Asthma is a chronic disease of the airways characterized by inflammation, structural and functional changes that are responsible for bronchial hyperresponsiveness (AHR) and limitation in airflow. While asthma symptoms can be controlled in the majority of patients, in around 5-10% of asthmatics, the disease remains symptomatic and inadequately controlled. For these reasons, additional therapeutic approaches are urgently required to provide better life conditions for these individuals. The present study is focused on the role of inducible Nitric Oxide Synthase (iNOS) and poly(ADP-ribose) polymerase-1 (PARP-1) in the pathophysiology of asthma and their potential interaction and cooperation. The role of iNOS in asthma has been examined in numerous studies with conflicting results. Its potential as a viable therapeutic target for the treatment of the disease was severely hampered by the negative results of a clinical trial showing that a selective iNOS inhibitor, although reducing effectively exhaled Nitric Oxide (eNO), did not affect AHR or airway inflammation after allergen challenge in human subjects with asthma. We believe that such conclusion is premature and that more careful studies are necessary to fully understand the role of iNOS in asthma and whether the enzyme can be adequately targeted at least as an adjuvant therapy for the treatment of this disease. In the current study I report that iNOS inhibition, pharmacologically, using the relatively selective inhibitor L-NIL or by gene knockout, in our animal experimental models provides an excellent protection against AHR upon an acute, but not upon a chronic, exposure to allergens (OVA or HDM) as assessed using full body plethysmography. These results correlate with the differential protection provided by iNOS inhibition against airway inflammation observed in a previous study. Considering all known beneficial effects of NO through its contribution to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium, I speculate that complete inhibition of iNOS, achieved by L-NIL administration or even more efficiently by gene knockout may be deleterious for some aspects of asthma such as AHR, which highly involve one of the major targets of NO, the smooth muscle cells. The fascinating aspect of my study is that the loss of protection observed in the chronic asthma model after L-NIL treatment was reversed by NO supplementation through administration of nitrite, a moderate NO source. The anion nitrite (NO2–) can be reduced to NO upon a reaction with deoxyhemoglobin. Our group, in previous studies, also established a reciprocal relationship between iNOS and PARP-1 with a special connection to oxidative DNA damage and IL-5 in the process of eosinophilia during allergen-induced lung inflammation. Such relationship is also based on the fact that PARP-1 exerts a decisive role on the interaction between p65 NF-κB and the exportin protein Crm1. PARP-1 activity, decrease the interaction between the transcription factor and Crm1, increasing the quantity of p65 NF-κB detectable in the nuclei of cells upon TLR4 stimulation. This increase leads to an upregulation of NF-κB–dependent gene expression, which includes iNOS. PARP inhibition, pharmacologically by Olaparib (AZD2281) or by gene knockout, protects against inflammation and AHR both upon single and multiple exposures to OVA. Similar protection was observed upon chronic exposure to HDM, demonstrating that the protective effect is not limited to the type of allergen used. Such protection may occur as a consequence that iNOS expression is markedly reduced upon PARP inhibition. However, it is interesting that PARP-1 inhibition does not completely abrogate expression of iNOS leaving the possibility that the protective effect of PARP inhibition against inflammation and AHR may be associated with the reduction but not the complete inhibition of iNOS and with consequent production of moderate levels of NO. A further confirmation of our hypothesis is that administration of iNOS inhibitor (L-NIL) to chronically HDM-exposed mice that received a PARP inhibitor (Olaparib-AZD2281) reversed the protection against AHR provided by PARP inhibition. Overall, our results may explain why clinical studies focused on the complete inhibition of iNOS failed to protect against different aspects of asthma. However, further efforts to investigate the exact role of the enzyme in asthma may provide a clearer view on how to utilize iNOS as a therapeutic target. But what seems to be plausible is that moderate levels of NO achieved through partial inhibition of iNOS could represent a good therapeutic strategy at least against some aspect of the disease, such as AHR. Additionally, our results revealed that targeting PARP-1 may constitute a better alternative therapeutic approach. Such strategy seems to be very efficient at reducing the most important asthma traits (e.g. Th2 cytokines, mucus, and IgE production) but also partially reduces expression of iNOS with a consequent moderate production of NO that provides protections against AHR. It is important to note that the beneficial effects of PARP inhibition could be correlated with the capacity of PARP-1 to positively influence GATA3, the mast regulator of Th2 population, the paramount cells in the manifestation of the allergic disease. This is, of course, without omitting the potential impact of PARP inhibition on the T-reg population, which is responsible for the control and reduction of inflammation. Finally, it is noteworthy that L-NIL (L-N6-(1-Iminoethyl)lysine dihydrochloride) and AZD2281 (olaparib) are two clinically tested iNOS and PARP inhibitors, respectively, which increases considerably the clinical relevance of this study.

COMMON AND DIFFERENTIAL ROLES OF INDUCIBLE NO SYNTHASE AND POLY (ADP-RIBOSE) POLYMERASE IN ALLERGEN-INDUCED INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS: A POTENTIAL CONNECTION TO NO LEVELS / S.v. Ibba ; tutore: D. Trabattoni ; co-tutore: H. Boulares. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE E CLINICHE "L. SACCO", 2015 Dec 16. ((28. ciclo, Anno Accademico 2015. [10.13130/ibba-salome-valentina_phd2015-12-16].

COMMON AND DIFFERENTIAL ROLES OF INDUCIBLE NO SYNTHASE AND POLY (ADP-RIBOSE) POLYMERASE IN ALLERGEN-INDUCED INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS: A POTENTIAL CONNECTION TO NO LEVELS

S.V. Ibba
2015-12-16

Abstract

Asthma is a chronic disease of the airways characterized by inflammation, structural and functional changes that are responsible for bronchial hyperresponsiveness (AHR) and limitation in airflow. While asthma symptoms can be controlled in the majority of patients, in around 5-10% of asthmatics, the disease remains symptomatic and inadequately controlled. For these reasons, additional therapeutic approaches are urgently required to provide better life conditions for these individuals. The present study is focused on the role of inducible Nitric Oxide Synthase (iNOS) and poly(ADP-ribose) polymerase-1 (PARP-1) in the pathophysiology of asthma and their potential interaction and cooperation. The role of iNOS in asthma has been examined in numerous studies with conflicting results. Its potential as a viable therapeutic target for the treatment of the disease was severely hampered by the negative results of a clinical trial showing that a selective iNOS inhibitor, although reducing effectively exhaled Nitric Oxide (eNO), did not affect AHR or airway inflammation after allergen challenge in human subjects with asthma. We believe that such conclusion is premature and that more careful studies are necessary to fully understand the role of iNOS in asthma and whether the enzyme can be adequately targeted at least as an adjuvant therapy for the treatment of this disease. In the current study I report that iNOS inhibition, pharmacologically, using the relatively selective inhibitor L-NIL or by gene knockout, in our animal experimental models provides an excellent protection against AHR upon an acute, but not upon a chronic, exposure to allergens (OVA or HDM) as assessed using full body plethysmography. These results correlate with the differential protection provided by iNOS inhibition against airway inflammation observed in a previous study. Considering all known beneficial effects of NO through its contribution to vessel homeostasis by inhibiting vascular smooth muscle contraction and growth, platelet aggregation, and leukocyte adhesion to the endothelium, I speculate that complete inhibition of iNOS, achieved by L-NIL administration or even more efficiently by gene knockout may be deleterious for some aspects of asthma such as AHR, which highly involve one of the major targets of NO, the smooth muscle cells. The fascinating aspect of my study is that the loss of protection observed in the chronic asthma model after L-NIL treatment was reversed by NO supplementation through administration of nitrite, a moderate NO source. The anion nitrite (NO2–) can be reduced to NO upon a reaction with deoxyhemoglobin. Our group, in previous studies, also established a reciprocal relationship between iNOS and PARP-1 with a special connection to oxidative DNA damage and IL-5 in the process of eosinophilia during allergen-induced lung inflammation. Such relationship is also based on the fact that PARP-1 exerts a decisive role on the interaction between p65 NF-κB and the exportin protein Crm1. PARP-1 activity, decrease the interaction between the transcription factor and Crm1, increasing the quantity of p65 NF-κB detectable in the nuclei of cells upon TLR4 stimulation. This increase leads to an upregulation of NF-κB–dependent gene expression, which includes iNOS. PARP inhibition, pharmacologically by Olaparib (AZD2281) or by gene knockout, protects against inflammation and AHR both upon single and multiple exposures to OVA. Similar protection was observed upon chronic exposure to HDM, demonstrating that the protective effect is not limited to the type of allergen used. Such protection may occur as a consequence that iNOS expression is markedly reduced upon PARP inhibition. However, it is interesting that PARP-1 inhibition does not completely abrogate expression of iNOS leaving the possibility that the protective effect of PARP inhibition against inflammation and AHR may be associated with the reduction but not the complete inhibition of iNOS and with consequent production of moderate levels of NO. A further confirmation of our hypothesis is that administration of iNOS inhibitor (L-NIL) to chronically HDM-exposed mice that received a PARP inhibitor (Olaparib-AZD2281) reversed the protection against AHR provided by PARP inhibition. Overall, our results may explain why clinical studies focused on the complete inhibition of iNOS failed to protect against different aspects of asthma. However, further efforts to investigate the exact role of the enzyme in asthma may provide a clearer view on how to utilize iNOS as a therapeutic target. But what seems to be plausible is that moderate levels of NO achieved through partial inhibition of iNOS could represent a good therapeutic strategy at least against some aspect of the disease, such as AHR. Additionally, our results revealed that targeting PARP-1 may constitute a better alternative therapeutic approach. Such strategy seems to be very efficient at reducing the most important asthma traits (e.g. Th2 cytokines, mucus, and IgE production) but also partially reduces expression of iNOS with a consequent moderate production of NO that provides protections against AHR. It is important to note that the beneficial effects of PARP inhibition could be correlated with the capacity of PARP-1 to positively influence GATA3, the mast regulator of Th2 population, the paramount cells in the manifestation of the allergic disease. This is, of course, without omitting the potential impact of PARP inhibition on the T-reg population, which is responsible for the control and reduction of inflammation. Finally, it is noteworthy that L-NIL (L-N6-(1-Iminoethyl)lysine dihydrochloride) and AZD2281 (olaparib) are two clinically tested iNOS and PARP inhibitors, respectively, which increases considerably the clinical relevance of this study.
TRABATTONI, DARIA LUCIA
Asthma; iNOS; PARP-1; NO; Airway hyperresponsiveness; Inflammation
Settore BIO/14 - Farmacologia
Settore BIO/11 - Biologia Molecolare
COMMON AND DIFFERENTIAL ROLES OF INDUCIBLE NO SYNTHASE AND POLY (ADP-RIBOSE) POLYMERASE IN ALLERGEN-INDUCED INFLAMMATION AND AIRWAY HYPERRESPONSIVENESS: A POTENTIAL CONNECTION TO NO LEVELS / S.v. Ibba ; tutore: D. Trabattoni ; co-tutore: H. Boulares. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE E CLINICHE "L. SACCO", 2015 Dec 16. ((28. ciclo, Anno Accademico 2015. [10.13130/ibba-salome-valentina_phd2015-12-16].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/351284
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