All animals’ experimental procedures were strictly performed according to the Guidelines for Animal Experiments of Xuzhou Medical University and the National Guide for the Care and Use of Laboratory Animals. The study protocol was reviewed and approved by the Committee on Ethics of Animal Experiments, Xuzhou Medical University (201701w007).

Bone marrow (BM) cells were flushed from femur and tibia of Adrb2^+/+ and Adrb2^−/− mice and cultured using DMEM supplemented with 20% (v/v) FBS, 1% (v/v) P/S, 20 ng/ml mouse recombined murine M-CSF (Proprotech, USA) for 7 days (17). The purity of BM-macrophage cultures was confirmed by FACS using CD11b (1:100, BioLegend, San Diego, CA, USA) and F4/80 (1:100, BioLegend, San Diego, CA, USA) antibodies. Seven days later, BM-derived macrophages were cultured in a DMEM medium (Invitrogen, Carlsbad, CA, USA) for 24 h to remove the residual M-CSF. M0 macrophages (non-differentiated macrophages) were then stimulated with recombinant IL-4 (rIL4) (20 ng/ml, Proprotech, USA) with or without mTORC1 inhibitor rapamycin (100 ng/ml, MCE, USA) for 24 h. After that, the rIL-4 was removed and the cells were washed to remove the remaining rIL-4 and collected for further analysis. For measuring IL-4, the cells were washed and the DMEM medium (Invitrogen, Carlsbad, CA, USA) was added for another 24 h.

C. sinensis metacercariae were obtained as previously described (18, 19). Briefly, the meat from Pseudorasbora parva (a kind of freshwater fish) containing C. sinensis metacercariae was minced and digested with artificial gastric juice, a solution of 0.7% pepsin A and 0.1% HCl, at 37°C in a shaking water bath for 12 h. The digested mixture was filtered through a sieve with a mesh size of 100 μm. Then the pellet was sedimented in a phosphate-buffered solution (PBS) in a sedimentation jar until the supernatant was clear. C. sinensis metacercariae were identified, collected under a dissecting microscope, and stored in PBS at 4°C until use.

Males FVB wild-type mice (Adrb2^+/+ ) were purchased from the Beijing Vital River Laboratory Animal Technology Co. Ltd. β2-AR knockout mice (Adrb2^−/− ) were kindly gifted by Prof. Hong Sun from the Department of Physiology, Xuzhou Medical University. All mice were bred and maintained at the Animal Center of Xuzhou Medical University (Xuzhou, Jiangsu, China). All the mice were group-housed in a specific pathogen-free condition with the temperature-control 24°C with a 12 h dark/light cycle and permitted free access to food and water at the Animal Center of Xuzhou Medical University (Xuzhou, Jiangsu, China). The experimental procedure was carried out on 6–10-week-old mice as follows: two main groups including FVB wild-type (Adrb2^+/+ ) and β2-AR knockout mice (Adrb2^−/− ) were each divided into the control group and the infected group. In each mouse of the infected group, 45 metacercariae were intragastrically administered, and the irrigating solution was observed under the microscope to ensure that all metacercariae were completely infused; the mice of the non-infected group were given the same volume of normal saline. Four weeks later, the metacercariae were able to develop into adult worms and release eggs, with the development of liver fibrosis (20). On the 28th day of infection in mice, serum and liver tissue were collected for further study.

The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total bile acid (TBA), and Cellular cholesteryl ester (CHE) in plasma (Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, China) were assayed to indicate liver function in infected and uninfected mice.

To evaluate the histopathological changes in C. sinensis–infected mice and their capacity to induced cholangiopathy, the liver tissue was serially sectioned at 4 μm for H&E staining according to the manufacturer’s instructions (Jiangsu Beyotime Biotechnology Research Institute, China). After sealing the slides with neutral adhesive, the pathological changes of stained histological sections were observed by microscope (Olympus, Japan). Tissue sections were stained with H&E; and hyperplasia, epithelial damage, infiltration of inflammatory cells, and histological scoring were determined based on the histological findings.

For IHC and IF staining, serial sections of embedded tissue from each mouse were used for the staining of cytokeratin 19 (CK-19), CD206, CD68, Arginase-1, and β2-AR. The liver tissue was deparaffinized, hydrated, and heated in citric acid buffer at 95°C for 15 min and then blocked with 5% BSA for 30 min. The slides were then incubated overnight with primary Anti-Cytokeratin 19 (1:200, ab133496, Abcam, Cambridge, USA), Anti-CD206 (1:400, CST 24595 Danvers, MA, USA), Anti-CD68 (1:200, ab201973, Abcam, Cambridge, USA), Anti-Arginase-1 (1:400, 16001-1-AP, Proteintech, USA), and anti-β2-AR antibody (1:200, ET1703-04, Huabio, China). After washing with PBS, DAB (1:200, ZSGB–BIO, Beijing, China) as an enzyme-substrate was added. Five high-power fields (×400 magnifications, Olympus, Japan) were randomly selected from each mouse-staining section. CK19 and β2-AR-positive expressions integrated optical density IOD (Integral optical density) was calculated by Image-Pro Plus 6.0 software. A higher IOD value indicates a stronger positive expression.

Four percent paraformaldehyde was used to fix the liver tissue from each strain mice, and then the liver was embedded in paraffin. Four-μm-thick sections were prepared and stained with Masson’s trichrome, according to the manufacturer’s instructions (Jiancheng, Nanjing, Jiangsu, China). The sections were observed under the microscope and digitized using an imaging system (Olympus, Japan). Five high-power visual fields (200× and 100× magnifications, Olympus, Japan) were randomly selected from the staining sections of each mouse, and Image Pro Plus6.0 software was used to calculate the Integral optical density (IOD) of fibrous tissue. Higher IOD value means stronger positive expression

To quantify liver fibrosis, hepatic hydroxyproline content was determined according to the instructions of the manufacturer (Jiancheng Institute of Biotechnology, Nanjing, China) according to the manufacturer’s recommendations. Hydroxyproline content was measured at 570 nm using a hydroxyproline standard curve.

Total RNA was extracted from liver tissue and bone marrow–derived macrophage (BMDM) cells by using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA was reverse transcribed to complementary DNA. Quantitative real-time PCR was performed using SYBR green real-time PCR master mix according to the manufacturer’s instructions (Roche Applied Science, Mannheim, Germany). Relative quantification of each gene expression was calculated in terms of the comparative cycle threshold (Ct) normalized by beta-actin (Shanghai General Biotech Co. China).

Serum and mouse liver homogenate from each mouse were immediately subjected to evaluate the concentrations of IL-4, IL-6, IL-10, IL-13, TNF-α by a commercial ELISA Kit with Plates (Thermo Scientific, USA). All procedures were performed according to the instructions provided by the kit. Concentrations of cytokines in the sera were calculated using standard curves as references.

Total protein was extracted from liver tissue and BMDM cells by using a homogenizer and analyzed with a bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Anti-phospho-PKA (p-PKA, 1:1000, SC54-04, Huabio, China), anti-phospho-PI3K (p-PI3K, 1:1000, AP0854, ABclonal, Cummings Park, USA), anti-phospho-ERK1/2 (p-ERK1/2, 1:1000, AP0490, Bioworld, Bloomington, USA), anti-phospho mTOR (p-mTOR, 1:1000, AP0115, Abcam, Cummings Park, USA), anti-mTOR (1:1000, ET1608-5, Huabio, China), anti-phospho-AKTs473 (p-AKTs473, 1:1000, BS4006, Bioworld, Bloomington, USA), anti-AKT (1:1000, ET1609-47, Huabio, China) anti-phospho-Stat6 (p-Stat6, 1:1000, Ab263947, Abcam, Cambridge, USA), anti-b2-AR (1:1000, ET1703-04, Huabio, China), anti-alpha SMA (1:1000, 14395-1-AP, Proteintech, USA), anti-beta-actin (1:1000, 66009-1-Ig, Proteintech, USA), and anti-GAPDH (1:1000, AC033, ABclonal, Cummings Park, USA) were used as primary antibody after separation by electrophoresis in 7.5 and 10% SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). The secondary antibodies (anti-rabbit IgG, 1:5000, HRP-S0001, Affinity Biosciences, Cincinnati, USA and anti-mouse IgG, 1:5000, 7076S, CA, USA) were incubated for 2 h at room temperature. The membrane containing antibody-protein complexes was visualized with an enhanced chemiluminescence detection system on radiographs film (Bio-rad, Hercules, CA, USA). The bands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of the protein in each sample was normalized by GAPDH or beta-actin (Santa Cruz Biotechnology, CA, USA).

The cell suspensions were blocked with an Fc blocker (Anti-mouse CD16/32, 101319, Biolegend, USA) before incubation with surface marker antibodies. The following antibodies were used to stain cell surface markers: Briliant Violet 510 anti-mouse/human CD11b (101236, Biolegend, USA), CD206 Alexa 647 MR5D3 (565250, Biolegend, USA), and PerCP/Cy5.5 anti-mouse F4/80 (123127, Biolegend, USA). For the characterization of M2 macrophages, CD11b and F4/80 were used to define the population of macrophages. After that, CD206 was used to characterize the population of M2 macrophages in the total population of macrophages. The results were analyzed using FlowJo software.

All the data were expressed as mean ± s.e.m. The data were analyzed by SPSS 23.0 software (SPSS Inc, Chicago, IL, USA). The student t-test was used for comparison between the two groups. One-way ANOVA with Tukey’s post hoc test was used for comparison for more than two groups. Statistical significance was ranked *P<0.05, **P<0.01, and ***P<0.001.

Previous studies have shown that beta 2 adrenergic receptors control several functions such as cell proliferation, apoptosis, and immune response (21, 22). In the liver, several types of cells expressed β2-AR (23). In this study, our results showed that the β2-AR are highly expressed in the liver of Adrb2^{+/ +} (wild-type) mice after C. sinensis infection ( Figures 1A, B ). Moreover, there was almost no expression of β2-AR in the liver of Adrb2^−/− regardless of infection or Non-infection in β2-AR-deficient mice ( Figures 1A, B ).

Next, the role of β2-AR in hepatobiliary damage in mice caused by infection with C. sinensis was determined. The liver weight/body weight ratio increases significantly in the infected Adrb2^+/+ group compared with the non-infected Adrb2^+/+ group. However, it decreased in infected Adrb2^−/− mice, compared with the infected Adrb2^+/+ mice ( Figures 1C, D , P<0.01). As shown by HE staining in Figures 1E, F , the liver structure showed more damages in the infected Adrb2^+/+ mice, compared with the non-infected Adrb2^+/+ mice, whereas the Adrb2^−/− mice, after C. sinensis infection, showed alleviation of liver damages compared to the infected Adrb2^+/+ mice. Similarly, observation of the cross-section of the bile duct shows dilatation of the bile duct in infected Adrb2^+/+ mice compared to the control group. Also, the dilatation of the bile duct was more evident in the infected Adrb2^+/+ than in the infected Adrb2^−/− mice ( Supplementary S1A ). Also, in the infected Adrb2^+/+ mice, the liver and biliary damages were significantly increased compared to the control Adrb2^+/+ mice as indicated by the measurements of ALP (P<0.01), AST (P<0.001), ALT (P<0.01), CHE (P<0.001), and TBA (P<0.01), confirming the liver damage caused by C. sinensis infection ( Figure 2A ). However, the infected Adrb2^−/− mice significantly decrease the production of ALP (P<0.01), AST (P<0.001), ALT (P<0.01), CHE (P<0.05), and TBA (P<0.01), compared with the C. sinensis–infected wild-type mice. In general, cholangiocytes proliferate in response to an injury, which can act as an indicator of biliary injuries. As shown in Figures 2B, C , the infected mice showed a significant increase in the proliferation of cholangiocytes compared to the non-infected control group where the proliferation of cholangiocytes was limited. Also, compared with Adrb2^+/+ mice infected with C. sinensis, the proliferation of cholangiocyte reduced obviously in Adrb2^−/− mice with infection, as indicated by IHC staining of CK19 ( Figures 2B, C , P<0.001).

Since C. sinensis can induce liver fibrosis in mammals, we firstly investigated whether the knocking out of β2-AR has effects on liver fibrosis caused by the worm or not. Masson’s staining, which allows the observation of ECM, is a stain used to detect liver fibrosis and liver cirrhosis. The results of this study showed that the control group (non-infected mice) did not show accumulation of collagen fibers, as shown by the lack of blue strips. In contrast, as shown in Figure 3A , the accumulation of collagen fibers represented by the blue color was significantly decreased in infected Adrb2^−/− mice, compared with infected Adrb2^+/+ mice. Similarly, compared to the control group without infection, alpha-smooth-muscle actin (α-SMA), which is an indicator of fibroblast activity, increased in both Adrb2^+/+ and Adrb2^−/− mice infected with C. sinensis. However, the β2-AR-deficient mice infected by C. sinensis showed a significant decrease in α-SMA expression compared to the Adrb2^+/+ infected group ( Figure 3B , P<0.001). Besides, hydroxyproline content, which is a common marker of liver fibrosis, was much higher in the infected group compared to the non-infected control group. C. sinensis infection induced a significant increase in hydroxyproline content in Adrb2^+/+ mice, while the knocking out of β2-AR in mice (Adrb2^−/− mice) significantly decreased hydroxyproline content ( Figure 3C , P<0.01). These data together indicate that β2-AR deficiency abolished liver fibrosis caused by worm infection.

A characteristic of helminth infection is the upregulation of type 2 immune response. Type 2 immune response characterized by type 2 cytokines plays an important role in liver fibrosis as IL-4, IL-13, and TGF-β1 produced by macrophages can activate HSCs leading to liver fibrosis (24). Since some studies have shown that C. sinensis infection leads to type 2 immune response in FVB mice (25), we decided to check whether β2-AR can regulate type 2 immune response during liver fluke infection or not. Not surprisingly, the results showed that C. sinensis infection increases type 2 cytokine production, especially IL-4 and IL-13, in infected Adrb2^+/+ compared to the non-infected Adrb2^+/+ ( Figures 3D, E ). Furthermore, our results revealed that Adrb2^−/− mice infected with C. sinensis showed significantly lower production of type 2 cytokines, in particular, IL-4 ( Figure 3D , P<0.001) and IL-13 ( Figure 3E , P<0.01), compared to that observance in Adrb2^+/+ mice with worm infection. The level of tgfb1 mRNA, which indicates type 2 immune response, was also significantly decreased in infected Adrb2^−/− mice compared to Adrb2^+/+ mice ( Figure 3F , P<0.01).

AAMs are one of the most important sources of IL-4 and IL-13 during helminth infection, and liver fibrosis is associated with AAMs infiltration (26). In our present study, we further investigated the effects of the β2-AR on the AAMs in C. sinensis–infected mice. We co-stained CD68 with CD206 to define the AAMs in the liver section (27). Our data showed that the infiltrated macrophages highly expressed CD206 ( Figures 4A, B , P<0.001). We also found that AAMs infiltration was higher in the infected group than that observed in the control group ( Figures 4A, B , P<0.001). In addition, our data revealed that β2-AR-deficient mice significantly decreased the activation and infiltration of AAMs. We also found that arginase-1, which is highly expressed by AAMs, was increased in the liver of infected mice, but there was a very low expression of arginase-1 in the liver of control mice. Moreover, arginase-1 was highly expressed in infiltrated macrophages of infected Adrb2^+/+ mice, indicating that the infiltrated macrophages involved in C. sinensis–induced liver fibrosis are AAMs ( Figures 4C, D , P<0.001).

The previous results of this work have demonstrated that β2-AR-deficient (Adrb2^−/− ) mice show a decrease in liver fibrosis following C. sinensis infection, which may be associated with M2 macrophages. To investigate the mechanism by which β2-AR regulates AAMs, we isolated and cultured bone marrow–derived macrophages from Adrb2^−/− mice and Adrb2^+/+ mice. In this study, we generated AAMs by IL-4 stimulation to highlight the role of β2-AR in the regulation of AAMs. Thus, we tested whether macrophages stimulated by IL-4 in this study express M2 markers. The expression of M2 marker genes such as Arginase-1, Fizz-1, Ym-1 was significantly upregulated in IL-4-stimulated macrophages ( Figures 5A–C ). Similarly, the percentage of CD206, which is a surface marker of M2 macrophages, was also significantly upregulated ( Figures 5E, F , P<0.001). Type 2 cytokine IL-4 was also significantly upregulated in M2 macrophages compared to M0 (unstimulated macrophages) ( Figure 5D ). These data suggest that we successfully induced AAMs with the stimulation of IL-4. We then asked whether β2-AR regulated the M2 macrophages response in vitro or not. M2 Adrb2^−/− macrophages stimulated by IL-4 downregulated the expression of M2 markers such as Arginase-1, Fizz, and Ym-1, compared to M2 Adrb2^+/+ macrophages ( Figures 5A–C ). Also, the percentage of CD206 in β2-AR-deficient (Adrb2^−/− ) macrophages was significantly decreased compared to that of Adrb2^+/+ macrophages ( Figures 5E, F , P<0.01). IL-4, which is one of the most important type 2 cytokines, was significantly upregulated in Adrb2^+/+ M2 macrophages compared to Adrb2^−/− M2 macrophages ( Figure 5D , P<0.001).

It has previously been demonstrated that mTOR plays an important role in the metabolic reprogramming of the M2 macrophages (17). To investigate the pathway by which β2-ARs regulate type 2 immune response in macrophages, we firstly investigated β2-AR-mediated canonical signaling. Our results revealed that M2 macrophages (IL-4-stimulated macrophages) did not result in a difference in PKA phosphorylation in Adrb2^+/+ macrophages and Adrb2^−/− macrophages compared to M0 macrophages (unstimulated macrophages) ( Supplementary S1B ). More interestingly, no differences in the phosphorylation of AKT and PI3K, which are downstream of PKA and upstream of mTOR, were observed between M2 Adrb2^+/+ and M2 Adrb2^−/− macrophages, indicating that canonical signaling was not involved in the control of inflammation by β2-AR ( Supplementary S1B ). Subsequently, we investigated whether non-canonical signaling through MAPK was involved in this process or not. Our results showed that IL-4 stimulation increased the phosphorylation of ERK1/2 in M2 Adrb2^+/+ macrophages compared to M2 Adrb2^−/− macrophages ( Figure 6A ). The phosphorylation of ERK1/2 in M2 Adrb2^+/+ macrophages was associated with an increase in mTOR phosphorylation ( Figure 6A ), indicating that the control of inflammation by β2-AR occurred along the non-canonical signaling pathway. Moreover, M2 Adrb2^−/− macrophages showed a significant decrease in the phosphorylation of mTOR compared to M2 Adrb2^+/+ macrophages. In addition, compared to M2 macrophages, M0 (unstimulated macrophages) did not induce phosphorylation of mTOR, indicating that mTOR was essential for IL-4 to induce AAMs.

To investigate the mechanism by which β2-AR controls type 2 inflammation in macrophages by mTOR, we checked whether the inhibition of mTORC1 by Rapamycin can decrease the M2 markers. Compared to the M2 control macrophage, the inhibition of mTORC1 in M2 macrophage significantly decreased the phosphorylation of mTOR in Adrb2^+/+ ( Figure 6B ). Moreover, Rapamycin completely abolished the increase of M2 macrophage markers in Adrb2^+/+ ( Figures 6C–G ). Compared to M2 Adrb2^−/− macrophages, the decrease of M2 markers in M2 Adrb2^−/− macrophages inhibited with Rapamycin was not significant ( Figures 6C–G ). The above results indicated that the inhibition of mTORC1 by Rapamycin inhibited mTOR phosphorylation and the decrease in type 2 inflammatory response in macrophages. However, inhibition of mTORC1 by Rapamycin does not induce any change in Stat6 phosphorylation, indicating that the signaling by which β2-ARs regulate M2 macrophages is through mTORC1 and independent of Stat6 ( Figure 6B ).