β2-Adrenergic Receptor Enhances the Alternatively Activated Macrophages and Promotes Biliary Injuries Caused by Helminth Infection

The autonomic nervous system has been studied for its involvement in the control of macrophages; however, the mechanisms underlying the interaction between the adrenergic receptors and alternatively activated macrophages (M2) remain obscure. Using FVB wild-type and beta 2 adrenergic receptors knockout, we found that β2-AR deficiency alleviates hepatobiliary damage in mice infected with C. sinensis. Moreover, β2-AR-deficient mice decrease the activation and infiltration of M2 macrophages and decrease the production of type 2 cytokines, which are associated with a significant decrease in liver fibrosis in infected mice. Our in vitro results on bone marrow–derived macrophages revealed that macrophages from Adrb2−/− mice significantly decrease M2 markers and the phosphorylation of ERK/mTORC1 induced by IL-4 compared to that observed in M2 macrophages from Adrb2+/+ . This study provides a better understanding of the mechanisms by which the β2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.


INTRODUCTION
Macrophages are a key element in the initiation of the immune response (1). Macrophages can modify their phenotype and the type of response according to different types of stimuli. Thus, macrophages exposed to stimuli arising from the type 1 immune response such as interferongamma (IFN-g) or lipopolysaccharide (LPS) differentiate into classically activated macrophages (M1) characterized by the high production of pro-inflammatory cytokines (2,3), whereas macrophages exposed to stimuli such as interleukin (IL)-4 and IL-13 (type 2 immune cytokine) differentiate into alternatively activated macrophages (AAMs) or M2 with the high expression of Arginase-1, Fizz-1, chitinaselike protein 3 (Chil3) [Ym1], scavenger receptors such as CD163, and mannose receptors (CD206), as well as the production of profibrotic cytokines (3)(4)(5)(6).
The activation of macrophages is a process that is highly regulated. The involvement of the autonomic nervous system in the control of macrophages has been studied in recent years, but mechanisms underlying the interaction between the nervous system and activation of macrophages in the context of type 2 immune response remain obscure. Catecholamines produced by neurons of the sympathetic nervous system and those released into the bloodstream by the pituitary gland activate different effectors located on the membranes of macrophages and binding with adrenergic receptors allowing their control (7). Beta 2 adrenergic receptor (b2-AR) is such kind of receptor that can control the activation of macrophages (8,9). In macrophages, many data have shown that activation of b2-AR has immuno suppressive properties by reducing the pro-inflammatory response via the canonical cAMP/PKA pathway (10). In addition, Grailer et al. (2) showed that the activation of b2-AR by norepinephrine not only reduces the production of proinflammatory cytokines such as TNF-a, IL-12, and CXCL chemokines but also increases the production of IL-10 in the condition of LPS-induced acute lung injury. However, the regulatory mechanism by which b2-AR orchestrates AAMs in the context of type 2 immune response has so far not been investigated.
Clonorchis sinensis (C. sinensis) is a helminth that dwells in the bile ducts for a long time (almost 20~30 years if not treated) (11). Infection with C. sinensis can lead to cholestasis, cholangitis, and biliary fibrosis, which finally cause liver cirrhosis and possibly cholangiocarcinoma (12,13). During infection with C. sinensis, the contact of the parasite with its host triggers the activation of immune cells such as macrophages, eosinophils, mast cells, basophils, Th2 cells, and group 2 innate lymphoid cells (ILCs) (14). The resulting production of type 2 cytokines (IL-4 and IL-13) induces the differentiation of macrophages into AAMs (M2), which then begins the repair process and the restoration of the homeostasis (15,16). However, the signaling by which b2-AR orchestrates AAMs and promotes biliary fibrosis caused by helminth infection is still unknown.
In the present study, we used FVB wild-type mice (Adrb2 +/+ ) and b2-AR knockout mice (Adrb2 −/− ) with FVB background because of their susceptibility to helminth infection and their ability to elicit a predominant type 2 immune response. This study allowed us to explore and understand the mechanism by which b2-AR regulates M2 macrophages and promotes liver fibrosis induced by C. sinensis infection. In our present study, we found that b2-AR can regulate the response of alternatively activated macrophages during liver fibrosis induced by C. sinensis infection. Moreover, the b2-AR-orchestrated activation of M2 is mediated by the mTORC1. This study provides a better understanding of the mechanisms by which the b2-AR enhances type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their role in liver fibrosis.

Ethics Statement
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).
Preparation of C. sinensis Metacercariae 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 mm. 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.
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 6-10week-old mice as follows: two main groups including FVB wildtype (Adrb2 +/+ ) and b2-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.

Liver Function Test
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.

Histology, Immunohistochemistry, and Immunofluorescence Staining
To evaluate the histopathological changes in C. sinensis-infected mice and their capacity to induced cholangiopathy, the liver tissue was serially sectioned at 4 mm 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 b2-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-

Masson's Staining
Four percent paraformaldehyde was used to fix the liver tissue from each strain mice, and then the liver was embedded in paraffin. Four-mm-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

Measurement of Hepatic Hydroxyproline Content
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.

RNA Isolation and Reverse Transcription
Total RNA was extracted from liver tissue and bone marrowderived 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).

Enzyme-Linked Immunosorbent Assay
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-a 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.

Flow Cytometry
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.

Statistical Analysis
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.

b2-AR Deficiency Alleviates Hepatobiliary Damage in Mice Infected With C. sinensis
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 b2-AR (23). In this study, our results showed that the b2-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 b2-AR in the liver of Adrb2 −/− regardless of infection or Non-infection in b2-AR-deficient mice ( Figures 1A, B).
Next, the role of b2-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 noninfected 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 crosssection 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 Deficiency Abates Liver Fibrosis Caused by Infection With C. sinensis Since C. sinensis can induce liver fibrosis in mammals, we firstly investigated whether the knocking out of b2-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 (a-SMA), which is an indicator of fibroblast activity, increased in both Adrb2 +/+ and Adrb2 −/− mice infected with C. sinensis. However, the b2-ARdeficient mice infected by C. sinensis showed a significant decrease in a-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 b2-AR in mice (Adrb2 −/− mice) significantly decreased hydroxyproline content ( Figure 3C, P<0.01). These data together indicate that b2-AR deficiency abolished liver fibrosis caused by worm infection.
Knocking Out of b2-AR Prevents the Increase in Type 2 Cytokines After C. sinensis 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-b1 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 b2-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).
Adrb2 −/− Significantly Decreased Alternatively Activated Macrophages Infiltration in C. sinensis Infection 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 b2-AR on the AAMs in C. sinensisinfected 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 b2-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).

b2-AR Enhances Alternatively Activated Macrophages In Vitro
The previous results of this work have demonstrated that b2-ARdeficient (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 b2-AR regulates AAMs, we isolated and cultured bone marrowderived macrophages from Adrb2 −/− mice and Adrb2 +/+ mice. In this study, we generated AAMs by IL-4 stimulation to highlight the role of b2-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-4stimulated 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 b2-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 b2-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).

b2-AR Controls M2 Macrophage via ERK/mTORC1 Signaling In Vitro
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 b2-ARs regulate type 2 immune response in macrophages, we firstly investigated b2-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 b2-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 b2-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 b2-AR controls type 2 inflammation in macrophages by mTOR, we checked whether The alpha SMA expression in the liver of control and infected mice. The liver expression of alpha SMA in the control group was significantly lower than that observed in the infected group (P < 0.001). Also, the expression of alpha SMA in the infected group was significantly higher in infected Adrb2 +/+ than infected Adrb2 −/− (P < 0.001). (C) The measurement of Hydroxyproline content showed an increase in the infected group than the control group, and the increase in the hydroxyproline content was higher in infected Adrb2 +/+ compared to infected Adrb2 −/− (P < 0.01). (D-F) Measurement of the production of type 2 cytokines IL-4 and IL-13 and the relative mRNA expression of TGF-beta in liver tissue of control and infected mice. b2-AR-deficient mice prevented the increase in the production of IL-4, IL-13, and tgf-beta after Clonorchis sinensis infection. The infected Adrb2 +/+ showed a significant increase in the production of IL-4 (P < 0.001), and IL-13 (P < 0.01), and the increase in the mRNA expression of tgf-beta (P < 0.001), compared to the control group and infected Adrb2 −/− group. Compared with indicated groups, **P < 0.01, ***P < 0.001.
Koda et al.

b2-Adrenergic Receptor and Macrophages
Frontiers in Immunology | www.frontiersin.org October 2021 | Volume 12 | Article 754208 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 do es not induce an y change in Stat6 phosphorylation, indicating that the signaling by which b2-ARs regulate M2 macrophages is through mTORC1 and independent of Stat6 ( Figure 6B).

DISCUSSION
The autonomic nervous system regulates the activity of immune cells through a set of signals. Macrophages possess receptors that allow them to respond to signals from the autonomic nervous system. Alternatively activated macrophages (M2) are important in the process of tissue repair and the maintenance of homeostasis; however, little is known about the regulatory roles of b2-AR in the activation of M2. In the present study, compared with Adrb2 +/+ mice, we found that Adrb2 −/− mice showed alleviated hepatobiliary injuries and fibrosis after C. sinensis infection, which is associated with reduction of M2 activation and its relative cytokines (IL-4 and IL-13). In vitro, we also found that b2-AR orchestrated the activation of M2 associated with mTOR. Our present study provides a good understanding of the mechanisms by which b2-AR is involved in the regulation of M2 macrophages. The immunofluorescence co-staining of CD68 and CD206 indicating the alternatively activated macrophages revealed that there was a strong infiltration of alternatively activated macrophages in infected mice than that observed in control mice; however, the knocking out of b2-AR induced a decrease in alternatively activated macrophages infiltration. (C, D) In vivo Immunofluorescence co-staining of CD68 and Arginase-1. The co-expression of CD68 and Arginase-1 indicating AAMs showed that there was a very low expression of CD68 and Arginase-1 in the liver of control mice. The infection of mice with C. sinensis resulted in a significant increase in the expression of arginase in the liver. Moreover, activated macrophages expressed arginase-1. However, Adrb2 −/− mice infected with C. sinensis decreased the expression of positive CD68 and Arginase-1. Compared with indicated groups, ***P < 0.001. FVB mice by their ability to elicit predominant type 2 immune response during helminth infection are a widely used model for the study of type 2 immune response (25,28). We found that the infection of mice with C. sinensis results in strong cholangiocyte proliferation ( Figure 2B). Cholangiocyte activation has been reported to play an important role in Kupffer cell activation and hepatic stellate cells as well, and the infiltration of macrophages promotes cholangiocyte proliferation (29,30). In general, cholangiocytes proliferate in response to an injury. The control of cholangiocyte proliferation is an important element in liver fibrosis because the activation of cholangiocytes leads to the activation of hepatic stellate cells, which are the main source of tumor growth factor-beta 1 (TGF-b1).
In the liver, resident macrophages (Kupffer cells) play an important role in maintaining homeostasis, orchestrating tissue remodeling in ontogenesis, and regulating metabolic functions (31), while infiltrated bone marrow-derived macrophages play a role in the fighting of the parasite (31). During the parasite infection, an elevated type 2 immune response can limit the excessive inflammatory reaction followed by fibrosis and ultimately cirrhosis. It has previously been reported that during C. sinensis infection, macrophages are the main source of type 2 immune response (32). The results of this study revealed that the infection of mice with C. sinensis results in significant macrophages activation and infiltration. In the liver, activated and infiltrated macrophages play a very important role in the expulsion of the parasite; however, the excessive inflammatory reaction can lead to liver damage (31). Moreover, there was significant infiltration of AAMs, which are the principal contributor to liver fibrosis. Here we have shown that the b2-AR-deficient mice significantly show a decrease in the activation and infiltration of macrophages as well as the prevention of liver fibrosis.
AAMs promote fibrosis by the upregulation of type 2 cytokines such as IL-4, IL-13, and TGF-b, leading to activation of hepatic stellate cells (33)(34)(35). The role of b2-AR in the regulation of inflammatory response has been the subject of several works. In classically activated macrophages, the activation of b2-AR prevents the increase of type 1 immune response by the increase in the cAMP/PKA level (36). In our present study, we found that the activation of b2-AR in AAMs promotes type 2 immune response. Our results are consistent with previous studies that have shown that an increase in the inflammatory response in macrophages is associated with the activation of ERK1/2 rather than cAMP/PKA (37). M2 macrophages are characterized by a significant increase in M2 markers such as Arginase-1, Fizz-1, Ym-1, CD206, and can be induced by type 2 cytokines such as IL-4 and IL-13 (4,5,38). The mechanism by which AAMs are fine-regulated during their differentiation has not yet been investigated. Although it is known that the autonomic nervous system regulates the activity of immune cells, no investigation has been carried out into the mechanism by which the autonomic nervous system regulates the activity of AAMs. In this study, we did not observe the production of IL-13 in M2 macrophages stimulated by IL-4. This might be because we used IL-4 to induce the differentiation of macrophages mediated by IL-4Ra but not IL-13. b2-ARdeficient AAMs showed a significant reduction of the M2 markers compared to b2-AR wild-type macrophages. The increase in M2 markers may be associated with an increase in metabolism as shown by the increase in mTOR in Adrb2 +/+ compared to Adrb2 −/− macrophages. Previous studies have suggested that IL-4 acts synergistically with M-CSF to induce M2 macrophages through PI3K/AKT/mTORC2 and Stat6 signaling (17). It has previously been demonstrated that the differentiation of macrophages from M0 to M2 macrophage requires mTORC2, but not mTORC1 signaling since nonselective mTORC2 inhibitor "Torin," but not "Rapamycin," a selective mTORC1 inhibitor, inhibits Stat6, induces M2 macrophages differentiation (17). In our study, M2 Adrb2 +/+ macrophages and M2 Adrb2 −/− macrophages show an increase in AAMs marker compared to M0 macrophages (in which there The inhibition of mTORC1 also decreases the percentage of CD206 in M2 Adrb2 +/+ macrophages. Compared with indicated groups, **P < 0.01, ***P < 0.001; ns, not significant. were not any M2 markers). However, the M2 Adrb2 +/+ macrophages show a significant upregulation of AAMs marker compared to M2 Adrb2 −/− macrophages. In addition, we did not observe any changes in AKT and PI3K phosphorylation, which are upstream of mTORC2, between Adrb2 +/+ and Adrb2 −/− macrophages, which might suggest that mTORC2 is not involved in the regulation of inflammatory response by b2-AR. Moreover, the inhibition of mTORC1 significantly decreases M2 activation in Adrb2 +/+ compared to that observed in M2 Adrb2 +/+ without mTOR inhibition, indicating that mTORC1 is implicated in b2-AR-induced alternatively activated macrophages regulation. This result showed that the inhibition of mTORC1 does not inhibit the differentiation of macrophages from M0 to M2, but decreases M2 macrophages response. The control of inflammatory response by b2-AR involves the canonical and non-canonical signaling pathways. In our present study, AAMs enhanced the M2 phenotype by the activation of ERK1/2-mTORC1 signaling, indicating that the regulation of AAMs by b2-AR occurred along with the non-canonical signaling. ERK1/2-mTORC1 has also been shown to induce epithelial-mesenchymal transition and fibrosis in retinal pigment epithelial cells (39). Also, stimulation of macrophages with LPS has been shown to enhance type 1 cytokines in ERK1/2 signaling (40). However, further investigations are needed to highlight the downstream as well as the mechanism by which mTORC1 controls AAMs.

CONCLUSION
This study provides a better understanding of the mechanisms by which the b2-AR regulates type 2 immune response through the ERK/mTORC1 signaling pathway in macrophages and their roles in liver fibrosis. We found that b2-AR-deficient mice showed a significant decrease in the activation and infiltration of AAMs, which is associated with the reduction of liver fibrosis after C. sinensis. Our results also showed that the control of AAMs occurs along with the non-canonical ERK1/2 and mTORC1 signaling. All these data together suggest that b2-AR regulates AAMs in the ERK/mTORC1 signaling pathway during liver fibrosis induced by helminth infection.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.