Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of

myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells

as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and

nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of

their infiltration into the lung. The nitric oxide-producing cells were Ly-6C+Ly-6G− and they downmodulated T-cell activation, recruited Treg cells, and dramatically downregulated

antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C−Ly-6G+ and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a

superoxide-dependent fashion. A smaller population of Ly-6C+Ly-6G+ cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells

represent important targets for asthma therapy.

Asthma is a disorder of respiratory function characterized by persistent T helper cell type 2 (Th2)-predominant inflammation and reversible airway obstruction, associated with airway

hyperresponsiveness (AHR).1 Studies in experimental models of asthma indicate that both innate and adaptive immune cells contribute to asthma pathogenesis.2, 3 Although lymphoid and myeloid

cell-derived cytokines and chemokines are recognized to contribute to the asthmatic phenotype, the mediators that induce AHR remain incompletely defined.2, 3 Reactive free radicals such as

nitric oxide (NO) and superoxide (O2·−), which can either augment or suppress inflammatory processes, have been identified in the airways of asthmatic subjects;4, 5, 6, 7 however, the

cellular sources of these molecules and their relationship to the major features of the asthmatic phenotype remain unknown.

The bioavailability of NO is regulated in vivo at the level of its production from L-arginine (L-Arg) by nitric oxide synthases (NOSs), particularly the inducible NOS (iNOS), and its

consumption in downstream chemical reactions.8, 9, 10 The ability of eosinophil cationic proteins to inhibit the transport and availability of L-Arg, thereby reducing the production of

bronchodilatory NO in the airway, has been implicated in the induction of AHR;11 however, increased levels of NO in exhaled breath condensate and bronchoalveolar lavage (BAL) fluid from

asthmatics5, 12 suggest that NO can have proinflammatory effects. Peroxynitrite (ONOO−), generated by reaction of NO and O2·−, is also a biomarker of airway inflammation.7, 13 Studies with

inhibitors of NO and O2·− also highlight NO and O2·− as drivers of AHR in asthma.14, 15 NO, with its dual potential, supporting physiological functions and tissue homeostasis as well as

activating inflammation, has remained a paradox in the context of asthmatic inflammation.

Pharmacological inhibition of arginase has also been reported to both improve and worsen inflammation in experimental asthma.10, 16, 17 Arginase catalyzes the conversion of l-Arg to urea and

polyamines, which can contribute to airway remodeling in chronic asthma.10, 17 Activation of arginase depletes l-Arg, not only reducing bioavailable NO because of limitations of

substrate,10, 17 but also uncoupling the NOS enzymes leading to increased production of O2·−.18 Thus, competition between arginase and iNOS for l-Arg can upset the important NO/O2·− balance

in asthmatic lungs.17 Regulated expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase also controls O2·− production in the local tissue environment.19

Populations of immature myeloid cells called myeloid-derived suppressor cells (MDSCs) can produce free radicals using the iNOS, arginase, and NADPH oxidase pathways.20, 21 MDSCs, broadly

characterized in mice by their surface expression of the Gr-1 and CD11b antigens, are immunosuppressive in cancer,20, 22 autoimmune and viral encephalitis,23 inflammatory bowel disease,24

and other conditions,25, 26, 27 where they inhibit both CD4 and CD8 T-cell proliferative responses via their production of NO and O2·−.28 Their free radical products also contribute to the

recruitment, maintenance, and activation of the MDSCs themselves.20, 21 The participation of MDSCs in determining the balance of the iNOS, arginase, and the NADPH oxidase pathways in a Th2

cell-dominant inflammatory disorder like asthma is undefined. We hypothesized that asthmatic inflammation and AHR are regulated by mechanisms that involve accumulation of free

radical-producing MDSC-like cells in lungs.

In this study, we identify three populations of Gr-1+CD11b+ myeloid cells that infiltrate the lung in a mouse model of allergic airway inflammation where they differentially generate the

reactive free radicals NO and O2·−. The Ly-6C+Ly-6G− subset (predominant NO producer) and the Ly-6C+Ly-6G+ subset suppress T-cell proliferation in vitro. The O2·−-generating Ly-6C−Ly-6G+

subset, in contrast, enhances T-cell responses. We refer to these Gr-1+CD11b+ myeloid cells as myeloid-derived regulatory cells (MDRCs) to acknowledge their potential to either suppress or

augment immune and inflammatory responses. Using pharmacological inhibitors and/or mouse strains with genetic knockout of the iNOS, arginase, or NADPH oxidase pathways, we show an integral

role for these enzymes and their metabolites in the accumulation of the MDRCs in the lungs. Importantly, intratracheal (i.t.) adoptive transfer of MDRC subsets dramatically modulates AHR.

Our studies thus highlight the functional significance of enzymes that produce free radicals and their metabolites in the induction and suppressive potential of the myeloid lineage in

experimental allergic airway disease. Identifying MDRCs as important sources of free radicals in the inflamed lung provides a new perspective on the role of NO in asthma.

This study was undertaken to test whether the iNOS, arginase, and NADPH oxidase pathways participate in antigen-driven inflammatory responses of the airways and whether the free radical

products of these pathways are central to these responses. We first examined the levels of the NO metabolite nitrite and the end product of the arginase pathway urea in the BAL fluid of

ovalbumin (OVA)-sensitized and -challenged mice. In the OVA-challenged C57BL/6 (wild-type (wt)) mice, nitrite was increased in BAL fluid at day 2 (d2), with a modest but significant

reduction at d3 and a progressive rise from d5 to d10 (Figure 1a, cross-hatched bars). This increase in nitrite was not observed in OVA-challenged iNOS−/− mice (Figure 1a, open bars)

suggesting that iNOS-derived NO is the source of nitrites in the BAL fluid of asthmatic wt mice. Levels of urea were elevated in BAL fluid at d3 following intranasal (i.n.) OVA challenge

(Figure 1b, cross-hatched bars), indicating that arginase activity was also increased in the antigen-driven airway inflammatory response. The iNOS and arginase enzymes compete for the same

substrate l-Arg, and metabolites of the arginase pathway increase in the absence of functional iNOS.29 Consistent with this, analysis of OVA-sensitized and -challenged iNOS−/− mice showed a

significant elevation of the levels of urea in BAL fluid during the inflammatory response (Figure 1b, open bars), compared with wt animals, indicating that activation of the arginase pathway

is enhanced in the absence of functional iNOS.

The inducible nitric oxide synthase (iNOS) and arginase pathways are induced in a mouse model of allergic airway inflammation. (a) Measurement using the Griess assay of nitric oxide (NO;

measured as the NO metabolite, nitrite) in bronchoalveolar lavage (BAL) fluid harvested at the indicated number of days after challenge. **P