Arginine metabolic control of airway inflammation

All NOS isoforms are present in the airways: neuronal NOS (nNOS; NOS1 ) in nerves, iNOS ( NOS2 ) in airway epithelium, and endothelial nitric oxide synthase ( NOS3 ) in lung endothelium and in very small amounts in the airway epithelium. Genetic deletion of each of the 3 genes in mice provides information of their individual contributions. iNOS is the only NOS that is induced in the OVA allergen airway inflammation model and is associated with increased expired NO. More than 90% of F E NO is attributable to iNOS using the NOS-deleted mice (41), and this parallels findings in patients in which iNOS induction occurs in allergen challenge of human asthmatics that also leads to increased F E NO (42). iNOS -deleted mice have less eosinophilic inflammation than WT in the OVA model, yet continue to have airway hyperresponsiveness (43). Conversely, nNOS-deleted mice have less airway hyperreactivity but no decrement in airway inflammation. In humans, airway inflammation of many asthmatics is typified by high F E NO, which decreases with corticosteroids (14, 15). Respiratory exposure of WT mice to HDME induces a combined eosinophilic and neutrophilic airway inflammation, along with angiogenic remodeling of the lungs. Recently, we have shown that mice lacking ARG2 have worsened Th2-driven eosinophilic airway inflammation compared with WT mice in an OVA mouse model (4). The OVA model, unlike HDME, is eosinophil dominated with little to no neutrophilic inflammation. iNOS –/– mice in the OVA model had lower eosinophilic inflammation compared with WT animals (41). In contrast, our data show that iNOS –/– and WT mice in the HDME model had similar levels of airway inflammation,


Introduction
Genetic and environmental factors are important to the genesis and progression of asthma (1).Over the past few years, accumulating evidence suggests that changes in metabolism regulates inflammation and T cell function (2).Current concepts suggest that arginine metabolism affects immune cell function via both mitochondrial and nonmitochondrial mechanisms of action (3,4).Recently, our group identified that arginine metabolism is a key driver of Th2 airway inflammation (4).Arginine is a semiessential amino acid metabolized by several enzymes, including the inducible nitric oxide synthase (iNOS) in the cytosol and by arginase-2 (ARG2) in the mitochondria (5).Arg1, another enzyme in arginine metabolism, is primarily expressed in the cytosol of hepatic cells as part of the urea cycle (6,7).There are abundant data linking both iNOS and ARG2 enzymatic pathways to asthma pathophysiology.ARG2 gene variants were some of the earliest and continue to be the most consistent single nucleotide polymorphisms (SNPs), discovered in genome-wide association studies (GWAS) of asthma, strongly linked to asthma origins and severity (ARG2 rs7140310 [T/G], rs742869 [G/A], rs3742879 [A/G]) (8)(9)(10).The relationship between ARG2 SNPs and the expression or activity of arginase is currently unknown.ARG2 lies within an asthma linkage region on chromosome 14q24 (11).Arginine metabolism via iNOS and ARG2 is higher in asthmatics than in healthy individuals (12,13).Loss of arginine metabolism in ARG2 -/-mice is also associated with more severe airway inflammation in the OVA mouse Th2 model of allergic respiratory inflammation.
High fractional exhaled NO (F E NO) in human asthma is generated by iNOS expressed in the airway epithelium (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), where it catalyzes the conversion of arginine to NO and citrulline (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).Despite greater iNOS and F E NO, arginine levels in asthma epithelium are 3-fold higher than normal, and citrulline levels are very low (21).This, together with the inconsistent effects of NOS inhibitors in asthma (23) and linkage of ARG2 to asthma, suggests that an NO-centric view of arginine metabolic effects in asthma is incomplete (23).Here, we investigate the concept that arginine regulates pathways of inflammation important in asthma Inducible nitric oxide synthase (iNOS) and arginase-2 (ARG2) share a common substrate, arginine.Higher expression of iNOS and exhaled NO are linked to airway inflammation in patients.iNOS deletion in animal models suggests that eosinophilic inflammation is regulated by arginine metabolism.Moreover, ARG2 is a regulator of Th2 response, as shown by the development of severe eosinophilic inflammation in ARG2 -/-mice.However, potential synergistic roles of iNOS and ARG2 in asthma have not been explored.Here, we hypothesized that arginine metabolic fate via iNOS and ARG2 may govern airway inflammation.In an asthma cohort, ARG2 variant genotypes were associated with arginase activity.ARG2 variants with lower arginase activity, combined with levels of exhaled NO, identified a severe asthma phenotype.Airway inflammation was present in WT, ARG2 -/-, iNOS -/-, and ARG2 -/-/iNOS -/-mice but was greatest in ARG2 -/-.Eosinophilic and neutrophilic infiltration in the ARG2 -/-mice was abrogated in ARG2 -/-/iNOS -/-animals.Similarly, angiogenic airway remodeling was greatest in ARG2 -/-mice.Cytokines driving inflammation and remodeling were highest in lungs of asthmatic ARG2 -/-mice and lowest in the iNOS -/-.ARG2 metabolism of arginine suppresses inflammation, while iNOS metabolism promotes airway inflammation, supporting a central role for arginine metabolic control of inflammation.
origins and severity.To test this idea, asthmatics with genetic variants of ARG2 were evaluated for severity of asthma in the context of high and low levels of F E NO.In mechanistic studies, WT, iNOS -/-, ARG2 -/-, and in-house-generated iNOS -/-/ARG2 -/-animals were evaluated in a house dust mite extract (HDME) model of airway inflammation, which is typified by eosinophilic and neutrophilic inflammation.

Results
ARG2 variant genotypes are associated with arginase activity.Characteristics of the study participants have been previously described (27,28) and are shown in Supplemental Table 1 (Supplemental material available online with this article; https://doi.org/10.1172/jci.insight.127801DS1),grouped by asthma versus control and by asthma with high and low F E NO. Asthmatics were older than healthy controls (age [yr]: control 30 ± 1; asthma 39 ± 1; P < 0.0001) but similar in sex (sex [M/F]: control, 59/97; asthma, 188/337; P = 0.6).Among the several ARG2 SNPs linked to asthma, rs742869 is a known common variant (major/minor allele: A/G), allowing us to have sufficient power among heterozygotes and homozygotes for comparisons (8)(9)(10).Compared with healthy controls, asthmatics tended to have the highest frequency of A/A (homozygous for the minor A allele) (GG/GA/AA: control, 38/71/47; asthma, 87/239/199; P = 0.05 ANOVA; GG vs. AA, P = 0.01) (Table 1).Among asthmatics, the comparison of AA vs. GG was significant (P = 0.015).Adjusted OR for age, sex, and smoking history was 1.82, with P = 0.024.Arginase activity tended to be the lowest in asthmatics with A/A allele (arginase activity, μmol/mL/h: GG, 0.63 ± 0.13; AA, 0.37 ± 0.05; P = 0.04) (Table 1).There were no differences in severity, lung function, inflammation, and F E NO across different alleles, but BMI and IgE trended to be higher in the AA genotype asthmatics (Table 1).ARG2 rs742869 genotypes have been related to phenotypes of obesity, airflow obstruction, hyperreactivity, and blood eosinophils and IgE levels.We further analyzed asthmatics dichotomized by high and low F E NO levels (Figure 1, A−F).A subphenotype group of patients with low F E NO and ARG2 G/G allele (with highest arginase activity) had the highest BMI (P < 0.001, 2-tailed ANOVA, Figure 1A), the highest PC 20 (provocative concentration of methacholine causing a 20% fall in FEV 1 ; P = 0.01, 2-tailed ANOVA, Figure 1C), and the lowest blood eosinophil levels (P < 0.0001, 2-tailed ANOVA, Figure 1D).Asthmatics with high F E NO and the ARG2 A/A genotype (with low arginase activity) had the highest blood IgE levels (P = 0.0002, 2-tailed ANOVA, Figure 1E).No sex differences were observed in our cohort.IgE levels, percent eosinophils, and F E NO were higher in male asthmatics compared with female, while lung function was lower (male vs. female asthmatics: IgE, 249 ± 406 male, 177 ± 263 female, P < 0.001; percent eosinophils, 4.3 ± 2.9 male, 3.6 ± 3.0 female, P < 0.001; F E NO, 44.2 ± 38 male, 39.2 ± 43 female, P < 0.01; baseline forced expiratory volume in 1 second (FEV 1 ) percent predicted, 67.4 ± 22.9 male, 76.6 ± 21.9 female, P < 0.001; FEV 1 /forced vital capacity (FVC), 0.64 ± 0.12 male, 0.72 ± 0.12 female, P < 0.001).Overall, the findings suggest that genotypes associated with arginase activity may interact with F E NO levels to manifest as different asthma phenotypes.These findings prompted us to study ARG2 and iNOS pathway interactions in a mouse model of airway inflammation.
The greater airway inflammation in ARG2 -/-mice is modulated by iNOS.Inflammatory cells were not observed in naive or saline exposed WT, ARG2 -/-, iNOS -/-, and iNOS -/-/ARG2 -/-in lung tissue sections stained with H&E (Figure 2A).All 4 genotypes developed airway inflammation upon exposure to HDME, but ARG2 -/-mice consistently had the highest numbers of inflammatory cell influx into the lungs (Figure 2A).Quantification of bronchoalveolar lavage (BAL) cellular content showed that total cell number (Figure 2B) and number of eosinophils (Figure 2C) and neutrophils (Figure 2D) were higher in HDME-exposed ARG2 -/-mice than other genotypes.There were no differences in inflammatory cells between WT and iNOS -/-animals (Figure 2, B-D).Macrophages and lymphocytes were similar across genotypes in allergen exposed groups (Figure 2, E and F).Deletion of iNOS attenuated the airway inflammation in ARG2 -/-mice so that levels were similar to WT levels (Figure 2, A-D).These data show that deletion of the mitochondrial arginine-consuming enzyme ARG2 results in exaggerated eosinophilic and neutrophilic airway inflammation, which was nullified to WT levels by the deletion of the other major arginine metabolizing enzyme iNOS.

Discussion
This study shows that arginine metabolism is a critical modulator of severity of inflammation and remodeling in eosinophilic and neutrophilic asthma.Early GWAS showed a strong association of asthma and ARG1 and ARG2 gene variants (8,10,38,39).Increased serum arginase activity was also quantitatively related to airflow limitation, as measured by FEV 1 (40).Here, we show that arginase activity depends on specific ARG2 SNPs.Common ARG2 variants that were associated with lower arginase activity, combined with high levels of F E NO, identified a more severe asthma phenotype.We modeled this severe asthma phenotype in the HDME ARG2 -/-mouse models to investigate the underlying arginine metabolic pathways.ARG2 -/-, iNOS -/-, and crossbreeds of these KOs were used to decipher the roles of iNOS and mitochondrial ARG2-dependent arginine metabolism.ϕ P < 0.05 between WT and ARG2 -/-.φ P < 0.05 between ARG2 -/-and ARG2 -/-iNOS -/-.σ P < 0.05 between iNOS -/-and ARG2 -/-iN-OS -/-.# P < 0.05 between iNOS -/-and ARG2 -/-.λ P < 0.05 between WT and iNOS -/-or WT and ARG2 -/-iNOS -/-.Each dot represents data from 1 mouse, and mean ± SEM values are shown.Wilcoxon test was used in A-D, and Student's t test was used in F-L for group comparisons.
All NOS isoforms are present in the airways: neuronal NOS (nNOS; NOS1) in nerves, iNOS (NOS2) in airway epithelium, and endothelial nitric oxide synthase (NOS3) in lung endothelium and in very small amounts in the airway epithelium.Genetic deletion of each of the 3 genes in mice provides information of their individual contributions.iNOS is the only NOS that is induced in the OVA allergen airway inflammation model and is associated with increased expired NO.More than 90% of F E NO is attributable to iNOS using the NOS-deleted mice (41), and this parallels findings in patients in which iNOS induction occurs in allergen challenge of human asthmatics that also leads to increased F E NO (42).iNOS-deleted mice have less eosinophilic inflammation than WT in the OVA model, yet continue to have airway hyperresponsiveness (43).Conversely, nNOS-deleted mice have less airway hyperreactivity but no decrement in airway inflammation.In humans, airway inflammation of many asthmatics is typified by high F E NO, which decreases with corticosteroids (14,15).Respiratory exposure of WT mice to HDME induces a combined eosinophilic and neutrophilic airway inflammation, along with angiogenic remodeling of the lungs.Recently, we have shown that mice lacking ARG2 have worsened Th2-driven eosinophilic airway inflammation compared with WT mice in an OVA mouse model ( 4).The OVA model, unlike HDME, is eosinophil dominated with little to no neutrophilic inflammation.iNOS -/- mice in the OVA model had lower eosinophilic inflammation compared with WT animals (41).In contrast, our data show that iNOS -/-and WT mice in the HDME model had similar levels of airway inflammation, suggesting that iNOS metabolism of arginine has lesser effects on inflammation generated via IL-17 pathways.ARG2 deficiency increased both eosinophilic and neutrophilic airway inflammation and angiogenic remodeling.While the deletion of iNOS alone had no major effect on the asthma phenotype, double deletion of iNOS and ARG2 attenuated eosinophilic/neutrophilic airway inflammation and angiogenic remodeling.The findings reveal that (a) arginine metabolism regulates eosinophilic and neutrophilic airway inflammation and remodeling, and (b) ARG2 is a critical checkpoint in modulating inflammation and angiogenesis.
ARG2 and iNOS have a common substrate arginine, but the interactions between these 2 enzymes are more complex than substrate competition (4).We previously showed that iNOS and ARG2 interactions are not controlled by the competition for arginine but through interaction of arginine metabolism and the TCA cycle (4).
Prior studies of asthmatic patients suggested that mitochondria were more oxidative (12).ARG2 gives rise to ornithine from arginine, which can be converted to glutamate that is transformed to α-ketoglutarate (αKG) to enter the TCA cycle.Arg2 is increased in asthmatics (4).Increased TCA cycle activity in asthma increases ATP production and levels of TCA cycle intermediates to other pathways (4).For example, αKG transported outside of the mitochondria serves as substrate for prolyl hydroxylase domain enzymes and inhibits hypoxia-inducible factor (HIF), an upstream regulator of Th2 cytokines (4).ARG2 deletion has previously been linked to higher HIF levels and increased Th2-driven eosinophilic inflammation and Th2 cytokine levels of IL-13 and eotaxin-1, along with increasing activation of pSTAT6 (4).Eotaxin-2, IL-5, and angiogenic remodeling of the airways in the HDME model are regulated by HIF (44).Human asthma involves Th2 (eosinophilic) with IL-17 (neutrophilic) inflammation, and IL-17 inflammation is increasingly recognized as an endotype of severe asthma (45).Eotaxin-2, mainly expressed by alveolar macrophages and proangiogenic hematopoietic progenitors, is a key driver in airway eosinophila in mouse models of airway inflammation (7,36).The induction of eotaxin-2, but not eotaxin-1, by arginine metabolism suggests a specific effect on these cells.Overexpression of eotaxin-2 and IL-5 in mice resulted in spontaneous severe eosinophilic asthma (35).IL-17 is a key mediator of severe neutrophilic asthma (46).Eotaxins and IL-17 are also potent angiogenic factors in asthma, cancer, and other inflammatory disorders (36,38,(47)(48)(49)(50)(51)(52)(53).IL-17 levels were increased in ARG2 -/-lungs, but Th17 differentiation was lower in these mice, suggesting a non-Th17 source of IL-17.Several other immune cells, including innate lymphoid cells, B cells, neutrophils, NKT cells, and γδ T cells, also produce IL-17 (54).In obesity-associated asthma, type III innate lymphoid cells (ILC3) is a significant source of IL-17 (55).A recent report demonstrated that alveolar macrophages, and not Th17 cells, are the major source of pulmonary IL-17 in the OVA mouse model and in patients with allergic asthma (56).The non-Th17 source of the increased IL-17 levels in lungs of ARG2 -/-mice is tipping the balance toward an unfavorable phenotype.Alternatively, splenic T cells may not represent pulmonary T cell polarization.The findings are in keeping with our previous publication, showing that WT BM transplantation reduces IL-17 levels in the BAL in an OVA model (4).
The asthma population in this study was exclusively White, which limits extrapolation of the findings to the broader asthmatic population in general.Another limitation of this study is that SNP rs742869 is associated with lower arginase activity, but whether this results from reduced protein expression and/or lower enzymatic activity is unknown.
Collectively, our findings reveal that eosinophilic and neutrophilic immune pathways that control severity of inflammation are regulated by arginine metabolism (Figure 4).Arginine metabolism via ARG2 suppresses inflammation and, via iNOS, promotes airway inflammation.New therapeutic strategies to enhance arginine metabolism by the mitochondrial ARG2 may provide benefit to patients with severe asthma.
BAL fluid collection and processing.Animals were euthanized by an overdose of pentobarbital (Akorn), and the lungs were lavaged with 700 μL sterile saline via a cannula inserted into the trachea.The BAL fluid (BALF) was centrifuged at 300 g for 10 minutes.The supernatant was collected and stored at -20°C for cytokine analyses, and the cell pellet was resuspended in 500 μL of 2% FBS (Atlanta Biologicals) in PBS (prepared in house).BAL cell concentration was determined by counting a mixture of 10 μL cell suspension, 10 μL of Live/Dead and nuclear stain (20 μg/mL Ethidium Bromide [Sigma-Aldrich], and 20 μg/mL Acridine Orange [Sigma-Aldrich] dissolved in PBS) under a fluorescent microscope (Olympus CKX41) using a hemocytometer (Hausser Scientific).Cytospins were prepared by centrifugation of 20,000 cells in 100 μL of 2% FBS loaded into Shandon Cytospin 3 instrument (400 g for 4 minutes).Air-dried cytospins were stained with Kwik-Diff (Dade Behring) and mounted using Cytoseal mounting medium.A differential count of hematopoietic cells, including macrophages, neutrophils, eosinophils, and lymphocytes, was performed under a microscope.At least 200 cells were counted based on standard morphologic criteria.
Lung tissue harvest and IHC.The lung vascular bed was perfused with warm PBS via the right heart to flush out remaining blood cells.The dissected left lobe was fixed in 10% formalin and processed for paraffin embedding and tissue sectioning.The right lung was snap frozen, stored in liquid nitrogen, and later used for cytokine quantification.Left lung tissue sections were stained with H&E.vWF staining and microvessel quantification were performed as described previously (36,63).
Cytokine quantification.Tissues of the right lobes were homogenized in PBS and subjected to repeated freeze/ thaw cycles.The suspension was centrifuged to pellet debris and supernatant -the latter was used for lung protein extract.IL-17 and GM-CSF (both from Abcam), along with CCL20, IL-1α, KC, MIP-2, and LIX (all from R&D Systems), levels in BAL and lung protein extract were measured using quantitative ELISA kits.Eotaxin-1/2, IL-5, and IL-13 levels in lung and BAL were quantified using Super-X Plex cytokine assay (Antigenix America) according to the instructions of the manufacturer on a LSRII flow cytometer (Becton Dickinson).
Amino acid analysis.Amino acids were measured by high-performance liquid chromatography (HPLC) using precolumn derivatization with o-phthaldialdehyde (OPA).OPA is a fluorophore, which -when combined with an amino acid -forms an isoindole that can easily be detected and quantified.Plasma

R E S E A R C H A R T I C L E
samples were deproteinized with an equal volume of 6% sulfosalicylic acid containing 50 μM ethionine as an internal standard.After 5 minutes of centrifugation at 1,000 g, the supernate was placed into an autosampler, where it was mixed with OPA and injected onto the column according to an injector program.
The HPLC system was an Agilent 1100 with ChemStation (Agilent Technologies) consisting of a binary pump, degasser, fluoresence dectector, and autosampler.Chromatographic separations were carried out using a Supelcosil LC-18 column 150 cm × 4.6 mm, 5 μm (Sulpelco), which was protected by an appropriate guard column.All reagents were HPLC grade and prepared with 18 MΩ water.The mobile phase consisted of 20 mM sodium acetate, pH 5.7, with 4.0% tetrahydrofuran.Buffer B was methanol.All chemicals are from Sigma-Aldrich.A gradient was used with percent B increasing to 100% over 44 minutes at 280°C.Twenty-one amino acid standards at appropriate concentrations were used for calibration and measured by fluorescence detector using excitation 340 nm and emission 455 nm (64).CD4 T cell polarization.Splenocytes were harvested to analyze Th cell differentiation.IL-4-, IL-17A-, IFN-γ-, and Foxp3-expressing CD4 + T cells were quantified by flow cytometry using the mouse Th1/Th2/ Th17/Treg phenotyping kit (BD Biosciences).
Statistics.Logistic regression analyses were used to estimate ORs of asthma patients for a comparison of AA with GG.A multivariable logistic regression model provided an adjusted OR estimate, with age, sex, and smoking history as covariates.Males and females were compared with respect to the frequency of asthma and other categorical characteristics using χ 2 tests, and they were compared with respect to quantitative characteristics using the Wilcoxon rank sum test.One-way ANOVA was used for comparison across groups.
Data are shown as mean ± SEM unless described differently.Statistical analysis was performed using JMP 7 software program.Two-tailed Student's t test for parametric and a Wilcoxon test for nonparametric data were used as appropriate.A log transformation was performed on skewed data for statistical analysis.P values smaller than 0.05 were considered significant.Bonferroni method was used to account for multiple comparisons.
Study approval.All animal experiments were approved by the Cleveland Clinic IACUC (Cleveland, Ohio).Local IRB approval and informed consent from all human participants was obtained.

Figure 1 .
Figure 1.ARG2 rs742869 single nucleotide polymorphism (SNP) related to phenotypes of obesity, airflow obstruction, hyperreactivity, and blood eosinophils and IgE levels in asthmatics dichotomized by high and low fractional exhaled nitric oxide (F E NO) levels.Each dot represents data from 1 patient; gray indicates low F E NO, and black represents high F E NO. (A) BMI was significantly different among genotypes of ARG2 rs742869 SNP in asth matics dichotomized by high F E NO (≥ 35 ppb) and low F E NO (< 35 ppb).*P = 0.0008 low F E NO and G/G vs. low F E NO and A/A, # P = 0.002 low F E NO and G/G vs. high F E NO and G/G, and & P < 0.0001 low F E NO and G/G vs. high F E NO and A/A.(B) Ratio of forced expiratory volume in 1 second (FEV 1 )/forced vital capacity (FVC) tended to be different among genotypes in asthmatics dichotomized by F E NO. *P = 0.03 low F E NO and A/A vs. high F E NO and G/G, and # P = 0.03 high F E NO and G/G vs. high F E NO and A/A.(C) PC 20 was significantly different.*P = 0.002 low F E NO and G/G vs. high F E NO and G/G, # P = 0.0007 low F E NO and G/G vs. high F E NO and A/A, Δ P = 0.04 low F E NO and A/A vs. high F E NO and G/G, and & P = 0.01 low F E NO and A/A vs. high F E NO and A/A.(D) Blood eosinophil levels were significantly different.*P = 0.01 low F E NO and G/G vs. high F E NO and G/G, # P = 0.005 low F E NO and G/G vs. high F E NO and A/A, Δ P = 0.01 low F E NO and A/A vs. high F E NO and G/G, and & P = 0.003 low F E NO and A/A vs. high F E NO and A/A.(E) Blood IgE levels were significantly different.*P = 0.0001 low F E NO and G/G vs. high F E NO and A/A, # P < 0.0001 low F E NO and A/A vs. high F E NO and A/A, Δ P = 0.02 low F E NO and A/A vs. high F E NO and G/G, and & P = 0.02 high F E NO and G/A vs. high F E NO and A/A.(F) Serum arginase activity tended to be different among genotypes in asthmatics dichoto mized by F E NO. *P = 0.04 low F E NO and G/G vs. low F E NO and A/A, # P = 0.03 low F E NO and G/A vs. high F E NO and G/A, and & P = 0.04 low F E NO and A/A vs. high F E NO and G/G.Student's t test was used for group comparisons and 1way ANOVA was used for comparisons of more than 2 groups.

Figure 2 .
Figure 2. Increased airway inflammation in ARG2 -/-mice.WT, ARG2 -/-, iNOS -/-, and iNOS -/-/ARG2 -/-mice were exposed to HDME or saline as control.(A) Lung tissue sections stained for H&E demonstrated that all 4 genotypes exposed to HDME developed airway inflammation, but the influx of inflammatory cells was the highest in ARG2 -/-mice.Black arrow heads indicate foci of inflammatory cells around the airways; a, airway.Representative tissue sections are shown.Scale bar: 100 μm.(B-D) Differential hematopoietic cell counts in BAL showing increased total cell count (B), eosinophils (C), and neutrophils (D) in HDME exposed ARG2 -/-mice were inhibited in the iNOS -/-/ARG2 -/-mice.(E and F) Macrophages (E) and lymphocytes (F) were not different across HDME exposed groups.*P < 0.05 between HDME and saline groups.Student's t test was used for group comparisons.Each dot represents data from 1 mouse and mean ± SEM values are shown.

Figure 4 .
Figure 4. Arginine metabolism regulates exaggerated eosinophilic/neutrophilic asthma.Less arginase2 activity, such as associated with by Arg2 SNP rs74869, produces an exaggerated asthmatic phenotype with higher levels of IL5, eotaxin2, and IL17 resulting in severe eosinophilic/neutrophilic airway inflammation and angiogenic airway remodeling.The loss of ARG2 activity, but intact iNOS arginine metabolism, worsens the airway inflammation, suggesting that a balance among the pathways for arginine metabolism is essential in regulating inflammation.Illustration by David Schumick, BS, CMI.Reprinted with the permission of the Cleveland Clinic for Medical Art & Photography © 2019.All rights reserved.

Figure 5 .
Figure 5. Mouse models.(A) Generation and genotyping of iNOS -/-/ARG2 -/-mice.Interbreeding of mice carrying the iNOS mutant allele or ARG2 mutant allele was performed to generate iNOS ARG2-double mutant mice.Genotypes were confirmed by PCR on genomic DNA utilizing WT and mutant allelespecific primers.Top gel is ARG, and lower gel is iNOS allele PCR.(B) Schematic representation of the HDME model of airway inflammation.

Table 1 . Study participants with ARG2 rs742869 single nucleotide polymorphism (SNP) genotyping
Results are presented as mean ± SEM.M, male; F, female; W, White; AA, African American; FEV 1 , forced expiratory volume in 1 second; FVC, forced vital capacity; PC 20 , provocative concentration of methacholine causing a 20% fall in FEV 1 ; F E NO, fractional exhaled nitric oxide.A P value, ANOVA of A/A, G/A, G/G in controls.B P value, G/G vs. A/A in controls.C P value, ANOVA of A/A, G/A, G/G in asthmatics.D P value, G/G vs. A/A in asthmatics.E Nonreactive.FEosinophils as a percentage of total WBCs.Student's t test was used.