Reza, M.I.

Abstract Rationale: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor and cellular chemical sensor. AhR maintains cellular homeostatsis in respons to endogenous and exogenous organic molecules. Airway smooth muscle (ASM) cells are major structural cells in airway which is involved in characteristic features of asthma such as increased airway wall thickness, airway remodeling and airway hyperresponsiveness. Previously, we found notable expression of AhR in human ASM and its increased expression during inflammation and asthma. However, the mechanisms of enhanced AhR expression in ASM is unknown. Therefore, we examined the role of pro-inflammatory signaling pathways in enhanced AhR expression. Methods: Primary human non-asthmatic and asthmatic ASM cells isolated from surgical lung resections (Mayo IRB approved) were cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. At ∼80% confluency, cells were serum deprived and treated with TNFα (20 ng/mL), IL13 (50 ng/mL) with or without PI3K/Akt inhibitor (Wortmannin: 50 nM), p38 inhibitor (SB203580: 600 nM), NFkB inhibitor (SN50 20 μM), and AP1 inhibitor (SR11302 10 μM). Subsequently, expression of AhR was determined using RNA sequencing, confocal microscopy, real-time qPCR, and Western blotting. AhR expression in isolated ASM fron lung tissue section was determined using laser capture microdissection (LCM). Moreover, role of AP1 in inducing AhR expression was confirmed using c-JUN (AP1) siRNA transfection in ASM cells. Additionally, binding of AP1 to the AhR promoter region was demonstrated by chromatin-immunoprecipitation (ChIP-qPCR) technique. Results: AhR expression was significantly high in asthmatic ASM cells and tissue than non-asthmatics. Exposure to pro-inflammatory cytokine TNFα increased AhR mRNA and protein expression, whereas IL-13 failed to do so. Furthermore, inhibition of p38 significantly reduced AhR expression whereas PI3K inhibition had no effect, indicating a role for p38 in AhR increased expression. AP1 inhibition significantly downregulated AhR, while NFkB inhibition showed no effect. Similarly, siRNA mediated silencing of c-JUN (AP1) also downregulated AhR, indicating AP1 role in AhR upregulation. Furthermore, transcription factor binding study suggested AhR promoter region has binding site for AP1 and NFkB. Interestingly, ChIP-qPCR study confirmed binding of AP1 at AhR promote region in response to TNFα. Conclusions: Collectively, the current study show that pro-inflammatory cytokine TNFα increases AhR expression through p38-AP1 pathway. The data shows that pro-inflammatory cytokines can regulate AhR expression in ASM, suggesting AhR in ASM has a role in asthma.

Abstract Rationale: Within the asthmatic lung, inflammatory signaling alters the contractile capacity of airway smooth muscle (ASM). In chronic inflammation, ASM become hypercontractile and are a key contributor toward airway hyperresponsiveness (AHR). As a heterogenous disorder, several asthmatic endotypes have been defined. A severe ASM endotype is partly defined as being glucocorticoid insensitive. We have reported the role of Kisspeptin (Kp)/KISS1R signaling in the lung in the context of airway fibrosis. In this study, KISS1R and Kp-10 were investigated for a potential interactions with intracellular calcium [Ca2+]i signaling and ASM contractility in the in vitro setting of severe asthmatic endotypes. Methods: Primary ASM were obtained from non-pathological samples from the Mayo Clinic. ASM cells were treated with inflammatory cytokines (TNFα 20 ng/ml, or IL-13 50 ng/mL)± Kp-10 (1 μM). The steroid-insensitive asthma endotype was investigated using pro-inflammatory cytokines (TNFα 20 ng/ml and IFN-γ 25 ng/mL) ± a corticosteroid (Fluticasone Propionate 100 nM). Β2-Adrenergic Receptor (β2-AR) desensitized endotypes were developed by treating cells with a β2-AR agonist (Salbutamol 1 μM) for 24 hours prior to and along with cytokine treatments (TNFα 20 ng/ml and IFN-γ 25 ng/mL or IL-13 50 ng/mL) ± Kp-10. [Ca2+]i was measured via Fura-2AM (4 μM) labeling on an Olympus Fluoview 300 microscope. Myosin light chain (MLC) phosphorylation studies were used to quantify downstream mechanics of ASM contractility. Results: KISS1R activation in IL-13 treated cells reduced [Ca2+]i in response to histamine. In contrast, TNFα treated cells were responsive to Kp-10 for acute treatments, however this effect was lost after 24 hours of Kp-10 treatment. TNFα and IFN-γ treated cells showed a robust [Ca2+]i response to histamine, and both fluticasone (∼20% reduction) and Kp-10 (∼10% reduction) alone had a minimal effect in reducing [Ca2+]i. Fluticasone and Kp-10 co-exposure significantly reduced [Ca2+]i and significantly reduced the effect of TNFα/IFN-γ (∼45% reduction). In B2-AR desensitized cells, Kp-10 exposure maintained the effect of reducing [Ca2+]i, suggesting Kp-10/KISS1R signaling works through an alternate or independent pathway. Kp-10 treatments on cytokine exposed cells reduced p-MLC in response to histamine, suggesting a role in ASM contractility. Conclusion: The increase of [Ca2+]i in inflamed cells is associated with AHR; Kp-10/KISS1R signaling showed varying efficacy in reducing [Ca2+]i within inflammatory endotypes. These results also highlight that Kp-10 acts synergistically with the current therapeutics available for asthma.