Low-dose nanotherapy-mediated site-specic inhibition of neutrophil extracellular traps for immunoregulatory treatment of asthma

Asthma, one of the most common lung diseases, remains a serious global healthy problem. Currently there is still an urgent need for effective and safe therapies against severe asthma. Neutrophil extracellular traps (NETs) have emerged as a new therapeutic target for different diseases, while precision regulation of NETs is highly challenging. Here, we report site-specic attenuation of oxidative stress in lung neutrophils via a cyclic oligosaccharide-derived nanotherapy (termed as TPCN) effectively inhibited the development of mouse neutrophilic asthma, a typical phenotype of severe asthma. Therapeutically, TPCN delivered via either intravenous injection or inhalation can distribute in lung neutrophils of asthmatic mice, thereby signicantly mitigating oxidative stress, suppressing inammatory responses, reversing airway remodelling, and improving pulmonary function. Notably, TPCN is effective even at an actual inhalation dose as low as 0.063 mg/kg. Mechanistically, therapeutic benets of TPCN are achieved by inhibiting reactive oxygen species (ROS)-induced formation of NETs, which further promotes immune homeostasis via regulating balance between regulatory T (Treg) and T helper 17 (Th17) cells. Thus, TPCN holds great promise for precision therapy of neutrophilic asthma, while the ROS-NETs-Treg/Th17 pathway can function as intriguing therapeutic targets for the treatment of severe asthma and other neutrophil-mediated inammatory diseases.

like extracellular structures of chromatin laments coated with histones, proteases, and cytosolic/granule proteins 16 . 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 in ammatory and immune diseases 16,17 . 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 diseases 17,18 . However, it remains unclear whether this NETosis inhibition strategy is e cacious for the treatment of asthma, in particular neutrophilic asthma.
Here we report that site-speci c inhibition of NETosis in the lungs can effectively suppress local in ammation, 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 bene cial 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-speci c strategy for the prevention and treatment of severe asthma.
Site-speci c 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 uorescence in the lungs of mice with induced asthma (Fig. 2b). Of note, asthmatic mice displayed signi cantly 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 asthma 21 , which may cause barrier dysfunction and epithelial permeability to different molecular and particulate substances 22-h after i.v. injection of Cy5-labeled TPCN (Fig. 2c). Further immuno uorescence analysis revealed considerable uorescence co-localization of Cy5 and Ly6G (Fig. 2d), a typical neutrophil biomarker. Collectively, these results demonstrated that i.v. delivered TPCN can site-speci cally distribute in pulmonary neutrophils of asthmatic mice. Targeted therapy of asthma by i.v. delivery of TPCN Then we evaluated in vivo e cacy 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 uid (BALF) collected from diseased mice (Fig. 2e). Also, TPCN effectively reduced typical proin ammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-17 ( Fig. 2f-h), which are implicated in the pathogenesis of asthma 25 . 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 asthma 20 . 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, in ammatory cell in ltration in airway mucosa, enlargement of bronchial smooth muscle, and mucus hypersecretion for diseased mice (Fig. 2k-l). After therapy with TPCN, we observed a pseudostrati ed ciliated columnar epithelium, an indistinct reticular basement membrane, few in ammatory 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 (R L ), similar to that of normal mice.
Site-speci c 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 ow cytometry revealed 93±3% lung neutrophils were Cy5-positive at 24 h after inhalation of Cy5/TPCN in asthmatic mice (Fig. 3d). This neutrophil-speci c distribution was con rmed by immuno uorescence 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, signi cantly 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 signi cantly 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 in ammatory cell in ltration, 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 signi cant bene ts, with respect to attenuating oxidative stress and suppressing in ammation in lungs. This is consistent with the previous nding that neutrophilic asthma is generally steroid-resistant 2 . 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 e ciency of inhalation was 6.3%, by comparing Cy5 uorescence intensities in lungs after direct pulmonary administration and aerosol inhalation ( Supplementary Fig. 5). This suggested that TPCN is e cacious 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 e cacy by site-speci cally 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 triphenylphosphonium 26 , giving rise to a mitochondrial-targeting nanotherapy (TTPCN). TEM and SEM observation revealed that TTPCN displayed well-de ned spherical morphology and narrow size distribution ( Fig. 4a-c), with the mean diameter of 105 nm and ζ-potential of -10.6 mV.
Interestingly, Cy5/TTPCN showed signi cantly 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 bene ts of TTPCN were tested, following the similar i.v. treatment procedures (Fig. 2a, Fig. 9). Also, overexpression of typical pro-in ammatory cytokines (TNF-α, IL-1β, and IL-8) in neutrophils was remarkably suppressed by TPCN (Fig. 5e-g). In addition, migration of neutrophils was signi cantly 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 in ammatory response in neutrophils after e cient 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 pro les of pulmonary tissues were identi ed by RNAsequencing. Among all detected mRNAs, there were 197 and 9 differentially up-regulated and downregulated genes in asthmatic mice (relative to healthy mice) and TPCN-treated asthmatic mice (relative to asthmatic mice), respectively ( Supplementary Fig. 12). We identi ed 20 signi cantly up-regulated mRNAs in asthmatic mice compared to healthy mice (Fig. 5i). In particular, neutrophil-associated genes Elane and Mpo were expressed at signi cantly higher levels in asthmatic mice than those of healthy mice, which were recovered to the normal levels after TPCN therapy. qRT-PCR analysis con rmed the abnormal expression levels of these two candidate transcripts and signi cant 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 NETs 27, 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 doublestranded DNA (dsDNA), NE, MPO, and citrullinated histone H3 (citH3, a modi ed form of histone H3 implicated in chromatin decondensation and NETs formation) signi cantly 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 immuno uorescence, as illustrated by release of extracellular dsDNA and citH3 that appeared as brous strands ( Fig. 6f and Supplementary Fig. 13). By contrast, TPCN treatment signi cantly 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 signi cantly 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 a rmed by immuno uorescence analysis (Fig. 6n and Supplementary  Fig. 14), as indicated by citH3 and NE positive uorescence 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 airways 29 . Particularly, both animal and clinical studies indicated that Treg/Th17 cell imbalance considerably contributes to the pathogenesis of asthma 30,31 . To explore whether inhibition of NETs formation by TPCN and its antiasthmatic 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 signi cantly 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 pro les were observed for the numbers of splenic Treg and Th17 cells as well as their ratios (Fig. 7f-j). By contrast, treatment with TPCN signi cantly 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 in ammatory and immune responses in asthma 13,14 , 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 signi cant effects (Fig. 7k-l). Further SEM observation revealed that naïve T cells could be covered by NETs (Fig. 7m, left). Accordingly, NETsinhibited 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 signi cantly 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 pro les 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 in ammatory cell in ltration in TPCN groups (Supplementary Fig. 16). Moreover, TPCN treatment did not lead to signi cant changes of oxidative stress-related factors and pro-in ammatory cytokines in lung tissues, such as H 2 O 2 , 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 e cient 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 100fold higher than those examined in therapeutic studies.

Discussion
Approximately 3-10% of people with asthma suffer from severe asthma 5, 32 . This debilitating and treatment refractory disease places a large physical, psychological, social, and economic burden on patients. In addition to other daily medications, high doses of inhaled corticosteroids or long-term oral corticosteroids are generally necessary for the treatment of severe asthma. However, long-term corticosteroids frequently cause serious medication side-effects, such as obesity, diabetes, osteoporosis, hypertension, adrenal suppression, and depression/anxiety 6,7 . Add-on treatments for severe asthma, including tiotropium, leukotriene receptor antagonist, macrolides, and biologic agents, also show varied degrees of limitations. In particular, the inconvenience and high cost of long-term administration, variation in responses, and possible severe allergic reactions are major concerns of biologics used for severe asthma 33,34 . Consequently, great challenges remain in the treatment of severe asthma.
About half of patients with severe asthma have neutrophilic in ammation 14 , and airway neutrophilia is closely associated with asthma severity 11 . NETs released by activated neutrophils show important roles in the pathogenesis of severe asthma and other asthma phenotypes 13-16, 35, 36 . These ndings implied that NETs can serve as a promising therapeutic target for asthma. Indeed, blocking NETosis by inhibitors of NE and protein-arginine deiminase 4 or degrading NETs with DNase can attenuate airway hyperresponsiveness [13][14][15] . However, currently existing molecules used for NETosis inhibition display real problems, such as speci city and safety concerns 4,17,18 . Here we found that an antioxidative and antiin ammatory nanotherapy TPCN can notably accumulate in the in amed lungs of mice with neutrophilic asthma, after either i.v. or inhalation administration. The enhanced accumulation of TPCN in lung tissues is mainly due to the abnormal structure and function of the respiratory epithelium in asthmatic mice 21,22 . Of note, i.v. delivered or inhaled TPCN was mainly distributed in lung neutrophils, showing above 90% TPCN-positive neutrophils. Accordingly, the nanotherapy TPCN can be site-speci cally delivered to lung neutrophils. Consistently, treatment with TPCN via either i.v. injection or inhalation in asthmatic mice afforded bene cial therapeutic effects, as manifested by signi cantly decreased oxidative stress, attenuated in ammatory reactions, inhibited mucus hypersecretion, suppressed airway remodeling, and improved airway hyperresponsiveness. Notably, bronchial hyperresponsiveness of asthmatic mice was almost completely reversed after TPCN therapy. Compared to BDN, a commonly used drug for asthma, inhaled TPCN more effectively alleviated asthma symptoms. Moreover, surface functionalization of TPCN with a mitochondrial-targeting moiety further improved its antioxidative activity and lung accumulation e ciency, thereby leading to additionally potentiated anti-asthmatic e cacies in mice. Collectively, the nanotherapy TPCN can effectively improve asthma symptoms, lung function, and airway hyperresponsiveness, by site-speci cally attenuating oxidative stress in lung neutrophils. It is worth noting that TPCN is e cacious at very low doses (especially inhalation at 0.063 mg/kg), which are dramatically lower than those of currently used drugs (varying from 0.26 to 78 mg/kg). In addition, preliminary in vivo tests in mice suggested that TPCN was safe after 7-day continuous inhalation at a dose 100-fold higher than those used in therapeutic assessment.
In line with in vivo e cacies, TPCN effectively inhibited oxidative stress and in ammatory responses in neutrophils in vitro. RNA-sequencing analysis of lung tissues revealed that TPCN treatment normalized abnormally up-regulated genes relevant to NETs, agreeing with the previous nding that oxidative stress has been implicated in the formation and release of NETs 37,38 . Furthermore, both in vitro and in vivo studies demonstrated that TPCN signi cantly inhibited the formation of NETs. These results substantiated that TPCN can site-speci cally block NETosis by eliminating ROS in lung neutrophils.
On the other hand, increasing evidence has revealed the intimate relationship between immune dysfunction and asthma 1 . Th17 cells are associated with the neutrophilic in ammation and more severe phenotypes of asthma 39,40 , while Treg cells display indispensable effects in anti-in ammation, maintaining self-tolerance, and preventing allergic diseases 41 . Also, previous studies indicated that Treg cells may interfere with asthma at different stages, such as sensitization, allergic in ammation, airway remodeling, and airway hyperresponsiveness 42,43 . Our results showed that asthmatic mice exhibited signi cantly increased Th17 cells in the spleen and lungs as compared to healthy mice, concomitant with notably decreased Treg cells and a reduced Treg/Th17 cell ratio. By contrast, treatment with TPCN normalized Treg and Th17 cell counts and their ratios to the levels comparable to those of healthy mice. This nding is consistent with the fact that Treg/Th17 cell imbalance plays a pivotal role in the development, exacerbation, and severity of asthma, especially in neutrophilic asthma 30,31,[44][45][46] . We further found that TPCN can rescue NET-attenuated Treg cell differentiation from naïve T cells, probably by decreasing NETs coating on T cells and inhibiting detrimental effects of the DNA/protein components of NETs on T cells. These results implied that TPCN therapy can also maintain immune homeostasis by regulating NET-mediated Treg/Th17 cell imbalance.
In summary, our ndings demonstrated nanotherapy-mediated site-speci c attenuation of oxidative stress and in ammatory responses in neutrophils is a promising strategy for effective treatment of neutrophilic asthma, by precisely inhibiting NETosis and promoting immune homeostasis. Our results also suggested that the ROS-NETs-Treg/Th17 axis may be potentially new therapeutic targets for severe asthma and other neutrophilic in ammation-associated noninfectious and infectious lung diseases such as adult respiratory distress syndrome, chronic obstructive pulmonary disease, cystic brosis, and coronavirus disease , in view of the important pathological effects of neutrophils.

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Any methods, Nature Research reporting summaries, source data, Supplementary, supplementary information, acknowledgements; details of author contributions and competing interests; and statements of data and code availability are available.  Statistical signi cance was performed by independent-samples t-test for data in (b,c), one-way ANOVA with post-hoc LSD tests for data in (e-j,m). *P < 0.05, **P < 0.01, ***P < 0.001.  f,h-m, n = 5). Statistical analysis was conducted by one-way ANOVA with post-hoc LSD tests for data in (e,h-j,l-m), by the independent-samples t-test for data in (f), and by the Kruskal-Wallis H test for data in (k). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5
In vitro bioactive effects of TPCN in neutrophils and RNA-sequencing analysis of pulmonary tissues from healthy or asthmatic mice with or without TPCN treatment. a-b, Fluorescence images (a) and ow cytometry quanti cation (b) of time-dependent endocytosis of Cy5/TPCN in neutrophils. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. c, Fluorescence images indicate ROS production in neutrophils after different treatments and staining with a uorescent probe DCFH-DA. The normal group was treated with fresh medium, while the model group was induced with PMA. Before stimulation with PMA, the TPCN groups were treated with TPCN at varied doses. Scale bars, 20 μm. d, Relative ROS levels in neutrophils quanti ed by ow cytometry after staining with DCFH-DA. e-g, Expression levels of TNF-α (e), IL-1β (f), and IL-8 (g) in neutrophils after different treatments. h, TPCN inhibits in vitro migration of peritoneal neutrophils. i, Clustering analysis showing top 20 regulated genes in lungs of healthy or asthmatic mice after different treatments. j-k, Elane and Mpo mRNA levels quanti ed by qRT-PCR. Data are mean ± s.e.m. (b, n = 6; d, n = 4; e-h, n = 5; j-k, n = 3). Statistical analysis was conducted by one-way ANOVA with post-hoc LSD tests. *P < 0.05, **P < 0.01, ***P < 0.001. The effects of TPCN on NETs-mediated differention of regulatory T cells. a-e, Representative ow cytomeitric pro les (a-b), quantiative analysis of Treg and Th17 cells (c-d), and Treg/Th17 cell count ratios (e) in lung tissues of healthy or asthmatic mice with or without TPCN treatment. f-j, Flow cytometric pro les (f-g), quanti cation of Treg and Th17 cells (h-i), and calculation of Treg/Th17 cell count ratios (j) in splenic tissues from healthy or asthmatic mice after different treatments. k-l, Flow cytometric pro les (k) and quantitative analysis (l) of in vitro Treg cell differentiation in the presence of neutrophils (control) or NETs (model). Naïve T cells in the normal group were treated with medium alone. m, SEM images show attenuated NETs coating on naïve T cells by TPCN. Non-trapped T cells were largely removed