolution and healing of the lesion was paralleled by an increase of tissue oxygenation (30). Altogether, these findings indicate that low oxygen levels prevail in Leishmania-infected tissue.

Potential Factors Regulating Tissue Oxygen levels in Leishmania-infected Tissues
The mechanisms that drive low oxygen levels in Leishmania infected tissue are, however, largely unknown. Recent studies demonstrate that influx of granulocytes results in increased O2 demand which ultimately results in low tissue oxygen levels (31). Low tissue oxygenation in L. major-infected tissue might result from either (i) infection-induced disturbance of local tissue perfusion or (ii) an increased local O2 consumption by the infectious agent and/or infiltrating immune cells (Figure 1). However, the contribution of infiltrating granulocytes, monocytes, NK cells, and T cells on tissue oxygen levels in Leishmania infected tissues is unexplored. Moreover, Leishmania might interfere with inflammation induced angiogenesis. For instance, Leishmania is able to scavenge angiogenic factors such as VEGF-A. Thereby Leishmania might interfere with inflammation-associated angiogenesis and disturb local oxygen supply (32). Nevertheless infection with L. major eventually triggers a vascular endothelial growth factor (VEGF)-A/VEGF receptor (VEGFR)-2-dependent proliferation of endothelial cells (33). Underscoring the potential important role of VEGF-A/VEGFR-2 signaling, Araújo and Giorgio suggest that VEGF-A levels upon infection might predict the outcome of L. amazonensis-infection. They recorded higher VEGF-A levels in healer mice compared to non-healer mice (34). Since VEGF-A is a known HIF-1α target (35) and HIF-1α is present in L. amazonensis-and L. major-infected lesions in humans and in preclinical models (26,33,34,36), it is possible that HIF-1α plays a role in infection-induced proliferation of the endothelial cells. However, to the best of our knowledge, this has not been tested in L. major and L. amazonensis mouse models yet. In addition to maintaining oxygenation of infected tissue, endothelial cell proliferation might curtail L. major induced-inflammatory responses (37). The activity of endothelial NO synthase plays an important role in endothelial cell mediated anti-inflammatory activity (38)(39)(40). Endothelial cell mediated anti-inflammatory activity might reduce the influx of immune cells and/or their O2-consumption, thereby increase tissue oxygen levels and facilitate subsequent resolution of disease. Since endothelial NO synthase expression is linked to HIF-1α-accumulation (41), endothelial HIF-1α might promote anti-inflammatory properties in endothelial cells as well. Further studies, however, are required to investigate this issue. Earlier findings demonstrate that the "myeloid cell differentiation antigen carcinoembryonic antigen-related cell adhesion molecule 1"  (CEACAM1) is involved in angiogenesis and antileishmanial control (42). Whether CEACAM1-dependent signaling affects VEGF-A/VEGFR and/or HIF-1α signaling in this state of affair is unknown. Further investigations are required to understand the interplay of inflammation-triggered angiogenesis, enhanced O2 demand in inflamed tissues, and its impact on overall tissue oxygenation in leishmaniasis.

iMPACT OF HYPOXiA ON HOST-PATHOGeN iNTeRACTiON internalization of Leishmania and Hypoxia
After transmission of Leishmania by its vector, myeloid cells are the main host cell [reviewed in Ref. (43)(44)(45)]. Hypoxic conditions are known to modulate the phagocytic/endocytic uptake of mononuclear phagocytes (46,47). However, to the best of our knowledge, the impact of oxygen levels below 4% O2 on Leishmania uptake or internalization is unexplored.

Polymorphonuclear Granulocytes (PMN), Hypoxia, and Leishmania
Polymorphonuclear granulocytes are quickly attracted to the site of infection. Dependent on the Leishmania species and the host's genetic background, they play a rather detrimental role in cutaneous leishmaniasis. PMN are inefficient in killing promastigotes and undergo apoptosis which hampers the proper activation of recruited dendritic cell (DC) [reviewed in Ref. (44,48)]. In general, PMN are relatively short-lived cells, whose survival, however, is prolonged in NF-κB-and HIF-1α-dependent manner under hypoxic conditions. Moreover, hypoxia induces the production of macrophage inflammatory protein-1β which further facilitates neutrophil survival (49 limited reports on the contribution of hypoxia on DC-mediated establishment of antileishmanial T cell immunity. Hypoxia might impact on DC viability (52), migratory capacity (53), maturation, and antigen presentation (54)(55)(56).

NK Cells, Hypoxia, and Leishmania
In early phase of Leishmania infection, NK cells are activated by IL-12 and IL-18. These activated NK cells contribute to parasite control through production of IFN-γ [reviewed in Ref. (57)]. NK cells respond to hypoxia by downregulation of surface markers NKp46, NKp30, NKp44, NKG2D, and CD16, decreased cytotoxic and antiviral activity, and reduced IFN-γ expression (58)(59)(60). In contrast to these findings, Krzywinska et al. observed no mitigated IFN-γ secretion by NK cells under hypoxia (61). However, the impact of hypoxia on NK-mediated antileishmanial defense is unexplored and requires further studies.

Monocytes/Macrophages, Hypoxia, and Leishmania
A few days after L. major and L. donovani infection, recruited monocytes are highly abundant in afflicted tissues (62,63). Inflammatory monocytes are able to upregulate their ROS production in the early stage of infection with L. major and L. donovani (64,65). However, in inflammatory monocytes infected with L. major, ROS production does eliminate the parasites (63). Moreover, L. donovani, for instance, has the ability to overcome the ROS-mediated apoptosis of host macrophages via induction of suppressor of cytokine signaling proteins (66). It is established that IFN-γ and TNF are two key cytokines that empower macrophages (67-69) and monocytes (63) to clear Leishmania. In the mouse system this results in a robust induction of the antileishmanial effector enzyme, the inducible or type 2 NO synthase (iNOS or NOS2). A high level of NO production is prerequisite of the cutaneous defense against Leishmania major [reviewed in Ref. (70,71)]. Moreover, in mouse models of visceral leishmaniasis, Nos2 expression is important for control of L. donovani in the liver (72), and contributes to the containment of L. donovani (73) in the spleen. For induction of NOS2 and production of high level of leishmanicidal NO, costimulation of macrophages with TNF plus IFN-γ is required (63,68,72,(74)(75)(76)(77)(78). Low oxygen conditions, however, impair NO production of activated macrophages because the inducible NO synthase activity critically hinges on the availability of O2 as a substrate (79)(80)(81). Accordingly, under oxygen conditions below 4% O2 NO production of activated macrophages is diminished (30). L. major possesses a globin-coupled heme containing oxygen sensor soluble adenylate cyclase which allows for its adaption to hypoxic conditions (82). Hence, while L. major is able to adjust to hypoxia, low oxygen conditions impair the NO-dependent antileishmanial potential of macrophages resulting in impaired clearance and survival of L. major in activated macrophages under low oxygen conditions. However, leishmanicidal activity is restored when the cells are reoxygenated (30). These data demonstrate that local tissue oxygen levels-at least transiently-do not match the macrophages' O2 demand to fight against L. major.
In visceral leishmaniasis, parasites express high tissue tropism toward monocytes in the spleen and bone marrow. L. donovani, e.g., induces expansion of hematopoietic stem cell-like mononuclear cells from bone marrow which serve as host cells for the parasite (62). Compared to the role of Nos2 in the defense against visceral leishmaniasis in the liver (72), the contribution of Nos2 in the control of parasite load in the spleen in visceral leishmaniasis is less pronounced. While there was no induction of Nos2 in spleen macrophages upon infection of hamsters with L. donovani (83), Nos2 expression, nevertheless, contributed to the parasitecontainment in the spleen of L. donovani and L. infantum-infected mice (73,84). In preclinical models of visceral leishmaniasis, the impact of local oxygen levels on Nos2-mediated antileishmanial defense and NO production in the spleen and liver is, however, to the best of our knowledge unexplored. It is tempting to speculate that low oxygen levels found in L. donovani-infected spleen tissue (29) incapacitate the Nos2-dependent antileishmanial defense in the spleen and, thus provide a safe niche for persistence and proliferation of L. donovani.
In contrast to L. major, exposure of L. amazonensis-infected macrophages to O2 levels of 5% and 1% O2 resulted in enhanced clearance of intracellular parasites (85,86). These findings suggest that either (1) NO-independent mechanisms are involved in control of L. amazonensis under hypoxic conditions and/or (2) L. amazonensis is not able to adjust to low oxygen conditions. For instance, the divergent effect of hypoxia on the survival of members of the genus Leishmania in mononuclear phagocytes under hypoxia might be due to different metabolic requirements and O2-demands of the respective Leishmania species investigated. While it is known that L. major and L. donovani are able to adjust to hypoxic conditions by increasing their rate of glycolysis (87,88), to the best of our knowledge detailed investigations on the intermediary metabolism of L. amazonensis upon exposure to low oxygen conditions are lacking.

Leishmania and Stabilization of HiF-1α in the Presence of Ample Oxygen
Currently, there is no consensus on whether Leishmania on its own is able to induce HIF-1α accumulation under normoxic conditions ( Table 1). Infection of mouse macrophages in vitro with L. amazonensis and L. donovani triggers HIF-1α accumulation under normoxic conditions without requiring any additional Hypoxia, HIF and Leishmania Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 216 inflammatory signals (90,91). Blockade of this Leishmaniainduced HIF-1α accumulation with cadmium chloride or RNAi results in impaired Leishmania recovery under normoxic conditions (90)(91)(92)(93). In contrast to these findings and in contrast to stimulation with Toll-like receptor ligands [reviewed in Ref.
(1, 8,9)] or infection with various pathogens such as S. pyogenes (35,94), M. tuberculosis (95), Histoplasma capsulatum (96), infection of mouse macrophages with L. major does not induce HIF-1α accumulation on its own but requires exogenous stimulation by IFN-γ and/or LPS (36). In line with this, Hammami et al. demonstrated that upon L. donovani-infection HIF-1α accumulation in CD11c + mouse mononuclear phagocytes in vivo requires interferon regulatory factor-5-dependent inflammatory signaling (97). This suggests that L. donovani infection on its own is not sufficient to induce HIF-1α accumulation in mouse mononuclear phagocytes in vivo as well (97). The different potential of various Leishmania species to induce HIF-1α accumulation in mononuclear phagocytes is unclear. HIF-1α accumulation in mononuclear cells might, for instance, help Leishmania to fuel its metabolic needs (98,99). Mechanistically, the differential ability of certain Leishmania species to interfere with NF-κB activation (100-102) might underlie the divergent ability of different Leishmania species to block infection-associated inflammatory HIF-1α-accumulation under normoxic conditions. However, further studies are required to assess this issue.

Role of HiF-1α expression in Mononuclear Phagocytes
Earlier findings demonstrate a requirement of HIF-1α for NOS2-induction (89,94,(103)(104)(105). In line with this, LPS/INFγcoactivated HIF-1α-deficient macrophages displayed a diminished NOS2-dependent production of NO under normoxic conditions and impaired killing of L. major in vitro. Conditional targeting of HIF-1α in macrophages revealed that HIF-1αactivation is required for antileishmanial defense against cutaneous L. major-infection under normoxic conditions (36). This was paralleled by a blunted induction of NOS2 in lesional myeloid cells (36). However, it remains unclear whether in addition to Nos2 other HIF-1α-regulated targets are involved in cutaneous containment of Leishmania as well.
While HIF-1α in mononuclear phagocytes is protective in L. major-induced cutaneous leishmaniasis, HIF-1α expression in these cells emerged as detrimental factor in visceral leishmaniasis. Hammami et al. used CD11c Cre-deleter mice to target HIF-1α in Cd11c + mononuclear phagocytes and to test the contribution of HIF-1α in a L. donovani-induced preclinical model of visceral leishmaniasis (29). In line with an inhibitory role of HIF-1α in DC-mediated T cell responses (106), Hammami et al. provide evidence that in L. donovani-infected mice HIF-1α in CD11c + cells limits the expansion of short lived effector CD8 + T cells and thereby exacerbates disease at early time points after infections (29). In a recently published follow-up study, they demonstrate that HIF-1α in CD11c + cells limits the frequency and numbers of granulocyte-monocyte progenitors and inflammatory monocytes. Moreover, HIF-1α in CD11c + cells enhances the inhibitory function of splenic mononuclear phagocytes while this TF is not involved in tissue neovascularization and splenomegaly (29). Of note, Hammami et al. demonstrate that HIF-1α negatively impacts on the expression of Nos2 in L. donovani-infected mononuclear phagocytes. Discrepancies of this finding to the published literature on the requirement of HIF-1α on Nos2-expression and NO production might be caused by the different context of HIF-1α stabilization (29). This issue, however, demands further detailed investigation. Overall, the data suggest that HIF-1α in CD11c + cells favors a myeloid-derived suppressor cell-like (MDSC) behavior of these cells. These findings were paralleled by an impaired antileishmanial control in the bone marrow of L. donovani-infected mice at late time points (29).
Taken together, these findings demonstrate that HIF-1α expression in CD11c + mononuclear phagocytes favors development of visceral leishmaniasis in spleen and bone marrow, while HIF-1α in LysM + myeloid cells supports defense against cutaneous L. major infection ( Table 1). These findings underscore that the role of HIF-1α in myeloid cells is highly dependent on the respective context. HIF-1α is known to promote proinflammatory function of mononuclear phagocytes (35,36,94,95,105). However, the very same TF is able to induce regulatory and immunosuppressive functions in mononuclear phagocytes as well (107)(108)(109). Therefore, it is very likely that the differential context of HIF-1α stabilization in visceral and cutaneous leishmaniasis impacts on the functional consequence of HIF-1α activation. Further studies are needed to uncover the distinct signals that ultimately result in differential HIF-1α-dependent innate immune cell function.