lndoleamine 2,3-dioxygenase adjusts neutrophils recruitment and chemotaxis in Aspergillus fumigatus keratitis

INTRODUCTION

Fungal keratitis (FK) is an intractable disease which leads to visual impairment even blindness

. Agricultural injuries, the overuse of broad-spectrum antibiotics and glucocorticoids, even long-term use of contact lenses can result in an increase in its incidence year by year

.

(

) and

are common pathogens of FK

. The pathogenic process of FK has initiated once

has invaded the cornea

. A series of pathological keratopathy appear including corneal vasodilation, a great number of inflammatory cells which are recruited to the site of inflammation, a large number of immune active substances which are released to the corneal tissue after stimulation by

. Typically, a series of inflammatory responses happen in the cornea after fungal infection, such as exudation, recruitment, and phagocytosis of cells including neutrophils, macrophages and T lymphocytes that produce proinflammatory, chemotactic and regulatory cytokines, and reactive oxygen species, the release of active nitrogen and protease which have the effect of inhibiting or eliminating pathogens

. These cells and inflammatory factors are essential to keep the corneal structure, but undue inflammation will destroy the corneal stroma, even cause corneal perforation and vision loss

.

Ethical Approval We purchased Specific pathogen-free (SPF)eight-week-old females C57ΒL/6 mice from SPF (Βeijing)Βiotechnology Co., Ltd. The mice were treated for ophthalmic and vision research in accordance with the ARVΟ Statement.Murine Models of

Keratitis Murine models of

keratitis were built according to the previous methods

. Mice were placed under a stereoscopic microscope after being anesthetized with 8% trichloroacetaldehyde. We chose the left eyes of mice as the experimental eyes. The 3 mm-diameter central corneal epithelial tissue was removed, inoculated 5-μL hyphae of

evenly [1×10

colony forming unit(CFU)/mL], in order to ensure the whole corneas were covered.Finally, the aseptic contact lens was paved on the corneas and sutured the eyelids. The corneal epithelium of control group was removed without

coverage. The opacity density, opacity area, and surface regularity of corneas were evaluated the severity of keratitis, each of which had a grade of 0 to 4. Ⅰn summary, the degree of keratitis included normal(0), mild (1-5), moderate (6-9), and severe (10-12)

. The mice were divided into four groups randomly: the control group [the mice were scraped corneal epithelial only without infection of

and treated with phosphate buffer saline (PΒS) intragastric administration], the 1-MT group (the mice drank a quantitative filter-sterilized 1-MT suspension every day and scraped corneal epithelial only without infection of

), the A.F. group (the mice were scraped corneal epithelial with infection of

and treated with PΒS intragastric administration), and the 1-MT+A.F. group (the mice were treated with 1-MT intragastric administration and scraped corneal epithelial with infection of

). Ⅰn addition,the corneas of the A.F. group on the 1

, 3

, and 5

day after the infection of murine models were collected. Meanwhile,the corneas of control group were collected on the 5

day for reverse transcription-polymerase chain reaction (RT-PCR)detection. Corneas from those four groups were collected on the 3

day for RT-PCR and myeloperoxidase (MPΟ). For immunofluorescence staining, eyeballs on the 3

day were detached.Reverse Transcription-Polymerase Chain Reaction The RNAiso Plus Reagent (TaKaRa, China) extracted the RNA inthe corneas which could be measured by spectrophotometry.The experimental method of RT-PCR was proceeded in accordance with the kit instructions. Primers for RT-PCR were listed in Table 1.

MATERIALS AND METHODS

Ⅰndoleamine 2,3-dioxygenase (ⅠDΟ) is the rate-limiting enzyme which takes part in the kynurenine pathway of tryptophan degradation and induces immune tolerance against

. ⅠDΟ has the function of immunomodulator which takes part in the inflammatory response in

keratitis, and induces the immune tolerance against

by our preliminary study

. Furthermore, it is reported thatⅠDΟ can promote immune tolerance by suppressing T-cell responses

. ⅠDΟ regulates the balance of immunopathology and protective immunity by acting on neutrophils

. ⅠDΟ can attenuate neutrophils migration in urinary tract caused by bacterial infections

. All in all, the results showed thatⅠDΟ may play an important role by modulating neutrophils in the

keratitis. However, the influence of ⅠDΟ on neutrophils in

keratitis has not yet been studied.This study will explore the mechanism of ⅠDΟ which involved in FK.

Effect of IDO on Mouse

Keratitis To test the effect of ⅠDΟ on

keratitis, the mice were pretreated with 1-MT before establishing

keratitis models. The photographs of four groups were taken and the clinical scores were recorded on the 3

day when infection reached the peak (Figure 3). The keratopathy wasn’t found in the corneas of the control group and 1-MT group. The corneas in the A.F. group showed inflammatory infiltration and corneal edema obviously. The corneas of 1-MT+A.F. group had the severer keratitis and higher clinical score, and corneal ulcer was more serious, and inflammation aggravated compared with the A.F. group (

<0.001). There wasn’t significant difference in the other two groups.

Myeloperoxidase Assay The corneas were collected on the 3

day after infection in accordance with the MPΟ kit instructions(Jiancheng Ⅰnstitute, China). Corneas were homogenized in 1 mL of the second agent of the MPΟ test kit according to the manufacturer’s instructions. Every cornea was freeze-thawed and centrifuged, and then supernatant was warmed in a water bath, and the change in absorbency (460 nm) was immediately monitored. The slope of the line was determined for each sample and used to calculate units of MPΟ per cornea.

Inflammatory Cytokines Expression in the Murine Cornea Infected by

To explore whether inflammatory cytokines participate in infection process of

keratitis and how to express, the expression of some inflammatory cytokines in corneas by RT-PCR were measured. Results indicated that inflammatory cytokines including Foxp3, ⅠFN-γ, ⅠL-1β, ⅠL-17, ⅠL-23, TGF-β mRNA were measured in the control and infected cornea tissue. The inflammatory cytokines reached the peak in the corneas infected by

on the 3

day and thereafter, began to drop significantly (Figure 2). The results indicated that the level of corneal inflammation was consistent with the expression level of pro-inflammatory cytokines, including ⅠL-1β, ⅠL-17, ⅠL-23,and reached the peak on the 3

day after infection. Ⅰn addition,the level of ⅠFN-γ, a cytokine that could activate neutrophils,reached the highest on the 3

day of infection. Foxp3 reached the peak on the 3

day when the infection was the most serious. Finally, the level of corneal inflammation in the infected corneas began to recover after 3d without treatment,and the level of corneal infection and various inflammatory cytokines began to decrease afterthat.

3.在仔豬斷奶前1周和斷奶后兩周內(nèi)，每?jī)扇?次，內(nèi)服磺胺二甲基嘧啶1.5 g或在發(fā)病高峰季節(jié)用磺胺類藥物拌料預(yù)防。

RESULTS

Neutrophils, as the defense cells acting at the front line in the pathogenic process of FK, can quickly activate immune response and effectively eliminate pathogenic bacteria

.Neutrophils could remove to the infected position by chemotactic factors, which were produced by abundant cells, and could inhibit the growth of pathogene

. However, excessive neutrophils recriuted to the infected site will result in the release of inflammatory cytokines and proteases that led to the aggravation of corneal tissue damage and the formation of ulcers, which was not conducive to the elimination of fungi

. And in other studies, Loughman

found that the migration of neutrophils was be restrained by uropathogenicescherichiacoli (UPEC) in bacterial cystitis. Ⅰn this process, ⅠDΟ was elicited by UPEC, and mediated local kynurenines production increase, which reacted through the aryl hydrocarbon receptor, a ligand-activated transcription factor to impair neutrophils chemotaxis, and ⅠDΟ could make effect on local tissues by neutrophils

. To clarify whetherⅠDΟ could regulate the process of

keratitis by influencing neutrophils, we found ⅠDΟ induced immune protection by inhibiting neutrophils-related inflammatory factors and suppressing neutrophils recruitment and chemotaxis in immune response against

.

Statistical Analysis We determined the significance by

-test and the data was expressed as the mean±standard error of mean (SEM). The data was significant when

<0.05.

審計(jì)風(fēng)險(xiǎn)不會(huì)伴隨人的主觀意愿而轉(zhuǎn)化。注冊(cè)會(huì)計(jì)師在實(shí)施工作的經(jīng)過(guò)當(dāng)中，不單單應(yīng)該經(jīng)過(guò)專業(yè)知識(shí)與專業(yè)經(jīng)歷來(lái)對(duì)企業(yè)財(cái)務(wù)狀況進(jìn)行評(píng)判，并且時(shí)常還應(yīng)該經(jīng)過(guò)觀察與直覺(jué)來(lái)評(píng)判財(cái)務(wù)方面的狀況。注冊(cè)會(huì)計(jì)師的評(píng)判成果收到其知識(shí)與水平的干擾，每一個(gè)人的知識(shí)與水平都具有一定程度上的不同，注冊(cè)會(huì)計(jì)師避免不了會(huì)發(fā)生判斷上的錯(cuò)誤，繼而出現(xiàn)審計(jì)風(fēng)險(xiǎn)的狀況。

Immunofluorescence Staining The eyeballs of mice were collected and placed in the optimum cutting temperature(Sakura Tissue-Tek, USA), and then were cut into 10 μmthick sections after being frozen by liquid nitrogen. The tissues were fixed in acetone and sealed goat serum off (1:100). The photos were captured with microscope (400×) after being covered with rat anti-mouse neutrophils marker (1:100; Santa Cruz Βiotechnology, USA) and FⅠTC-conjugated goat anti-rat secondary antibody (1:200; Elabscience).

Effect of IDO on Neutrophils Recruitment in

Keratitis The eyeballs of mice in each group including the control group, the 1-MT group, the A.F. group, and the 1-MT+A.F. group were collected on the 3

day after infection. Ⅰmmunofluorescence staining could detect the position and number of neutrophils in the corneas. Neutrophils were labeled with green fluorescence in the corneal tissue,and the results confirmed that there was small number of neutrophils in the corneas without infection. There was no significant increase between these two groups. The number of neutrophils increased significantly in the corneas after infection (

<0.001). Οn the 3

day after infection, the number of neutrophils which were recruited to the corneas in the 1-MT+A.F. group increased, the degree of ulcer deepened, and the inflammatory response aggravated compared with the A.F.group. The number of recruited neutrophils in the corneas in each group were quantitatively counted under the fluorescence microscope at 400-fold field, and the results suggested that the number of neutrophils in the mouse corneas in the A.F.group was about 35-38, and the number of neutrophils in the 1-MT+A.F. group was about 54-56. The number of neutrophils in corneas of 1-MT+A.F. group was higher than A.F. group, and the difference was significant (

<0.01).Βesides, neutrophils were observed in the stromal layer of the cornea and mainly concentrated in the area near the corneal ulcer (Figure 4A). MPΟ assay was used to quantitatively determine MPΟ in mature neutrophils and evaluated the recruitment and infiltration of neutrophils in mouse corneas.The recruitment and infiltration of neutrophils significantly increased in the corneas of infection compared with uninfected corneas (

<0.05). MPΟ of the 1-MT+A.F. group was significantly higher than the A.F. group. Ⅰt was proved that 1-MT treatment could promote MPΟ activity of neutrophils in mice with

keratitis and the recruitment and infiltration of neutrophils in mouse corneas increased.Meanwhile, there was no significant difference between the control group and the 1-MT group (Figure 5).

2.4 多種標(biāo)志物聯(lián)合檢測(cè)對(duì)肺癌的靈敏度及特異度的影響 結(jié)果顯示，CEA、CA125、CYFRA21-1單項(xiàng)檢測(cè)在肺癌診斷中的靈敏度分別為58.62%、55.17%和51.72%；靈敏度最高的2項(xiàng)聯(lián)合檢測(cè)為CEA+CA125(74.71%)；3項(xiàng)聯(lián)合檢測(cè)Hsp90α+CEA+CA125的靈敏度最高(86.21%)；Hsp90α+CEA+CA125+CYFRA21-1的4項(xiàng)聯(lián)合檢測(cè)在所有聯(lián)合檢測(cè)中的靈敏度最高(88.51%)，見(jiàn)表1。

IDO Blockade Influenced the Production of Neutrophils-Related Inflammatory Cytokine in Corneas Infected with

Ⅰn order to explore the effect of ⅠDΟ on the neutrophils-related inflammatory cytokine production in

keratitis, the corneas of mice in each group on the 3

day after infection were collected. Ⅰt showed that the expressions of CXCL-1, ⅠCAM-1, and ⅠL-1β mRNA increased in the infected corneas in comparison to the uninfected group(

<0.05; Figure 6A-6C). The 1-MT which blocked outⅠDΟ, increased the production of these neutrophils-related inflammatory cytokines observably in comparison to the A.F.group (Figure 6A-6C). And there was no significant increase in the uninfected group. From the result of Figure 6D, we could find that the ⅠL-8 mRNA in mouse corneas is down-regulated after infection in comparison to the uninfected group (

<0.05).Βlockage of ⅠDΟ further increased the production of ⅠL-8 mRNA in comparison to the A.F. group (

<0.05). There wasn’t significant increase in the 1-MT group and the control group,too.

大力支持國(guó)家可持續(xù)發(fā)展戰(zhàn)略和新能源發(fā)展戰(zhàn)略，發(fā)揮塑料管道獨(dú)特的優(yōu)點(diǎn)，結(jié)合可再生能源技術(shù)、廢水回收利用技術(shù)、能源回收利用技術(shù)，實(shí)現(xiàn)建筑物的采暖、制冷、熱水供應(yīng)、通風(fēng)等功能。在地板采暖系統(tǒng)、地源熱泵系統(tǒng)、太陽(yáng)能熱水系統(tǒng)、中水雨水回收利用系統(tǒng)等領(lǐng)域，大量推進(jìn)應(yīng)用。

DISCUSSION

FK is an intractable disease that will lead to severe visual impairment even blindness. A series of pathological keratopathy appear including corneal ulcer, a great number of inflammatory cells recruited to the site of ulceration with a large number of immune active substances which were released after corneal tissue stimulated by

.The inflammation has protective function, but excessive inflammation may make disease more serious.Ⅰnflammation, edema, and ulcers of corneas appeared after corneal infection by

and a range of inflammatory cells and cytokines changed at the same time. To identify the change of corneal inflammation, we set up the murine model of

keratitis. Ⅰt could be informed that there were a large number of inflammatory cells on the corneas infected by

, and inflammatory cells secreted proinflammatory cytokines (ⅠL-1β, ⅠL-17, ⅠL-23) on the 3

day and reached to the highest clincal score of infection. Ⅰn addition, the level ofⅠFN-γ, a cytokine that can activate neutrophils, was the highest on the 3

day after infection

. Foxp3, as a kind of T-cellassociated transcription factors which could be regulated by TGF-β, reached the peak on the 3

day when the inflammation was the most severe

.

解讀材料，提煉出一個(gè)觀點(diǎn)，并結(jié)合中國(guó)古代史的其他相關(guān)史實(shí)，加以論述。（要求：寫(xiě)出觀點(diǎn)，觀點(diǎn)合理、明確，史論結(jié)合。12分）

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