Asthma, characterized by airway inflammation and structural remodelling, is the second most prevalent chronic lung disease worldwide1-3. People affected by asthma increased by 29.4% from about 211 million in 1990 to more than 272 million in 20173, leading to a major public health problem with a substantial socioeconomic burden. Drugs including corticosteroids, long-acting β2-agonists and muscarinic antagonists, leukotriene receptor antagonists, and biologic therapies have been clinically used for asthma therapy1, 4, 5. In particular, inhalation of corticosteroids is the most frequently employed strategy for the management of asthma. However, long-term use of corticosteroids is generally accompanied with multiple adverse effects, such as adrenal suppression, osteoporosis, and glaucoma6, 7. Moreover, high doses of oral or inhaled corticosteroids can result in airway neutrophilia, mainly by inducing recruitment and inhibiting apoptosis of neutrophils in the airways8-10. Notably, severe asthma, characterized by neutrophilic inflammation, responds poorly to corticosteroids and shows insufficient benefits after standard treatment4, 11, 12. Candidate therapies for neutrophilic asthma, such as macrolides and antagonists/antibodies to key pro-inflammatory cytokines, pose safety issues and unfavourable risk-benefit profiles4, 11. Consequently, there is an unmet need for new therapeutic strategies for severe asthma. In addition, personalized approaches for both prevention and cure of poorly controlled asthma are still required1.
Recent studies have revealed critical roles of neutrophils in the pathogenesis of asthma. In addition to mediating severe asthma, neutrophils have crucial roles in type 2 allergic asthma induced by lipopolysaccharide, viruses, or ozone13-15, by releasing neutrophil extracellular traps (NETs) that are web-like extracellular structures of chromatin filaments coated with histones, proteases, and cytosolic/granule proteins16. Although NETosis, known as the formation of NETs by neutrophils, can prevent dissemination of different pathogens by immobilizing and catching them, dysregulated NETs are responsible for the pathogenesis of numerous inflammatory and immune diseases16, 17. Therefore, inhibiting NETosis or eliminating excessive NETs has been implicated as promising therapeutic targets for different diseases varying from autoimmune diseases to infectious and non-infectious diseases17, 18. However, it remains unclear whether this NETosis inhibition strategy is efficacious for the treatment of asthma, in particular neutrophilic asthma.
Here we report that site-specific inhibition of NETosis in the lungs can effectively suppress local inflammation, reduce airway remodelling, and improve lung function, thereby alleviating neutrophilic asthma in mice, which was achieved by selectively attenuating oxidative stress via either intravenous or inhaled nanotherapies (Fig. 1a). Furthermore, the suppressed NETs formation also afforded beneficial effects on the balance of T helper 17 (Th17) and regulatory T (Treg) cells by reducing NET trapping of naïve T cells. Thus, targeted inhibition of NETosis in the lungs via antioxidant nanotherapies represents a promising, translational, and pathway-specific strategy for the prevention and treatment of severe asthma.
Preparation and characterization of a bioactive nanotherapy
A reactive oxygen species (ROS)-scavenging bioactive material (i.e., TPCD) was synthesized by covalently conjugating 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol) and 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) onto β-cyclodextrin (β-CD), a cyclic oligosaccharide19. The chemical structure of TPCD was confirmed by Fourier Transform infrared and 1H NMR spectroscopy (Supplementary Fig. 1). Calculation according to the 1H NMR spectrum revealed 2 Tempol and 5 PBAP units in each TPCD. Then TPCD nanoparticles (defined as TPCN) were produced by a nanoprecipitation/self-assembly method (Fig. 1b). Thus obtained TPCN displayed well-defined spherical shape, as illustrated by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images (Fig. 1c-d), with a narrow size distribution and a mean diameter of 80 nm (Fig. 1e).
Site-specific delivery of TPCN to pulmonary neutrophils via intravenous injection
A mouse model of neutrophilic asthma was established by stimulation with ovalbumin (OVA) and Al(OH)3 via intraperitoneal (i.p.) injection, in combination with intranasal (i.n.) administration of lipopolysaccharide (LPS) and OVA (Fig. 2a)20. Subsequently, in vivo targeting capability of TPCN was examined, using Cy7.5-labeled TPCN (Cy7.5/TPCN) (Fig. 2a, the upper panel). At 24 h after intravenous (i.v.) injection, ex vivo imaging revealed notable Cy7.5 fluorescence in the lungs of mice with induced asthma (Fig. 2b). Of note, asthmatic mice displayed significantly higher pulmonary accumulation of Cy7.5/TPCN than that of normal animals (Supplementary Fig. 2). This passive targeting was mainly attributed to the structurally and functionally abnormal respiratory epithelium in asthma21, which may cause barrier dysfunction and epithelial permeability to different molecular and particulate substances22-24. In a separate study, flow cytometry showed 92±3% Cy5-positive neutrophils in pulmonary tissues at 24 h after i.v. injection of Cy5-labeled TPCN (Fig. 2c). Further immunofluorescence analysis revealed considerable fluorescence co-localization of Cy5 and Ly6G (Fig. 2d), a typical neutrophil biomarker. Collectively, these results demonstrated that i.v. delivered TPCN can site-specifically distribute in pulmonary neutrophils of asthmatic mice.
Targeted therapy of asthma by i.v. delivery of TPCN
Then we evaluated in vivo efficacy of TPCN in asthmatic mice (Fig. 2a, the lower panel). We found that i.v. treatment with different doses of TPCN notably decreased the ROS level in bronchoalveolar lavage fluid (BALF) collected from diseased mice (Fig. 2e). Also, TPCN effectively reduced typical pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-17 (Fig. 2f-h), which are implicated in the pathogenesis of asthma25. Consistently, we detected notable decrease in the myeloperoxidase (MPO) level and neutrophil count in BALF and lungs after therapy with TPCN (Fig. 2i-j and Supplementary Fig. 3), while their high expression is a common feature of the OVA/LPS-sensitized model of neutrophilic asthma20. Further inspection on histological sections of intrapulmonary bronchi stained with either hematoxylin and eosin (H&E) or periodic acid Schiff (PAS) revealed sloughing of epithelial cells, reticular basement membrane thickening, inflammatory cell infiltration in airway mucosa, enlargement of bronchial smooth muscle, and mucus hypersecretion for diseased mice (Fig. 2k-l). After therapy with TPCN, we observed a pseudostratified ciliated columnar epithelium, an indistinct reticular basement membrane, few inflammatory cells, and considerably reduced bronchial smooth muscle. Mice treated with 1 mg/kg TPCN displayed nearly unaltered microstructures, which are even comparable to the normal group. Further, pulmonary function was examined by a methacholine challenge test. Whereas asthmatic mice showed high airway response to increased doses of methacholine (Fig. 2m), treatment with 1 mg/kg TPCN completely attenuated pulmonary resistance (RL), similar to that of normal mice.
Site-specific delivery of TPCN to lung neutrophils via aerosol inhalation
Since inhalation therapy is the most preferred strategy for the treatment of pulmonary diseases, we assessed whether inhaled TPCN can be delivered to lung neutrophils. Asthmatic mice were induced as aforementioned (Fig. 3a, the upper panel), which were transiently accommodated in a chamber with aerosolized aqueous solution of TPCN (Fig. 3b). At 24 h after inhalation, effective accumulation of Cy7.5/TPCN in the lungs was observed by ex vivo imaging (Fig. 3c). A separate analysis by flow cytometry revealed 93±3% lung neutrophils were Cy5-positive at 24 h after inhalation of Cy5/TPCN in asthmatic mice (Fig. 3d). This neutrophil-specific distribution was confirmed by immunofluorescence analysis of lung cryosections (Fig. 3e). Consequently, TPCN can also be effectively delivered to pulmonary neutrophils by aerosol inhalation.
Treatment of asthma by aerosol inhalation of TPCN
Then therapeutic effects of inhaled TPCN were evaluated in asthmatic mice. In this case, budesonide (BDN), a commonly used drug, was employed as a positive control. All formulations were administered by inhalation after nebulization. Treatment with TPCN at theoretical doses of 0.1 and 1 mg/kg, particularly the high dose, significantly reduced the levels of ROS, TNF-α, IL-1β, and IL-8 in BALF, compared to the model group administered saline (Fig. 3f-i). Also, the MPO level and neutrophil count in BALF and pulmonary tissues notably decreased after TPCN inhalation (Fig. 3j-k and Supplementary Fig. 4). In particular, inhalation of TPCN at 1 mg/kg resulted in significantly lower levels of serum immunoglobulin E (IgE, an antibody that mediates allergic reactions and plays a critical role in allergic asthma) (Fig. 3l). Examination on H&E or PAS sections showed notable attenuation of inflammatory cell infiltration, pulmonary edema, and mucous secretion after inhalation of 1 mg/kg TPCN (Fig. 3m-n). By contrast, except notably reduced MPO and neutrophil levels, inhalation of BDN at a clinically relevant dose afforded no significant benefits, with respect to attenuating oxidative stress and suppressing inflammation in lungs. This is consistent with the previous finding that neutrophilic asthma is generally steroid-resistant2. Correspondingly, TPCN inhalation at 1 mg/kg effectively improved pulmonary function (Fig. 3o). Accordingly, TPCN delivered by inhalation is also effective in the treatment of asthma. It should be noted that, the estimated pulmonary delivery efficiency of inhalation was 6.3%, by comparing Cy5 fluorescence intensities in lungs after direct pulmonary administration and aerosol inhalation (Supplementary Fig. 5). This suggested that TPCN is efficacious even at an actual inhaled dose of 0.063 mg/kg.
Treatment of asthma in mice by a mitochondrial-targeting TPCN nanotherapy
Since TPCN is expected to exert its efficacy by site-specifically eliminating oxidative stress in lung neutrophils, we further examined whether mitochondrial-targeting of TPCN can enhance its therapeutic effects. As a proof of concept, TPCD nanoparticles were prepared by nanoprecipitation/self-assembly in the presence of stearyl triphenylphosphonium26, giving rise to a mitochondrial-targeting nanotherapy (TTPCN). TEM and SEM observation revealed that TTPCN displayed well-defined spherical morphology and narrow size distribution (Fig. 4a-c), with the mean diameter of 105 nm and ζ-potential of -10.6 mV. Using Cy5-labeled TTPCN, confocal microscopic observation demonstrated excellent mitochondrial-targeting capability of TTPCN in neutrophils (Fig. 4d), which was also confirmed in human pulmonary epithelial cells (Supplementary Fig. 6).
Interestingly, Cy5/TTPCN showed significantly higher pulmonary accumulation than Cy5/TPCN, after i.v. delivery in asthmatic mice (Fig. 4e). Of note, a high neutrophil distribution in lung tissues was detected (Fig. 4f-g). Subsequently, therapeutic benefits of TTPCN were tested, following the similar i.v. treatment procedures (Fig. 2a, the lower panel). Compared to the same dose of TPCN (1 mg/kg), TTPCN more effectively inhibited the expression levels of ROS, TNF-α, IL-1β, IL-8, and MPO as well as the neutrophil count in BALF (Fig. 4h-m). Inspection on histological sections stained with H&E or PAS also revealed desirable efficacy of TTPCN (Fig. 4n-o). Consequently, surface decoration with a mitochondrial-targeting unit can simultaneously enhance pulmonary delivery efficiency and in vivo efficacy of TPCN.
In vitro and in vivo mechanistic studies
In vitro antioxidative stress and anti-inflammatory effects of TPCN in neutrophils. After in vitro incubation, microscopic observation and flow cytometric quantification showed effective endocytosis of Cy5/TPCN by mouse peritoneal neutrophils, in both time- and dose-dependent manners (Fig. 5a-b and Supplementary Fig. 7-8). Correspondingly, treatment with various doses of TPCN significantly inhibited phorbol 12-myristate 13-acetate (PMA)-induced ROS generation in neutrophils (Fig. 5c-d and Supplementary Fig. 9). Also, overexpression of typical pro-inflammatory cytokines (TNF-α, IL-1β, and IL-8) in neutrophils was remarkably suppressed by TPCN (Fig. 5e-g). In addition, migration of neutrophils was significantly attenuated after TPCN treatment (Fig. 5h and Supplementary Fig. 10). Of note, the mitochondrial-targeting nanotherapy TTPCN more effectively reduced ROS production in neutrophils, as compared to TPCN (Supplementary Fig. 11). These results suggested that TPCN can markedly inhibit oxidative stress and inflammatory response in neutrophils after efficient cellular internalization, in line with in vivo results.
TPCN inhibits the formation of neutrophil extracellular traps. To further examine mechanisms underlying anti-asthmatic effects of TPCN, mRNA expression profiles of pulmonary tissues were identified by RNA-sequencing. Among all detected mRNAs, there were 197 and 9 differentially up-regulated and down-regulated genes in asthmatic mice (relative to healthy mice) and TPCN-treated asthmatic mice (relative to asthmatic mice), respectively (Supplementary Fig. 12). We identified 20 significantly up-regulated mRNAs in asthmatic mice compared to healthy mice (Fig. 5i). In particular, neutrophil-associated genes Elane and Mpo were expressed at significantly higher levels in asthmatic mice than those of healthy mice, which were recovered to the normal levels after TPCN therapy. qRT-PCR analysis confirmed the abnormal expression levels of these two candidate transcripts and significant inhibitory effects of TPCN (Fig. 5j-k). As well demonstrated, neutrophil elastase (NE) and MPO, separately encoded by Elane and Mpo genes, are the main components of NETs27, 28. Therefore we hypothesize that the development of neutrophilic asthma is closely related to NETs, while therapeutic effects of TPCN are mainly mediated by inhibiting NETs formation.
As expected, expression levels of the characteristic components of NETs, such as extracellular double-stranded DNA (dsDNA), NE, MPO, and citrullinated histone H3 (citH3, a modified form of histone H3 implicated in chromatin decondensation and NETs formation) significantly increased in PMA-activated neutrophils (Fig. 6a-e), which were remarkably reduced by TPCN in a dose-response pattern. Further, in vitro NETs formation by PMA-stimulated neutrophils was directly observed by immunofluorescence, as illustrated by release of extracellular dsDNA and citH3 that appeared as fibrous strands (Fig. 6f and Supplementary Fig. 13). By contrast, TPCN treatment significantly reduced the degree of NETosis and citH3 area (Fig. 6f-h). These results substantiated that TPCN can effectively inhibit in vitro NETs formation.
Moreover, asthmatic mice showed significantly higher levels of dsDNA and NE in BALF as well as higher NE and citH3 in lungs, compared to normal mice (Fig. 6i-m). In addition, NETs formation in pulmonary tissues of asthmatic mice was affirmed by immunofluorescence analysis (Fig. 6n and Supplementary Fig. 14), as indicated by citH3 and NE positive fluorescence signals. Treatment with TPCN at either 0.1 or 1 mg/kg remarkably reduced expressions of dsDNA, NE, and citH3 in BALF and lungs. These results demonstrated that TPCN can attenuate NETs formation in the lungs of asthmatic mice.
TPCN regulates NETs-mediated Treg/Th17 cell imbalance. Previous studies have demonstrated that Treg cells play a crucial role in the maintenance of immune homeostasis in the airways29. Particularly, both animal and clinical studies indicated that Treg/Th17 cell imbalance considerably contributes to the pathogenesis of asthma30, 31. To explore whether inhibition of NETs formation by TPCN and its anti-asthmatic effects are related to Treg/Th17 homeostasis, we analyzed numbers of pulmonary and splenic Treg/Th17 cells. Compared with those of healthy mice, the proportion of pulmonary Treg cells in asthmatic mice significantly decreased, while an opposite change was observed for Th17 cells in lungs (Fig. 7a-d), resulting in a remarkably decreased ratio of Treg/Th17 cells (Fig. 7e). Similar changing profiles were observed for the numbers of splenic Treg and Th17 cells as well as their ratios (Fig. 7f-j). By contrast, treatment with TPCN significantly increased Treg cells and decreased Th17 cells, and therefore effectively reversed the proportion of Treg cells in both lungs and spleens. These data substantiated that TPCN can regulate the Treg/Th17 balance in asthmatic mice.
Since recent studies have revealed an important role of neutrophils and NETosis in mediating inflammatory and immune responses in asthma13, 14, we speculate that Treg/Th17 imbalance is induced by NETs. Our in vitro studies indicated that NETs notably suppressed the differentiation of naïve T cells into Treg cells, whereas normal neutrophils showed no significant effects (Fig. 7k-l). Further SEM observation revealed that naïve T cells could be covered by NETs (Fig. 7m, left). Accordingly, NETs-inhibited Treg differentiation might be resulted from the NETs coating-derived physical barriers on naïve T cells that may impair effective diffusion of Treg-inducing molecules and their close contact with T cells. Treatment with TPCN significantly restored Treg differentiation from naïve T cells in the presence of NETs (Fig. 7n-o). Consistently, the coverage of T cells by NETs considerably decreased after TPCN treatment (Fig. 7m, right). In this case, only a small proportion of T cells were trapped by NETs.
Safety studies on TPCN after inhalation
Because TPCN is expected to be administered by aerosol inhalation, acute toxicity evaluation was performed in mice after nebulized inhalation at 50 or 100 mg/kg for 7 consecutive days (Supplementary Fig. 15a). During treatment, all treated mice remained healthy, without any behavioral abnormalities. In addition, all animals displayed similar change profiles in body weight gain and showed comparable organ indices for major organs at day 15 after treatment (Supplementary Fig. 15b-c). Examination on H&E-stained sections of major organs revealed no discernible destruction of tissue microstructure or inflammatory cell infiltration in TPCN groups (Supplementary Fig. 16). Moreover, TPCN treatment did not lead to significant changes of oxidative stress-related factors and pro-inflammatory cytokines in lung tissues, such as H2O2, MPO, TNF-α, and IL-1β (Supplementary Fig. 17a-d). Likewise, there were no abnormal changes in typical hematological parameters and biomarkers relevant to liver/kidney functions in TPCN-treated mice (Supplementary Fig. 17e-l). In line with these results, we detected efficient hydrolysis and/or metabolism of TPCN in neutrophils and mouse lung homogenates (Supplementary Fig. 18), resulting in parent β-CD and other water-soluble molecules that can be cleared by the kidneys. Collectively, these preliminary data revealed good safety of TPCN after inhalation at a dose that is 100-fold higher than those examined in therapeutic studies.