Glutamine deficiency shifts the asthmatic state toward neutrophilic airway inflammation

Abstract

Background: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model.

Methods: We depleted endogenous Gln levels using L-γ-glutamyl-p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2 and glutamine synthetase small interfering siRNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit.

Results: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A₂ (cPLA₂ ) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA₂ inhibitor and a leukotriene B4 inhibitor but not by dexamethasone.

Conclusion: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA₂ , resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.

Keywords: MAPK phosphatase-1; cPLA2; endogenous glutamine; neutrophilic airway inflammation; p38 MAPK.

FIGURE 1

Effects of Gln deficiency on MKP‐1 induction and p38 phosphorylation. (A) Representative immunoblots and densitometric analyses of GS. GS siRNA was injected intravenously from days 7 to 1, and control siRNA was administered at day 5 (n = 6, three independent experiments). (B) Measurement of Gln levels in the lungs using a Gln assay kit. Normal mice were treated either with siRNAs 5 and 3 days before or with GPNA 1 h before sacrifice (n = 12–15, three independent experiments). (C–D) Representative immunoblots and densitometric analyses of the MKP‐1 protein and p38 phosphorylation in the lungs of mice pretreated with GPNA (1 h before the second airway challenge; (C) n = 10–13, three independent experiments) or GS siRNA (5 and 3 days before the second airway challenge; (D) n = 10–13, three independent experiments). Data are presented as the mean ± SEM. A‐B, ns p > .05 vs. normal control, *p < .05 vs. normal control, **p < .01 vs. normal control. (C–D) *p < .05 vs. saline control, **p < .01 vs. saline control, ns p > .05 vs. OVA group, #p < .05 vs. OVA group, ##p < .01 vs. OVA group. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; Gln, glutamine; GPNA, L‐γ‐glutamyl‐p‐nitroanilide; GS, glutamine synthetase; MKP‐1, MAPK phosphatase‐1; ns, not significant; OVA, ovalbumin; SEM, standard error of the mean; siRNA, small interfering RNA

FIGURE 2

Effects of Gln deficiency on the development of airway inflammation. (A–D) Time kinetics of the levels of neutrophils from 0 to 36 h (A, n = 15–25, three independent experiments), Th1 cytokines from 0 to 4 h (B, n = 15–20, three independent experiments), eosinophils from 24 to 84 h (C, n = 15–25, three independent experiments), and Th2 cytokines from 12 to 36 h (D, n = 15–25, three independent experiments) in BALF after the second airway challenge. (E) Airway responsiveness assessed by invasive measurements (n = 60–75, three independent experiments). (F) Representative H&E‐stained sections of the lung (n = 10–15, three independent experiments). The bars indicate 100 μm. (G) Mucus secretion revealed by ELISA (n = 15–25, three independent experiments) (F–G) The parameters were assessed at 48 h after the second airway challenge. Data are presented as the mean ± SEM. *p < .05 vs. saline control, **p < .001 vs. saline control, ns p > .05 vs. OVA group, #p < .05 vs. OVA group, ##p < .001 vs. OVA group. BALF, bronchoalveolar lavage fluid; ELISA, enzyme‐linked immunosorbent assay; Gln, glutamine; GPNA, L‐γ‐glutamyl‐p‐nitroanilide; GS, glutamine synthetase; H&E, hematoxylin and eosin; IL, interleukin; MUC5AC, musin‐5 subtype A and C; ns, not significant; OVA, ovalbumin; Rrs, resistance of the respiratory system; SEM, standard error of the mean; siRNA, small interfering RNA; TNF, tumor necrosis factor

FIGURE 3

Lentiviral overexpression of MKP‐1 abrogates Gln deficiency‐induced neutrophilic inflammation. (A–B) Representative immunoblots (A) and immunohistochemical stainings (B) for MKP‐1 protein. Normal mice were injected with varying virus doses and sacrificed 2 days thereafter for western blots (A, n = 9–12, three independent experiments) or immunohistochemistry of the lung (B, n = 6, three independent experiments). The bars indicate 100 μm. C, MKP‐1 and phosphorylation of p38 and cPLA₂ (n = 10–15, three independent experiments) in the lungs of mice pretreated with lentiviral MKP‐1. (D–E) The levels of neutrophils at 10 h (D, n = 15–20, three independent experiments) and Th1 cytokines at 1.5 h (E, n = 15–20, three independent experiments) in BALF. (C–E) GPNA and viruses (4 × 10⁷ IU) were injected 1 h and 2 days before the second airway challenge, respectively. Data are presented as the mean ± SEM. (A) ns p > .05 vs. normal control, *p < .05 vs. normal control, **p < .001 vs. normal control. (C–E) *p < .05 vs. saline control, **p < .001 vs. saline control, #p < .05 vs. OVA group, ##p < .001 vs. OVA group, ns p > .05 vs. OVA + GPNA group, $p < .05 vs. OVA + GPNA group, $$p < .001 vs. OVA + GPNA group. BALF, bronchoalveolar lavage fluid; cPLA₂, cytosolic phospholipase A₂; eGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; Gln, glutamine; GPNA, L‐γ‐glutamyl‐p‐nitroanilide; IL, interleukin; MKP‐1, MAPK phosphatase‐1; ns, not significant; OVA, ovalbumin; SEM, standard error of the mean; TNF, tumor necrosis factor

FIGURE 4

Association between the p38/cPLA₂/LTB4 pathway and neutrophilia in Gln deficiency. (A–C) cPLA₂ phosphorylation (A, n = 11, three independent experiments), activity (B, n = 10–15, three independent experiments), and levels of LTB4 in BALF from 1 to 8 h (C, n = 10–15, three independent experiments). (D) Number of neutrophils in BALF at 12 h. (E) Levels of LTB4 in BALF at 1 h post‐challenge (D and E, n = 15–18, three independent experiments). Data are presented as the mean ± SEM. ns p > .05 vs. saline control, *p < .05 vs. saline control, **p < .001 vs. saline control, #p < .05 vs. OVA group, ##p < .001 vs. OVA group, ns p > .05 vs. OVA + GPNA group, $p < .05 vs. OVA + GPNA group, $$p ≤ .001 vs. OVA + GPNA group. BALF, bronchoalveolar lavage fluid; cPLA₂, cytosolic phospholipase A₂; Gln, glutamine; GPNA, L‐γ‐glutamyl‐p‐nitroanilide; GS, glutamine synthetase; LTB4, leukotriene B4; ns, not significant; OVA, ovalbumin; SEM, standard error of the mean; siRNA, small interfering RNA

FIGURE 5

Steroid resistance of neutrophilia in Gln deficiency. (A) Representative immunoblots and densitometric analyses of MKP‐1 protein and phosphorylation of p38 and cPLA₂ (n = 5–10, three independent experiments) at 1 h post‐challenge. (B–C) Number of eosinophils at 48 h (B, n = 10–15, three independent experiments) and Th2 cytokine levels at 18 h (C, n = 10–15, three independent experiments) in BALF. (D) Number of neutrophils in BALF at 12 h (n = 10–15, three independent experiments). (E) Levels of LTB4 in BALF at 1 h (n = 10–15, three independent experiments). A‐E, Gln (2 g/kg) was orally administered 30 min before the second airway challenge. Data are presented as the mean ± SEM. *p < .05 vs. saline control, **p < .001 vs. saline control, #p < .05 vs. OVA group, ##p < .001 vs. OVA group, ns p > .05 vs. OVA + GS siRNA group, $p < .05 vs. OVA + GS siRNA group, $$p < .001 vs. OVA + GS siRNA group. BALF, bronchoalveolar lavage fluid; cPLA₂, cytosolic phospholipase A₂; DEX, dexamethasone; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; Gln, glutamine; GS, glutamine synthetase; IL, interleukin; LTB4, leukotriene B4; MKP‐1, MAPK phosphatase‐1; ns, not significant; OVA, ovalbumin; SEM, standard error of the mean; siRNA, small interfering RNA