The infiltration of monocytes aggravates liver fibrosis in mice infected with Echinococcus granulosus

DOI: https://doi.org/10.21203/rs.3.rs-2071128/v1

Abstract

Background

Echinococcus granulosus (E. granulosus) infection involves multicellular inflammatory responses and fibrous repair. The study aimed to observe the effect of monocytes’ infiltration on hepatic fibrosis in mice infected with E. granulosus.

Methods

The pathological changes and fibrosis changes in the liver of mice infected with E. granulosus were observed at different time points (DAY2, 8, 30, 90, 180, 300). Chemokines, fibrosis related cytokines were detected. LAMP-1 as a marker of phagosome maturation and PKC-α regulating diverse cellular responses including immune responses.

Results

Change of chemokines indicated monocytes infiltrated into the liver of E. granulosus-infected mice. The increased α-SMA and Desmin indicated the continuous aggravation of fibrosis. The expression of LAMP-1 increased in the early stage, then decreased gradually in the middle and late stages; PKC-α was significantly higher than before after 300 days infection.

Conclusions

Our study facilitated clarification of molecular mechanisms of E. granulosus infection and contributed to the development of novel therapies.

Highlights

  1. E. granulosus infection involves multicellular inflammatory responses and fibrous repair.
  2. It indicates an immune evasion strategy of E. granulosus to ensure its survival.
  3. It might contribute to the development of novel therapies in E. granulosus infection.

Background

Echinococcosis, also known as hydatid disease, is a zoonosis caused by the parasitic larvae (protoscolex) in the liver, lung and other organs. It is mainly divided into cystic echinococcosis (CE) caused by Echinococcus granulosus (E. granulosus) and alveolar echiococcosis (AE) caused by Echinococcus multilocularis (E. multilocularis) [1]. With the highly adaptability of E. granulosus to a variety of intermediate hosts, CE is a major public health problem worldwide.

Acute or chronic liver injury caused by E. granulosus infection involves multicellular inflammatory responses and fibrous repair. During that, a large number of HSCs is activated and transforms into myofibroblasts (MFBs), leading to imbalance in the proliferation and degradation of collagen and other extracellular matrix (ECM), as a result, abnormal deposition of fibrous connective tissue performs in liver. In the early stage of liver fibrosis, if the inflammatory damage is controllable, fibrosis could be reversible[2, 3]. Labsi et al observed the fibrous tissue continuously increased in the course of CE [4] and agreed with the pericyst might lead to immune escape by hindering the host’s immune response against the parasite [5]. Monocytes are distributed around MFBs in the process of liver fibrosis [6], a variety of chemokine receptors, CX3CR1, CXCR4 and CCR2, are all expressed on the surface of mature monocytes. Most of them are transmembrane proteins and participate in the infiltration of monocytes. Compared with WT mice, CX3CR1 deficient mice had more severe inflammatory reaction, over activation of HSCs and aggravated liver fibrosis [7]. In the study of HCV-induced cirrhosis, the expression of CXCR4 was also significantly increased along with the activation of HSCs [8], suggesting that it might be involved in the process of liver fibrosis. CCR2 mainly expresses on the surface of monocytes and HSCs. In the inflammatory reaction of persistent liver injury, CCR2 mainly induced monocytes in blood circulation to the injured organs [9], and promoted the development of liver fibrosis [10]. Pharmacological inhibition of CCR2+ monocyte recruitment efficiently ameliorated insulin resistance, hepatic inflammation, and fibrosis in NASH [11]. Matrix metalloproteinase-8 (MMP-8) belongs to matrix metalloproteinase family (MMPs), is a kind of protease that mainly degrades ECM. It plays a very important role in maintaining ECM homeostasis and expresses in monocytes / macrophages. Therefore, MMP8 could be a used marker of monocyte infiltration and differentiation in the regulation of liver fibrosis.

In this study, we investigated the effect of monocytes’ infiltration by dynamically expression of chemokines and fibrosis cytokines in the liver of E. granulosus-infected mice, then contributed to the development of novel therapies for prevention and treatment of liver fibrosis in E. granulosus-infected mice.

Materials And Methods

Animals and infected-model establishment

BALB/c mice (female, 8-week-old) purchased from the Experimental Animal Science Research Department were maintained in specific pathogen-free (SPF) conditions. All animals met the International Guiding Principles for Biomedical Research Involving Animals as issued by the Council for the International Organizations of Medical Sciences. This study was performed according to the Declaration of Helsinki and approved by the ethic committee of Xinjiang Medical university (NO.20160218-14).

Histopathology

Mouse liver tissue was formalin-fixed and paraffin-embedded. Three µm thick sections were stained with Hematoxylin-Eosin (H&E), Masson staining and Sirius red according to standard protocols. HE staining was carried out by means of preparations deparaffinized with xylol, rehydration by in decreasing graded alcohol (100%, 96%, 80%, and 70%). The preparations were then incubated in hematoxylin solution, then washed with running water for a short time, and continued with eosin incubation [12]. Masson staining was carried out by deparaffinizing preparations with xylol and rehydration by soaking in decreasing graded alcohol (100%, 96%, 80%, and 70%). The preparation was then put into Bouin’s fixative solution and soaked in Weigert hematoxylin solution and Biebrich scarlet acid fuchsin. The preparation is then immersed in phosphomolybdic acid and incubated in aniline blue.12 Sirius Red staining the samples were immersed by soaking in decreasing graded alcohol (100%, 96%, 80%, and 70%), then stained with Sirius Red solution for 8 min and quickly dehydrated with 100% alcohol. Finally, the samples were dehydrated with xylene for 5 min and sealed with neutral gum [13].

Quantitative Real-time PCR

Total RNA from liver tissue was extracted by TRIzol™ (Invitrogen, Carlsbad, USA) following the manufacturer’s instructions. Average of 500 ng total RNA was reverse-transcribed by Primer Script RT kit (Takara Bio, Dalian, China) following the manufacturer’s instructions. Along with SYBR Green Realtime PCR Master Mix and Permix Ex Taq (Takara Bio), following the manufacturer’s instructions, Quantitative real-time reverse transcriptase-polymerase chain reaction (quantitative real-time PCR) was performed on the ABI Prism 7500 Sequence Detection System (BioRad, Life Science Research, Hercules, CA, USA). Each reaction mix was processed: one cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, at the annealing temperature of 60°C for 30 min. Gene expression was normalized to GAPDH and quantified by the 2-△△Ct method. All reactions were performed in duplicates for each sample. Primer sequences were synthesized by Sangon Biotech (Shanghai, China) and details showed as follows,

GAPDH: F-AACTTTGGCATTGTGGAAGG, R-CACATTGGGGGTAGGAACAC;

CX3CR1: F-AGCTCACGACTGCCTTCTTC, R-GTCCGGTTGTTCATGGAGTT;

CCR2: F-CTCAGTTCATCCACGGCATA, R-TGACAAGGCTCACCATCATC;

CXCR4: F-GCTGGCTGAAAAGGCAGTCT, R-TGTCATCCCCCTGACTGATG;

α-SMA: F-ATCCGATAGAACACGGCATC, R-GTGCCTCTGTCAGCAGTGTC.

Immunofluorescence

After deparaffinization and antigen unmasking, liver sections were permeabilized with 0.2% Triton X-100 in PBS for 10min at room temperature and blocked in PBS supplemented with 1% BSA and 0.1% Triton X-100 for 1h at room temperature. For double immunofluorescence, slices were incubated overnight at 4°C with anti-CX3CR1 antibody (1:1000, ab8021, Abcam). Antibody was prepared in PBS supplemented with 1% BSA and 0.3% Triton X-100. Goat Anti-Rabbit IgG H&L Alexa Fluor 488 (1:200, ab150077, Abcam) was used as secondary antibody. Nuclei were counterstained with Hoechst for 20 min at room temperature.

Immunohistochemistry

The liver tissue slides (3 µm) were deparaffinized and hydrated, then the soaked slides were inactivated in 5% H2O2 / methanol to block endogenous peroxidase activity. Next, the slides were incubated with goat serum for 10 min and incubated with MMP8 antibody (1:250, ab53017, Abcam), Desmin antibody (1:200, AF5334, Affinity) and α-SMA antibody (1:400, AF1032, Affinity) overnight at 4℃. The slides were washed the next day and incubated with biotinylated secondary antibody at 37℃ for 60 min. Then, the slides were washed again and incubated with horseradish peroxidase-labeled streptavidin at 37℃. The samples were developed with diaminobenzidine (DAB) and stained with haematoxylin. After the slides were washed with distilled water and dehydrated, they were made transparent and mounted under a microscope for examination. Image J software was employed to evaluate the mean optical density value of the images after immunohistochemical analysis.

Western blot

Snap-frozen liver samples were homogenized in RIPA buffer (150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl, pH = 8) supplemented with Protease Inhibitor Cocktail (Solarbio, China). Cells were homogenized in lysis buffer containing 1% Triton, 150mM NaCl, 20mM Tris-HCl, pH = 7.5. Protein concentration was determined using a bicinchoninic acid assay kit (Termo Scientifc Pierce BCA Protein Assay Kit, Termo Scientifc, Rockford, USA). Equal amounts of protein lysate or equal volumes of conditioned media were mixed with Laemmli loading buffer and heated at 100°C for 10 min. Proteins were subjected to SDS-PAGE on 10% acrylamide gel and electro-transferred to a nitrocellulose membrane (Sartorius AG, Goettingen, Germany), incubated with primary antibody overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 90 min at room temperature. Detection was performed using a chemiluminescence substrate with Alliance system (UVItec, Cambridge, UK). Primary antibodies were used: anti-mouse LAMP-1 (1:100, DF7033, Affinity), anti-mouse PKC-α (1:2000, AF6196, Affinity).

Statistical analysis

Data was statistically performed by SPSS19.0 software, and the results were expressed as mean ± standard deviation (͞x ± s). The statistical difference between different time points in E. granulosus-infected mice was analyzed using the two-tailed paired or unpaired t-test and one-way analysis of variance, with P < 0.05 as the significant difference.

Results

Pathological observations in liver tissue of E. granulosus-infected mice at different stages

We showed the mice general condition in different E. granulosus-infected stages (Fig. 1). In HE staining, the liver tissue structure of mice in different periods of the control group was normal. In E. granulosus-infected model group, 2 days after infection, the hepatic lobular structure of the puncture site was destroyed, a large number of red blood cells and inflammatory cells aggregated and infiltrated, the surrounding hepatocytes were edema. 8 days after infection, red blood cells around the puncture site decreased, inflammatory cells increased, hepatocellular edema worsened, and some cells have appeared vacuoles and balloon-like degeneration. 30 days after infection, inflammatory cell-encapsulated hydatid infections appeared in the liver, and fibrous connective tissue layer formed between the lesion and inflammatory cells. 90 days after infection, the hepatic lobule structure around the foci was unclear, with fibrous connective tissue proliferation and infiltration. 180 days after infection, cyst fluid increased in some vesicles, and the fibrous layer was thickened. 300 days after infection, the liver tissue structure was disordered, inflammatory cell infiltration was obvious, and pseudo lobular formation around the lesion. Whereas both Masson staining and Sirius red staining showed that a wrapping of collagen fibers around the lesion at 30 days after infection. At 90 days after infection, the collagen layer around the lesion gradually thickened and expanded, affecting the surrounding liver tissue. At 180 or 300 days after infection, the collagen fiber layer around the focus further thickened and the collagen fibers penetrated deeper into the surrounding liver tissue. (Fig. 2) 

Chemokine expression and HSC activation in liver tissue of E. granulosus-infected mice at different stages

The expression of CX3CR1 and CCR2 is closely related to the function of monocyte-macrophages. The expression of CX3CR1 and CCR2 mRNA in the liver of the model group was significantly variant at different stages, and the trends were similar (Fig. 3a,b), which indicated that there were a large number of monocyte-macrophages infiltrating into the liver. However the expression of CXCR4 mRNA increased significantly at the 30 days after infection and reached its peak (Fig. 3c) and significantly higher than that in the control group (P＜0.05), it is mainly expressed in monocytes and neutrophils, which is closely related to inflammatory response, liver fibrosis, tumor progression and metastasis [14,15]. The expression of α-SMA mRNA in the model group at the 8 days after infection was higher than the control group was slightly higher with no significantly different (P2d=0.495 and P8d=0.591 compared with control group) (Fig. 3d).

Changes in expression of monocytes-related molecules in liver tissues of E. granulosus-infected mice at different stages

At different stages of E. granulosus infection model, CX3CR1 and MMP8 were positively expressed in liver tissues. The expression of CX3CR1 presented as significantly higher in the early stages, with a tendency of decreasing gradually (P＜0.05, compared with control group) (Fig. 4a,b), the expression of MMP8 was increased in the middle and late stages (Fig. 4c,d). 

The expression and distribution of fibrosis related cytokines in liver tissues of E. granulosus-infected mice at different stages

At different time periods, Desmin had a positive expression in the liver tissue of E. granulosus-infected model group at the 2 days and 300 days after infection, and expressed greatly higher at the 180 days after infection (P180d＜0.05, compared with control group), details showed in Fig. 5a,b, while α-SMA was collected positive signal at all stages in the liver of E. granulosus-infected model (Fig. 5c,d).

Protein LAMP-1 and PKC-α expression change might indicated an immune evasion strategy of E. granulosus to ensure its survival

The lysosomal protein (LAMP-1) as a marker of phagosome maturation and protein kinase C-α (PKC-α) regulating diverse cellular responses including immune responses were detected. LAMP-1 protein expression in liver tissues of E. granulosus-infected model group showed a high expression at the early stage and then gradually decreased with a tendency of inhibition in the middle and late stages (Fig. 6a). PKC-α protein expression showed no significant difference in the early stage of infection, but tended to increase in the middle and late stages, and expressed greatly higher at 300 days after infection (P300d＜0.05, compared with control group), details showed in Fig. 6b.

Discussion

Liver fibrosis is the main protective pathological process of liver tissue in continuous stimulation E. granulosus infection. The early stage of liver fibrosis can be reversed, the damaged liver tissue can be repaired by collagen tissue and the growth of worm can be limited. As the lesion is continuously expand, the degree of fibrosis in the middle and late stages becomes more serious, even irreversible, which can further develop into cirrhosis and liver cancer.

Monocyte-macrophage system is a subgroup of leukocyte system, which originates from bone marrow hematopoietic cells and exists in blood circulation. After the pathogen infected the body, Kupffer Cells (KCs) in the liver firstly recognized it, then recruited many circulating monocyte-macrophages to play a role at the infection site [16]. In the early stage of parasite infection, the liver microenvironment was dominated by Th1 type immune response. With the deposition of parasite eggs, the immune response gradually changed to Th2 type in the liver [17]. Human and mouse studies indicated that KCs and MoMFs both had tissue-destructive and pro-restorative/tissue repair functions in APAP injury [18, 19].

The expression of CX3CR1 and CCR2 is closely related to the function of monocyte-macrophages. In our study, in the early stage of E. granulosus infection, the non-parenchymal cells in the liver expressed CX3CR1 and CCR2, which indicated that there were a large number of monocyte-macrophages infiltrating into the liver, and many CX3CR1 positive cells mainly located in the hepatic sinusoids; in the middle and late stages, the positive expression of CX3CR1 in the hepatic lobules gradually weakened, and the positive expression mainly existed around the lesions, which proved that after receiving the chemokine signal from KCs cells, the peripheral blood derived monocytes were collected from the blood to the liver, and then migrated to the lesions from the liver sinusoid and other hepatocyte spaces, indicating the infiltration of monocytes [20]. As for CXCR4, it is mainly expressed in monocytes and neutrophils, which is closely related to inflammatory response, liver fibrosis, tumor progression and metastasis [14, 15]. In this study, the expression of CXCR4 first showed a significant difference at the 30 days after infection, while pathology showed that at the 30 days after infection, lesions caused by E. granulosus infection gradually occurred in the liver. Whether there is a correlation between the two remains to be further studied.

α-SMA and Desmin, as indicators of HSCs activation [21], had a significant difference at the 180 days after infection, and a large number of HSCs were activated. Masson and Sirus red staining both showed the collagen fiber layer around the focus thickened and the collagen fibers penetrated deeply into the surrounding liver tissue with the aggravation of E. granulosus infection, however, the increased geen-expression only happened at the 8 days after infection, this might be due to the small number of infection models in each group, which led to statistical difference. IHC results showed that the expression level of HSCs in different stages of infection was higher than that in the control group, and mainly located around the lesion, it indicated that cytokines secreted by monocytes might activated HSCs with α-SMA expression was increased.

MMP-8 belongs to MMPS family, is a kind of protease that mainly degrades ECM components and mainly expressed on monocyte-macrophages [22]. In this study, we found that the expression of MMP8 in the model group was significantly higher than that in the control group at all stages, which indicated that the stimulation of E. granulosus promoted the release of chemokines, recruited a large number of monocytes to activate HSCs, and the fibrogenic microenvironment reversely promoted monocytes, it produced MMP-8 and other secreted proteins to degrade the ECM. In the middle and late stages, the expression of MMP-8 was mainly concentrated around the hepatic lesions, located in the wall layer of the fibrous sac, which proved that its main role was to degrade the matrix protein components of the fibrous sac wall, however the balance of ECM generation and degradation was destroyed, gradually leading to fibrosis around the lesions [23].

Phagosomal recruitment of the lysosomal protein LAMP-1 is a widely used marker of phagosome maturation [24]. In order that phagocytes can exert microbicidal activities, their phagosomes must undergo a process called phagosome maturation, if the proteins are lacking, the maturation process is impaired [24]. In this study, the results showed that the expression of LAMP-1 in the liver of mice infected with E. granulosus was significantly higher than that in the control group from 2 to 8 days, and it seemly inhibited with a low level after 30 days of infection, it seemed that phagocytic lysosomes were activated in the early stage of infection, and played a role of phagocytic clearance of pathogens, which was inhibited in the late stage of infection, it seemed to evade its microbicidal activity along with the aggravation of E. granulosus infection. This conclusion seems to be consistent with Leishmania promastigotes infection [25, 26].

Protein kinase C (PKC) is a serine/threonine kinase that regulates diverse cellular responses including immune responses [27]. PKC isoforms was shown to regulate immune cell signaling, and their inhibition was shown to alter immune cell effector functions in Leishmania infection [28]. And Mukherjee et al demonstrated that as an immune evasion strategy, Leishmania modulated those responses to ensure its survival [29]. After activation PKC-α translocated toward the plasma membrane and co-localized with surrounding collagen fiber within the contact areas of the cells and PKC-α directly bound and phosphorylated filamin, and was responsible for cell adhesion, cell migration and actin arrangement in the cells [30, 31]. In our study, we found that PKC-α expression was always higher than that in the control group at the 300 days infection. We speculated that, due to the influence of E. granulosus infection on liver microenvironment, the recruitment of monocyte-macrophages upregulated PKC-α expression after receiving external signals and PKC-α also regulated fibrotic markers to promote the process of fibrosis. The expression change of LAMP-1 and PKC-α in our study might indicate an immune evasion strategy of E. granulosus to ensure its survival in the liver.

Conclusion

In a word, our study focused on inflammatory and fibrotic markers occurred during the development of E. granulosus infection, and facilitated clarification of molecular mechanisms of E. granulosus infection and contributed to the development of novel therapies.

Declarations

Conflict of Interest Statement 

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

Funding

This work was supported by the National Natural Science Foundation (81760372), Xinjiang Autonomous Region Regional Collaborative Innovation Project (2020E0277) and State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia Fund (SKL-HIDCA-2018-27, SKL-HIDCA-2020-50, SKL-HIDCA-2021-5). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Ethics statement

BALB/c mice (female, 8-week-old) purchased from the Experimental Animal Science Research Department (Urumqi, China) were maintained in an air-conditioned animal room with a 12-h light/dark cycle and provided with rodent chow and water. E. gultilocularis protocercaria were obtained from intraperitoneal lesions maintained in BALB/c mice. All animals met the International Guiding Principles for Biomedical Research Involving Animals as issued by the Council for the International Organizations of Medical Sciences, with protocols approved by the Institutional Animal Use and Care Committee of Xinjiang Medical University (No. 20160218-14).

Author contributions

L.Y.M. and M.X.M. designed, instructed and approved the final manuscript. Y.Y, T.F.M. analyzed the literature and wrote the manuscript. Q.X.W., L.B., and L.J. collected the patient data. Z.X., M.Y.Y. and Z.X. disposed the data. All authors reviewed the manuscript.

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