HU Wang，ZHANG Zhanhong，F(xiàn)ENG Haojie，CUI Yu，DU Qiupei，YU Wenhao，F(xiàn)AN Haining#

(1.Department of Hepatobiliary and Pancreatic Surgery，Qinghai University Affiliated Hospital，Xining，Qinghai Province，810001，China；2.Qinghai Provincial Key Laboratory of Hydatid Disease Research，Xining，Qinghai Province 810001 China)

Abstract Objective The purpose of this study was to investigate the expression of Peroxisome proliferation-activated receptorsβ&γ(PPARβ，γ)to regulate macrophage polarization in alveolar echinococcosis(AE).Methods The expression levels of PPARβ，γ in RAW264.7 macrophages were detected by qRT-PCR and Western Bolt in vitro co-culture.The expression levels of M1 and M2-related markers were detected by qRT-PCR to investigate the effects of PPARβ，γ on the polarization of macrophages.In addition，25 patients with multilocular echinococcsis were collected and the surface density of M1/M2 macrophages in normal liver tissues and liver lesion ranging was detected by immunofluorescence as well as the positive intensity of PPARβ，γ expression in M1/M2 macrophages to further verify the results of the in vitro experiment.Results M1 macrophage polarization markers increased first and then decreased，while M2 markers showed an increasing trend in the co-culture.Simultaneously，the expressions of PPARβ，γ were consistent with the variation of M1 and M2 markers.Meanwhile，the analysis of the clinical samples revealed that the expression of PPARβ，γ were in agreement with the trend of M1 and M2 polarization respectively.Conclusions It is showed that PPARβ，γ may regulate M1 and M2 polarization respectively in AE.

Keywords：PPARβ，γ；Macrophage；Polarization；Alveolar echinococcosis

Introduction

Alveolar echinococcosis(AE)is a zoonotic parasitic disease caused by the infection of echinococcus multilocularis larvae in the middle taenia stage[1].Transmitted by ingesting the parasite eggs，AE is excreted in the feces of the definitive host and then infected by oral ingestion of parasite eggs in ungulates and humans(intermediate hosts)[2].It is mainly prevalent in animal husbandry areas and is globally distributed，principally in Asia，Africa，South America，the Middle East，Central Europe，North America，Alaska，Hokkaido，Japan and western China[3，4].In 98% of the cases，the liver and lungs are the main infected organs presenting with tumor-like invasive and metastatic growth.Approximately 90% of the patients die within 10 to 15 years without treatment[5].It is estimated that there are more than 18，000 new AE cases worldwide each year with China accounting for 91%[6].Owing to the risk of the disease，AE has been listed by the World Health Organization(WHO)as one of the 20 neglected tropical diseases.AE is a food borne parasite that ranks third in the global impact among the 24 parasitic diseases[4].

Macrophages are highly plastic which can respond to the microenvironmental signals by changing their phenotypes to obtain corresponding functions.There are two major subpopulations of macrophages with different functions，including classically activated macrophage (M1)and alternatively activated macrophage (M2)[7].M1 and M2 phenotypes are the two extremes of macrophage polarization which jointly regulate the homeostasis of its internal environment.The two phenotypes are reversible and interconvertible.M1 has proinflammatory，antigen-presenting，host immune clearance of pathogens and tumor cells，while M2 mainly inhibits inflammation and promotes tumor growth，invasion and metastasis[8，9].

Peroxisome proliferation-activated receptors(PPARs)are a family of adopted orphan nuclear receptors involved in lipid metabolism and inflammation，including PPARα，PPARβ/δ，and PPARγ.Each of species has distinct ligands，target genes and biological functions[10-12].PPARs are involved in the metabolism and homeostasis of fat and carbonate in the organism as well as cell proliferation and differentiation，vascular biology，inflammation and carcinoma[13].PPARs are mainly expressed in the kidney，liver，small intestine，heart and other tissues to activate fatty acid catabolism[14].PPARs play an important role in regulating immune response which affect mononuclear cells，macrophages，neutrophils，peripheral blood lymphocytes，dendritic cells，T cells，NK cells and eosinophils[15，16].Besides，it is found in these studies that PPAR β，γ are also involved in the process of macrophage polarization in metabolic and infectious diseases[7].Since the effect of PPAR β，γ on macrophage polarization in AE has not been reported yet，the purpose of this study was to analyze the effect of PPAR β，γ on macrophage polarization in AE.

1.Material and Methods

1.1 Collection of echinococcus multilocularis protoscolex(PSC)

PSC was extracted from the infected gerbils in the laboratory，while PSC in gerbils was inoculated from the naturally infected wild voles at Guoluo of Qinghai province in China.The gerbils were sacrificed by cervical dislocation.Then， they were placed in the cell plates on the benchtop to open the abdominal cavity and the lesions were separates and removed.Next，the lesions were cut into a paste with scissors，and the funnel was placed at the mouth of a 50 mL centrifuge tube for filtration with an 80-molybdenum cell screen.PSC was allowed to sediment for 15 mins in the 50 mL centrifuge tube. After PSC settled at the bottom，the supernatant was sucked out with a straw and normal saline was added to the centrifuge tube.The above was repeated 3-5 times.Finally，a little PSC was stained with Trypan blue to observe the activity and count of the nodes.PSC was incubated in RPMI 1640(Gibco)culture medium containing 100 U/mL penicillin，100 μg/mL streptomycin，20% heat-inactivated fetal bovine serum(FBS)，0.45% yeast extract and 0.4% glucose in a 5% CO2atmosphere at 37 ℃ for 10 h.Then，PSC was used in the subsequent experiment.

1.2 Co-culture experiment of RAW264.7 macrophages with PSC

RAW264.7 macrophages(Procell Life Science & Technology Co，.Ltd)were cultured in Dulbecco′s modified eagle media(DMEM)solution containing 10% FBS，100 U/mL penicillin and 100 μg/mL streptomycin in a 5% CO2atmosphere at 37 ℃.All the experimental interventions were carried out on the third passage of cells.The cell suspension was dropped onto the cell count plate and counted under the light microscope.The cells were placed in the plates of DMEM solution containing 10% FBS.The non-adherent cells were removed after incubation for 4 h，and the adherent RAW264.7 macrophages were added into 1 mL DMEM containing 1 000 viable PSC for co-culture.The cells were collected for total RNA and protein extraction on days 1，3，5 and 7 after co-culture.

1.3 Total RNA isolation and real-time polymerase chain reaction(PCR)

Total RNA was extracted using RNA Simple Total RNA Kit(TIAN GEN?)，and the purity of RNA was determined by calculating the absorbance ratio at 260 nm and 280 nm.It was showed that the ratio of 1.8～2.0 was suitable for cDNA synthesis.cDNA was synthesized by reverse transcription of 1 μg of total RNA using the PrimeScript RT Reagent kit(TaKaRa).For real-time polymerase chain reaction(PCR)，80 ng cDNA was added in a 15 μL reaction system containing each primer and TB Green PreMix(TaKaRa).The reaction conditions were 95 ℃ for 30 s，followed by 40 cycles of 95 ℃ for 5 s，60 ℃ for 30 s and the melting curve analysis at 63 ℃～95 ℃.18S rRNA was used as a housekeeping gene.

Relative mRNA amounts were quantified by the 2-ΔΔCtmethod.The mice gene primers(provided by Sangon Biotech)were as follows (Table 1).

1.4 Western blot analysis

The levels of PPARβ、γ and ACTIN protein expression were measured by the Western blot analysis.The whole protein extracted from RAW264.7 macrophage was separated by 10% SDS polyacrylamide gel electrophoresis(SDS-PAGE).The protein samples were transferred to polyvinylidene difluoride membranes and incubated with PPARβ(1：1000，Abcam)，PPAR γ(1：800，Cell Signaling Technology)，and ACTIN(1：8000，protein tech?)antibody overnight at 4 ℃.Next，the membranes were washed thrice using a mixture of tris-buffered saline and polysorbate 20 and incubated with horseradish peroxidase-conjugated secondary antibody(1：10000，Abcam)at room temperature for 1 h before signal detection by the chemiluminescent substrate.

1.5 Liver histology and immunofluorescence analysis

For confirming the normal liver tissue and liver lesions ranging，the liver tissue samples were fixed to 10% formalin，paraffin-embedded，hematoxylin and eosin.Immunofluorescence was performed to detect the area density of M1/M2 polarization and the positive intensity of PPARβ，γ in macrophages about the normal liver and liver lesions ranging which were obtained from clinically resected specimens of patients(Table2)with hepatic alveolar echinococcosis(HAE).The nucleus turned blue by labeling with DAPI.In immunofluorescence mono-staining，M1 marker CD86(Servicebio)and M2 marker CD206(Servicebio)turned red by labeling with CY3(Servicebio).

Table 2 General clinical data of the patients(n=25)

In immunofluorescence double staining，M1/M2 markers were all green-labeled with FITC(Servicebio)，and PPARβ，γ were red-labeled with CY3.Expression quantification of markers was performed with the Image-Pro plus 6.0 open-sources developed at Media Cybemetics in USA.

1.6 Statistical analysis

Data were represented as mean ± standard error of the mean(SEM)using Student′s t-test.For experiments of more than three groups，statistical analyses were performed with analysis of variance followed by the post hoc Tukey pairwise comparisons.It is considered to be dramatically changed when theP-value<5%.The specified p-values were showed in the figures as follow：*P<0.05；**P<0.01；***P<0.005；****P<0.001.

2.Results

2.1 RAW264.7 macrophage mainly polarized toward M1 in the early stage and M2 in the late stage after in vitro PSC stimulation

To further understanding the polarization of RAW264.7 macrophages during co-culture with PSC，the polarization trend of macrophages was observed by detecting M1/M1 polarization markers through real-time PCR.In comparison with the control group，the mRNA levels of tumor necrosis factor-α(TNF-α)and interferon-γ receptor1(INF-γ R1)peaked on the third day after treatment，showing a trend of rising first and then decreasing(Figure 1A～B).As shown in Figure 1C，the mRNA level of granular macrophage-colony stimulating factor(GM-CSF)increased significantly on the third day until it reached a peak on day 5 and then began to decline.Figure 1D demonstrates that the changes in M1 markers(TNF-α，INF-γ R1，and GM-CSF)increased first and then decreased after RAW264.7 co-cultured with PSC.Compared with the controls，the increased mRNA levels of Arg-1，transforming growth factor-β(TGF-β)and GM-CSF were increasing significantly on day 3 after treatment and then gradually reached peaking on day 7(Figure 2A～C).It was showed in Figure 2D that the change of M2 markers illustrated an overall upward trend after RAW264.7 co-cultured with PSC.The above results suggested that macrophage co-culture with PSC is mainly polarized toward M1 in the early-stage and M2 in the late stage.

A～C：The mRNA expression of M1 markers genes(TNF-α，IFN- γ R1，and GM-CSF)in RAW264.7 macrophage are measured by qRT-PCR.D：The overall variation of mRNA expression of TNF-α，IFN-γ R1 and GM-CSF genes at different time points.*P<0.05；**P<0.01；***P<0.005；****P<0.001 vs.control.

A～C：The mRNA expression of M2 markers genes(Arg-1，TGF-β，and M-CSF)in RAW264.7 macrophage are measured by qRT-PCR.D：The overall variation trend of mRNA expression of Arg-1，TGF-β，and M-CSF genes at different time points.*P<0.05；**P<0.01；***P<0.005；****P<0.001 vs.control.

2.2 PPARβ，γ play an important role in the polarization of macrophages co-cultured with PSC

For a better understanding of the effect of PPARβ，γ on macrophage polarization in AE，the expression of PPARβ，γ in macrophages after co-culture with PSC was detected by real-time PCR and Western blot analysis.Figure 3A reveals that the expression of PPARβ mRNA reached its peak on day 3 after RAW264.7 co-cultured with PSC and then began to decline.However，F(xiàn)igure 3B showed that PPARγ mRNA expression of macrophages was significantly lower than the control at one day of co-culture with PSC and significantly increased after five days of co-culture.The overall change in the trend of PPARβ，γ in RAW264.7 co-cultured with PSC was that PPARβ first increased and then decreased，while PPARγ continued to increase(Figure 3C).Moreover，the protein expression level of PPARβ in the macrophages co-cultured with PSC also showed a trend of increase，followed by the decrease.The level of the protein was the highest on day 3.The expression level of PPARγ showed an overall upward trend with the highest expression on day 7(Figure 3D).Based on the results mentioned above，it was found that the expression of PPARβ in RAW264.7 co-cultured with PSC was consistent with the changing of M1 polarization marker，and that of PPARγ was consistent with the changing of M2 polarization marker.Therefore，it was considered that PPARβ mainly regulated the polarization of M1 macrophages in the early stage of infection in AE disease，while PPARγ mainly regulated the polarization of M2 macrophages in the late stage.

A～B：mRNA expression of PPARβ，γ genes in RAW264.7 macrophages is measured by qRT-PCR.C：Overall variation of mRNA expression of PPARβ，γ genes at different time points.D：Level of PPARβ，γ protein expression in RAW264.7 macrophages measured by Western blot analysis.ACTIN is used as a loading control.*P<0.05；**P<0.01；***P<0.005；****P<0.001 vs.control.

2.3 The macrophages in liver lesions ranging were mainly M2 polarization in patients with HAE

Macrophage polarization was verified with clinical samples to determine its tendency in HAE.The progression of HAE is slow with no obvious clinical symptoms.The herdsmen in western China are primarily in the middle and late stages after being diagnosed with the disease.Therefore，all the clinical surgical samples were collected from patients with advanced stage.

First，hematoxylin and eosin(HE)staining were performed on all samples before immunofluorescence detection for confirming the normal liver tissue and liver lesions ranging.The liver lesions ranging was defined as the peripheral inflammatory infiltration zone that away from the central necrotic tissue 0.5～1 cm(Figure 4A).As shown in Figure 4B，there are prominent hepatic lobules in the normal liver tissues with extensive infiltration of granulocytes and lymphocytes in liver lesions ranging. Immunofluorescence staining was performed on the markers of M1/M2 in the normal liver and liver lesions ranging(Figure 5A～B).The results showed that M2(CD206)polarization macrophages were significantly increased in the liver lesions ranging，while M1(CD86)polarization macrophages were significantly decreased compared with the normal liver(Figure 5C～D).These data demonstrate that macrophage polarization in the middle and late stages of HAE was mainly M2 polarization in the liver lesions ranging，which also verified the results of the in vitro experiment of macrophage polarization toward M2 in the late stage.

Hematoxylin and eosin(HE)staining was performed on all clinical samples before immunofluorescence detection to confirm the normal liver tissue and liver lesion ranging

A～B：Immunofluorescence staining of M1(CD86)and M2(CD206)markers in the normal liver and liver lesion ranging.C～D：Areal density values of M1(CD86)/M2(CD206)markers in the normal liver and liver lesion ranging are analyzed.Data represented mean ±standard error of the mean(SEM)，n=25 clinical samples，*P<0.05 vs.control

2.4 PPARβ，γ are involved in the regulation of macrophage polarization in HAE

In the in vitro experiments，PPARβ，γ were considered to regulate the polarization of macrophages M1 and M2 respectively.For further verification of this result，the expression of PPARβ，γ in macrophages was detected by immunofluorescence double staining.As shown in Figure 6A～B，the M1，M2 macrophages and PPARβ in the normal liver and liver lesions ranging were simultaneously stained by immunofluorescence.By analyzing the average intensity of PPARβ expression in M1 and M2 macrophages，it was found that the PPARβ expression in M1 macrophages was significantly decreased compared with that in normal liver，and the expression of PPARβ in M2 macrophages was not significantly different from that in normal liver(Figure 6C～D).Meanwhile，immunofluorescence staining and analysis of PPARγ in M1 and M2 macrophages showed that the expression of PPARγ in M2 was significantly increased compared with the control.However，the expression of PPARγ in M1 was not significantly different compared with that in the normal liver(Figure 7A～D).It is showed in the experimental detection of clinical samples that the expression trends of PPARβ，γ in M1 and M2 macrophages were consistent with those in vitro experiments.Therefore，it can be concluded that PPARβ mainly regulates M1 polarization，while PPARγ mainly regulates M2 polarization in HAE.

A～B：Immunofluorescence staining of PPARβ in M1(CD86)and M2(CD206)macrophages in the normal liver and liver lesion ranging.C～D：Average intensity analysis of PPARβ expression in M1(CD86)and M2(CD206)macrophages.Data represented mean ±standard error of the mean(SEM)，n=25 clinical samples，*P<0.05 vs.control

A～B：Immunofluorescence staining of PPARγ in M1(CD86)and M2(CD206)macrophages in the normal liver and liver lesion ranging.C～D：Average intensity analysis of PPARγ expression in M1(CD86)and M2(CD206)macrophages.Data represented mean ±standard error of the mean(SEM)，n= 25 clinical samples，*P<0.05 vs.control

3.Discussions

It is observed in this study that the macrophage polarization could be induced during the co-culture of PSC and macrophages，and the increased expression of PPARβ，γ in macrophages was related to the polarization of M1 and M1 macrophages respectively.The data also confirmed that the infection of larval echinococcus multilocularis leads to the polarization of macrophages.This co-culture model has its limitations as it is not a primary infection owing to the ingestion of oncospheres，nor is it a non-natural host.Most importantly，this study suggests that the regulation of macrophage polarization may be a potential target for AE therapy.The findings provide new clues to the basic pathogenesis and progression of AE so as to greatly advance our understanding of AE.Thus，it is significantly advancing our understanding of AE.

AE is an alarming clinical zoonotic parasitic disease with the chronic progressive liver damage caused by the continuous proliferation of multilocular echinococcosis in the larval stage as its main characteristic[17].Additionally，immune tolerance and/or immune downregulation is a remarkable feature that is increasingly observed as AE disease progresses to the chronic stage of infection[18].Studies have revealed that the pathological changes of AE are related to the infiltration of macrophages at specific stages of various functional types[19].

Macrophages are a critical component of the innate immune system and a key regulator of normal homeostasis and pathology[20，21].They not only mediate the innate and specific immune responses of the body but also participate in the process of immune tolerance[22].Macrophages have different functions owing to their different subtypes.M1 macrophages mainly have the function of proinflammatory，antigen presentation，host immune clearance of pathogens and tumor cells，while M2 macrophages have the function of anti-inflammatory，promoting wound healing，fibrosis，tissue repair，promoting tumor growth and infiltration.Through the co-culture of macrophages with PSC，it is observed that the macrophages were primarily M1 polarization before day 3，followed by M2 polarization.Therefore，it is speculated that the M1 polarization macrophages in the early stage of larval multilocular echinococcus infection were mainly responsible for the clearance of pathogens in the body.Other studies have indicated that the macrophages from mice infected with larval multilocular echinococcosis exhibited a lower ability to present an antigen to specific T lymphocytes compared with that in mice without infection[23].It was found in this study that PSC could induce the polarization of macrophages.However，macrophages are mainly polarized toward M2 with the progression of the disease，which is more conducive to the growth and infiltration of the pathogen，leading to the aggravation of the disease.Thus，macrophage polarization，mainly toward M2，may be an important mechanism for immune tolerance to pathogens in the late stage of HAE.

Currently，it is critical to explore the molecular mechanism of macrophage polarization in AE disease.Several other studies revealed that PPARs play an important role in the regulation of macrophage polarization in the signal pathway.The expression of PPARβ/δ in macrophages can be increased by activation of STAT6，thereby inhibiting the activation of JNK to regulate the transformation between M1 and M2[24].Additionally，STAT6/PPARγ pathway regulates the generation of many M2-type markers[25].Also，some cytokines(such as TNF-α，IL-12，IL-23，IL-27，and IFN-γ)activate PPARs，which will inhibit the production of proinflammatory cytokines.They are and are essential for the formation，activation，and maintenance of M2 macrophage[26].It is hypothesized that PPARβ，γ might mediate the regulation of M1 and M2 macrophages polarization in AE respectively.It is observed that the expression trend of PPARβ，γ in M1 and M2 was consistent with the expression trend of M1/M2 polarization markers in both the in vitro and clinical sample experiments.

The survival mechanism of echinococcus multilocularis larvae is to protect itself from the body′s immune response.The pathology of AE is characterized by intense infiltration of the immune cells[27].After natural infection in the intermediate host，the immune system should encounter different stages of parasite development[17].When the parasite is not effectively suppressed by the surrounding protective immune response，it eventually becomes a fully mature metacestode that will continue to grow until the host dies[28].This study suggests that PPARβ，γ are involved in the regulation of macrophage polarization in HAE.Therefore，it is considered that the direction of M1/M2 polarization could be regulated by regulating the signaling pathways of PPARβ and PPARγ in macrophage polarization. In the complete process of disease infection，more induction of macrophage polarization toward M1 and reduction of M2 polarization may slow the progression of the disease.

In conclusion，we have demonstrated the role of PPARβ，γ on macrophage polarization in AE.The results of this study are important implications for further understanding of the AE pathogenesis，providing a potential target for AE treatment.

Disclosureofconflictofinterest

The authors declare no competing conflicts of interest.