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See related article on pg 2339

The Virtues of Oxygenation: Low Tissue Oxygen Adversely Affects the Killing of Leishmania Lopa M. Das1 and Kurt Q. Lu1 Hypoxia contributes to the persistence of infections through altered immune responses. Studies examining skin O2 changes at the site of a lesion are limited. The prevailing methods require the use of electrochemical O2 sensors or radiolabeled electrodes that utilize O2 and may interfere with the precision at low O2 levels. In this issue, Mahnke et al. (2014) demonstrate, using a novel fluorescence-based imaging technology, that low oxygen tension (pO2) impairs NO-mediated anti-leishmanial immunity, leading to increased parasite burden. Replenishing tissue oxygen profoundly enhanced NO-mediated leishmanial killing, underscoring the need to accurately assess oxygenation in infected tissues as a novel strategy to challenge intracellular infection. The technology presented here may have clinical-translational potential in noninvasively assessing disease burden and response to treatment. Journal of Investigative Dermatology (2014) 134, 2303–2305. doi:10.1038/jid.2014.232

Hypoxia in medicine, inflammation, and infection

O2 therapy is emerging as one of the most effective yet inexpensive forms of alternative medicine for several pathological states. This notion evolves from studies demonstrating that improved tissue oxygenation combined with antimicrobials has achieved greater efficacy in pathogen clearance (Knighton et al., 1986). During infection and disease, O2 demand can increase because of various factors including increased metabolism of infiltrated immune cells and microbial proliferation, resulting in a hypoxic environment (Lewis et al., 1999). Hypoxia has a pleiotropic role in tissue inflammation and infection and may exacerbate or attenuate disease. Depending on the pathogen, low pO2 can evoke heightened antimicrobial innate immune responses or, conversely, support pathogen adaptation, proliferation, and survival, resulting in propagation of infection (Degrossoli et al., 2011; Sen Santara et al., 2013). Areas of inadequate O2 tension in the lung of cystic fibrosis patients support

colonization of Pseudomonas bacteria to establish infection and effectively block host immune responses (Callaghan and McClean, 2012). Eukaryotic organisms, including Leishmania parasites, express a heme containing globin protein that functions as a natural O2 sensor to detect hypoxia and adapt accordingly to prevent cell death (Sen Santara et al., 2013). These and other protozoan, bacterial, and viral infections are modulated by hypoxia, either directly or through the activation of other antimicrobial responses such as defensins. Effect of hypoxia on NO-dependent regulation of Leishmania infection

Leishmania are intracellular protozoan parasites that reside within professional antigen-presenting cells (macrophages and dendritic cells (DCs)) to cause cutaneous leishmaniasis, characterized by skin lesions and intracellular parasite load. Although significant advances in drug therapy have allowed for control of intracellular proliferation of Leishmania, the drugs in use have severe side effects,

1

Department of Dermatology, Case Western Reserve University, Cleveland, Ohio, USA

Correspondence: Lopa M. Das, Case Western Reserve University, 2109 Adelbert Road, BRB 529, Cleveland, Ohio 44106, USA. E-mail: [email protected]

urging alternative strategies to be considered. Potentiating a robust host immune response through macrophagemediated NO production may permit a lower dose of drug administration and thus a more effective combinatorial approach to reduce parasite burden. In this issue, Mahnke et al. (2014) present an elegant study demonstrating a critical role for transcutaneous O2 in modulating the course of infection in a murine model of cutaneous leishmaniasis. Host immune responses to Leishmania include macrophage infiltration, activation, phagocytosis, and NO-mediated killing. Using a self-resolving model of cutaneous leishmaniasis to study the effect of pO2 on immune response, the authors demonstrate that low O2 tension in leishmanial skin lesions renders mice susceptible to infection due to insufficient O2-dependent NO production. This model allowed them to delineate using NOS2  /  mice that NO-driven antileishmanial activity is fundamentally linked to tissue O2 levels and can be inhibited in spite of normal NOS2 expression. The Mahnke findings may be strainspecific as two separate in vitro studies of Leishmania-parasitized macrophages demonstrate that activated macrophages under hypoxic conditions are less susceptible to L. amazonensis infection (Colhone et al., 2004) and that hypoxia renders infected macrophages unresponsive to anti-leishmanial drugs (Ayres et al., 2008). In addition, macrophages show enhanced infiltration and increased microbicidal activity (through the expression of mediators such as hypoxia-inducible factor-1a (HIF-1a)) under hypoxic conditions, suggesting that macrophages are most adaptable to a hypoxic microenvironment. The study reported by Mahnke builds on earlier studies demonstrating that hypoxia enhanced cytokine and Tolllike receptor (TLR) ligand–induced mRNA and protein expression of NOS2 in macrophages, yet it inhibited NO production (Albina et al., 1995; Melillo et al., 1996), partially because of an impaired mitochondrial respiratory chain (Wiese et al., 2012). A study by Daniliuc et al. (2003) elucidates the mechanism, demonstrating that low pO2 causes prolonged inactivation of the NOS2 protein by disrupting its interwww.jidonline.org 2303

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Clinical Implications 

Exacerbation of many skin infections and diseases has roots in dysregulated transcutaneous O2 levels; thus, accurate assessment of pO2 using a noninvasive sensor will improve appropriate methods of treatment.



Oxygenation arms immune cells to mobilize their arsenal against pathogens and confers protection from chronic infection.



As tissue oxygenation potentiates host immune response against microbial challenge, restoring pO2 at the site of infection may allow for designing improved combinatorial therapy at a lower dose of the drug.

action with the cytoskeleton. Together, these results emphasize the profound influence of hypoxia on anti-leishmanial activity, especially in macrophages, and supports the concept that hypoxia induces NOS2 protein but impairs NO production. Importantly, in recent studies the same authors have demonstrated that TLR ligation of DCs under hypoxia induces and stabilizes HIF-1a, a molecule required for adaptation and induction of an inflammatory response (Jantsch et al., 2011). This is important because NO production in the PC-12 cell line varies inversely with HIF-1a expression and function (Agani et al., 2002). The fact that NO-mediated leishmanial killing is compromised by hypoxia as demonstrated by Mahnke et al. (2014) in this issue can be rationalized by increased HIF-1a induction that favored parasite survival (Singh et al., 2012). Hypoxia modulation of NO-independent pathways of macrophage infection and parasite survival

Studies indicate that hypoxia at the site of leishmanial infection causes macrophage infiltration and bolsters immune activation to modulate leishmanial killing via NO-independent mechanisms. In a mouse model of L. amazonensis infection, low O2 tension improves macrophage control of Leishmania infection through expression of HIF-1a. The fact that poor O2 levels in skin lesions enhance adhesion and extravasation of macrophages to the endothelium and subsequent infiltration into tissues (Dietz et al., 2012) supports the notion that macrophages are the predominant cells to phagocytose and eliminate Leishmania. However, certain

strains of Leishmania utilize HIF-1a to their advantage so that it serves as a survival factor for parasitized macrophages, and inhibition of HIF-1a reduces parasite survival. The critical role of hypoxia in parasite survival is in agreement with work by Sen Santara et al. (2013) demonstrating that hypoxia compromises Leishmania and Trypanosoma promastigote survival by impairment of an O2-dependent cAMP signaling pathway via protein kinase A. These studies unify the theme that lack of adequate tissue oxygenation at the site of infection or infected lesion mobilizes the expression of critical factors and modulates several pathways that interfere with the establishment of infection and parasite survival. Assessment of transcutaneous pO2 and its effect on infection

Measurement of transcutaneous O2 at the site of infection is a popular method of evaluating microbial persistence or its clearance. In the study by Mahnke et al. (2014), the authors measure transcutaneous O2 levels using planar sensor films to obtain luminescence-2D-in vivo oxygen imaging. Other strategies to quantify skin O2 levels include hyperspectral imaging that allows for evaluation and visualization of acute changes in oxygenation and perfusion in skin following irradiation (Chin et al., 2012). Their study reveals that parasite load is maximized at a point in time when skin pO2 has reached its nadir. Selecting a self-resolving mouse model of cutaneous leishmaniasis allowed Mahnke et al. (2014) to monitor transcutaneous O2 from maximum parasite burden to the time until infection has cleared. pO2 ranged from acute hypoxia to normoxia, and NO production reflec-

2304 Journal of Investigative Dermatology (2014), Volume 134

ted a similar pattern. The model also allowed the authors to explore further whether the resolving phenotype, corresponding to normoxia, could be altered by modulating pO2 at the site of infection. Through gain and loss of function approaches, Mahnke et al. (2014) establish that NO-mediated L. major killing depends on an adequate supply of tissue O2. To advance our understanding that accurate assessment of pO2 tension can predict the status of an immune response against L. major infection in an NO-dependent manner, the authors methodically eliminate the possibilities that reduced NO level is a result of impaired protein synthesis, or insufficient iNOS substrate arginine, and establish that reduced NO production is a direct consequence of inadequate skin O2 levels. CONFLICT OF INTEREST

The authors state no conflict of interest.

REFERENCES Agani FH, Puchowicz M, Chavez JC et al. (2002) Role of nitric oxide in the regulation of HIF-1alpha expression during hypoxia. Am J Physiol Cell Physiol 283:C178–86 Albina JE, Henry WL Jr., Mastrofrancesco B et al. (1995) Macrophage activation by culture in an anoxic environment. J Immunol 155: 4391–6 Ayres DC, Pinto LA, Giorgio S (2008) Efficacy of pentavalent antimony, amphotericin B, and miltefosine in Leishmania amazonensisinfected macrophages under normoxic and hypoxic conditions. J Parasitol 94:1415–7 Callaghan M, McClean S (2012) Bacterial host interactions in cystic fibrosis. Curr Opin Microbiol 15:71–7 Chin MS, Freniere BB, Lo YC et al. (2012) Hyperspectral imaging for early detection of oxygenation and perfusion changes in irradiated skin. J Biomed Opt 17:026010 Colhone MC, Arrais-Silva WW, Picoli C et al. (2004) Effect of hypoxia on macrophage infection by Leishmania amazonensis. J Parasitol 90:510–5 Daniliuc S, Bitterman H, Rahat MA et al. (2003) Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4. J Immunol 171:3225–32 Degrossoli A, Arrais-Silva WW, Colhone MC et al. (2011) The influence of low oxygen on macrophage response to Leishmania infection. Scand J Immunol 74:165–75 Dietz I, Jerchel S, Szaszak M et al. (2012) When oxygen runs short: the microenvironment drives host-pathogen interactions. Microbes Infect 14:311–6

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Jantsch J, Wiese M, Schodel J et al. (2011) Toll-like receptor activation and hypoxia use distinct signaling pathways to stabilize hypoxia-inducible factor 1alpha (HIF1A) and result in differential HIF1A-dependent gene expression. J Leukoc Biol 90:551–62 Knighton DR, Halliday B, Hunt TK (1986) Oxygen as an antibiotic. A comparison of the effects of inspired oxygen concentration and antibiotic administration on in vivo bacterial clearance. Arch Surg 121:191–5

Melillo G, Taylor LS, Brooks A et al. (1996) Regulation of inducible nitric oxide synthase expression in IFN-gamma-treated murine macrophages cultured under hypoxic conditions. J Immunol 157:2638–44 Sen Santara S, Roy J, Mukherjee S et al. (2013) Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia. Proc Natl Acad Sci USA 110:16790–5

Lewis JS, Lee JA, Underwood JC et al. (1999) Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol 66: 889–900

Singh AK, Mukhopadhyay C, Biswas S et al. (2012) Intracellular pathogen Leishmania donovani activates hypoxia inducible factor-1 by dual mechanism for survival advantage within macrophage. PLoS One 7:e38489

Mahnke A, Meier RJ, Schatz V et al. (2014) Hypoxia in Leishmania major skin lesions impairs the NO-dependent leishmanicidal activity of macrophages. J Invest Dermatol 134:2339–46

Wiese M, Castiglione K, Hensel M et al. (2012) Small interfering RNA (siRNA) delivery into murine bone marrow-derived macrophages by electroporation. J Immunol Methods 353: 102–10

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A New Player on the Psoriasis Block: IL-17A- and IL-22-Producing Innate Lymphoid Cells Nicole L. Ward1 and Dale T. Umetsu2 Innate lymphoid cells (ILCs) are a recently discovered family of innate immune cells belonging to the lymphoid lineage, yet lacking antigen-specific receptors. ILCs were first identified in the intestinal tract, where they contribute to epithelial barrier integrity and host responses to commensal microbes. Teunissen et al. (in the current issue) and Villanova et al. (2014) now suggest an important role for type 3 ILCs (ILC3s) in the skin, particularly in psoriasis. Both groups found an increased frequency of IL-22- and/or IL-17A-producing ILCs in psoriatic skin and blood. These cells are activated in response to IL-1b and IL-23, correlate with disease severity, and are decreased following antitumor necrosis factor-a (antiTNFa) treatment. The presence of a novel ILC population in psoriatic skin, one that responds to biologic therapeutics, suggests that dysregulation of ILCs is a contributing factor to psoriasis pathogenesis. Journal of Investigative Dermatology (2014) 134, 2305–2307. doi:10.1038/jid.2014.216

Innate lymphoid cells (ILCs) represent a family of multifunctional, lymphoidlineage cells that lack B- and T-cell receptors and display innate immune effector functions. These cells often reside at mucosal interfaces where they rapidly produce cytokines in response to environmental challenges. At least three

groups of ILCs have been identified, based upon unique transcription factor utilization, cytokine profiles, and effector functions. Groups 1 and 2 produce type 1 and 2 cytokines, respectively, whereas group 3 has a unique capacity to produce IL-17A and IL-22. Dysregulation of ILCs has been implicated in

1

Department of Dermatology, Case Western Reserve University, Cleveland, Ohio, USA and 2Genentech, San Francisco, California, USA Correspondence: Nicole L. Ward, Department of Dermatology, Case Western Reserve University, BRB519, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA. E-mail: [email protected] or Dale T. Umetsu, Genentech, San Francisco, California 94080, USA. E-mail: [email protected]

autoimmune and inflammatory diseases, thus broadening our understanding of how innate immune cells contribute to disease pathogenesis. However, less well understood until now is the potential role of ILCs in psoriasis. Two papers, both published in the Journal of Investigative Dermatology in 2014 (Teunissen et al., 2014; Villanova et al., 2014), provide compelling evidence for a pathogenic role of ILCs in psoriasis. Teunissen et al. (2014) show the upregulation of group 3 ILCs (ILC3s) in nonlesional and lesional skin and in blood of psoriasis patients, and that these cells are novel cellular sources of IL-22 in the skin. Nestle and colleagues (Villanova et al., 2014) demonstrate a close association between disease improvement and the number of IL-17A- and IL-22-producing ILC3s in the skin and in circulation following antitumor necrosis factor-a (anti-TNFa) therapy, suggesting that ILC3s contribute to the pathogenesis of psoriasis. ILCs

ILCs lack T-cell receptors and B-cell receptors, and therefore are antigen nonspecific; however, they respond rapidly to environmental challenges, such as tissue injury or infection, via cytokine secretion. Type 1 ILCs, or ILC1s, require the transcription factor T-bet, are induced in response to IL-12, produce type 1 cytokines such as IFNg, and have been observed in the intestines of patients with inflammatory bowel disease. Type 2 ILCs, or ILC2s, require the transcription factor RORa and GATA3, respond to the innate cytokines IL-33, IL25, and thymic stromal lymphopoietin (TSLP), produce IL-5 and IL-13 but not IL-4, and have been found in the intestines in the context of helminth infection (Neill et al., 2010), in the lungs in the context of asthma (Chang et al., 2011), and in the skin in the context of atopic dermatitis (Salimi et al., 2013). Type 3 ILCs, or ILC3s, require the transcription factor RORgt, respond to IL-23 and IL1b, and produce IL-17A and/or IL-22. ILC3s producing IL-17A have been observed in the intestines of patients with Crohn’s disease (Coccia et al., 2012) and in the lungs of patients with nonallergic asthma (Kim et al., 2014). www.jidonline.org 2305

The virtues of oxygenation: low tissue oxygen adversely affects the killing of Leishmania.

Hypoxia contributes to the persistence of infections through altered immune responses. Studies examining skin O2 changes at the site of a lesion are l...
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