Inhibition of histone-deacetylase activity rescues inflammatory cystic fibrosis lung disease by modulating innate and adaptive immune responses

The loss of the membrane-localized cystic fibrosis transmembrane conductance regulator (CFTR) protein due to a loss-of-function mutation in the Cftr gene, leads to cystic fibrosis disease that severely affects the respiratory, gastrointestinal and reproductive organs [1‐3]. The CF-related airway disease is a major contributor of the morbidity and mortality in CF patients. Although average survival age of CF subjects has substantially risen due to better clinical management of disease and availability of potent therapeutics that primarily target the symptoms of the disease, correction of the underlying genetic defect is needed to achieve the potential cure [4]. Clinically, CF-lung disease initiates as sustained airway inflammation and other complex abnormalities such as persistent infections, chronic inflammatory-oxidative stress and mucus hyper-secretion all contribute to the chronic airway obstruction [1, 2, 5]. The chronic exacerbation of CF involves repeated or stable infection with Pseudomonas aeruginosa (Pa) that results in chronic airway inflammation involving IL-8 mediated neutrophil chemotaxis resulting in irreversible pulmonary damage and respiratory failure [1, 2, 4, 5].

The most common genetic defect in CF is the deletion of phenylalanine-508 (ΔF508) in CFTR that results in misfolding of mutant CFTR protein and failure of CFTR ion channel to reach the plasma membrane; that results in ion channel dysfunction [1, 6]. This misfolded non-functional CFTR protein is either degraded by the ubiquitin proteasome system (UPS) [7] or aggregated as aggresome-bodies [8, 9], as autophagy is impaired [8, 10, 11]. The recent studies have shown that defective CFTR itself leads to ROS-mediated autophagy-impairment that contributes to the CFTR dysfunction and chronic inflammatory-oxidative stress observed in CF airways [8, 9]. Thus, the absence of CFTR on the cell surface results in chronic inflammation geared primarily by NFκB mediated pro-inflammatory signaling and IL-8-dependent neutrophil chemotaxis [1, 5]. The dominant pathogenic role of neutrophils in CF-lung disease is specifically attributed to the unopposed deleterious effects of neutrophil elastase on the structural lung epithelial cells that significantly contributes to lung damage and decreased pulmonary function in CF subjects [5, 12]. Apart from neutrophils, the other innate and adaptive immune cells also contribute to the pathophysiology of CF-lung disease by elevating levels of several pro-inflammatory cytokines and chemokines such as IL-1β, IL-6, IL-8, TNFα, IL-33 and IL-17 [13]. In addition, intriguing recent findings describe that an imbalance of Th17/T-reg cells contributes to the chronic inflammatory state seen in CF airways [14, 15], where pharmacological augmentation of T-reg numbers and/or their function can be used to control pathogenesis and/or progression of chronic lung disease.

It is important to note that, in addition to its classical channel function, lipid-raft or membrane-CFTR controls NFκB mediated inflammatory signaling that impacts both innate and adaptive immune responses in the CF airways [1, 16]. This suggests that future therapeutics targeting CF-lung disease should focus on rescuing misfolded ΔF508-CFTR to the plasma membrane while concurrently suppressing NFκB mediated hyper-inflammatory responses [1]. To this end, the pharmacological modulation of histone deacetylases (HDACs) seems promising, as HDACs regulate the expression of genes involved in inflammation and apoptosis through deacetylation of histone or non-histone proteins, in addition to rescuing the underlying CF defect. In support of this concept, the therapeutic potential of HDAC inhibitors (or proteostasis-modulators), in rescuing ΔF508-CFTR to the plasma membrane is clearly demonstrated [3, 17]. In addition, the therapeutic and clinical efficacy of HDAC-inhibition (HDACi) in numerous other disease states such as inflammatory bowel disease (IBD), cancer, AIDS, graft-versus-host disease and rheumatoid arthritis warrants its further preclinical evaluation in controlling pathogenesis of CF-lung disease.

Thus, we aimed to evaluate the therapeutic utility of HDACi in controlling Pa-LPS induced neutrophilic lung inflammation in CF-preclinical murine models (Cftr ^+/+ and Cftr ^−/− mice). Our data suggests that inhibition of HDAC activity can control Pa-LPS induced lung disease by modulating NFκB-mediated inflammatory signaling, neutrophil chemotaxis and T-reg-activation by CFTR-dependent and/or independent mechanisms. The HDACi-governed mechanisms of innate and adaptive immune response modulation described in this study highlight the scope of a novel intervention strategy for controlling CF-lung disease or other chronic conditions.

Cystic fibrosis lung disease is a hereditary condition caused due to the presence of ΔF508-CFTR, the misfolded form of the CFTR-protein, and the resulting diminished expression of functional WT-CFTR on the plasma membrane that leads to autophagy-impairment and elevated chronic inflammatory-oxidative stress responses [1, 6, 8, 9, 16]. These factors contribute significantly to the pathophysiological manifestations of CF-lung disease causing chronic and irreversible airway inflammation geared by invasion of several immune-inflammatory cells into the CF-airways [13]. Moreover, the pathogenic CF-Pseudomonas aeruginosa exacerbation further complicates the disease by recruitment and activation of additional inflammatory cells, especially neutrophils, into the airways [5, 12]. Thus, given the prominent role of IL-8-dependent neutrophil recruitment and activation in inflicting structural damage to the lung parenchyma, controlling neutrophilia is an important strategy for controlling CF-lung disease progression [12, 35]. Additionally, other immune cells such as T-helper type 2 (Th2) cells and macrophages also play a vital role in governing the chronic inflammatory response-mediated lung disease progression in the CF-airways [13]. Moreover, an imbalance between Th17 (inflammatory cells) and regulatory-T cells (T-regs, immunosuppressive cells) has been implicated in CF pathogenesis [14], which also provides a potential therapeutic strategy for suppressing CF lung inflammation. In the present study, we demonstrate the utility of SAHA (suberoylanilide hydroxamic acid, a HDAC inhibitor), in modulating innate and adaptive immune responses by controlling NFκB-IL-8-mediated neutrophil-chemotaxis, and T-reg activation, which may control the progression of chronic CF-lung disease by balancing the inflammatory response.

Numerous studies have documented the therapeutic benefits of SAHA in controlling tumorigenesis and inflammatory or auto-immune disease states such as RSV infections, septic shock, ventilator-induced lung injury, and inflammatory bowel disease (IBD) [23, 24]. Mechanistically, SAHA’s inflammation quenching function is due to its ability to control NFκB-mediated pro-inflammatory cytokine (TNFα, IL-1β, IL-8) response and myeloperoxidase (MPO, neutrophil specific enzyme) activity (Fig. 1a) [23, 24]. Our present data validates these findings as treatment with SAHA suppresses TNFα- induced IL-8 and NFκB activities (Fig. 1b, c). We used Pa-LPS instillation into the murine lungs to trigger inflammatory-oxidative stress as observed in the CF-airways and demonstrated here the pharmacological potential of SAHA in controlling the Pa-LPS induced inflammatory cytokine, IL-6 and MPO (neutrophil activation marker, Fig. 2a-d) levels, suggesting its anti-inflammatory potential. Moreover, SAHA is also reported to act as an antioxidant, as it suppresses LPS-mediated reactive oxygen species (ROS) activity and elevates glutathione levels, which is a potent anti-oxidant [36]. Our data supports this hypothesis as we observed that SAHA modulates Pa-LPS mediated changes in the anti-oxidant Nrf2 (in Cftr ^+/+ mice) and NFκB (inflammation) (Fig. 2e, f and Fig. 4c, d), which indicates towards its potential utility in controlling inflammatory-oxidative stress in the CF-airways that requires further experimental validation. In fact, some previous studies have clearly shown that the CF-epithelial cells have diminished Nrf2 protein levels (especially in the nucleus), reduced transcriptional activity, as well as insufficient expression of Nrf2 target genes, as compared to non-CF cells [22, 37‐39]. This supports our notion that SAHA-mediated increase in Nrf2 levels might be beneficial in controlling inflammatory-oxidative stress in CF. Moreover, infection-mediated airway inflammation in CF is also known to trigger the unfolded protein response (UPR), a form of endoplasmic reticulum stress (ER stress) that is implicated in the pathology of CF-lung disease [40]. In the present study, we used cigarette smoke extract (CSE) - induced in vitro model of ER-stress to demonstrate that HDAC inhibition by SAHA, Tubacin (a specific HDAC6 inhibitor) or HDAC6-knockdown, controls CSE-induced ER-stress (Additional file 1: Figure S1A and B) and resulting inflammatory response, verifying its added pharmacological potential in controlling the CF-lung disease.

Since chronic inflammation involves aberrant activation of immune cells that governs CF-disease pathogenesis [13], it is not surprising that the immunosuppressive regulatory T cells (T-regs) that maintain immune homeostasis, are suggested to play a crucial role in CF lung pathophysiology [41]. A recent study shows that in CF patients with chronic Pa infection, there is an age-dependent, qualitative and quantitative impairment of T-regs [14]. Moreover, in children with CF, a decreased expression of CD4⁺CD25⁺FOXP3⁺ T cells (T-regs) correlates with lung function decline (FEV1) [42] suggesting that deficiency of T-reg numbers and/or function contributes to the chronic inflammatory state of CF-airways. Here, we verify this hypothesis by showing that SAHA treatment augments the number of FOXP3⁺ T cells (Fig. 3a, Additional file 1: Figure S1C). Our data verifies that SAHA-mediated T-reg-induction could act as a potential mechanism of action (MOA) for controlling inflammatory CF-lung disease.

Although, the use of anti-inflammatory treatment strategies seems promising, their efficacy is restricted to symptomatic control of CF airway inflammation rather than targeting the basic genetic defect that leads to the loss of the functional membrane localized CFTR-protein. Previous studies have indicated that SAHA also acts as a proteostasis-modulator that may help restore the membrane expression and function of misfolded ΔF508-CFTR [3, 17, 34], warranting its further preclinical evaluation for treating chronic CF-lung disease. In support of this concept, we demonstrate that HDACi by SAHA and Trichostatin-A, indeed delays the degradation of ΔF508-CFTR protein, compared to the untreated control (Fig. 5c). Although, a single recent study suggests that SAHA may not significantly rescue membrane CFTR levels but data from this study is inconclusive due to insuffiecient number of replicates and experimental variation, where SAHA mediated CFTR increase was observed in some cellular models but not other [43]. Moreover, the same study also suggests that SAHA treatment decreases CFTR mRNA levels and thus cannot be used as an efficient CFTR corrector. In contrast, in our observation and studies from other groups [3, 17], neither SAHA nor any other HDACi decreases CFTR mRNA levels. Additionally, we also evaluated the pre-clinical therapeutic efficacy of SAHA in controlling Pa-LPS induced neutrophil activity and lung inflammation in Cftr ^+/+ and Cftr^−/−-gut-corrected (Figs. 4, 5a and b, Fig. 6) murine models of airway-inflammation and chronic-CF lung disease [16]. Our finding that SAHA controls Pa-LPS induced lung inflammation and neutrophil activity in Cftr ^+/+ mice might have interesting implications in ΔF508-CF and COPD subjects with acquired CFTR-dysfunction, where SAHA can augment CFTR function as well as control inflammatory response by CFTR dependent and independent mechanisms (Fig. 6). In support of these findings, previous studies have clearly demonstrated the key role of membrane-localized WT-CFTR in controlling chronic inflammatory signaling in CF and other chronic inflammatory lung diseases [1, 2, 8, 16, 22, 44]. Moreover, it is also important to note that both Pa-infection and neutrophil elastase can negatively affect CFTR expression and/or function [45‐47]. Thus, it is plausible that rescue of functional CFTR-activity is one of the mechanisms by which SAHA protects against Pa-LPS induced lung inflammation in Cftr ^+/+ mice. This suggests an additional potential therapeutic application of SAHA in controlling intermittent and stable Pa-exacerbations in both ΔF508-CF and COPD subjects [46], that warrants further clinical evaluation. In addition, we used Pa-LPS induced chronic lung inflammatory (Cftr ^−/− ) murine model to verify CFTR-independent anti-inflammatory potential of SAHA, which demonstrates its therapeutic effectiveness in controlling CF-inflammatory lung disease, irrespective of mutant-CFTR rescue or correction. To the best of our knowledge, this is the first report demonstrating the efficacy and the mechanism of action of HDACi, such as SAHA in controlling CF-related lung inflammation. Finally, identification of specific HDACs that control CFTR processing [3, 17] and lung disease, would lead to the development of selective HDAC-inhibitor drugs. As a proof of concept, HDAC6 (Tubacin) [48] and HDAC7 [3, 17, 48] inhibitors have known potential application in rescuing misfolded-CFTR protein from proteasomal degradation or aggresome-accumulation.

In conclusion, proposed SAHA-mediated CF-lung disease intervention can benefit from multiple mechanism of actions such as (1) an anti-inflammatory, (2) T-reg inducer and (3) ΔF508-CFTR corrector drug (Fig. 6). Thus, further clinical evaluation of this potent pharmaceutical strategy is warranted, and we anticipate that using the specific HDAC-inhibitors [3, 17] and novel nano-based drug delivery system [49], that we recently described, can further enhance its therapeutic potency in reverting the CF-lung disease.