Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Abstract Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45+CD19+ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45+CD3+ T lymphocytes and CD45+F4/80+ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of Bnull HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.


| INTRODUC TI ON
Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide and is an independent risk factor for type 2 diabetes mellitus (T2DM), cardiovascular disease, etc. 1,2 Visceral adipose tissue (VAT) is implicated in the development of NAFLD. 3,4 As a depot of VAT, mesenteric adipose tissue (MAT) is located between the gut and liver, which makes it possible to affect the liver through the portal vein by secreting pro-inflammatory cytokines, adipocytokines and releasing free fatty acid (FFA), etc. 5,6 Mesenteric adipose tissue is now thought to play a more important role in the pathogenesis of obesity-related liver diseases or in insulin resistance than other depots of VAT. 8,9 Rytka et al found that mice receiving a portal drained fat transplant, similar to MAT, had worse hepatic insulin resistance compared to mice receiving vena cava drained fat. 6 Intervention strategies targeting MAT have demonstrated that MAT can affect hepatic metabolism, specifically inhibiting MAT inflammation and resulting in less lipolysis and better hepatic insulin resistance and steatosis. 10 Mesenteric adipose tissue inflammation contributes to NAFLD, but what promotes MAT inflammation is still not clear. In obesity-related diseases, various kinds of lymphocytes such as macrophages, natural killer (NK) cells and CD8 + T cells are infiltrated and activated in MAT, 11,12 promoting inflammation and lipolysis and eventually aggravating metabolic disorders. 13 In recent years, increasing studies have suggested that B lymphocytes are also involved in adipose tissue inflammation. B lymphocytes in epididymal adipose tissue (EAT) can promote EAT inflammation through modulation of T lymphocytes and production of immunoglobulin G (IgG) antibody in HFD-fed mice. 14 Transferred B2 cells in EAT of HFD-fed mice to HFD-fed B null recipient mice result in increased adipose tissue inflammation. 15 B null mice show improved insulin resistance and glucose metabolism compared to control mice under the stimulation of HFD, 14,15 and B2 cell depletion in mice resulted in alleviated NAFLD. 16 However, the role of B lymphocytes in the inflammation of MAT is poorly understood. Here, we aimed to explore the role of MAT B cells in MAT inflammation.
Recently, immune dialogue between the gut and liver has drawn researchers' attention. Studies have shown the existence of enterohepatic lymphocyte circulation. 17 In our previous study, we proved that immune cells including T and B lymphocytes could shuttle from mesenteric lymph nodes to the liver and promote NAFLD. 18 As mentioned above, MAT is rich in lymphatic and blood vessels draining in the portal vein, laying the structural and physiological foundation of lymphocyte circulation between MAT and the liver. In the current study, we also investigated whether MAT B cells could migrate to the liver and their effects on NAFLD.
Our results showed that during the development of NAFLD, B lymphocytes infiltrated earlier than macrophages in MAT and might promote MAT inflammation through modulation of macrophages.
Besides, the migration of MAT B lymphocytes to the liver increased, promoting hepatic inflammation in NAFLD.

| Animals and NAFLD model
Six-to-eight-week-old male C57BL/6J wild-type and B6.129S2 muMT mice (homozygous mutant mice that lack mature B cells, B null mice) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and Jackson Laboratory (Bar Harbor, ME, USA) respectively. Mice were raised in a specific pathogen-free (SPF) facility at Peking University People's Hospital. Thirtysix wild-type male mice were randomly divided into six groups. Half of them were fed with 45% kcal high-fat diet (HFD) (Mediscience Ltd., Yangzhou, China), and the others with normal diet (ND). Six mice from each group were killed in time gradients (4, 8 and 12 weeks of feeding). Six 6-8-week-old muMT mice and control mice were killed after 12 weeks of HFD feeding. All protocols were approved by the Peking University People's Hospital Ethics Committee.

| Isolation of lymphocytes in the liver and adipose tissue
Liver was perfused with PBS via the portal vein to eliminate the effects of lymphocytes in blood. Single-cell suspensions were prepared by mechanically disrupting liver and went through a 100-µm filter. Intrahepatic lymphocytes were isolated using 40% and 80% discontinuous Percoll (GE healthcare Bio-Sciences AB, Uppsala, Sweden) density gradient centrifugation. The cells in the middle layer were collected and washed with PBS three times.
Fresh adipose tissues were cut into pieces and digested with 1 mg/ mL type-Ι collagenase (Sigma-Aldrich, Merck Life Science (Shanghai) Co., Ltd. Shanghai, China) and 0.4 g/mL bovine serum albumin (BSA) dissolved in PBS for 30 minutes at 37°C with shaking at 200 rpm.
Undigested impurities were excluded using a 70-µm strainer, and soluble supernatants were centrifuged for 10 minutes at 300× g to acquire precipitated stromal vascular fractions (SVFs) that were used for flow cytometry after washing with PBS three times.

| Macrophage and adipocyte co-culture experiments
3T3-L1 cells were cultured in DMEM with 10% FBS and differentiated into mature adipocytes using insulin, dexamethasone and 3-isobutyl-1-methylxanthine. Mature adipocytes (2 × 10 5 cells/mL) were cultured with an equal number of RAW264.7 macrophages for 24 hours in the co-culture system, in which macrophages were pre-treated with an equal number of ND and HFD B cells or were not pre-treated for 24 hours. Adipocytes were harvested for RNA extraction.

| Liver homogenate and transwell assay
Liver sample (100 mg) was homogenized and diluted in 5 mL RPMI 1640 medium to a 20% concentration. All the procedures were performed on ice. Cell debris was removed by centrifugation, and supernatant homogenates were stored. Afterwards, 0.2 mL 20% liver homogenates and 0.4 mL RPMI 1640 medium were added to the lower chamber of the transwell system (24-well plates; 5.0 µm pore size; Costar, Corning), and RPMI 1640 medium (0.2 mL) with 10 5 B cells was added to the upper chamber. After culture at 37°C for 6 hours, the number of B cells in the lower chamber was counted, and chemotactic index was calculated. Three wells per group were set, and the experiment was repeated three times.

| B lymphocyte and primary hepatocyte coculture experiments
Primary hepatocytes (2 × 10 5 cells/mL), either from ND-or HFDfed mice, were cultured with an equal number of MAT B cells for 24 hours in the same co-culture system. Hepatocytes and cultural supernatant were harvested.

| RNA extraction, cDNA synthesis and realtime polymerase chain reaction
Total RNA was extracted using RNeasy Plus Mini kit (QIAGEN Co., Ltd., Hilden, Germany), and cDNAs were synthesized using RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Vilnius, Lithuania). Real-time polymerase chain reaction (PCR) was performed with a StepOne Plus Real-Time PCR System (Applied Biosystems, Waltham, MA) using SYBR Premix Ex Taq (Toyobo, Kita-ku, Osaka, Japan). The sequences of primers are listed in Table S1. The 2 −△△Ct method was used to compare relative mRNA expressions.

| Statistical analysis
Data are presented as mean ± SEM. Two-tailed student's t test or one-way ANOVA were used to compare values between the groups. spss 20.0 (IBM, New York, NY, USA) was used to perform statistical analyses. P < 0.05 was considered statistically significant.  Figure 1B. CD45 + CD19 + lymphocytes were considered as B cells, CD45 + CD3 + lymphocytes were considered as T cells and CD45 + F4/80 + lymphocytes were considered as macrophages. Compared with ND-fed mice, intra-hepatic CD45 + CD19 + B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted up to the 12th week (P < 0.05). CD45 + CD3 + T lymphocytes decreased in the 8th week (P < 0.05) in HFD-fed mice, and CD45 + F4/80 + macrophages did not increase in HFD-fed mice until the 12th week ( Figure 1C). Similarly, B lymphocytes within MAT increased from the 4th week to the 12th week in HFD-fed mice (P < 0.01). T lymphocytes showed no significant differences between the HFD-fed and ND groups at three time points. Macrophages increased in the 12th week (P < 0.01) ( Figure 1D). These data indicated that B lymphocytes infiltrated earlier than macrophages in HFD-fed mice.

| Macrophages and MAT inflammation decreased in B null mice
muMT mice, lacking mature B lymphocytes, were used to confirm the role of B lymphocytes in MAT inflammation. Immunofluorescence staining evidenced the lack of B cells in HFD-fed muMT mice ( Figure 2A). Histological assessment showed that adipocytes were smaller in B null mice than in wild-type mice with HFD feeding ( Figure 2B), and the mRNA expression of TNF-α (P < 0.01), IL-6 (P < 0.05) and MCP-1 (P < 0.01) decreased, and that of IL-10 (P < 0.05) increased in MAT of B null mice ( Figure 2C). Compared to HFD-fed wild-type mice, immunohistochemical staining showed lesser F4/80 + macrophages (P < 0.01) in MAT as well as decreased mRNA expression of F4/80 (P < 0.05) in HFD-fed muMT mice ( Figure 2B,D).

| Infiltrated B lymphocytes might relate to macrophage inflammation and their differentiation into M1 macrophage
As mentioned above, B lymphocytes infiltrated earlier than macrophages in MAT. Besides, immunofluorescence staining showed that some B lymphocytes were located next to macrophages ( Figure 3A). In

| Infiltrated B lymphocytes might contribute to MAT inflammation by regulating macrophage
As far as we know, there was no B cell receptor on the surface of adipocytes; thus, we investigated whether B lymphocytes could affect adipocytes by regulating macrophages. The protocols of co-culture experiments are shown in Figure 4A. Macrophages promoted mRNA expression of IL-6 in mature adipocytes (P < 0.05), and TNF-α or IL-10 levels did not change ( Figure 4B). The mRNA expression of leptin increased (P < 0.05) and that of adiponectin decreased (P < 0.01) after simple macrophage stimulation in mature adipocytes ( Figure 4C).
Compared with simple macrophages, increased mRNA level of TNF-α (P < 0.01) and adiponectin (P < 0.05) in adipocytes were observed in macrophages pre-treated with ND MAT B cells, as well increased IL-6 (P < 0.05), TNF-α (P < 0.01) and adiponectin (P < 0.05) in adipocytes in macrophages pre-treated with HFD MAT B cells, and IL-10 or leptin levels were not significantly different ( Figure 4B,C). Compared

| B lymphocyte within inflamed MAT tended to migrate to the liver
To verify whether B lymphocytes in inflamed MAT could migrate to the liver, we used near-infrared scanning in vivo and transwell chemotaxis assay in vitro. B cells isolated from MAT of HFD-fed mice accumulated in the liver more than those from ND-fed mice, whether the recipients were HFD-or ND-fed mice ( Figure 5A,B).
Besides, B cells from HFD-fed mice seemed to stay longer in the liver than those from ND-fed mice, and this trend did not reduce at 24 hours after injection ( Figure 5A,B). Compared with ND MAT B cells, a higher chemotaxis index (P < 0.01) of HFD MAT B cells to liver homogenates was observed, whether liver homogenates were made from ND or HFD-fed mice (P < 0.01) ( Figure 5C). The results of the transwell chemotaxis assay were consistent with near-infrared scanning in vivo. All these results demonstrated that B lymphocytes in inflamed MAT tended to migrate to the liver.

| B lymphocyte within inflamed MAT promoted inflammation of primary hepatocyte
To test whether B lymphocytes that migrated to the liver had effects on hepatocytes, we conducted a co-culture experiment of MAT B lymphocytes and primary hepatocytes. B lymphocytes had no effects on the ALT or AST level in the cultural supernatant, neither B cells were isolated from ND-fed nor HFD-fed mice ( Figure 6A).

| D ISCUSS I ON
The theory of 'multiple hits' has been proposed to clarify the pathogenesis of NAFLD. 20  In EAT, B lymphocytes were found to be infiltrated early, followed by an increase in T cells and macrophages and finally by the appearance of insulin resistance in HFD-fed mice. 28 Winer et al also evidenced that B cells accumulated in EAT as early as in 3 weeks of HFD feeding. 14  with HFD showed attenuated inflammation of EAT, although hepatic steatosis was similar to that in normal HFD mice. 29 We found that MAT inflammation alleviated and numbers of macrophages decreased in HFD-fed B null mice compared with HFD-fed wild-type mice. It was reported that B6.129 mice seemed to be less susceptible to HFD than C57BL mice. 30,31 Meanwhile, improved adipose tissue inflammation was also found in B cell depletion mice using anti-CD20 antibody, compared with control mice. 14 33 In our previous study, we evidenced that intrahepatic B lymphocytes promoted NAFLD by secreting pro-inflammatory F I G U R E 6 B lymphocyte within inflamed MAT promoted inflammation of primary hepatocyte. A, Aminotransferase of the culture supernatant in the co-culture system of primary hepatocytes and MAT B cells, either from ND-or HFD-fed mice. B, The relative mRNA expression of inflammatory cytokines in hepatocytes, specifically, TNF-α, MCP-1 and IL-10. Values represent means ± SEM, *P < 0.05, **P < 0.01. HFD, high-fat diet; IL, interleukin; MAT, mesenteric adipose tissue; ND, normal diet; TNF, tumour necrosis factor F I G U R E 7 Schematic model for the role of MAT B cell in local inflammation and hepatic inflammation. B lymphocytes increased early in MAT of HFD-fed mice and promoted macrophage differentiation into type I macrophages as well as secretion of more pro-inflammatory cytokines. The effects of B lymphocytes on macrophages resulted in MAT inflammation. Besides, B lymphocytes within MAT could migrate to the liver and promote hepatic inflammation. HFD, high-fat diet; MAT, mesenteric adipose tissue cytokines, enhancing the activation of CD4 + T cells and their differentiation into Th1 cells. 34 Apart from intrahepatic B cells, the effects of B cells from circulation or EAT on metabolic syndrome have also been described in several studies, 35,36 but whether B cells from spleen or EAT can migrate to the liver is unknown. Here, we found that early infiltration of B cells occurred both in the liver and MAT during the development of NAFLD; thus, we speculated that MAT B cells could migrate to the liver and participate in liver injury. In fact, the cycle of immune cells among various organs is the fundamental guarantee of a normal immune response. The capability of gut-and blood-derived B cells to migrate to the liver has been demonstrated, and this phenomenon promotes the development of NAFLD. 18,37 Using adoptive transfer of MAT B cells and near-infrared scanning in vivo as well as transwell assay, we showed that B cells within inflamed MAT that migrated to the liver increased, promoting the inflammation of hepatocytes. Although B cells within MAT of NDfed mice also promoted the hepatocytes to secrete cytokines, nearinfrared scanning in vivo showed few B cells migrating to livers in ND-fed mice.
In this study, we showed that B lymphocytes within MAT participated in NAFLD not only by partly promoting MAT inflammation by regulating macrophages, but also by migrating to the liver and inducing the inflammation of hepatocytes (Figure 7). Considering the early infiltration of B lymphocytes in longitudinal development of NAFLD, we believe that B lymphocytes may play a more important role than that already elucidated. All these results highlight the importance of B cells in the communication between the immune system and inflammation as well as MAT and liver dialogue.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Fund of China (No.81470798, 81500404, 81670787)