Elsevier

Free Radical Biology and Medicine

Volume 91, February 2016, Pages 293-301
Free Radical Biology and Medicine

Original Contribution
Role of NOS2 in pulmonary injury and repair in response to bleomycin

https://doi.org/10.1016/j.freeradbiomed.2015.10.417Get rights and content

Highlights

  • Loss of iNOS function attenuates the inflammatory cell recruitment early (day3–8) in the response to bleomycin.

  • Loss of iNOS functionleads to a failure to resolve the inflammatory response to bleomycin.

  • .

    iNOS is important in balancing classical and alternative inflammatory signaling.

Abstract

Nitric oxide (NO) is derived from multiple isoforms of the Nitric Oxide Synthases (NOSs) within the lung for a variety of functions; however, NOS2-derived nitrogen oxides seem to play an important role in inflammatory regulation. In this study, we investigate the role of NOS2 in pulmonary inflammation/fibrosis in response to intratracheal bleomycin instillation (ITB) and to determine if these effects are related to macrophage phenotype. Systemic NOS2 inhibition was achieved by administration of 1400 W, a specific and potent NOS2 inhibitor, via osmotic pump starting six days prior to ITB. 1400 W administration attenuated lung inflammation, decreased chemotactic activity of the broncheoalveolar lavage (BAL), and reduced BAL cell count and nitrogen oxide production. S-nitrosylated SP-D (SNO-SP-D), which has a pro-inflammatory function, was formed in response to ITB; but this formation, as well as structural disruption of SP-D, was inhibited by 1400 W. mRNA levels of IL-1β, CCL2 and Ptgs2 were decreased by 1400 W treatment. In contrast, expression of genes associated with alternate macrophage activation and fibrosis Fizz1, TGF-β and Ym-1 was not changed by 1400 W. Similar to the effects of 1400 W, NOS2−/− mice displayed an attenuated inflammatory response to ITB (day 3 and day 8 post-instillation). The DNA-binding activity of NF-κB was attenuated in NOS2−/− mice; in addition, expression of alternate activation genes (Fizz1, Ym-1, Gal3, Arg1) was increased. This shift towards an increase in alternate activation was confirmed by western blot for Fizz-1 and Gal-3 that show persistent up-regulation 15 days after ITB. In contrast arginase, which is increased in expression at 8 days post ITB in NOS2−/−, resolves by day 15. These data suggest that NOS2, while critical to the development of the acute inflammatory response to injury, is also necessary to control the late phase response to ITB.

Introduction

Nitric oxide (NO) is a pluripotent molecule with a wide range of bioactivity and chemical reactivity [1]. Within the lung it is both an important physiological regulator and a key agent within pulmonary disease [2], [3], [4], [5], [6]; it regulates airway and vascular tone as well as pulmonary surface tension. However, under pathologic conditions, excessive expression of iNOS results in high efflux of NO, leading to post-translational modification of proteins, such as surfactant protein-D (SP-D), and mediates lung inflammation [7]. NO, and NO-mediated modifications, have been identified in pulmonary pathology in animal and human studies [2], [8], [9], [10], [11], [12]). NO is produced by alveolar macrophages and pulmonary epithelial cells upon exposure to pro-inflammatory stimuli such as LPS, TNF-α, IL-1β and interferon-γ [13], [14], [15], [16], [17], [18], [19], [20], [21]. These inflammatory mediators induce NOS2 expression through a series of signal transduction pathways including NF-κB and interferon regulatory factors (IRFs). Induction of iNOS, the product of NOS2, results in high efflux of NO, which further accelerates pulmonary inflammation. It has been suggested that inhibition or ablation of NOS2 inhibits persistent injury of the lung including pulmonary inflammation and fibrocystic processes [22].

Pulmonary inflammation and the subsequent transition to fibrosis is a complicated process involving multiple cellular activators and growth factors including such well known cytokines as TNF-α, IL-1β and TGF-β. A key step in the transition from inflammation to fibrosis is the development of a re-organized extracellular matrix. Both reactive oxygen and nitrogen species appear to play a role in the inflammation-fibrosis transition. Recently, Pini et, al reported that inhibition of cyclooxygenase and NO-donation attenuates pulmonary fibrosis induced by bleomycin [23]. To further address the potential role of iNOS-derived NO in pulmonary inflammation and fibrosis, we have examined the effects of genetic and pharmacological inhibition of iNOS function in the well-established lung injury model of intratracheal instillation of bleomycin (ITB). ITB in rodents has relevant clear phases of pulmonary pathology including sub-acute lung injury, inflammation, and fibrosis [24], [25], [26], [27]. The production of free radical species such as nitric oxide is considered as a factor resulting in endothelial and epithelial cell damage, the appearance of DNA-damage inducible proteins, increased micro-vascular permeability, and respiratory distress with surfactant dysfunction. ITB produces an initial inflammatory response marked by peak levels of TNF-α and TGF-β 7–10 days after injury mediated by increased activity of NF-κB that corresponds with maximal inflammatory cell infiltrate and respiratory distress. From 14 to 21 days, a transition from inflammation to either extracellular matrix production or to tissue healing and repair ensues [24], [25], [26], [27].

The increased NOS2 expression and altered NO metabolism observed within bleomycin-induced acute inflammation in mice indicate that NOS2 is a critical mediator of the inflammatory process seen within these animals [24], [25], [26], [27], [28]. We have previously demonstrated that selective inhibition of NOS2 can reverse chronic pulmonary inflammation [10]. In the present study, we investigated the effect of systemic NOS2 inhibition using the specific inhibitor 1400 W or genetic ablation of the NOS2 gene upon the indices of inflammation and fibrosis seen following acute injury with ITB. In this paper we demonstrate that 1400 W treatment attenuated bronchoalveolar lavage (BAL) inflammatory cell counts, macrophage size, SNO-SP-D formation and inflammatory cytokine/chemokine gene expression. However, the fibrotic precursors were not altered by systemic NOS2 inhibition. Similarly, NOS2 ablation while reducing inflammation early in the response to ITB does not improve the fibrotic endpoint, evidence is shown that indicates that macrophage phenotype may play a role in determining the outcome of ITB.

Section snippets

Preparation and implantation of micro-osmotic pumps and 1400 W treatment

Before instillation, the specific NOS2 inhibitor 1400 W (Cayman Chemicals), or control saline, was filtered through a 0.22- µM filter to ensure the sterility of the infusate [10]. Alzet micro-osmotic pumps (model 1002) were filled under sterile conditions with 100 µl (10 mg/kg/h) of 1400 W or saline. Loaded pumps were submerged overnight in sterile saline at 37 °C before implantation. C57 BL6/J mice were each anesthetized with 50 mg/kg i.p. injected pentobarbital. Under sterile conditions a small

1400 W attenautes NO metabolite formation in response to ITB.

NOS2 expression and increased production of NO have both been observed in response to ITB. In this study we delivered a continuous dose of the selective iNOS inhibitor 1400 W via osmotic pump. Previously, we demonstrated that we could inibit lung NOS function by this method in a long term inflammatory model [31]. To ensure that this method was effective in an acute injury model, such as ITB, we measured NO metabolites within the BAL. 8 days after ITB the concentration of nitrogen oxides (NOx)

Discussion

In the present study we show that loss of iNOS function, either through pharmacologic inhibition or through genetic deletion, attenuates the acute pulmonary inflammatory process in response to ITB, however, it appears to exacerbate the long term inflammation/fibrosis. Evidence from both the use of 1400 W and NOS2−/− mice shows that inflammation at 3 days post ITB is attenuated (Fig. 1, Fig. 3, Fig. 4). However, these same studies demonstrate that lack of iNOS function exacerbates the late phase

Grant and funding information

This work was supported by NIH HL086621 (to AJG). Additional support has been provided by the NIEHS sponsored Center for Environmental Exposures and Disease (CEED-Grant Number NIEHS P30E5S005022) at EOHSI (to CJG and AJG).

Authors' contributions

Conceived and designed the experiments: CJG, ENAV and AJG. Performed the experiments: CJG, HA, BG, VM and PS. Analyzed the data: CJG, ENAV and AJG. Wrote the paper: CJG and AJG.

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