Brown adipose tissue ameliorates autoimmune arthritis via inhibition of Th17 cells

The functions of adipose tissue are associated with autoimmune diseases, such as rheumatoid arthritis (RA). Some studies have shown that the three compositions of adipose tissue (white, brown, and beige) have different functions. Brown adipose tissue (BAT) is known to secrete several factors that differ from those in white adipose tissue. This suggests that BAT might have potential positive advantages in the physiology of autoimmune diseases. We compared the functions of collagen-induced arthritis mice-derived BAT (CIA BAT) with normal mice-derived BAT. DBA/1J mice (6–7 weeks of age) were immunized by intradermal injection at the base of the tail with 100 μg of bovine type II collagen (CII) emulsified in complete Freund’s adjuvant. Immunized mice then received booster immunizations by intraperitoneal injection with 100 μg of CII in incomplete Freund’s adjuvant. We transplanted CIA BAT and normal BAT into CIA recipient mice. After transplantation, we measured the functions of CIA BAT and normal BAT in mice. Normal BAT-transplanted mice showed significantly lower scores of bone damage, inflammation, and cartilage damage. The proinflammatory cytokines in normal BAT-transplanted mice, such as IL-12, IL-17, IL-6, and tumor necrosis factor-α (TNF-α), tended to decrease. Microarray analysis showed that the PI3K-AKT signaling pathway and IL-17 levels of CIA BAT tissues were significantly higher than those of normal BAT tissues. These results suggest that the transplantation of normal brown fat may have a therapeutic effect in RA patients.

Recent studies have shown that adipose tissue is composed of three types of tissue: white, brown, and beige 25 . However, because beige fat derives from white adipose tissue (WAT), through a process known as browning of WAT 26 , adipose tissue is generally classified as white or brown 27 . WAT is an energy reservoir for other organs that is capable of adjusting its storage and secretory role to the nutritional state of the organism 28 . The size and quantity of WAT increase to accommodate excess energy when the energy intake exceeds the energy expenditure 29 . Furthermore, WAT releases heat shock protein (Hsp72), macrophage chemoattractant protein (MCP-1), IL-6, and IL-10 when it becomes stressed 30 . In contrast, brown adipose tissue (BAT) is a heat-producing adipose tissue, a specialized organ responsible for thermogenesis, which is primarily found in infants; its abundance decreases with age, such that it is lost in adulthood 31,32 . Unlike WAT, BAT is equipped with abundant mitochondria and uniquely expresses uncoupling protein (UCP)-1 33 , which allows protons produced by lipolysis and glycolysis to reenter the mitochondrial matrix, while producing heat instead of adenosine triphosphate 34 . In addition, BAT is highly innervated by the sympathetic nervous system 35 and contains developed vasculature 36 . BAT is known to secrete adiponectin and IL-6, similar to WAT 37 . However, several factors may be secreted by BAT alone, such as fibroblast growth factor 21 (FGF-21) 38 . FGF-21 is expected to play a vital role in anti-inflammation by downregulating mRNA expression of IL-17, TNF-α, IL-1β, IL-6 39 . Taken together, endocrine factors produced by BAT might have beneficial effects in autoimmune diseases.
We hypothesized that BAT can be associated with treating RA by reducing inflammatory cytokines. IL-23-IL-17 axis is thought to have a critical role in RA 40 . Therefore, we thought that by reducing IL-17, further deterioration of RA could be prevented. Hence, we compared the functions of collagen-induced arthritis (CIA) mice-derived BAT (CIA BAT) with normal mice-derived BAT in this study. We injected CIA mice with CIA BAT or normal BAT and evaluated the therapeutic role of normal BAT in CIA mice.

Results
Experimental RA requires normal BAT activity for therapy. The scheme of the transplantation of BAT was presented (Fig. 1A). Epididymal white adipose tissues (eWAT), normal BAT which was acquired to control mouse and BAT which was derived from CIA mouse (CIA BAT) were showed (Fig. 1B). Compared to mice that received sham surgery or transplantation of CIA BAT, normal BAT transplantation mice had significantly lower arthritis scores and incidence of arthritis throughout the experimental period (Fig. 1C). The joint tissues were presented using H&E and safranin O staining for the disruption of tissues (Fig. 1D). Histological analysis showed that the average scores of bone damage, inflammation, and cartilage damage of the normal BAT group were significantly lower than those of the control and CIA BAT groups (Fig. 1E).
Transplantation of normal brown adipose tissue ameliorates proinflammatory cytokines and impacts anti-inflammatory cytokine secretion. Inflammatory cytokines were observed by immunohistochemistry in murine joint tissues. Levels of proinflammatory cytokines, such as IL-12, IL-17, IL-6, and TNF-α, tended to decrease in normal BAT-transplanted mice. IL-12 significantly increased in CIA BAT-transplanted mice, compared to its levels in normal BAT-transplanted mice. Levels of IL-10, an anti-inflammatory cytokine, significantly differed between sham-and CIA BAT-transplanted mice. However, there were no significant differences between sham-and normal BAT-transplanted mice (Fig. 2). Treg cells did not significantly differ in each group. However, Th17 cells in the normal BAT group tended to be fewer than those in the control and CIA BAT groups (Fig. 3A). Confocal imaging showed significantly fewer CD4 + IL-17 + cells in the spleen tissues of normal BAT-transplanted mice than in the spleen tissues of shamor CIA BAT-transplanted mice. Although CD4 + CD25 + Foxp3 + cells did not significantly differ in each group, CD4 + CD25 + Foxp3 + cells showed a tendency to increase in the normal BAT-transplanted group, compared to the sham-or CIA BAT groups (Fig. 3B).

Brown adipose tissue inhibits IL-17 secretion and strongly induces IL-10 expression.
To determine the expression of IL-17 and IL-10 in each adipose tissue, BAT, epididymal white adipose tissue (eWAT) and CIA BAT were cultured for 72 h. IL-17 and IL-10 levels were detected by ELISA in BAT culture medium and eWAT culture medium, respectively. In prevalent condition, IL-17 of brown fat tissues was lower expressed compared with eWAT, however, IL-10 which is anti-inflammatory cytokine were highly expressed in brown fat tissues. (Fig. 4A). Furthermore, IL-17 expression of eWAT was higher than BAT and IL-10 expression of eWAT was lower than the BAT in the Th17 differentiation condition (Fig. 4B). The level of IL-17 of CIA BAT was more elevated than normal BAT and the level of IL-10 was decreased in CIA BAT compared with normal BAT (Fig. 4C). To investigate BAT decreases the population of Th17, the differentiated-Th17 cells derived from CD4 positive splenocytes were cultured with BAT and eWAT respectively. Although the population of Treg cells showed no significant differences, the population of Th17 cells was decreased in BAT co-culture condition (Fig. 4D,E).

Comparison of gene expression between normal BAT with CIA BAT in murine.
We compared normal mice-derived BAT with CIA mice-derived BAT using a microarray (Fig. 5A). Differentially expressed genes, identified using an absent/present (A/P) classification and ≥ 2-fold difference as cut-offs, constituted 252 of 24,532 genes ( PI3K-AKT signaling-associated genes and IL-17 signaling-associated genes were highly expressed in CIA BAT, compared to normal BAT. According to the Kyoto Encyclopedia of Genes and Genomes, the PI3K-AKT and IL-17 signaling pathways significantly differed between CIA BAT and normal BAT tissues (Table 1). In the PI3K-AKT pathway, Flt1, Pik3r1, Sk1, and Nr4a1 were highly expressed in CIA BAT. Western blotting showed the expressions of PI3K/AKT pathway proteins (Fig. 5F). The expressions of phosphorylated PI3K and AKT was increased in CIA BAT than normal BAT (Fig. 5G). However, Fn1, Col4a5, Col1a1, and Col1a2 (PIK3-AKT signaling-associated genes and focal adhesion molecules) were downregulated www.nature.com/scientificreports/ in CIA BAT. IL-17 signaling pathway-associated genes, such as Jun, S100a8, and S100a9, were highly expressed in CIA BAT.

Discussion
The role of adipose tissue was once presumed to be the storage of fat and protection of the body 41 . However, recent studies have identified other functions of adipose tissue and its association with autoimmune diseases, such as osteoarthritis and RA 42 . Brown adipose tissue, a subtype of adipose tissue, is well known for its heat-producing role 43 . In addition to this role, brown adipose tissue may be beneficial for the treatment of autoimmune diseases.
To determine whether functions differed between normal BAT and CIA BAT, we transplanted normal BAT or CIA BAT into CIA mice. The results showed that normal BAT-transplanted mice had lower arthritis scores and incidence of arthritis than control mice or CIA BAT-transplanted mice. This was confirmed by histological analysis. Safranin O staining showed that the cartilage of normal BAT-transplanted mice was healthier than that of sham-or CIA BAT-transplanted mice. The scores of bone damage, inflammation, and cartilage damage were significantly lower in the normal BAT-transplanted group. Furthermore, compared with the severity of arthritis in the normal CIA model and the sham CIA model, there were no notable differences (data not shown).
The proinflammatory cytokines of normal BAT-transplanted mice tended to decrease relative to those of other experimental groups, as evidenced by immunohistochemistry analysis of mouse joint tissues. IL-12, a Th1 cellstimulating factor 44 , was significantly higher in CIA BAT-transplanted mice than in normal BAT-transplanted mice. Conversely, the expression of IL-10 was significantly lower in CIA BAT-transplanted mice than in control mice. There were no significant changes between sham-and normal BAT-transplanted mice.
Ex vivo flow cytometry and immunofluorescence staining analyses showed that the populations of Foxp3 + Treg cells did not significantly differ among groups. However, the population of Th17 cells was significantly www.nature.com/scientificreports/ model 47,48 . In this study, Th1 cells were determined by flow cytometry in each mouse groups, there were no significant differences respectively (data not shown). Subsequently, IL-17 and IL-10 expression levels in the cell culture medium were measured by ELISA. Notably, the IL-17 expression level in eWAT was significantly higher than that in normal BAT, while the IL-10 expression level in eWAT was significantly lower than that in normal BAT. In Th17-inducing conditions, the expression levels of IL-17 in eWAT and CIA BAT were significantly higher than in normal BAT. IL-10 was highly expressed by normal BAT, but not by eWAT and CIA BAT. Taken together, these findings suggested that CIA BAT was similar to eWAT and distinct from normal BAT.
To determine differences between CIA BAT and normal BAT, we performed microarray analysis, which identified 252 differentially expressed genes. The 10 dominant GO terms of biological processes were identified; seven of these 10 terms were related to the regulation of cellular and biological processes (Fig. 5D). In the Kyoto Encyclopedia of Genes and Genomes enrichment data, 11 of the top 20 enrichment terms were related to human disease (data not shown). These data showed that brown fat in the RA mouse model differs from brown fat in normal mice.
Eight of the 10 dominant GO terms of molecular function were related to protein binding and molecular binding (Fig. 5E). PI3K signaling pathway-associated genes (Flt1, Pik3r1, Sgk1, and Nr4a1) showed increased expression in CIA BAT. Also, we assessed the expression of PI3K and AKT proteins by western blotting. The activated forms of PI3K and AKT were known to be phosphorylated 49,50 . Also, the activated form of PI3K and AKT were known to be increase of inflammation through secreting of pro-inflammatory cytokines such as IL-1β, IL-6 TNFα 51,52 . This study showed that the activated form of PI3K and AKT was increased in BAT of CIA mice than normal. The increased PI3K/AKT signaling is thought to aggravate inflammation in the CIA model. Other genes showed reduced expression in CIA BAT, such as Fn1, Col4a5, Col1a1, and Col1a2. These were both PI3k signaling pathway-associated genes and focal adhesion-associated genes.
The expression of genes in the IL-17 signaling pathway was higher in CIA BAT than in normal BAT (Table 1). Notably, proinflammatory genes (Jun, S100a8, and S100a9) were increased in CIA BAT, compared to normal BAT; these are closely related to inflammation. Taken together, these data indicate that the brown fat of the RA model mouse activates the PI3K pathway and promote inflammatory conditions. However, the reduction of focal adhesion molecules in CIA BAT can reduce cell survival.
Our study showed that normal brown fat had a therapeutic effect on the RA mouse model. However, brown fat derived from CIA mice did not have a therapeutic effect on other CIA mice. Normal brown fat reduced the pathological score of arthritis in CIA mice and reduced the levels of proinflammatory cytokines in tissues. In particular, brown fat tissue showed significant differences in proinflammatory cytokine (IL-17) and an antiinflammatory cytokine (IL-10), compared with epididymal white tissue. Microarray data showed that expression levels in some pathways significantly differed between normal BAT and CIA BAT. Taken together, the brown fat of RA mouse model showed a similar aspect with white adipose tissues. Since normal brown adipose tissue has the ability to regulate immunity, we suggested that the normal brown fat transplantation or the trans-differentiation of white fat to brown fat may have therapeutic effects in RA patients.

Materials and methods
Animals. Male DBA/1J mice (6-8 weeks of age) were purchased from Orient Bio (Seoul, Korea). All mice were reared under specific pathogen-free conditions. All experiments were approved by the Animal Research Ethics Committee of the Catholic University of Korea (permit number: CUMC: 2015-0128-01) and conducted in accordance with the guidelines of the National Institutes of Health.
Type II collagen (CII) immunization and induction of CIA. DBA/1J mice were immunized by intradermal injection at the base of the tail with 100 μg of bovine type II collagen (CII) emulsified in complete Fre-

Assessment of arthritis.
Mice were observed three times per week for the onset, duration, and severity of joint inflammation for 9 weeks after the primary immunization. The severity of arthritis in each paw was graded in accordance with an established scoring system, as follows: 0 = normal, 1 = mild swelling, 2 = moderately severe arthritis involving toes and ankle, 3 = severe arthritis involving an entire paw, and 4 = severe arthritis resulting in ankylosis and loss of the joint; the maximum cumulative score for each animal was 16 53 .
Co-culture of Th17 cells with adipocytes. Sorted Th17 cells were seeded in 24-well plates at 1 × 10 6 per well in 5% FBS added-RPMI and brown/white adipocyte was a layer on to Th17 cells. Cells were incubated at 37 °C for 72 h. 3 days later, the culture supernatants were collected. Transplantation of BAT. BAT was acquired from DBA1/J donor mice (age 7 weeks) and washed in sterile PBS. BAT of donor mice has coated with phenol red-free Matrigel (BD Biosciences). We then transplanted 0.2 g donor BAT into the subcutaneous dorsal region of CIA mice as quickly as possible. The spleen samples of recipient mice were obtained 5 weeks after the transplantation. The sham surgery group underwent the same procedure. The procedure was described in Fig. 1A.

Enzyme-linked immunosorbent assay (ELISA
Microarray analysis. We performed microarray analysis using an Affymetrix Mouse Gene 2.0 ST array for 24,532 genes (Illumina) by Macrogen Co. (Seoul, Korea), in accordance with the manufacturer's instructions.
Statistical analysis. All data were analyzed using the nonparametric Mann-Whitney U test to compare two groups or one-way analysis of variance with Bonferroni's post hoc test for multiple comparisons (≥ 3 groups). GraphPad Prism software (ver. 5.01) was employed for all analyses. A value of P < 0.05 was considered statistically significant. Data are expressed as means ± standard deviations.