NADPH oxidase 2 plays a protective role in experimental Aspergillus fumigatus keratitis in mice through killing fungi and limiting the degree of inflammation

INTRODUCTION

Changes in Clinical Performance After the Inhibition of NOX2 Mice were treated with different concentrations of DPI to inhibit the expression of NOX2. As shown in Figure 2A, corneal NOX2 mRNA levels were significantly reduced following treatment with 1 μmol/L DPI (

＜0.01). Western blotting also verified the inhibitory effect of DPI on NOX2 protein expression in corneas (Figure 2B). After verification of the inhibitory effect of DPI on NOX2, clinical performance was observed to detect the role of NOX2. As shown in Figure 2C, with DPI treatment, clinical performances were worse after

infection and marked by a larger ulcer area,more severe edema, and more neovascularization than those observed without infection. The analysis of the clinical scores is presented in Figure 2D (

＜0.01, ＜0.05, ＜0.01 at 1, 3, and 5d p.i., respectively).

MATERIALS AND METHODS

Ethical Approval Eight-week-old female C57BL/6 mice were purchased from Jinan Pengyue Laboratory Animal Co., Ltd.(Jinan, China) and treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.

Mice and Fungal Culture The central corneal epithelium(3 mm diameter) was removed from the right eye, and a 5 μL aliquot of

was dropped onto the ocular surface.Then, the cornea was covered with a soft contact lens, and the eyelid was sutured. The control groups were treated by removing the central corneal epithelium without introducing fungal infection and covering the cornea with a soft contact lens. At 24h post infection (p.i.), the suture was removed.Before each cornea was harvested, its clinical score was recorded according to the standard for evaluation.

As a critical member of the NOX family, NOX2 transports electrons across biological membranes and converts oxygen into superoxide to mediate antimicrobial reactions. CGD patients who have a genetic disorder that results in defects in NOX2 (or its associated subunits), showed impaired ROS production by phagocytes. They are highly susceptible to severe and invasive bacteria and fungi and endure excessive inflammatory reactions. The degree of NOX2 impairment is related to the severity of this disease. As the most common etiological agent of invasive aspergillosis, which is the leading cause of death for CGD patients,

is very sensitive to ROS and NOX2 defects

. After NOX2 knockout, the ability of

hyphae to kill is obviously decreased. Studies have shown that NOX is expressed in corneal epithelial and stromal cells in both rabbits and humans

. In experimental alkali burn-induced corneal injury mouse models, NOX2 participated in the inflammatory and neovascular responses, and the inhibition of NOX2 by DPI effectively attenuated alkali burn-induced ROS production by regulating oxidative stress,inflammatory responses and choroidal neovascularization(CNV)

. To date, there has been no report on the role of NOX2 in keratitis, especially its role in FK. Therefore, we chose

which was closely connecting to ROS and NOX2,to determine how NOX2 affects the process of FK in mice.

The inhibition of NOX2 was carried out by subconjunctivally injecting the experimental eyes with 5 μL DPI (1 μmol/L,Sigma Aldrich, Saint Louis, USA) 24h before infection with fungi. After infection, an additional 0.03 μg/100 μL of the DPI solution was immediately injected intraperitoneally(

.); control corneas were injected with dimethyl sulfoxide(DMSO) at the same concentration in phosphate buffered saline (PBS). After the sutures were removed, 5 μL DPI was subconjunctivally injected every day.

2.4 不同耕作方式对夏玉米农田土壤呼吸速率的影响 从表1可以看出，夏玉米整个生育期内，不同耕作方式下0～10 cm土层土壤呼吸速率均大于10～20 cm土层土壤呼吸速率，且不同耕作方式下10～20 cm土层无显著差异(P>0.05)。

Isolation of Neutrophils C57BL/6 mice were injected

.with 1 mL 9% casein (Sigma, China) and again after 24h.Three-hours after the second injection, we began to collect neutrophils in the abdominal cavity by injection of 10 mL Dulbecco’s modified Eagle’s medium (DMEM; Gibco, San Diego, CA, USA). After centrifugation at 300× g for 10min,the neutrophils were collected and purified using 64% and 80% Percoll solutions. Then, the cells were cultured in DMEM with 10% fetal bovine serum (FBS; Gibco) for 2h and used for subsequent experiments. To stimulate

, the fungal hyphae were added at a final concentration of 5×10

CFU/mL.One micromolar of DPI was added 30min before the fungus,and 0.3 mg/mL laminarin (Solarbio, Beijing, China), 5 μmol/L PRT062608 (MedChemExpress, New Jersey, USA) and 0.5 μmol/L rottlerin (Tocris, Bristol, UK) were added 1h before the fungus to carry out the inhibition test. DMSO at the same concentrations was added to control samples.

吸收液体循环的目的是降低尾气中的氨气浓度，最后的尾气吸收器兼有去除气体中雾沫的功能。再沸器汽化率约10%，塔顶回流比3～4，塔顶采出率D/F=0.035。

A small numbers of neutrophils exist in normal corneas, and upon fungal infection, the neutrophils rapidly enter into the site of inflammation from limbal blood vessels. Recent studies from our team regarding how neutrophils work were mainly focused on the role of various pathogen recognition receptors(PRRs) such as PAR-2 and LOX-1

. Less is known about the method by which neutrophils kill fungi. NOX is the main origin of ROS in phagocytes. The production of NOXderived ROS, also called an oxidative burst, can mediate many biological functions

. ROS, as inflammatory mediators,have been studied in the cornea, and their levels in the corneal epithelium increase in dry eyes and are the etiology of alterations in the corneal epithelium

.

现有的研究生新生入学教育主要存在两种现象，第一种为忽视研究生特点的“形式化”教育，入学教育基本采取学校开大会、学院开小会、集中进行讲座的形式。该种现象形成的根本原因是认识不到位，认为研究生有导师一对一的教育，无需再进行完整的入学教育。这种观点忽视了研究生入学教育的重要性，导致研究生入学教育只停留在表面，并没有形成真正合理完整的体系。

Western Blot Analysis Six corneas in each well in a 6-well plate were sampled for Western blot analysis. The samples were ground in radio immunoprecipitation assay(RIPA; Solarbio, Beijing, China) lysis buffer with 1 mmol/L phenylmethylsulfonyl fluoride (PMSF; Solarbio, Beijing,China) for 2h and then centrifuged, the supernatant was collected. The total protein was separated by 12% acrylamide SDS-polyacrylamide gelelectrophoresis (PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes (Solarbio,Beijing, China) and then blocked. The membranes were incubated with polyclonal antibodies against β-actin (1:6000;Bioss, Beijing, China), GADPH (1:7000; Elabscience,Wuhan, China), NOX2 (1 μg/mL; Abcam, Cambridge, UK)at 4°C overnight. Then, the membranes were incubated with the corresponding secondary antibodies (1:8000, Abcam,Cambridge, UK) at room temperature for 1h. Finally, the blots were developed by using chemiluminescence (ECL; Beyotime,Shanghai, China).

Enzyme-linked Immunosorbent Assay Each cornea(

=6/group/time) was homogenized in 500 μL PBS with 0.1% Tween 20 and a protease inhibitor cocktail (CWbiotech,Beijing, China). The neutrophil culture supernatants were collected 4h p.i., and 100 μL of each sample was assayed in duplicate to test for TNF-α and H3 according to the manufacturer’s instructions (Elabcience, Wuhan, China).

Immunofluorescent Staining Eyeballs were embedded in optimal cutting temperature (OCT) compound (Sakura Tissue-Tek

, USA), frozen in liquid nitrogen and sliced into 10 μm sections. After fixation for 5min with acetone and blocking,the sections were incubated with NOX2 (20 μg/mL) and NIMP-R14 (1:100; Santa Cruz Biotechnology, CA, USA)at 4°C overnight. Then, samples were incubated with FITCconjugated goat anti-rat secondary antibody (CWbiotech,Wuhan, China; 1:500) for 1h at room temperature. Digital images were captured with a Zeiss Axiovert microscope.

Myeloperoxidase Assay At 3d p.i., the corneas (

=6/group/time)were harvested and homogenized using an myeloperoxidase(MPO) test kit (Njjcbio, Nanjing, China) according to the manufacturer’s instructions and then monitored by their absorbance at 460 nm. The results were reported as U/wet weight of cornea. One unit of MPO activity is equivalent to ～2×10

neutrophils.

NOX2 Expression in the Corneas of C57BL/6 Mice We detected the mRNA and protein expression of NOX2 by realtime reverse transcription-polymerase chain reaction, Western blot and immunofluorescent staining. The results shown in Figure 1A indicated that relative NOX2 mRNA levels were significantly higher in the

-infected corneas than those in the normal and control groups (Figure 1A;

＜0.05,＜0.01, ＜0.01 at 1, 3, and 5d p.i., respectively). As shown in Figure 1B, NOX2 protein levels were low in normal corneas,but after

infection, NOX2 expression was increased at 1, 3, and 5d p.i. As shown in Figure 1C, there was little positive staining for NOX2 in the normal corneas, while 3d p.i.there was stronger staining for NOX2 in the corneal epithelial cells, and some positively stained cells could have been neutrophils and macrophages in the stroma of the cornea.

Measurement of Reactive Oxygen Species The detection of ROS was carried out both

in mouse corneas and

in peritoneal neutrophils (

=6/group/time). Corneal ROS were measured with 2’,7’-Dichlorodihydrofluorescein diacetate(DCFH-DA; Njjcbio, Nanjing, China) by flow cytometry.After corneal cell suspensions were prepared with Liberase(Sigma Aldrich, Saint Louis, USA), they were incubated with 10 mmol/L DCFH-DA 1h at 37°C. The cells were collected and washed with PBS, and the DCFH-DA fluorescence was measured on a FITC channel (CytoFLEX, Beckman Coulter).Data were recorded with the use of CytExpert software as the ‘P1 percentage’ fluorescence variation.

, ROS generation was detected in cultured 2h p.i. by incubation with 10 mmol/L DCFH-DA for 45min at 37°C.

Statistical Analysis All experiments were repeated once to ensure reproducibility, and data from each representative experiment are shown as the mean±standard error of the mean (SEM). A one-way ANOVA and an unpaired, twotailed Student’s

-test was used to determine the statistical significance. Data were considered significant at

＜0.05.

RESULTS

Colony Forming Units Corneas (

=5/group/time) were collected 3d p.i. and ground evenly in 1 mL 0.9% strokephysiological saline solution (NS). One hundred microliters of homogenate were painted onto the surface of Sabouraud dextrose agar and incubated at 29℃ for 48h. The number of fungal colonies were counted manually. The log10 CFU/cornea values were used for statistical analysis.

As a common blinding disease that mostly occurs in developing countries, fungal keratitis (FK) is difficult to diagnose and treat. Neutrophils play a critical role in the defense against invasive fungal infections

, and fungal infection can cause many effects in neutrophils, including reactive oxygen species (ROS) production by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)system, the formation of neutrophil extracellular traps (NETs), and the release of bactericidal and fungicidal peptides

. NOX is the major origin of ROS in phagocytes. NOXs can transport electrons between the intracellular and extracellular environment,leading to the reduction of oxygen to superoxide

. NOXs play diverse roles in different organisms through redox signaling

.In the genetic disorder chronic granulomatous disease (CGD),in which NOX2 is defective, patients are hypersensitive to lifethreatening bacterial and fungal infections due to their inability to kill invasive pathogens. In CGD patients,

(

) is the main etiological agent

.

is very susceptible to ROS, and phagocytes kill

by NOX-dependent mechanisms

.The role of NOX2 in

keratitis remains not well understood. The study showed that hyphae of

could activate neutrophil NOX through CD18 and NOX activation was essential for killing hyphae

. The effect of

gene deficiency on immune function has been confirmed by many experiments, but the effect of exogenous interference with NOX2 in normal mice on fungal infection is still unknown. Thus, the current study explored the expression of NOX2, its role in immunity against

infection in the cornea of C57BL/6 mice and the signaling pathway used in this process. Our data showed that NOX2 expression was upregulated after

infection in mouse corneas.Treatment with an inhibitor of NOX2, diphenyleneiodonium chloride (DPI), reduced NOX2 expression. Blocking NOX2 decreased clinical performance and resulted in the infiltration of more neutrophils, a decrease in the ability to clear fungi, the release of fewer ROS and the formation of NETs.

尽管涉诉信访制度在联系人民群众、维护司法公正方面发挥了一定的作用，但是其弊端也较为明显。最主要的就是涉诉信访统计通报考评等机制加强了上级法院在“审判系统内部的司法行政方面的权力”［1］386，进而导致对下级法院审判独立的损害。

Effect of NOX2 on Neutrophils

treatment with DPI increased the infiltration and intensity of neutrophil labeling with NIMP-R14 after

infection (Figure 3A). The expression of myeloperoxidase (MPO) in the corneas increased significantly 3d p.i., and with DPI treatment, its expression was enhanced (Figure 3B;

＜0.01, ＜0.05, respectively). DPI treatment increased the number of CFUs counted (Figure 3C;

＜0.05). Twenty-four hours after the corneas were infection with fungi, the concentration of H3 was increased, while after treatment with DPI, the concentration of H3 was decreased(Figure 3D;

＜0.01, ＜0.01, respectively). In neutrophils 4h p.i., the concentration of H3 in the culture medium increased significantly compared to that observed for normal cells, while following treatment with DPI, the H3 concentration decreased(Figure 3E;

＜0.01, ＜0.05, respectively).

Effect of NOX2 on ROS Generation The corneas were collected 24h p.i. to detect ROS expression

by flow cytometry. Compared with the

-infected group(43.98% ROS-positive) and the DMSO control group (47.04%ROS-positive), there were fewer ROS-positive cells (17.34%)in the DPI treatment group (Figure 4A-4C), which showed that the inhibition of NOX2 by DPI suppressed ROS expression in mouse FK. Then, immunofluorescent staining of ROS in neutrophils was performed (Figure 4D).

Effect of NOX2 on Cytokines After

Infection Three days p.i., the corneas were collected to detect mRNA levels of the following cytokines: Nrf2, NF-κB, IL-17A,IL-6, IL-10 and TGF-β (Figure 5). Compared with their levels in the normal corneas, the relative mRNA levels of Nrf2,NF-κB, IL-17A, IL-6, IL-10, and TGF-β increased following fungal infection (

＜0.05, ＜0.05, ＜0.05, ＜0.01, ＜0.05, ＜0.05,respectively). Treatment with DPI increased the mRNA levels of NF-κB, IL-17A, and IL-6 (

＜0.05, ＜0.05, ＜0.05, respectively)and decreased the mRNA levels of Nrf2, IL-10 and TGF-β(

＜0.05, ＜0.05, ＜0.01, respectively) compared to those observed in control corneas.

采用SPSS 20.0统计学软件进行统计分析，计量资料（±s）及计数资料[n（%）]分别利用 t检验和 χ2检验，P＜0.05为差异有统计学意义。

DISCUSSION

Real-Time Reverse Transcription-Polymerase Chain Reaction Each cornea or cell was placed in a well in a 12-well plate and was considered to be a single sample (

=6/group/time).After the collection of each sample, the total RNA was isolated with RNAiso plus reagent (Takara, Dalian, China), and then the RNA was transformed to cDNA using the PrimeScript RT reagent kit with gDNA Eraser according to the manufacturer’s instructions (Takara, Dalian, China). After dilution, a total reaction volume of 20 μL was used with SYBR Premix Ex Taq (Takara, Dalian, China). The cycling parameters were as follows: 95°C for 30s, followed by 40 cycles of 95°C for 5s,60°C for 30s, and a final stage of 95°C for 15s, 60°C for 30s,and 95°C for 15s. Relative transcription levels were calculated by using the relative standard curve method, which compares the amount of the target normalized to β-actin. The primer sequences used in this study are listed in Table 1.

(Number 3.0772, China General Microbiological Culture Collection Center, Beijing, China) was cultured at 29℃ for 5d. After shaking at 37℃ and 120 rpm for 3d in liquid medium, the hyphae were collected and cut into pieces 20-40 μm in length [1×10

colony forming units (CFU)/mL].

Similar to many other infectious diseases, NOX2 was significantly activated by fungal infection in corneas. DPI,an inhibitor of NOXs, reduced the activity of NOX2 and interdicted the production of ROS

. We suppressed NOX2 by DPI and found excessive inflammation with less ROS production both

and

. The clinical performances of NOX2-defective mice were more severe compared with those of control mice, and the mice did not show signs of improvement over time. More neutrophil infiltration was observed at the infection site in both the staining and MPO assays. The termination of neutrophil inflammation is necessary to inhibit excessive tissue damage. Although the direct effects of NOX2 activation such as ROS generation and protease activation appear to increase tissue damage, NOX2 activation can also limit the degree of acute inflammation by accelerating apoptosis and the clearance of neutrophils

. Impaired apoptosis and the increased recruitment of neutrophils together may lead to excessive inflammatory reactions in patients with CGD. NOX2-deficient neutrophils have impaired suppressive function (anti-inflammation)

. Similar to the results of our tests, NOX2-deficient mice showed strong pulmonary neutrophil inflammation and proinflammatory cytokine response stimulated by zymosan, and the inflammatory response became self-limited after transfection with NOX2

.The stimulation of

hyphae also resulted in strong and long-term pulmonary inflammation. Another new study showed that neutrophil infiltration in the intestinal mucosa led to severe epithelial injury and impacted wound healing,and MPO was an important regulator of this process

, which inspired us to investigate the role of neutrophil infiltration in the repair of corneal epithelial defects in FK in our next experiments.

除此之外，会议现场还进行了“年度魅力生态景区、文化景区、主题线路、乡村旅游目的地、魅力小镇、特色美食、旅游演艺项目、旅游文创产品、文旅扶贫城市、文旅发展潜力城市”的榜单发布及授牌，延安市副市长孙矿玲受邀出席活动。

在沈阳农大禾丰饲料有限公司车间进行试验料生产。对照组使用锤片粉碎机粉碎，试验组使用经改造的对辊粉碎机。对辊粉碎机，为布勒生产，型号为DFZL1500。

Neutrophils kill invasive fungi through ROS production, the formation of NETs and the release of fungicidal peptides. We found that their ability to clear fungi in corneas was weakened as more CFUs were observed after NOX2 inhibition and that the release of H3 was reduced both

and

compared to that measured without NOX2 inhibition. H3 normally resides in the nucleus. During the formation of NETs, H3 is released due to the rupture of the cellular and nuclear membranes. Therefore, the H3 concentration largely reflects the formation of NETs. The formation of NETs is now recognized as an important antifungal effector mechanism

,and NOX is necessary for NET formation in the innate immune response against

infection

.

By challenging NOX2-deficient mice with zymosan,lung inflammation was limited by the attenuation of proinflammatory NF-κB and the activation of Nrf2, a key redox-sensitive anti-inflammatory regulatory transcription factor

. NOX2 was necessary for Nrf2 activity, and Nrf2 was reduced while NF-κB activity was enhanced after NOX2 was blocked

. NADPH oxidase ROS impact multiple pathways triggered in response to fungal cell walls and other inflammatory stimuli, including attenuating activation of NF-κB

. Our data were consistent with these tests and showed that Nrf2 decreased while NF-κB increased with DPI treatment. In addition, following DPI treatment, the expression of the proinflammatory cytokines IL-6 and TNF-α increased,while the expression of the anti-inflammatory cytokines IL-10 and TGF-β decreased, which was evidence that the inhibition of NOX2 resulted in an excessive inflammatory response.However, there NOX2 inhibition had a completely opposite effect on NF-κB. The use of a high dose of another NOX2 inhibitor, apocynin, to reduce ROS production suppressed the NF-κB pathway and subsequent proinflammatory cytokine transcription, attenuated lung injury and improved survival in a rat hemorrhagic shock model

. And in a mouse experimental autoimmune encephalomyelitis (EAE) model, Nox2 deficiency attenuates EAE-induced neural damage and reduces disease severity, pathogenic immune cells infiltration

. The discrepancy between these results may be caused by the different disease models used and may be explained by the distinction between infectious and noninfectious inflammation.NOX2 also participates in adaptive immunity by regulating the differentiation of T cell subtypes. After NOX2 knockout,tryptophan catabolism was impaired, which led to Th17 upregulation, defective Treg activity and the dominant production of IL-17 in acute inflammatory lung injury

.We detected IL-17A levels and found that DPI treatment increased IL-17A expression in corneas and neutrophils. NOX2 may also limit inflammation by inhibiting the Th17 response. Next,we plan to test the differentiation of Th17/Treg cells by flow cytometry.

In summary, the data presented herein indicated that increased NOX2 levels in mouse corneas infected by

were inhibited by DPI treatment. The inhibition of NOX2 resulted in an excessive inflammatory response with increased neutrophil infiltration and decreased fungal clearance than that observed in control cells. Fewer ROS were produced, fewer NETs were formed. With DPI treatment, the expression of proinflammatory cytokines increased while the expression of anti-inflammatory cytokines decreased. These data suggest that NOX2 plays an important role in FK. Further studies are needed to investigate the role of NOX2 and its pathway in the pathogenesis of FK.

ACKNOWLEDGEMENTS

Authors’ contributions: Zhao GQ conceived of the research and participated in its design and coordination. Xu Q carried out the whole experiments and drafted the manuscript. Wang Q and Li C participated in the design of the research. Hu LT and Lin J performed the statistical analysis. Jiang N helped preparing

antigen. Peng XD helped taking mice experiment. All authors read and approved the final manuscript.

Conflicts of Interest: Xu Q, None; Wang Q, None; Hu LT,None; Lin J, None; Jiang N, None; Peng XD, None; Li C,None; Zhao GQ, None.

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