Attenuation of obesity and related metabolic disorders by the individual or combination treatment with IL-2/anti-IL-2 complex and hyperbaric oxygen

Obesity is the disease accumulating excessive fat in the body. The prevalence of obesity and related metabolic disorders is increasing every year worldwide. Immunologically, obesity is a chronic low-grade inflammatory state with the increase of M1 macrophages and decrease of regulatory T cells (Tregs). IL-2/anti-IL-2 complex (IL-2C) and hyperbaric oxygen (HBO) are known to expand Tregs in vivo and suppress inflammation. Therefore, in this study, IL-2C and HBO were investigated for the preventive effect of obesity and related metabolic disorders. Male C57BL/6 mice were fed with a high-fat diet (HFD) for 16 weeks, and counterparts were fed with a low-fat diet (LFD). At the end of the experiment, the body weight gain and impaired glucose metabolism, elevated levels of insulin and total cholesterol induced by HFD were improved by the individual or combination treatment with Il-2C and HBO. Histological examination of the epididymal white adipose tissue showed adipocyte hypertrophy and many crown-like structures in the HFD control groups. In addition, the liver showed the progression of non-alcoholic fatty liver disease (NAFLD) in the HFD control groups, but it was significantly improved by the individual or combination treatment with IL-2C and HBO. As for the underlying mechanism, inflammation induced by obesity was decreased, and HIF-1α expression by adipocyte hypertrophy was also reduced by the individual or combination treatment with IL-2C and HBO. In addition, adipose tissue browning was activated in brown and inguinal adipose tissue, and the expression of UCP-1 involved in the thermogenesis was increased by the individual or combination treatment with IL-2C and HBO. Overall, these results suggested that IL-2C and HBO might be a new promising immunotherapy for the treatment of obesity and related metabolic disorders by regulation of inflammation and activation of adipose tissue browning.

shivering thermogenesis [19]. Recently, the brown-like adipocytes were 97 discovered in the WAT and it was called as 'beige or brite' adipocytes [20]. 98 It implies the increase in metabolic activity, and therefore, browning of 99 WAT may be a new strategy of anti-obesity therapy [21]. As obesity  housed in a specific pathogen-free facility at Ewha Womans University.

157
For animal care, a room was controlled with a 12 hours light/dark cycle, 158 50% humidity and ad libitum access to food and water treated with γ-159 irradiation.

160
Animal experiment 161 Seven-week-old C57BL/6J male mice were used in the experiment.

165
The specific aim of this experiment is to investigate if IL-2C and/or 166 hyperbaric oxygen (HBO), that are known to expand Tregs in vivo, 167 attenuate HFD-induced obesity and related metabolic disorders.

168
Accordingly, the mice in the LFD and HFD groups were further divided into 169 5 sub-groups treated with IL-2C, HBO or both, and the control groups      The slides were rinsed again with 60% isopropanol and were stained with     combination of the IL-2C and HBO were significantly lower than those in the controls 307 of the HFD group (Fig 1A). In the LFD groups, at the end of the experiment, the body 308 weights in the group by individual treatment with IL-2C and combination treatment 309 were also significantly lower than those in the controls. The body weights in the 310 group by individual treatment with HBO was also lower but was not statistically 311 significant. There was no significant difference in the food intake between the 312 experimental groups (Fig 1B). At the end of the experiment, blood glucose levels after fasting for 15 hours 320 were significantly higher in the HFD control groups than in the LFD groups, 321 suggesting hyperglycemia was induced (Fig 2A). Individual treatment with IL-2C or 322 HBO decreased blood glucose levels, but without statistical significance, while 323 combination treatment decreased it significantly. IPGTT showed delayed glucose 324 clearance from the blood in the HFD control groups, suggesting dysregulation of 325 glucose metabolism (Fig 2B). In the combination treatment groups, the blood 326 glucose levels were significantly decreased starting from 30 minutes and from 60 327 minutes in each LFD and HFD compared to the control groups (Fig 2C and D).

328
Glucose clearance rate after glucose injection was shortened by the individual or 329 combination treatment with IL-2C and HBO (Fig 2E). In order to investigate if insulin 330 functions appropriately, blood glucose levels were measured after IP injection of 331 insulin. The blood glucose levels after fasting for 4 hours was significantly lower in 332 the HFD groups by individual treatment with HBO or combination treatment (Fig 2F).

333
The levels of blood glucose at 2 hours after insulin injection were showed 334 significantly higher and delayed glucose clearance in the HFD control groups, 335 therefore this result suggests the inappropriate function of insulin ( Fig 2G). Individual 2H and I). Therefore, glucose clearance rate after insulin injection was significantly 340 improved by the individual or combination treatment with IL-2C and HBO in the HFD 341 ( Fig 2J). In addition, serum insulin levels after fasting for 15 hours were significantly 342 higher in the HFD control groups (Fig 2K). Albeit at higher insulin levels,   from the ends of the dermis to muscle layer (Fig 3D and E). The depth of anterior 384 SAT was also significantly thicker in the HFD control groups compared with the LFD 385 groups. Meanwhile, it was significantly thinner in the LFD and HFD groups by 386 combination treatment with IL-2C and HBO compared with that in the control groups. with IL-2C and HBO (Fig 4B). According to the fat accumulation in the liver, the 411 weight of liver was significantly increased by HFD, which were also significantly 412 decreased by individual treatment with IL-2C and combination treatment, but not 413 significantly with individual treatment with HBO ( Fig 4C). There was no significant 414 difference between in the LFD each group. In the HFD control groups, hepatocyte 415 ballooning and Mallory-Denk bodies were also observed, suggesting degenerative 416 changes of the liver (Fig 4D). Lobular inflammation by infiltration of 417 polymorphonuclear leukocytes and lymphocytes also suggested ongoing hepatitis.

418
Masson's trichrome staining shows slight but evident fibrotic changes (Fig 4E). 419 Taken together, it could be argued that non-alcoholic steatohepatitis (NASH) has 420 been induced by HFD, and semi-quantitative analysis also showed the significant 421 increase of NAFLD activity score (NAS) in the HFD control groups (Fig 4F-I). proportion of Treg in the spleen of the HFD control groups was also decreased 468 compared with the LFD groups (Fig 5F). It was significantly increased in both LFD  Another cardinal manifestation of obesity is hypoxia in the WAT. IHC showed 487 scarce expression of HIF-1α in the eWAT of the LFD groups, which was significantly 488 enhanced in the HFD control groups, suggesting hypoxic state (Fig 6A and B). The in the HFD control groups, which was decreased by the individual or combination 494 treatment with IL-2C and HBO (Fig 6C and D).  Histological examination of the iBAT showed small and multilocular features in 506 the LFD groups (Fig 7A). In the HFD control groups, many unilocular hypertrophic 507 adipocytes were observed with infiltration of inflammatory cells, suggesting whitening 508 of adipose tissue. These histological features were less prominent by the individual 509 or combination treatment with IL-2C and HBO of the HFD. The weight of iBAT was 510 also slightly increased in the HFD control groups, compared with the LFD control 511 groups, but without statistical significance (Fig 7B). In the mice treated with the 512 combination of IL-2C and HBO, the weight of iBAT was significantly decreased in 513 both LFD and HFD groups compared with each control groups. IHC showed that the 514 uncoupling protein-1 (UCP-1) was intensely expressed in the iBAT of the LFD 515 groups, which became more intense by the individual or combination treatment with 516 IL-2C and HBO (Fig 7C and D). The UCP-1 expression in the iBAT of the HFD 517 control groups was slightly less intense, which was also strengthened by the 518 individual or combination treatment with IL-2C and HBO.

519
In addition, histological examination of the iWAT of the LFD groups showed 520 homogeneous unilocular adipocytes, it was almost similar to those of eWAT (Fig 7E).

521
However, many small multilocular adipocytes were observed by the individual or  week in the LFD group. At the end of the experiment, decrease of body weight gain 563 was as much as 21.2% and 20.1% in the HFD and LFD groups, respectively.

564
Individual treatment with IL-2C or HBO also reduced weight gain, but the significant 565 difference was always represented by combination treatment with IL-2C and HBO 566 (S1 Table).  individual treatment with IL-2C was different from the expectation (Fig 3B). 629 Nonetheless, glucose metabolism and insulin resistance were significantly improved 630 by individual treatment with IL-2C. Furthermore, the thickness of SAT was 631 significantly increased by HFD, and it was decreased by combination treatment with 632 IL-2C and HBO. It was traditionally considered that VAT was the important organ for   therapeutic strategy for obesity and related metabolic disorders (Fig 8).