Arginine metabolic control of airway inflammation

Abstract

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.

Keywords: Amino acid metabolism; Asthma; Inflammation; Metabolism; Nitric oxide.

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_ENO) levels.

Each dot represents data from 1 patient; gray indicates low F_ENO, and black represents high F_ENO. (A) BMI was significantly different among genotypes of ARG2 rs742869 SNP in asthmatics dichotomized by high F_ENO (≥ 35 ppb) and low F_ENO (< 35 ppb). *P = 0.0008 low F_ENO and G/G vs. low F_ENO and A/A, ^#P = 0.002 low F_ENO and G/G vs. high F_ENO and G/G, and ^&P < 0.0001 low F_ENO and G/G vs. high F_ENO and A/A. (B) Ratio of forced expiratory volume in 1 second (FEV₁)/forced vital capacity (FVC) tended to be different among genotypes in asthmatics dichotomized by F_ENO. *P = 0.03 low F_ENO and A/A vs. high F_ENO and G/G, and ^#P = 0.03 high F_ENO and G/G vs. high F_ENO and A/A. (C) PC₂₀ was significantly different. *P = 0.002 low F_ENO and G/G vs. high F_ENO and G/G, ^#P = 0.0007 low F_ENO and G/G vs. high F_ENO and A/A, ^ΔP = 0.04 low F_ENO and A/A vs. high F_ENO and G/G, and ^&P = 0.01 low F_ENO and A/A vs. high F_ENO and A/A. (D) Blood eosinophil levels were significantly different. *P = 0.01 low F_ENO and G/G vs. high F_ENO and G/G, ^#P = 0.005 low F_ENO and G/G vs. high F_ENO and A/A, ^ΔP = 0.01 low F_ENO and A/A vs. high F_ENO and G/G, and ^&P = 0.003 low F_ENO and A/A vs. high F_ENO and A/A. (E) Blood IgE levels were significantly different. *P = 0.0001 low F_ENO and G/G vs. high F_ENO and A/A, ^#P < 0.0001 low F_ENO and A/A vs. high F_ENO and A/A, ^ΔP = 0.02 low F_ENO and A/A vs. high F_ENO and G/G, and ^&P = 0.02 high F_ENO and G/A vs. high F_ENO and A/A. (F) Serum arginase activity tended to be different among genotypes in asthmatics dichotomized by F_ENO. *P = 0.04 low F_ENO and G/G vs. low F_ENO and A/A, ^#P = 0.03 low F_ENO and G/A vs. high F_ENO and G/A, and ^&P = 0.04 low F_ENO and A/A vs. high F_ENO and G/G. Student’s t test was used for group comparisons and 1-way ANOVA was used for comparisons of more than 2 groups.

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 3. Increased angiogenic airway remodeling, inflammatory, and angiogenic cytokines in ARG2^–/– mice.

(A–D) CD4⁺ T cell polarization in ARG2^–/– mice analyzed using splenic T cells. Splenocytes were isolated and analyzed for effector T cell polarization. (E) vWF staining for blood vessels in paraffin-embedded lung tissue sections. Representative low- and high-power lung images from WT mice exposed to saline or HDME as shown. Original magnification ×200. (F) Quantification of the microvessel density in WT, ARG2^–/–, iNOS^–/–, and iNOS^–/–/ARG2^–/– mice exposed to HDME. The number of vessels per 2500 μm² area is shown. (G) IL-5 levels in BALF. (H) Eotaxin-2 levels in BALF. (I–L) IL-17, KC, MIP-2, and LIX levels in lung tissue protein extract. *P < 0.05 between respective genotypes in HDME vs. saline groups. ^ϕP < 0.05 between WT and ARG2^–/–. ^φP < 0.05 between ARG2^–/– and ARG2^–/–iNOS^–/–. ^σP < 0.05 between iNOS^–/– and ARG2^–/–iNOS^–/–. ^#P < 0.05 between iNOS^–/– and ARG2^–/–. ^λP < 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.

Figure 4. Arginine metabolism regulates exaggerated eosinophilic/neutrophilic asthma.

Less arginase-2 activity, such as associated with by Arg2 SNP rs74869, produces an exaggerated asthmatic phenotype with higher levels of IL-5, eotaxin-2, and IL-17 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 Center for Medical Art & Photography © 2019. All rights reserved.

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 allele–specific primers. Top gel is ARG, and lower gel is iNOS allele PCR. (B) Schematic representation of the HDME model of airway inflammation.