Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Aryl hydrocarbon receptor-dependent retention of nuclear HuR suppresses cigarette smoke-induced cyclooxygenase-2 expression independent of DNA-binding.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR(-/-) ) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR(-/-) mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR(-/-) mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target

Aryl Hydrocarbon Receptor-Dependent Retention of Nuclear HuR Suppresses Cigarette Smoke-Induced Cyclooxygenase-2 Expression Independent of DNA-Binding

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR−/−) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR−/− mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR−/− mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target

Methionine exposure alters glutamate uptake and adenine nucleotide hydrolysis in the zebrafish brain

Hypermethioninemic patients may exhibit different neurological dysfunctions, and the mechanisms underlying these pathologies remain obscure. Glutamate and ATP are important excitatory neurotransmitters co-released at synaptic clefts, and whose activities are intrinsically related. Adenosine—the final product of ATP breakdown—is also an important neuromodulator. Here, we investigated the effects of long-term (7-day) exposure to 1.5 or 3 mM methionine (Met) on glutamate uptake in brain tissues (telencephalon, optic tectum, and cerebellum) and on ATP, ADP, and AMP catabolism by ecto-nucleotidases found in brain membrane samples, using a zebrafish model. Also, we evaluated the expression of ecto-nucleotidase (ntdp1, ntdp2mg, ntdp2mq, ntdp2mv, ntdp3, and nt5e) and adenosine receptor (adora1, adora2aa, adora2ab, adora2b) genes in the brain of zebrafish exposed to Met. In animals exposed to 3.0 mM Met, glutamate uptake in the telencephalon decreased significantly. Also, ATP and ADP (but not AMP) catabolism decreased significantly at both Met concentrations tested. The messenger RNA (mRNA) levels of ntpd genes and of the adenosine receptors adora1 and adora2aa increased significantly after Met exposure. In contrast, adora2ab mRNA levels decreased after Met exposure. Our data suggest that glutamate and ATP accumulate at synaptic clefts after Met exposure, with potential detrimental effects to the nervous system. This phenomenon might explain, at least in part, the increased susceptibility of hypermethioninemic patients to neurological symptoms

AhR retains HuR in the nucleus in response to CSE but does not contribute to HuR expression.

<p>(A) HuR and CUGPB2 are constitutively expressed and are unaffected by AhR expression or CSE exposure. Note that there was an increase in Cox-2 protein in CSE-exposed <i>AhR^−/−</i> fibroblasts but not <i>AhR^+/+</i> cells. (B) <i>AhR^−/−</i> and <i>AhR^+/−</i> lung fibroblasts were exposed to 1% CSE for 4 hours and IF performed for CUGBP2. Nuclei are visualized by Hoechst (blue) and the merged images are shown. CUGBP2 was localized predominantly in the nucleus in <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts (<i>arrowheads</i>), although cytoplasmic expression was detectable (<i>arrows</i>). Cytoplasmic CUGBP2 increased in both <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts exposed to 1% CSE (<i>arrows</i>). (C) In cells treated with media, HuR is predominantly localized in the nucleus both in <i>AhR^−/−</i> and <i>AhR^+/−</i> fibroblasts (<i>arrowheads</i>). CSE exposure (1%) for 4 hours in absence of AhR expression (<i>AhR^−/−</i>) induces HuR shuttling from the nucleus to the cytoplasm (<i>arrows</i>). When <i>AhR^+/−</i> fibroblasts are challenged with 1% CSE, HuR remains in the nucleus. Results are representative of three independent experiments. (D) There was an increase in cytoplasmic HuR only in the <i>AhR^−/−</i> cells beginning at one hour of exposure and continuing through 4 hours. The purity of the extraction was determined by Tubulin, which was not detectable in the nuclear fraction. Representative western blot is shown. (E) Densitometric analysis of cytoplasmic and nuclear extracts following exposure to CSE: there was a significant increase in cytoplasmic HuR in response to CSE in only <i>AhR^−/−</i> cells (2.5±0.3; *p<0.05 compared to media only; ^¶p<0.05 compared to respective <i>AhR^+/−</i> fibroblasts exposed to CSE at the indicated time-point). Results are expressed as mean ± SEM of three independent experiments. (F) Classic AhR ligands do not cause cellular HuR redistribution in mouse lung fibroblasts. HuR remained within the nucleus upon exposure to B[<i>a</i>]P. Images are representative of two independent experiments. Magnification = 40×. (G) There was no increase in cytoplasmic HuR in <i>AhR^+/+</i> or <i>AhR^−/−</i> cells exposed to B[<i>a</i>]P for 4 hours. Representative western blot is shown.</p

AhR activation by CSE does not increase COX-2 protein.

<p><i>AhR^−/−</i> and <i>AhR^+/+</i> lung fibroblasts were exposed to CSE or B[<i>a</i>]P (1 µM) for 3, 6 or 24 hours and whole cell lysates collected for protein or RNA analysis. (A) There was a significant increase in <i>Cyp1a1</i> mRNA in response to both CSE and B[<i>a</i>]P for 6 hours only in <i>AhR^+/+</i> cells (***p<0.0001). Results are expressed as the mean ± SEM of 3–6 independent experiments. (B) Basal levels of CYP1A1 protein were not detectable in primary lung fibroblasts. CYP1A1 was not increased by CSE or the AhR ligand TCDD. MLE-12 cells express basal CYP1A1 that was further increased by B[<i>a</i>]P treatment. Western blot is representative of three experiments. (C) There was significantly more <i>Cyp1b1</i> mRNA in lung fibroblasts exposed to 1% CSE or B[<i>a</i>]P compared to <i>AhR^−/−</i> cells. Results are expressed as the mean ± SEM of 3–8 independent experiments. (D) There is no CYP1B1 protein induction by CSE exposure for 24 hours; note the increase in COX-2 protein only in <i>AhR^−/−</i> fibroblasts. B[<i>a</i>]P increased CYP1B1 protein expression in <i>AhR^+/+</i> fibroblasts. Representative western blot is shown.</p