Inhibition of the IL-17A axis in adipocytes suppresses diet-induced obesity and metabolic disorders in mice


Ana Teijeiro1, Amanda Garrido1, Anna Ferre1, Cristian Perna2 and Nabil Djouder1, *

1Molecular Oncology Programme, Growth Factors, Nutrients and Cancer Group, Centro Nacional de Investigaciones Oncológicas, CNIO, Madrid, ES-28029, Spain, 2Department of Pathology, Hospital Universitario Ramón y Cajal, IRYCIS, Madrid, 28034, Spain.

*Correspondence:		Nabil Djouder
				Tel:	0034 91 7328000 (Ext.: 3830)
				Fax:	0034 91 2246914
				Email:	ndjouder@cnio.es




Abstract
Overnutrition causes obesity, a global health problem without any effective therapy, characterized by low-grade inflammation, which predisposes individuals to metabolic syndrome via unknown mechanisms. Here, we demonstrate that abolishing the IL-17A axis in mice by inhibition of RORt-mediated IL-17A production by digoxin, or by ubiquitous deletion of IL-17 receptor A (Il17ra) suppresses diet-induced obesity (DIO) and metabolic disorders, and promotes adipose tissue browning, thermogenesis and energy expenditure. Genetic ablation of Il17ra specifically in adipocytes is sufficient to completely prevent DIO and metabolic dysfunctions in mice. IL-17A produced in response to DIO induces PPAR phosphorylation at Ser-273 in adipocytes in a CDK5-dependent manner, thereby modifying expression of diabetogenic and obese genes, which correlates with IL-17A signalling in white adipose tissues from individuals with morbid obesity. These findings reveal an unanticipated role of IL-17A in adipocyte biology, in which its direct action pathogenically reprograms adipocytes, promoting DIO and metabolic syndrome. Targeting the IL-17A axis could be an efficient anti-obesity strategy.

Overweight and obesity (excessive fat accumulation normally caused by chronic overfeeding and/or inadequate physical activity) cause a common disorder called metabolic syndrome, with various comorbidities. These include type 2 diabetes, hypertension, non-alcoholic steatohepatitis (NASH), cardiovascular diseases and cancer, which may lead to death 1. 1.9 billion adults are overweight, 600 million are obese (WHO, 2016), and 51% of the population worldwide are predicted to be obese by 2030 2. Thus, obesity is a major global health problem with increasingly alarming incidence.
Despite efforts to find safe, effective obesity treatments, current options are limited and have not improved in the last 20 years, mainly due to insufficient knowledge about the pathophysiology and mechanisms of fat accumulation. Lifestyle intervention and therapeutic agents targeting appetite reduction or fat absorption typically result in just 10% and 2-7% loss of body weight, respectively. Moreover, many therapeutic agents have been withdrawn due to severe side effects 3. Bariatric surgical procedures are the most effective options for reducing body weight, but are restricted to patients with morbid obesity due to risks of surgical complications, mortality and reoperation 3. Increased understanding of the mechanisms involved is urgently needed to find new drug candidates that boost weight loss and ameliorate metabolic syndrome.
Depending on anatomical location, function and histology, adipose tissue responds to various nutritional inputs to control appetite, glucose homeostasis, insulin sensitivity and body temperature. White adipose tissue (WAT) stores lipids, important energy reserves for the body. Brown adipose tissue (BAT) is a major site of non-shivering thermogenesis and critical for maintaining body temperature regulated by the mitochondria uncoupling protein-1 (UCP-1) 4 which is activated by cold exposure. WAT has extraordinary physiological plasticity and can acquire BAT features characterized by appearance of brown-fat-like (also called beige or brite) adipocytes within white fat depots, namely ‘browning’, known to enhance thermogenesis, energy expenditure (EE) and potentially reduce obesity 5.
Activation of the adipocytes’ β3-adrenergic receptors can lead to UCP-1 expression and browning 4. Moreover, PPARγ phosphorylation at Ser-273 by CDK5 reportedly represses transcription of brown-fat-like genes and induces expression of diabetogenic and obesity genes involved in insulin resistance and obesity 6,7. Thus, modulating these pathways to promote WAT browning, thermogenesis and EE would combat obesity and associated metabolic dysfunctions.
Growing body of evidences indicate that nutrients are inflammatory by themselves 8, increasing IL-17A levels 9, which depend on the activation of the transcription factor Retinoic-acid-receptor (RAR)-related orphan receptor (ROR)  thymus (RORt) 10. IL-17A is a member of the IL-17 cytokine family composed by 6 members (IL-17A, B, C, D, E and F). IL-17A and IL-17F bind to ubiquitously-expressed heterodimeric A/C surface receptors (IL-17RA/IL-17RC) 11. However, IL-17RA shows 100 times more affinity to IL-17A than to IL-17F, but both cytokines bind to IL-17RC with the same affinity 11,12. Recent findings show that genetic ablation of IL-17F and its cognate receptor IL-17RC impair thermogenesis and promote diet-induced obesity (DIO) 13, indicating that IL-17RA activation by IL-17A, rather than IL-17RC ablation, may lead to obesity. IL-17A is also associated with morbid obesity in women 14 and, predisposes to various obesity-associated disorders such as NASH and liver cancer, and autoimmune disorders, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis 9,15-19. These findings suggest potential links between nutrient overload, the IL-17A axis, obesity and metabolic diseases but mechanisms of action remain unknown.

Digoxin prevents DIO and metabolic disorders
We first hypothesize that if the IL-17A axis is involved in DIO and metabolic syndrome, inhibitors of RORt-mediated IL-17A production may be prime therapeutic candidates for these disorders. Different compounds have been shown to bind to the ligand-binding domain of RORt, inhibiting its transcriptional activity, thereby suppressing differentiation of IL-17A-producing cells, and dramatically reducing IL-17A levels 20,21. One of the most specific RORt antagonists, digoxin, a plant-derived cardiac glycoside used to treat human heart failure 22 reportedly affects IL-17A production 9,20,21. The potential therapeutic value of inhibiting IL-17A signalling in obesity and associated metabolic disorders was explored by simultaneously treating C57BL/6 mice with a high-fat diet (HFD) and varying doses of digoxin in drinking water (Fig. 1a). HFD increased number of IL-17A-producing inflammatory cells in blood and WAT, which resulted in high levels of IL-17A, but not of IL-17C, IL-17E and IL17F. These increases were reduced after 1 week of digoxin treatment (Fig. 1b-d and Extended Data Fig. 1a, b). Moreover, measurements of target genes 20,23 confirmed that digoxin antagonizes RORt transcriptional activity without affecting ROR in WAT (Extended Data Fig. 1c).
Digoxin dose-dependently prevented body weight gain (Fig. 1e, f). Notably, 10 g/ml sufficed to completely prevent obesity, and the half-maximal effective concentration (EC50) was 1.57 g/ml (Fig. 1g). Decreased adiposity was accompanied by reduced fat mass and gonadal and inguinal WAT (gWAT and iWAT), and BAT weights (Fig. 1h-l). Lean mass was also slightly reduced, possibly due to reduced hepatomegaly since bone mineral density (BMD) was not affected (Extended Data Fig. 1d-f). 
Further characterization revealed that digoxin had other metabolic beneficial effects. Haematoxylin and Eosin (H&E) and Oil Red O (ORO) staining showed that it prevented HFD-induced hepatic lipid accumulation (Fig. 1m, n). It also decreased Sirius Red (SR) staining and alanine aminotransferase (ALT) levels (Fig. 1m, o, p), indicating reduced fibrosis and liver injury. Moreover, digoxin prevented HFD-induced hypercholesterolemia (Fig. 1q) and diabetes, manifested by reduced fasting glucose levels and improved glucose and insulin tolerance test results (Fig. 1r-t). Importantly, at 30 g/ml, it completely restored glucose homeostasis, indicating that digoxin dose-dependently reduces features of metabolic syndrome.

Digoxin suppresses DIO and metabolic disorders
HFD-fed C57BL/6 mice were therefore treated with 30 g/ml of digoxin in drinking water when obesity peaked 12 weeks later (Fig. 2a). Digoxin-treated obese C57BL/6 mice gradually, but strikingly, lost 41% of their body weight after 1 month of treatment, eventually reaching the basal body weight of normal diet (ND)-fed mice. ND-fed mice also showed a slight but significant reduction in body weight below the basal level with digoxin treatment (Fig. 2a, b). Moreover, digoxin-induced body weight loss was maintained until the treatment ended, suggesting that chronic treatment does not induce resistance. However, removal of digoxin from drinking water after 3 months of treatment (digoxin OFF) progressively but completely reversed body weight loss (Fig. 2a, b).
Importantly, injection of recombinant IL-17A (rIL-17A) in digoxin-treated HFD-fed C57BL/6 mice reinstated body weight gain (Fig. 2c), but rIL-17C, rIL-17E or rIL-17F did not show any effects (Extended Data Fig. 2a), supporting that digoxin effects are mediated through the inhibition of RORt and subsequent reduction of IL-17A production. To confirm this, Rorc-Cre mice 24 were crossed with Cre-inducible diphtheria toxin receptor (DTR) transgenic mice 25 (Extended Data Fig. 2b), generating offspring designated as Rorc-Cre and Rorc-Cre-DTR. Adult mice were treated with HFD and diphtheria toxin (DT) to induce obesity and deplete RORt-positive cells, respectively (Fig. 2d). Obesity was prevented in Rorc-cre-DTR mice (Fig. 2d). Moreover, Rorc-Cre-DTR mice treated with digoxin had similar body weights than non-treated Rorc-Cre-DTR mice (Fig. 2d), confirming that digoxin antagonizes RORt.
Another mechanism of action of digoxin is the inhibition of sodium-potassium adenosine triphosphatase (NA+/K+ ATPase) pump, which is mainly expressed in the myocardium. However, mice express a cardiac glycoside-resistant enzyme due to mutations in the NA+/K+ ATPase 1 subunit (Atp1a1) 26. Yet, to confirm that digoxin supresses weight gain by inhibiting RORt and not by inhibition of the NA+/K+ ATPase pump, we treated C57BL/6 mice with ouabain, a specific inhibitor of the NA+/K+ ATPase pump, which does not bind and inhibit RORt 21. Expectedly, ouabain did not reduce body weight gain in HFD-fed mice (Extended Data Fig. 2c). Thus, digoxin suppresses body weight gain by inhibiting RORt-mediated IL-17A production.
Decreased body weight in digoxin-treated HFD-fed mice was accompanied by reductions in adiposity with reduced fat mass, and gWAT, iWAT and BAT weights (Fig. 2e-i). Lean mass and liver weight were also reduced without altering BMD (Extended Data Fig. 2d-f). Moreover, digoxin treatment ameliorated HFD-induced steatosis, liver injury, hypercholesterolemia and diabetes in a time-dependent manner, and concomitantly to weight loss starting after 1 week of treatment (Fig. 2j-q, Extended Data Fig. 2g-m), suggesting that body weight loss and metabolic changes arise in parallel at early time, and hence might be controlled simultaneously by one common molecular event. Furthermore, genetic depletion of RORt-positive cells improved glucose tolerance and insulin sensitivity in HFD-fed mice (Fig. 2r, s). Conversely, injection of rIL-17A in digoxin-treated HFD-fed C57BL/6 mice restored glucose intolerance and insulin resistance (Fig. 2t, u). Thus, digoxin leads to early and simultaneous body weight loss and metabolic changes via inhibition of IL-17A axis.
Further histological characterization of key organs indicated no signs of pathology or tissue dysfunctions with chronic digoxin treatment (4.5 months of treatment) (Extended Data Fig. 2n). Moreover, measurement of creatinine and ammonia (NH3) levels suggested that digoxin did not induce renal or hepatic toxicity, respectively (Extended Data Fig. 2o, p). Finally, behavioural tests revealed that 1 month of digoxin treatment was sufficient to significantly improve motor coordination and balance, which were impaired in obese mice. However, no differences in cognitive functions were detected (Extended Data Fig. 2q-t). 

RORt inhibitors suppress DIO and metabolic disorders
We also checked the effects of GSK805 and TMP778, potent RORt inhibitors previously tested in mice 20. Like digoxin, GSK805 and TMP778 efficiently prevented or suppressed DIO and metabolic dysfunctions (Fig. 3 and Extended Data Fig. 3a-s). We also compared effects of digoxin, GSK805 and TMP778 with other anti-obesity drugs reported in preclinical mouse models of DIO (Extended Data Fig. 3t) 27-32. Digoxin prevention and treatment respectively resulted in a gradual, reversible 46% and 41% reduction in body weight to a weight similar to that of non-obese mice (ND-fed mice) after 1 month, and stabilization over 8 months with no toxic side effects. Likewise, GSK805 and TMP778 had potent effects on body weight loss. The CNIO PI3K inhibitor (CNIO-PI3Ki) also reportedly elicited reversible responses, but was less efficient for reducing body weight 28. Moreover, treatment with celastrol, considered the most efficient pharmacological agent for treating obesity in mice, induced a rapid, drastic body weight reduction of 68%, however, to below the normal weight of non-obese mice, after 21 days, and reversibility has not been reported (Extended Data Fig. 3t) 30. Therefore, digoxin seems to afford full remission of obesity with reversible effects and possibly no toxic side effects in mice, and may represent a quickly translatable drug for obesity.

Deletion of IL-17RA prevents DIO and metabolic disorders
Next, we ubiquitously suppressed IL-17RA signalling by crossing Il17ra-flox mice with hUBC-CreERT2 mice 33,34 (Extended Data Fig. 4a). Adult mice were fed a tamoxifen diet for 2 weeks to produce Il17ra(+/+) and Il17ra() offspring, which were later provided the HFD to induce obesity (Fig. 4a). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of various organs confirmed efficient ablation of Il17ra in Il17ra() mice (Extended Data Fig. 4b). Remarkably, HFD-fed Il17ra() mice were prevented against DIO, and no striking body weight differences were observed when compared to ND-fed Il17ra(+/+) mice (Fig. 4b-d). Moreover, digoxin-treated HFD-fed Il17ra() mice had similar body weights to digoxin-treated HFD-fed Il17ra() littermate controls and no significant additional effects were observed (Fig. 4b), confirming that digoxin action is mediated through inhibition of IL-17A signalling.
As seen in digoxin-treated HFD-fed mice, decreased adiposity in HFD-fed Il17ra() mice was accompanied with reductions in total body fat mass and, gWAT, iWAT, and BAT weights (Fig. 4e-i). Lean mass and hepatomegaly were also reduced, without changes in BMD (Extended Data Fig. 4c-e). Additionally, pervasive deletion of IL-17RA prevented HFD-induced steatosis, liver injury, hypercholesterolemia, glucose intolerance and insulin resistance (Fig. 4j-q). Thus, ubiquitous genetic ablation of Il17ra prevents DIO and metabolic disorders, recapitulating digoxin effects and suggesting that IL-17A signalling is critical for their onset.

Inhibition of IL-17A axis promotes browning and thermogenesis
To elucidate physiological mechanisms involved in body weight reduction resulting from inhibition of IL-17A signalling, we first examined energy intake and expenditure in our mouse models. HFD-fed C57BL/6 mice subjected to digoxin treatment or Il17ra gene ablation did not alter food intake during the first month of inhibition of IL-17A axis (Extended Data Fig. 5a, b). However, chronic treatment with digoxin (4.5 months of treatment) led to an increase of food intake, but no changes were observed in Il17ra() mice after long-term depletion (Extended Data Fig. 5c, d). Moreover, impaired drink intake or any other signs of dehydration were not detected in these mice (Extended Data Fig. 5e-g). In line with these findings, digoxin in drinking water prevented DIO with the same efficiency than its intraperitoneal administration (Extended Data Fig. 5h). Additionally, pharmacological or genetic inhibition of IL-17A signalling did not induce alterations in locomotor activity and respiratory exchange ratios (RERs) compared to non-treated obese mice or littermate controls, respectively (Extended Data Fig. 5i-l). Notably, digoxin-treated or IL17RA-deficient HFD-fed mice had a RER less than 0.8 (Extended Data Fig. 5k, l), indicating that the main energy sources are lipids in HFD-fed mice, and carbohydrates in ND-fed mice.
Mechanisms of hyperphagia observed with digoxin treatment after complete weight loss might reflect a properly functioning regulatory system, and hence, might be secondary and compensatory to increased EE, as previously reported 35. We therefore monitored EE in digoxin-treated and IL-17RA-deficient mice. When normalized to body mass, EE was increased at early (1 week) and late stages (4.5 months) of digoxin treatment (Extended Data Fig. 5m-p), as well as in IL-17RA-deficient mice (Extended Data Fig. 5q, r). Since body mass could influence energy balance in each group of mice, we reanalysed EE by applying the guidelines and analytical procedures proposed by Tschop et al.36 and Mina et al. 37. The regression plot representing EE and body weight for 1-week digoxin-treated mice combined with the use of analysis of variance (ANOVA) with interaction (also called analysis of covariance (ANCOVA) for non-parallel slopes) showed that each group had different associations between EE and body mass (Fig. 5a-c and Supplementary Table 1a), impeding the additional use of “conventional” ANCOVA. The direct application of Tukey’s post-hoc test revealed that 1-week digoxin-treated C57BL/6 mice had greater energy dissipation in comparison to non-treated mice (Fig. 5b and Supplementary Table 1b). Notably, at this stage, body weight differences between the groups were minimal (ND versus ND + digoxin = 2.87 grams; HFD versus HFD + digoxin = 3.14 grams). However, after long-term treatment with digoxin (4.5 months), the use of ANOVA with interaction showed that the different groups had similar association between EE and body mass (the slopes of EE on mass were the same for each group and no significant differences were detected) (Extended Data Fig. 5s-u and Supplementary Table 1a). In this case, the appropriate application of “conventional” ANCOVA using body weight as covariate followed by Tukey’s post-hoc test indicated that EE did not change between the different groups (Extended Data Fig. 5t and Supplementary Tables 1c, d), most likely due to the substantial body weight differences detected between the groups (ND versus ND + digoxin = 7.47 grams; HFD versus HFD + digoxin = 13.72 grams). Interestingly, HFD-fed Il17ra()  mice showed increased EE when compared to ND-fed Il17ra(), as evaluated by “conventional” ANCOVA followed by Tukey’s post-hoc comparison (Fig. 5d-f and Supplementary Tables 1a, c, d). Hence, digoxin-treated mice dissipate energy at early stages and before a complete and significant body weight loss.
H&E, immunohistochemistry and Western blot (WB) analysis of gWAT and iWAT from digoxin-treated and IL-17RA-deficient C57BL/6 mice revealed increased UCP-1 expression localized in regions of multilocular adipocytes, a characteristic feature of WAT-browning (Fig. 5g, h and Extended Data Fig. 5v). Increased UCP-1 staining and fatty acid oxidation were also detected in the BAT of digoxin-treated and IL-17RA-deficient mice fed or not with HFD (Fig. 5g-j and Extended Data Fig. 5w). Importantly, infrared thermography showed that 1-week digoxin treatment increased WAT, BAT and body temperatures (Fig. 5k-n), indicating enhanced thermogenesis. To further assess UCP-1-positive adipocyte functionality, ND- and HFD-fed C57BL/6 mice were treated (or not) with digoxin for 1 month then exposed to cold (4 ºC) for 24 h (Fig. 5o). After acute cold exposure, body temperatures of digoxin-treated mice increased (Fig. 5p), suggesting that digoxin has potent thermogenic effects. Predictably, thermogenesis after cold exposure was stronger in HFD-fed mice than in ND-fed mice, and further enhanced following digoxin treatment (Fig. 5p) 38. Cold-exposed digoxin-treated mice expressed more UCP-1 in WAT and BAT than cold-exposed controls (Extended Data Fig. 5x), suggesting non-shivering thermogenesis. Importantly, long-term cold exposure-induced adaptive thermogenesis accelerated body weight loss in digoxin-treated HFD-fed mice (Fig. 5q, r). These findings suggest that inhibiting IL-17A axis by digoxin or ablation of IL-17RA increases UCP-1 expression in adipose tissues, inducing thermogenesis and EE, potentially leading to body weight loss.

IL-17A mediates obesity independently of the leptin axis
Next, we investigated the mechanisms of action by which IL-17A signalling promotes obesity. Patients with obesity have high blood levels of leptin, an adipokine that inhibits food intake in response to nutrient stimuli, but these patients have defective leptin signalling, probably due to leptin resistance 39. Accordingly, both leptin-deficient (Lepob(-/-)) mice  and leptin-receptor-deficient (Leprdb(-/-)) mice are hyperphagic and obese 40,41. We therefore studied whether IL-17A signalling promotes obesity through regulation of the leptin signalling pathway. Strikingly, Lepob(-/-) or Leprdb(-/-) mice did not respond to digoxin (Extended Data Fig. 6a-d), and mice obtained by crossing Lepob(-/-) and Il17ra() mice had similar body weights to Lepob(-/-);  Il17ra() littermates (Extended Data Fig. 6e). In line with these findings, administration of recombinant leptin (rLeptin) to digoxin-treated, HFD-fed C57BL/6 mice induced unremarkable weight loss (Extended Data Fig. 6f), suggesting that inhibiting IL-17A signalling does not alleviate obesity-induced leptin resistance. Likewise, digoxin treatment did not increase weight loss in rLeptin-injected Lepob(-/-) mice (Extended Data Fig. 6g). Interestingly, HFD exacerbated obesity in Lepob(-/-) mice, which decreased their body weight with digoxin (Extended Data Fig. 6h). Therefore, IL-17A signalling promotes obesity independently of the leptin axis. These data are supported by previous findings showing that leptin-deficient mice display chronic thymic atrophy with reduced functions 42 and, leptin is required for T cell differentiation and IL-17A production 43,44, indicating that leptin deficiency induces IL-17A signalling-independent obesity.

Digoxin does not prevent DIO via myeloid cells’ activation
Low-grade inflammation is an obesity hallmark. CD8+ T-cells contribute to macrophage recruitment to cause WAT inflammation and insulin resistance in obese mice 45. Moreover, myeloid cell ablation normalizes insulin sensitivity in obese animals 46, and IL-17A recruits granulocytes to WAT, provoking insulin resistance via elastase secretion 9. Consequently, we checked whether IL-17A signalling inhibition reduces frequencies of inflammatory cells, particularly WAT macrophages and neutrophils, which might cause body weight gain and insulin resistance at obesity onset. B-cell, T-cell, macrophage, and granulocyte infiltration of WAT in HFD-fed mice decreased after digoxin treatment, suggesting that decreased obesity is accompanied by reduced general inflammation (Extended Data Fig. 6i-m). Therefore, we specifically blocked IL-17A signalling in myeloid cells by crossing Il17ra-flox 34 and LysM-Cre 47 mice. The offspring, designated Il17ra(+/+)Myeloid and Il17ra(Δ/Δ)Myeloid had similar body weights when fed with HFD, but HFD-fed Il17raMyeloid mice lost body weight in response to digoxin (Extended Data Fig. 6n-q). These findings indicate that IL-17RA signalling may not influence DIO onset via myeloid cells’ activation.

Genetic ablation of Il17ra in adipocytes prevents DIO
Further research indicated that digoxin treatment of HFD-fed C57BL/6 mice did not alter intestinal architecture (see Extended Data Fig. 2n) and permeability, faecal excretion, faecal caloric density or macronutrient absorption (Extended Data Fig. 7a-d), suggesting that digoxin does not perturb intestinal homeostasis or nutrient processing. Moreover, systemic free fatty acid levels and hepatic oxidation were not altered (Extended Data Fig. 7e, f). However, HFD-fed mice that were either treated with digoxin or had IL-17RA ubiquitously deleted showed significant reductions in adipocyte size, and improvement in adipocyte function according to leptin secretion measurements in fasting-refeeding experiments (Extended Data Fig. 7g-l), suggesting a direct action of IL-17RA on adipocytes, potentially impairing its plasticity and functionality.
We therefore generated mice with IL-17RA-deficient adipocytes by crossing Il17ra-flox and Adipoq-Cre mice 34,48 generating offspring designated Il17ra(+/+)Adipoq and Il17ra()Adipoq (Extended Data Fig. 7m). HFD-fed Il17ra()Adipoq mice strikingly prevented body weight gain, mimicking digoxin treatment and ubiquitous IL-17RA deletion, despite only ca. 50% efficiency of Il17ra elimination in their adipose tissues, as shown by qRT-PCR (Fig. 6a-c and Extended Data Fig. 7n, o). Notably, digoxin treatment did not further decrease body weight of Il17ra()Adipoq mice (Fig. 6a). Decreased body weight in HFD-fed Il17ra()Adipoq mice was accompanied by reductions in adiposity with reduced fat mass, gWAT, iWAT and BAT weights (Fig. 6d-h). As seen with other models, lean mass and hepatomegaly were slightly reduced, but no changes were detected in BMD (Extended Data Fig. 7p-r). Additionally, adipose tissue analysis of Il17ra()Adipoq mice showed increased browning gene expression, WAT browning and increased BAT activity as shown by UCP-1 expression (Fig. 6i, j and Extended Data Fig. 6s). Accordingly, WAT, BAT and body temperatures were increased in Il17ra()Adipoq mice (Fig. 6k-n). Moreover, as seen in digoxin-treated or IL-17RA-deficient obese mice, adipocyte-specific IL-17RA deletion prevented HFD-induced hepatic steatosis, hypercholesterolemia, glucose intolerance and insulin resistance (Fig. 6o-s). Thus, ablating IL-17RA on adipocytes prevents obesity and metabolic dysfunctions.

IL-17A phosphorylates PPAR in adipocytes to promote obesity
IL-17A axis inhibition causing reduction in adipocyte size, increased leptin secretion, and WAT browning suggests that PPAR signalling is affected in adipocytes 6,7. In agreement with recent indications that IL-17A contributes to propagation of inflammation but does not impair adipogenesis in individuals with obesity 49, PPAR target genes involved in lipogenesis or differentiation were not changed (Extended Data Fig. 7t, u). Therefore, we hypothesize that IL-17A activates adipocytes to regulate a subset of genes modulated by PPAR, presumably phosphorylation-dependently. We used 3T3-L1 fibroblast-differentiated adipocytes to further explore IL-17A’s potential role in PPAR signalling. Differentiated 3T3-L1 adipocytes expressed significantly higher levels of Il17ra than undifferentiated 3T3-L1 cells (Fig. 7a). Stimulating differentiated adipocytes with rIL-17A increased expression of the IL-17A target gene lipocalin-2 (Lcn2), dose- and time-dependently (Fig. 7b), suggesting that IL-17A signal transduction occurs in differentiated adipocytes. WB analysis showed that rIL-17A enhanced PPAR phosphorylation on Ser-273, but roscovitine-induced inhibition of CDK5 6,7 suppressed PPAR phosphorylation, without affecting ERK activity (Fig. 7c). Consequently, decreased target genes of phosphorylated PPAR involved in protection against obesity, insulin resistance and metabolic syndrome (adiponectin (Adipoq) and adipsin (Cfd)) 6,50-53 were detected in IL-17A-stimulated differentiated adipocytes (Extended Data Fig. 7v).
Moreover, HFD-induced PPAR phosphorylation on Ser-273 in WAT was substantially reduced in digoxin-treated HFD-fed C57BL/6, Il17ra()Adipoq, and roscovitine-treated HFD-fed Il17ra(+/+)Adipoq and Il17ra()Adipoq mice, but ERK phosphorylation was not affected (Fig. 7d-f). Remarkably, PPAR phosphorylation in WAT was completely suppressed already after 1 week of digoxin treatment, and rIL-17A treatment reinstated this phosphorylation (Fig. 7f). A subset of WAT genes which repression induces obesity and insulin resistance and depends on PPAR Ser-273 phosphorylation was also significantly decreased in HFD-fed mice 6. However, digoxin treatment and ubiquitous or adipocyte-specific IL-17RA deletion significantly reinstated expression of this subset in HFD-fed mice (Fig. 7g-i, Extended Data Fig. 7w). Pharmacological inhibition of CDK5-mediated PPAR phosphorylation on Ser-273 by roscovitine prevented obesity, and induced browning in HFD-fed Il17ra(+/+)Adipoq mice, as previously shown 7, but did not have additional effects in HFD-fed Il17ra()Adipoq mice (Fig. 7d, e, j, k). Taken together, these data reveal a previously undescribed function of IL-17A which reprograms metabolically adipocytes, and represses browning as well as modulates expression of diabetogenic and obese genes through CDK5-dependent PPAR phosphorylation on Ser-273 (Fig. 7l).

IL-17A signalling correlates with obese genes in human WAT
To assess our findings’ clinical relevance, we analysed visceral WAT from 75 individuals with morbid obesity (class III, body mass index, BMI > 40) (Supplementary Table 2). Levels of adiponectin (ADIPOQ) and adipsin (CFD) genes, which are downregulated by phosphorylated PPAR and implicated in protection against obesity and metabolic syndrome 6,50-53, were negatively correlated with patient BMI (Extended Data Fig. 8a, b). Moreover, expression of IL-17A and its target gene lipocalin-2 (LCN2), positively associated with body weight (Extended Data Fig. 8c-e), and negatively correlated with ADIPOQ and CFD expression (Extended Data Fig. 8f-i). Consistently with previous indications of enhanced IL-17A expression in WAT of women with morbid obesity 14, expression of ADIPOQ, CFD, IL17A, and LCN2 was also associated with class III obesity in women (Extended Data Fig. 8j-r). However, IL-17C, IL-17E and IL-17F expressions did not correlate with obesity (Extended Data Fig. 8s-u). Thus, IL-17A signalling is associated with a transcription signature of PPAR phosphorylation at Ser-273 in individuals with obesity, and targeting IL-17A axis might counteract adipokine dysregulation, and consequently fat mass gain and metabolic disorders.
We therefore conducted a retrospective population-based epidemiological study to assess the association between digoxin, body weights and metabolic syndrome. BMI and metabolic parameters, including cholesterol levels, of 6,352 individuals aged 35 to 79 years were recorded, 58 (0.91%) of whom were taking digoxin. Associations between digoxin use and both anthropometric and laboratory variables were assessed by multivariate linear regression analyses. Strikingly, digoxin use correlated negatively with total cholesterol and low density lipoprotein cholesterol (LDL-C) levels. Unfortunately, BMI was not significantly correlated with digoxin use, presumably because of high liquid retention in individuals with heart failure (> 20 litres) 54 (Extended Data Fig. 8v, w).

Discussion
We present here strong evidences that IL-17A plays a critical role in DIO and metabolic disorders. Nutrient overload increases IL-17A 9, and inhibiting the IL-17A axis in mice, by suppressing RORt activity by digoxin or by genetically ablating IL-17RA on adipocytes, prevents and suppresses DIO and metabolic alterations. IL-17A triggers CDK5-dependent PPAR phosphorylation at Ser-273 in adipocytes, thereby repressing WAT browning and BAT activity, as well as genes implicated in protection against obesity and insulin resistance. Digoxin treatment in obese mice completely suppresses phosphorylation of PPAR after 1 week, before complete body weight loss and restoration of glucose tolerance and insulin sensitivity. Moreover, 1 week-digoxin treatment simultaneously reduces body weight and improves metabolic dysfunctions, suggesting that metabolic changes are not a consequence of body weight loss, but both might be regulated by IL-17A-induced PPARphosphorylation in adipocytes. Altogether, our data reveal a previously undescribed function of IL-17A signalling in adipocyte biology, which direct action changes adipocyte metabolic genetic landscape, and promotes obesity and metabolic disorders in mice.
Digoxin and other RORt inhibitors prevent and suppress body weight at a level similar to the one detected by pervasive IL-17RA deletion or ablation in adipocytes. Additionally, genetic depletion of RORt-positive cells in mice leads to complete suppression of body weight gain when fed with HFD. Furthermore, the non-additive effects of digoxin on body weight loss in mice with genetic inhibition of IL-17A axis demonstrate that digoxin suppresses obesity and metabolic disorders by inhibiting RORt-mediated IL-17A production. Importantly, injection of rIL-17A in HFD-fed C57BL76 mice treated with digoxin restores body weight gain and metabolic dysfunctions.
Deficiencies of IL-17F and its receptor IL-17RC have been recently linked to defective thermogenesis and body weight gain, whereas pervasive ablation of IL-17RA had apparently no effects on body temperature, measured over 3 h maximum of cold exposure 13. Here, we show that inhibition of IL-17A signalling induces a thermogenic response after 6 h of cold exposure and no responses were detected during the first 3 h (Fig. 5p). Since IL-17A has more affinity to IL-17RA than IL-17F, activation of IL-17RA by IL-17A rather than deletion of IL-17RC impairs thermogenesis and promotes obesity and metabolic dysfunctions. These data are also supported by the fact that recombinant IL-17C, IL-17E and IL-17F do not reinstate body weight in digoxin-treated HFD-fed mice, and these cytokines are not correlated with obesity in human WAT (Extended Data Fig. 2a and Extended Data Fig. 8s-u).
Recent controversial data reported by Kohlgruber et al. show that mice with global deletion of IL-17A since embryonic stage (IL-17A-/- mice) have impaired thermoregulation 55. Our conclusions, which are supported by consistent data and phenotypes from a broad set of genetic mouse models and pharmacological treatments, highlight the complexity and dual roles of IL-17A axis during development and adult stage. Importantly, Kohlgruber et al. compare metabolic features of IL-17A-/- mice to wild-type C57BL/6 mice 55, and not to their littermate controls. Impaired thermoregulation in IL-17A-/- mice could be attributed to different metabolic phenotypes observed in different breeder lines, as previously reported 56.
Unfortunately, the population-based epidemiological study demonstrates that digoxin has no effects on BMI, despite a significant decrease in cholesterol levels. This lack of effects on body weight loss in human can be explained by the huge liquid retentions in individuals with heart failure (> 20 litres), most likely interfering with body weight measurements, and possibly influencing the final data  54. Moreover, we demonstrate that body weight loss in mice is dose-dependent, and perhaps increasing the digoxin dose in humans might induce weight loss. Indeed, according to current recommendations for dose conversion 57, digoxin doses used to treat humans are approximately 6-fold lower than doses used to treat mice (1-2 mg/kg), suggesting that they are suboptimal for weight loss (Supplementary Table 3 and https://www.pdr.net/drug-summary/Digoxin-digoxin-724). Although digoxin has a narrow therapeutic window, the human dose corresponding to the standard mouse dose is still below the toxic concentration. Additionally, digoxin titrations we conducted show that weight loss can be obtained even with a 3-fold reduction in the standard mouse dose, which still corresponds to a higher human dose than currently administered. Therefore, a 3-fold increase in digoxin doses could be considered to investigate the potential therapeutic effects of digoxin in human obesity and metabolic syndrome. Clearly, launching a new epidemiological study in a global population with obesity would be more informative and interesting to conduct. Importantly, analysis of WAT from patients with morbid obesity demonstrates a significant correlation between IL-17A signalling and expression of diabetogenic and obese genes targeted by phosphorylated PPAR, suggesting that IL-17A could be targetable in human obesity.
Since no effective medical treatments for obesity and metabolic syndrome are available, and development of new drugs may be time-consuming and costly, analogues of digoxin or other RORt/IL-17A axis inhibitors may represent metabolically potent and effective therapeutic options. Despite digoxin induces body weight loss in mice that have a normal body weight, it improves motor coordination and balance in mice, indicating that digoxin may be a safe treatment with no detectable toxic effects. It is also a readily-available and quickly translatable candidate drug, which could be considered for clinical trials for obesity. However, it is noteworthy to consider, that due to its reversible effects, patients with obesity might require a continuous therapy, which could lead to thymic involution and impair immune cell functions, increasing susceptibility to pathogenic infections. Hence, it is tempting to propose that digoxin should be taken for a short time period by patients with obesity, until body weight loss is stabilized, then followed by a healthy diet and interventional programs. Yet, digoxin could be also used to improve insulin sensitivity and/or hypercholesterolemia, independently of treating obesity.
Additional information
Materials are available upon request to N.D. and might be subjected to standard material transfer agreements. 

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Acknowledgments
We are very thankful to Drs. Roberto Elosua and Isaac Subirana for collecting and analysing the epidemiological data (Hospital del Mar Medical Research Institute, Barcelona). We particularly thank the biostatistician Claudia Coscia for discussing the statistical analysis of EE. We are grateful to the CNIO Biobank for helping us to collect WAT from patients and associated clinical data. We particularly acknowledge the patients enrolled in this study for their participation and the Aragon Health Sciences Institute in the framework of the Biobank of the Aragon Health System for its collaboration. We are also thankful to M. Malumbres for critical reading of this manuscript, and to the CNIO Mouse Genome Editing Core Unit and Animal Facility for the mouse re-derivation and maintenance, respectively. This work was funded by the European Foundation for the Study of Diabetes (EFSD) award supported by EFSD/JRDF/Lilly programme (EASD 96103), the Pfizer Foundation, and the State Research Agency (AEI, 10.13039/501100011033) from the Spanish Ministry of Science and Innovation (projects SAF2016-76598-R, SAF2017-92733-EXP and RTI2018-094834-B-I00), cofunded by European Regional Development Fund (ERDF). This work was developed at the CNIO funded by the Health Institute Carlos III (ISCIII) and the Spanish Ministry of Science and Innovation. The authors declare no conflict of interest.

Author contributions 
A.T. and N.D. designed the experiments. A.T. performed most of the experiments and statistical analyses. A.G. helped in processing and analysing human WAT samples. A.F. helped with some experiments performed in 3T3-L1 cells. C.P. analysed histopathologically mouse tissues. A.T. and N.D. analysed the data. N.D. conceived, developed, and wrote the project, study and manuscript with A.T.. N.D. secured all funding.

Competing interests 
The authors declare no competing financial interests.



Figure legends
Fig. 1 | Digoxin prevents DIO and metabolic disorders. a, Scheme of HFD and digoxin treatments since 8 weeks in C57BL/6 mice. b, c, Flow cytometry of IL-17A+ immune cells in blood (b) (n = 3, 4, and 3 mice; p = 0.0188; 0.0044, from left to right) and WAT (c) (n = 4 mice; p = 0.0175; 0.0239, from left to right) from ND- or HFD-fed-C57BL/6 mice treated with/without digoxin for 1 week. d, qRT-PCR in spleen of ND- or HFD-fed-38-week-old C57BL/6 mice treated with/without digoxin (n = 5 mice; p = 0.0091; 0.0016, from left to right). e, Weights of HFD-fed-C57BL/6 mice treated with/without digoxin (n = 5 mice; p = 0.0047; <0.0001, from left to right). f, Weights of HFD-fed-24-week-old C57BL/6 mice treated with/without digoxin, relative to the weight of HFD-fed-C57BL/6 mice (n = 5 mice; p = 0.0077; <0.0001; <0.0001, from left to right). g, Dose-response curve and EC50 of weight loss efficiency induced by digoxin in HFD-fed-C57BL/6 mice (n = 5 mice). h, Percentage of total fat in mice from (f) (n = 5 mice; p = 0.0125; <0.0001; <0.0001, from left to right). i, Total fat mass in mice from (f) (n = 5 mice; p = 0.0021; <0.0001; <0.0001, from left to right). j-l, gWAT (j) (p = 0.0067; <0.0001, from left to right), iWAT (k) (p = 0.0388; 0.0003; 0.0002, from left to right), and BAT (l) (p = 0.0194; 0.002; 0.0015, from left to right) weights in mice from (f) (n = 5 mice). m, Stainings in livers of mice from (d). n, o, Quantification of ORO (n) (p = 0.0002; 0.0004, from left to right) and SR (o) (p = 0.049; 0.0248, from left to right) in livers of mice from (d) (n = 5, 3, and 5 mice). p, q, ALT (p) (p = 0.0121; 0.0477; 0.0459, from left to right) and cholesterol (q) (p = 0.0172; 0.0003; <0.0001; 0.0008, from left to right) in plasma from mice from (f) (n = 5 mice). r, Fasting glucose in mice from (f) (n = 5 mice; p = 0.0497; 0.0431; 0.048, from left to right). s, t, GTT (s) (p <0.0001) and ITT (t) (p <0.0001; 0.0390, from left to right) in mice from (f) (n = 5 mice). Scale bar represents 50 m (m). Unpaired two-tailed Student’s t test (b-d, f, h-l, n-r) or two-way ANOVA (e, s, t) were used. Data are represented as means ± SEM.

Fig. 2 | Digoxin suppresses DIO and metabolic disorders. a, Weights of ND- or HFD-fed-C57BL/6 mice since 8 weeks, treated with/without digoxin since 20 weeks (n = 9, 9, 5, 15, 8, and 7 mice; p <0.0001). b, Weights of ND- or HFD-fed-38-week-old C57BL/6 mice since 8 weeks, treated with/without digoxin since 20 weeks, relative to the weight of ND-fed-C57BL/6 mice (n = 9, 9, 5, 15, 8, and 7 mice; p <0.0001; 0.0005; <0.0001; <0.0001; <0.0001, from left to right). c, Weights of HFD-fed-C57BL/6 mice since 8 weeks, treated with/without digoxin since 16 weeks and injected with rIL-17A since 21 weeks (n = 5 mice). d, Weights of HFD-fed-Rorc-Cre and -Rorc-Cre-DTR mice since 8 weeks, treated with DT and with/without digoxin since 8 weeks (n = 8, 5, 9, and 6 mice; p <0.0001). e, Percentage of total fat in mice from (b) (n = 10, 5, 8, and 15 mice; p <0.0001). f, Total fat mass in mice from (b) (n = 10, 5, 8, and 15 mice; p <0.0001). g-i, gWAT (g) (p <0.0001; 0.0045; 0.03, from left to right), iWAT (h) (p = 0.0043; <0.0001; 0.0043, from left to right), and BAT (i) (p <0.0001; 0.0002; 0.0005, from left to right) weights in mice from (b) (n = 9, 5, 10, and 8 mice). j, Stainings in livers of mice from (b). k, l, Quantification of ORO (k) (n = 5, 5, 8, and 5 mice; p = 0.0006; 0.0053; 0.0089, from left to right) and SR (l) (n =5, 5, 6, and 5 mice; p = 0.0499; 0.048, from left to right) from mice from (b). m, n, ALT (m) (p = 0.0459; 0.0263, from left to right) and cholesterol (n) (p = 0.0005; <0.0001; <0.0001, from left to right) in plasma from mice from (b) (n = 6, 5, 10, and 5 mice). o, Fasting glucose in mice from (b) (n = 4, 5, 5, and 5 mice; p = 0.0305; 0.033; 0.0093, from left to right). p, q, GTT (p) (p = 0.0006) and ITT (q) (p = 0.0024) in mice from (b) (n = 4, 5, 5 and 5 mice). r, s, GTT (r) (p = 0.0002) and ITT (s) (p <0.0001) in mice from (d) (n = 8, 5, 9, and 6 mice). t, u, GTT (t) (p = 0.0342) and ITT (u) (p = 0.0124) in mice from (c) (n = 5 mice). Scale bar represents 50 m (j). Unpaired two-tailed Student’s t test (b, e-i, k-o) or two-way ANOVA (a, d, p, q, r, s, t, u) were used. Data are represented as means ± SEM.


Fig. 3 | RORt inhibitors suppress DIO and metabolic disorders. a, Scheme of HFD and GSK805 treatments since 8 weeks in C57BL/6 mice. b, Weights of HFD-fed-C57BL/6 mice treated with vehicle or GSK805 (n = 5 and 3 mice; p = 0.0015). c, Weights of HFD-fed-20-week-old-C57BL/6 mice treated with vehicle or GSK805, relative to the weight of vehicle-treated HFD-fed-C57BL/6 mice (n = 5 and 3 mice; p = 0.0025). d, Fasting glucose in mice from (c) (n = 5 and 3 mice; p = 0.05) e, GTT (p = 0.0354; 0.0002) and ITT (p <0.0001) in mice from (c) (n = 5 and 3 mice). f, Scheme of HFD treatment since 8 weeks, followed by GSK805 treatment since 20 weeks in C57BL/6 mice. g, Weights of HFD-fed-C57BL/6 mice treated with vehicle or GSK805 (n = 5 and 3 mice; p = 0.0011). h, Weights of HFD-fed-26-week-old C57BL/6 mice treated with vehicle or GSK805, relative to the weight of vehicle-treated HFD-fed-C57BL/6 mice (n = 5 and 3 mice; p = 0.0093). i, Fasting glucose in mice from (h) (n = 5 and 3 mice; p = 0.0005). j, GTT (p = 0.041) and ITT (p = 0.041) in mice from (h) (n = 5 and 3 mice). k, Scheme of HFD and TMP778 treatment since 8 weeks in C57BL/6 mice. l, Weights of HFD-fed-C57BL/6 mice treated with vehicle or TMP778 (n = 5 mice; p = 0.0285). m, Weights of HFD-fed-18-week-old-C57BL/6 mice treated with vehicle or TMP778, relative to the weight of HFD-fed-C57BL/6 mice treated with vehicle (n = 4 and 5 mice; p = 0.0030). n, Fasting glucose in mice from (m) (n = 4 and 5 mice; p = 0.0185). o, GTT (p = 0.0004) and ITT (p = 0.0035) in mice from (m) (n = 4 and 5 mice). Unpaired two-tailed Student’s t test (c, d, h, i, m, n) or two-way ANOVA (b, e, g, j, l, o) were used. Data are represented as means ± SEM.



Fig. 4 | Deletion of IL-17RA prevents DIO and metabolic disorders. a, Scheme of tamoxifen treatment for 2 weeks at 6 weeks of age, followed by HFD since 8 weeks in Il17ra(+/+) and Il17ra() mice. b, Weights of ND- or HFD-fed-Il17ra(+/+) and -Il17ra() mice treated with/without digoxin (n = 5, 6, 9, 7, 6, and 7 mice; p = 0.0001). c, Pictures of ND- or HFD-fed-38-week-old Il17ra(+/+) and Il17ra() mice. d, Weights of mice from (c), relative to the weight of ND-fed-Il17ra(+/+) mice (n = 5, 6, 9, and 7 mice; p = 0.039; <0.0001; 0.0001, from left to right). e, Percentage of total fat in mice from (c) (n = 4, 4, 6, and 5 mice; p = 0.0028; <0.0001; 0.0069, from left to right). f, Total fat mass in mice from (c) (n = 4, 4, 6, and 5 mice; p = 0.0176; <0.0001; 0.0035, from left to right). g-i, gWAT (g) (p = 0.0231; 0.0044; 0.0056, from left to right), iWAT (h) (p = 0.0326; <0.0001; 0.0002, from left to right), and BAT (i) (p = 0.0458; 0.0002; 0.0004, from left to right) weights in mice from (c) (n = 5, 6, 9, and 6 mice). j, Stainings in livers of mice from (c). k, l, Quantification of ORO (k) (p = 0.0005; 0.011, from left to right) and SR (l) from mice from (c) (n = 5, 4, 6, and 3 mice). m, n, ALT (m) (p = 0.0457; 0.0355, from left to right) and cholesterol (n) (p = 0.05; 0.0025; 0.0016, from left to right) in plasma from mice from (c) (n = 6, 6, 9, and 6 mice). o, Fasting glucose levels in mice from (c) (n = 4, 4, 7, and 5 mice; p = 0.0003; 0.0033, from left to right). p, q, GTT (p) (p <0.0001) and ITT (q) (p = 0.0074) in mice from (c) (n = 4, 4, 7, and 5 mice). Scale bars represent 1 cm (c) and 50 m in (j). Unpaired two-tailed Student’s t test (d-i, k-o) or two-way ANOVA (b, p, q) were used. Data are represented as means ± SEM.



Fig. 5 | Inhibition of IL-17A axis promotes browning and thermogenesis. a, b, EE monitored for 3 days (a) and total EE (b) in ND- or HFD-fed-13-week-old C57BL/6 mice since 8 weeks, treated with/without digoxin since 12 weeks for 1 week (n = 10, 8, 9, 8 mice; p = 0.0334; 0.0321, from left to right). Grey squares denote night periods. c, Regression plot of total EE from (b) and body weight from each mouse from (a) (n = 10, 8, 9, 8 mice). d, e, EE monitored for 3 days (d) and total EE (e) in ND- or HFD-fed-38-week-old Il17ra(+/+) and Il17ra() mice (n = 7, 6, 7, and 6 mice; p = 0.0002). f, Regression plot of total EE from (e) and body weight from each mouse from (d). (n = 7, 6, 7, and 6 mice). g, H&E and IHC in adipose tissues of ND- or HFD-fed-38-week-old C57BL/6 mice treated with/without digoxin since 8 weeks. h, H&E and IHC in adipose tissues of mice from (d). i, UCP-1 quantification from BAT of mice from (g) (n = 7, 5, 9, and 6 mice; p = 0.0011; 0.0362; <0.0001, from left to right). j, UCP-1 quantification from BAT of mice from (d) (n = 3, 3, 3, and 8 mice; p = 0.0439; 0.0384; <0.0001, from left to right). k, Infrared thermography in mice from (a). Squares denote WAT and BAT in ventral and dorsal pictures, respectively. l-n, Temperature quantification in WAT (l) (p = 0.0008), BAT (m) (p = 0.048), and whole body (n) (p = 0.0012) based on infrared thermography in mice from (a) (n = 5 mice). o, Scheme of 24-h-cold exposure in ND- or HFD-fed 12-week-old C57BL/6 mice treated with/without digoxin since 8 weeks for 1 month. p, Body temperature of mice from (o) (n = 5 mice; 0.008; 0.035, from left to right). q, Scheme of cold exposure in HFD-fed-C57BL/6 mice since 8 weeks, treated with/without digoxin since 20 weeks for 1 month. r, Weights of mice from (q) (n = 5 mice; p = 0.0009). Scale bars represent 100 m in WAT, and 50 m in BAT (g, h) and 1 cm (k). Unpaired two-tailed Student’s t test (i, j, l-n), two-way ANOVA with interaction (c, f), two-tailed Tukey’s post-hoc test (b, e), “conventional” two-way ANCOVA (e), or two-way ANOVA (p, r) were used. Each dot in (c, f) represents one mouse. Data are represented as means ± SEM.


Fig. 6 | Genetic ablation of Il17ra in adipocytes prevents DIO. a, Weights of ND- or HFD-fed-Il17ra(+/+)Adipoq and Il17ra()Adipoq mice since 8 weeks, treated with/without digoxin (n = 6, 4, 5, 6, 5, and 5 mice; p = 0.0048). b, Pictures of ND- or HFD-fed-20-week-old Il17ra(+/+)Adipoq and Il17ra()Adipoq mice since 8 weeks. c, Weights of mice from (b), relative to the weight of ND-fed-Il17ra(+/+)Adipoq mice (n = 6, 4, 8, and 6 mice; p = 0.0311; <0.0001; 0.002, from left to right). d, Percentage of total fat in mice from (b) (n = 6, 4, 8, and 4 mice; p = 0.0437; <0.0001; 0.0202, from left to right). e, Total fat mass in mice from (b) (n = 6, 4, 8, and 4 mice; p = 0.0449; <0.0001; 0.005, from left to right). f-h, gWAT (f) (p = 0.043; 0.0002; 0.0232, from left to right), iWAT (g) (p <0.0001; 0.0038, from left to right), and BAT (h) (p = 0.05; 0.0071; 0.0134, from left to right) weights in mice from (b) (n = 6, 4, 8, and 6 mice). i, H&E and IHC in iWAT and BAT of mice from (b). j, Quantification of UCP-1 from BAT of mice from (b) (n = 5, 4, 8, and 4 mice; p = 0.0365; 0.0009, from left to right). k, Pictures of infrared thermography in mice from (b). Squares denote WAT and BAT in ventral and dorsal pictures, respectively. l-n, Quantification of WAT temperature (l) (p = 0.0301), BAT temperature (m) (p = 0.0175), and mouse body temperature (n) (p = 0.02) based on infrared thermography in mice from (b) (n = 5 mice). o, H&E in livers of mice from (b). p, Cholesterol in plasma from mice from (b) (n = 6, 4, 8, and 6 mice; p <0.0001). q, Fasting glucose levels in mice from (b) (n = 6, 4, 8, and 6 mice; p = 0.0347; 0.0007; 0.05, from left to right). r, s, GTT (r) (p = 0.0469) and ITT (s) (p <0.0001) in mice from (b) (n = 6, 4, 8, and 6 mice). Scale bar represents 1 cm (b, k), 50 m in BAT and 100 m in iWAT (i), and 50 m (o). Unpaired two-tailed Student’s t test (c-h, j, l-n, p, q) or two-way ANOVA (a, r, s) were used. Data are represented as means ± SEM.


Fig. 7 | IL-17A phosphorylates PPAR in adipocytes to promote obesity. a, Il17ra mRNA in 3T3-L1 cells (n = 3 independent experiments; p = 0.0391). b, Lcn2 mRNA in differentiated 3T3-L1 stimulated with rIL-17A for 12 h or 24 h (n = 9 samples from 3 independent experiments; p <0.0001; 0.0005; 0.0104; 0.0015; <0.0001, from left to right). c, WB of differentiated 3T3-L1 cells treated with rIL-17A and/or roscovitine for 15 min (n = 2 samples). d, WB of WAT from HFD-fed-20-week-old Il17ra(+/+)Adipoq and Il17ra()Adipoq mice treated with/without digoxin or roscovitine since 8 weeks (n = 2 samples). e, pPPARSer-273 quantification from (d) (n = 3 independent experiments; p = 0.0032; 0.0132; 0.0132; 0.0089; 0.0418, from left to right). f, WB of WAT from HFD-fed-C57BL/6 mice since 8 weeks, treated with/without digoxin since 20 weeks for 1 week, and injected with/without rIL-17A for 1 week (n = 2, 3, and 3 samples). g, Adipoq and Cfd mRNA in WAT from ND- or HFD-fed-38-week-old C57BL/6 mice since 8 weeks, treated with/without digoxin since 20 weeks (n = 6, 4, 6, and 7 mice; p <0.0001, 0.0012, from left to right). h, Adipoq and Cfd mRNA in WAT from ND- or HFD-fed-38-week-old Il17ra(+/+) and Il17ra() mice (n = 6, 7, 6, and 4 mice; p = 0.0018; 0.0433; 0.002; 0.011, from left to right). i, Adipoq and Cfd mRNA in WAT from ND- or HFD-fed-20-week-old Il17ra(+/+)Adipoq and Il17ra()Adipoq mice since 8 weeks (n = 4, 4, 7, and 3 mice, p = 0.0129; 0.0137; 0.0004; 0.0173, from left to right). j, Weights of ND- or HFD-fed-Il17ra(+/+)Adipoq and Il17ra()Adipoq mice since 8 weeks, treated with vehicle or roscovitine (n = 7, 5, 5, 4, and 4 mice; p = 0.0198).  k, UCP-1 from iWAT and BAT of mice from (j). l, Scheme of IL-17A action on adipocytes. Scale bar represents 50 m in BAT and 100 m in iWAT (k). Unpaired two-tailed Student’s t test (a, b, e, g-i) or two-way ANOVA (j) were used. Data are represented as means ± SEM.



METHODS
Antibodies
Antibodies used for immunoblotting and IHC were as followed: Rabbit polyclonal anti-UCP-1 (1:500 for IHC, 1:1000 for western blot, WB) (Ab10983) and Rat monoclonal anti-Tubulin (YL1/2) (Ab6160) (1:1000) were purchased from Abcam. Goat polyclonal anti-Mouse HRP conjugated (1:5000) (P0447), goat polyclonal anti-rabbit HRP conjugated (1:5000) (P0448), and rabbit polyclonal anti-MPO (1:500) (A0398) were obtained from Dako. Mouse monoclonal anti-Vinculin (1:10000) (V9131) was purchased from Sigma. Goat polyclonal anti-CD3(M-20) (1:500) (Sc-1127) was obtained from Santa Cruz Biotechnology. Rabbit polyclonal anti-PPAR Ser-273 (1:1000) (BS-4888R) was purchased from Bioss Antibodies. Rabbit monoclonal anti-PPAR (1:1000) (2435), rabbit polyclonal anti-ERK1/2 Thr202/Tyr204 (9101) (1:1000), and mouse monoclonal anti-ERK1/2 (4696) (1:1000) were obtained from Cell signaling. Rat monoclonal anti-F4/80 (1:1) was produced by CNIO Monoclonal Antibodies Unit. Rat monoclonal anti-CD45R/B220 clone RA3-3B2 (1:200) (557390), rat monoclonal anti-mouse CD45 BUV563 clone 30-F11 (1:200) (565710), rat monoclonal anti-mouse IL-17A PE clone TC11-18H10 (1:250) (559502) were obtained from BD Biosciences. Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block™) (1:200) (553141) was purchased from BD Pharmingen. Rat monoclonal anti-IL-17RA PE clone PAJ-17R (1:200) (12-7182-82) and Rat monoclonal anti-CD11b APC-eFluor 780 clone M1/70 (1:200) (47-0112-82) were obtained from eBioscience.

Materials
Materials are described in Supplementary Table 4.


Mouse models
C57BL/6 mice were provided by the CNIO Animal Facility. Rorc-Cre-DTR were generated in our lab by crossing Rorc-Cre mice 24 with the Cre-inducible diphtheria toxin receptor (DTR) transgenic mice 25. Inducible IL-17RA knock-out mice were obtained in our lab by crossing Il17ra-flox mice 34 and hUBC-CreERT2 mice 33. Lepob mice were obtained from Dr. Erwin F. Wagner’s lab 40. Leprdb mice 41 were acquired from Charles Rivers Laboratories. Il17ra()Myeloid mice were generated as previously described 9. Adipoq-Cre mice 48 were provided by Dr. Marcos Malumbres and crossed with Il17ra-flox mice to generate Il17ra()Adipoq mice (Supplementary Table 5). All mice have been housed in pathogen-free conditions, with a 12 h light/dark cycle between 8:00 and 20:00 in a temperature-controlled room (23 ± 1 °C) following the recommendations of the EUMORPHIA Consortium for animal housing, unless stated otherwise. All experiments were approved by the CNIO-ISCIII Ethics Committee and performed in accordance with the guidelines for ethical conduct in the care and use of animals as stated in the international guiding principles for biomedical research involving animals, developed by the Council for International Organizations of Medical Sciences. All genetic mouse models have been backcrossed to the C57BL/6 background for at least 7 generations (except for the Leprdb model, which is under C57BLKS/J (BKS) background). Only age-matched males were used for the study. Female mice were also used to check that gender disparity was not present upon digoxin treatment, but data were not included in this manuscript. Littermates of the same sex were randomly assigned to either experimental or control groups. Age/developmental stage of mice is included appropriately in the text and figure legends. Food (Harlan Laboratories and Research Diets Inc.) and water were provided ad libitum, unless described elsewhere.


Mouse diets and treatments
Cre-mediated recombination was activated by feeding mice with tamoxifen diet at the concentration of 400 mg/kg at 6 weeks of age and for the next 2 weeks. 
Mice were fed either with chow diet (here as normal diet, ND) (18% fat, 58% carbohydrates and 24% proteins) (Harlan Laboratories, 2018S) or HFD (45% fat, 35% carbohydrates and 20% proteins) (Research Diets Inc., D12451). Food and water were provided ad libitum unless stated otherwise.
For oral administration, digoxin (Abmole, M3935) was dissolved in drinking water at a concentration of 30 g/ml, unless specified. Drinking treatment was changed three times per week. For intraperitoneal administration, digoxin was dissolved in PBS at a concentration of 30 g/ml and 1 mg/kg was injected daily. PBS was injected as vehicle.
Diphtheria toxin (DT) (Sigma, 322326) was dissolved in PBS at a concentration of 2 g/ml and 10 ng/g were intraperitoneally injected three times per week.
rIL-17A (GenScript, Z03031-50), rIL-17C (Cloud-Clone Corp. RPD347Mu02), rIL-17E (Peprotech, 210-17E) or rIL-17F (Peprotech, 210-17F) were dissolved in PBS at a concentration of 0.006 mg/ml and 0.04 mg/kg were intraperitoneally injected every other day.
Ouabain (Abmole, M2904) was dissolved in PBS at a concentration of 0.112 mg/ml and 0.3 mg/kg were intraperitoneally injected three times per week.
GSK805 (DC chemicals, DC23184) was dissolved in 3% DMSO in 0.5% methylcellulose in PBS at a concentration of 3 mg/ml and 10 mg/kg were intraperitoneally injected every other day. 3% DMSO in 0.5% methylcellulose in PBS was injected as vehicle.
TMP778 (Aobious, AOB2009) was dissolved in 5% DMSO in 0.5% methylcellulose in PBS at a concentration of 1.5 mg/ml and 10 mg/kg were subcutaneously injected every other day. 5% DMSO in 0.5% methylcellulose in PBS was injected as vehicle.
Recombinant murine leptin (Peprotech, 450-31) was dissolved in PBS at a concentration of 1 mg/ml and intraperitoneally injected to HFD-fed C57BL/6 mice (2 mg/kg) or to Lepob (-/-) mice (1 mg/kg). PBS was injected as vehicle.
Roscovitine (Abmole, M1974) was dissolved in 5% DMSO in PBS at a concentration of 5 mg/ml and intraperitoneally injected to 8-week-old mice at a concentration of 50 mg/kg for 12 weeks three times per week. 5% DMSO in PBS was injected as vehicle.

Body weight (BW) measurement
Mice were weighed between 2 pm and 8 pm using an analytical balance. Mice were weighed weekly since weaning until the end of the experiments.
To estimate weight loss efficiency induced by the described compounds, weight loss was indicated in the appropriate figure legends and calculated as follows:
Body weight (BW) loss adjusted to ND-fed mice (%):



Body weight (BW) loss adjusted to HFD-fed mice (%):



Determination of weight loss efficiency and EC50
Weight loss achieved with different doses of digoxin (0,1; 1; 10 and 30 g/ml) was normalized to 100%, being 30 g/ml of digoxin the dose with the maximal efficiency. Then, the log of each concentration and the EC50 were calculated with GraphPad Prism V5.0 software.
Body mass composition
Animals were anesthetized with 2% isofluorane (Isovet, Braun Vetcare) and fat lean mass, and bone mineral density (BMD) were determined by Dual energy X-ray Absorptiometry (DXA) (Piximus, Lunar® Corporation). Analysis of fat and lean masses and BMD was performed using a Region of Interest (ROI) in the whole body using Lunar PIXImus 2.10 software.

Behavioural tests
The rotarod and the tightrope tests were used to evaluate motor coordination and balance in mice. In the rotarod test, mice were tested in a rotarod apparatus (Panlab model LE 8200) set up at 4 rpm. Mice were acclimated to the machine during 2 days. The third day, the time before the mice fell was recorded, and the average of three consecutive trials was used in the quantification. The maximum trial duration is 300 s. In the tightrope test, mice were placed onto a rod for 5 trials for 60 s. Each trial that the mouse did not fall was counted as a success, and the percent success for each mouse was determined.
The open-field test was used to evaluate cognitive function and willingness to explore. The open field chamber measured 50 cm (length) x 50 cm (width) x 38 cm (height). 10 x 10 cm blocks were labeled on the basement to delineate the outer zone (16 blocks surrounding the borders of the field) and the inner zone (9 blocks in the center of the field). Each mouse was placed in the open field and allowed to explore it for 10 min. Before and after each test, the chamber was cleaned with 75% ethanol. Movements of mice were recorded with a camera placed above the chamber mounted on top of a tripode and analyzed offline.
The object recognition test was used to evaluate cognitive function and memory. In the first session, two identical objects were paced in an open field and mice were allowed to explore for 10 min. 24 h later, one object was replaced by a novel object and mice were allowed to explore for 10 min. The amount of time taken to explore the new object was estimated as the ratio of time investigating the new object to the total time investigating both objects in the second session. Mice were recorded with a camera placed above the chamber mounted on top of a tripode and analyzed offline. Before and after each test, the chamber and the objects were cleaned with 75% ethanol.

Metabolic activity
Metabolic activity was studied with Oxylet System metabolic chambers (Panlab Harvard Apparatus), using 8 simultaneous metabolic chambers. Mice were individually caged for 1 week before the metabolic chamber studies. Then, animals were acclimatized to the measurement cages for three days prior to data recording. Measurement period consisted on 72 h. Mice had free access to food and water during the study. Mouse activity, food intake, and drink intake were recorded in time intervals of 20 min during the whole measurement period. Respiratory exchange ratio (RER) was calculated as RER = VCO2/VO2 from volumes of consumed O2 (VO2) and eliminated CO2 (VCO2) recorded every 24 min. Energy expenditure (EE) was calculated as EE = (3.815 + (1.232 ×RER)) × VO2 × 1.44). Data analysis was performed using Metabolism 3.0 software.

Measurement of cumulative food and drink intake
To monitor food and drink intake for a period of 1 month at early stages of inhibition, mice were individually caged and the weight of food pellets and water bottles was measured every day.
To monitor food and drink intake at late stages of IL-17A inhibition, food and water intake were recorded for 3 days with Oxylet System metabolic chambers as explained in the previous section.

Infrared thermal imaging
An infrared camera (HT-instruments, THT46) mounted on top of a tripod was used to acquire thermal images. Mice were anesthetized with 2% isofluorane (Isovet, Braun Vetcare) and placed on dorsal and ventral position to acquire static thermal images at a focal length of 40 cm. For each image, the whole body mouse, WAT or BAT area was delineated and the average temperature of each area was calculated using ImageJ.

Cold exposure
For acute cold exposure, C57BL/6 mice in pathogen-free conditions, in a temperature-controlled room (23 ± 1 °C) were simultaneously fed with/without HFD and treated with/without digoxin since 8 weeks for 1 month. Rectal temperature was measured using a rectal mouse probe (Bioseb, BIO-BRET3) connected to a rodent thermometer (Bioseb, BIO-TK9882) and then mice were transferred to a temperature-controlled room at 4 °C for 24 h. After cold exposure, mice were sacrificed for histological analysis.
For chronic cold exposure, C57BL/6 mice were fed with HFD for 3 months since 8 weeks in a temperature-controlled room (23 ± 1 ºC) and then transferred to a climate controlled chamber (HPP750life, Memmert). A 12 h light:dark cycle (8:00 – 20:00.) and humidity at 75% were maintained during the housing period in the climate chamber. After 1 week of acclimatization at 18 ºC, mice were exposed to cold temperature (5 ± 1 ºC) with/without digoxin for 1 month.

Estimation of the human equivalent dose (HED)
To estimate the HED, mouse dose in mg/kg was calculated by measuring drink intake with Oxylet System metabolic chambers (Panlab Harvard Apparatus) and weight of the mice. Mouse dose was converted to the HED following recommendations for dose conversion between animals and humans as previously described 57.
Fasting-refeeding experiments
C57BL/6 mice were fed with ND or HFD for 3 months since 8 weeks and then treated with/without digoxin in drinking water for 1 month. Mice were fasted for 24 h, when blood was collected. Then, mice were refed with ND or HFD. Blood was collected 8 h, 24 h, and 48 h post-refeeding. Mice had drink ad libitum during the performance of the experiment. Blood was transferred to an EDTA containing tube and plasma was obtained after 4000 g, 20 min centrifugation at 4 ºC and kept at - 80 ºC until further analyzes. 

FITC-dextran permeability assay
Mice were first fasted for 12 h and then given 200 l of FITC-dextran dissolved in water (Sigma-Aldrich) at a concentration of 60 mg/ml by oral gavage. After 4 h, blood was collected from the cheek and transferred to an EDTA containing tube for centrifugation at 4000 g for 20 min at 4 ºC. Plasma was collected and analyzed by spectrofluorometry at the 485 nm excitation wavelength and 528 nm emission wavelength as previously reported 58,59.

Faecal mass quantification
Faeces were collected from metabolic cages after the measurement period. Faecal mass was weighed with an analytical balance.

Bomb calorimetry of faeces
Bomb calorimetry was conducted by the Brigham and Women’s Hospital (BWH) Metabolic Core facility using a Parr 6725EA semimicro calorimeter and a 1109 oxygen bomb. Faecal samples were collected from colons during mouse necropsies and stored at -80 ºC until processing. Briefly and as processed by Brigham and Women's Hospital (BWH) Metabolic Core Facility (affiliated with Harvard Medical School in Boston), samples were heated at 60 °C for 48 h to remove water content. Faecal samples were combusted and the energy content of the faecal matter was measured as heat of combustion (kcal/g).

Extraction and quantification of faecal triglycerides
Faeces were collected from colons during mouse necropsies and stored at -80 ºC until processing to extract and measure triglycerides. 100 mg of faeces were dried for 1 h at 70 ºC and incubated with 1.5 ml of chloroform-methanol (2:1) for 30 min at 60 ºC with constant agitation. Solution was homogenized using Precellys 24 Bead Mill homogenizer (Bertin Technologies) (15 x 2 s, 5500 w) and then clarified by centrifugation for 10 min at 2300 g at room temperature (RT). Supernatant was collected and mixed with 750 l of water. After vortexing, phase separation was induced by spinning samples for 10 min at 400 g at RT. Upper layer was discarded and chloroform evaporated using a vacuum machine for 2 h.  Pellet was resuspended in 500 l of 1% triton X-100 in chloroform and evaporated to dryness with a vacuum machine for 2 h.
Finally, pellet was dissolved with 200 l of 1X NP40 substitute assay reagent from Triglyceride Colorimetric Assay Kit (Cayman, 10010303). Triglycerides were quantified with the kit according to manufacturer’s instructions.

Extraction and quantification of faecal carbohydrates
Faeces were collected from colons during mouse necropsies and stored at -80 ºC until processing to extract and measure carbohydrates. 50 mg of samples were homogenized with 200 l of distilled water and centrifuged at 13600 g for 10 min. 30 l of the supernatant were mixed with 120 l of anthrone reagent (0,2% Anthrone in 98% sulfuric acid) and incubated at 90 ºC for 15 min. After cooling down the samples, absorbance was read at 630 nm. The concentration of carbohydrates present in the samples was determined from the D-glucose standard curve.

Extraction and quantification of faecal proteins
Faeces were collected from colons during mouse necropsies and stored at -80 ºC until processing to extract and measure proteins. 50 mg of samples were homogenized with 500 l of RIPA lysis buffer containing: 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 10% glycerol, and supplemented with 10 g/ml protease inhibitor aprotinin and 0.5 mM PMSF. Samples were homogenized using Precellys 24 Bead Mill homogenizer (Bertin Technologies) (15 x 2 s, 5500 w) and then clarified by centrifugation at 4 °C and 10000 g for 10 min. Protein concentration was measured using Bio-Rad Bradford reagent (Bio-Rad) and BSA as standard protein.

Glucose tolerance test (GTT) and insulin tolerance test (ITT)
GTT and ITT were performed as previously reported 9,60. Briefly, mice were fasted for 6 h. For GTT, mice were injected intraperitoneally with 2 g/kg glucose in PBS. For ITT, 0.75 U/kg insulin in 0.9% NaCl was intraperitoneally administered. Blood from the tail vein was obtained before the injection and at 15, 30, 60 and 90 min after the injection for determination of blood glucose using the Accu-Check Compact test strips (Roche, 13072). 

Blood parameters
Blood was collected from the heart and transferred to an EDTA containing tube for haemogram analysis using Abacus Junior Vet analyzer (Diatrom). Plasma was collected after 4000 g, 20 min centrifugation at 4 ºC and kept at -80 ºC until further analyzes. Alanine transaminase (ALT), cholesterol, and creatinine were quantified using ABX Pentra 400 and following manufacturer’s instructions. Ammonia (NH3) was measured using Ammonia Assay Kit (Sigma, AA0100-1KT). Free fatty acids were quantified using Free Fatty Acid Quantification Kit (BioVision, #K612-100) and following manufacturer’s instructions. Leptin was measured using Mouse Leptin ELISA kit (Crystal Chem, 90030).

Tissue preparation
Necropsies were performed from 2 pm to 8 pm. Mice were sacrificed using CO2 chambers. Tissues were quickly removed from mice and weighed using an analytical balance. Then tissues were washed in PBS and incubated overnight in 10% formalin at room temperature and processed for paraffin embedding, or snap frozen, or embedded in O.C.T and kept at -80 ºC until further analyzes, or freshly processed for flow cytometry analysis.

Isolation of immune cells
C57BL/6 mice were fed with ND or HFD for 2 months since 8 weeks and then treated with or without digoxin in drinking water for 1 week. To enhance the detection of IL-17A, mice were intraperitoneally injected with 10 mg/kg of Brefeldin A (Focus Biomolecules, 21910-1071) 12 h before immune cell isolation. To isolate immune cells from WAT, gonadal adipose tissue was collected and washed with PBS. WAT was minced and digested with 1 mg/ml of collagenase in DMEM supplemented with 10% FBS (digestion buffer) for 30 min at 37 °C. Digested tissue was transferred to a GentleMACS C tube (Miltenyl Biotech, 120-005-331) in presence of 10 ml of digestion buffer and further homogenized with GentleMACS dissociator. Dissociated WAT was filtered through a 70m nylon cell strainer and centrifuged at 500 g for 10 min at 4 °C to separate adipocytes and stromal vascular cells. The stromal vascular fraction was resuspended in 0.5 ml of RBC lysis buffer (Gibco, A10492-01) and reaction was stopped by adding 10 ml of 0.5% BSA, 2 mM EDTA in Ca2+/Mg2+-free PBS (MACS buffer). After centrifugation at 500 g for 10 min at 4 °C, supernatant was discarded and pellet was resuspended in MACS buffer.
To isolate immune cells from spleen, spleen was minced, dissociated and filtered through a 70 m nylon cell strainer and centrifuged at 500 g for 10 min at 4 °C. Cells were resuspended in 0.5 ml of RBC lysis buffer (Gibco, A10492-01) and reaction was stopped by adding 10 ml of MACS buffer. After centrifugation at 500 g for 10 min at 4 °C, supernatant was discarded and pellet was resuspended in MACS buffer.

Flow cytometry
To study IL-17A expression in WAT, after final wash, cells were resuspended in MACS buffer containing 1:200 BD Fc block (BD BioSciences, 553141) and incubated for 20 min at 4 ºC. Discrimination of live/dead cells was done using 3l/ml Zombie Aqua Fixable Viability Dye (Biolegend, 423101), for 30 min at 4 ºC. Following washing with MACS buffer, surface markers were stained for 20 min at 4 ºC in the dark. Stromal vascular cells pooled from multiple mice were used to prepare Fluorescence Minus One (FMO) controls. To stimulate IL-17A production, cells were then treated for 3 h at 37 ºC with 20 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Sigma, P1585), 1 g/ml Ionomycine (Sigma, I9657) and 0.6 l/ml Golgi Stop (BD Biosciences, 554724) in DMEM supplemented with 10% FBS. After washing with MACS buffer, cells were fixed with BD Cytofix (BD BioSciences, 51-2090KZ) for 15 min at RT. Cells were further permeabilized using BD Perm/Wash (BD BioSciences, 51-2091KZ) for 15 min at RT and followed by staining using the indicated intracellular markers for 30 min at 4 ºC. After washing, cells were resuspended in 500 µl of MACS buffer and analyzed using LSR Fortessa (BD BioSciences). 
To test the deletion of Il17ra in myeloid cells, splenocytes were resuspended in MACS buffer containing 1:200 BD Fc block (BD BioSciences, 553141) and incubated for 20 min at 4 ºC. Discrimination of live/dead cells was done using 1 μl/ml Aqua Live Dead (Life Technologies, L34957), for 30 min at 4 ºC. Following washing with MACS buffer, surface markers were stained for 20 min at 4 ºC in the dark. After washing, cells were resuspended in 500 μl of MACS buffer and analyzed using FACS Canto II (BD BioSciences).
FMOs were used to gate cell populations and compensation beads (UltraComp eBeads™, Thermofisher 01-2222-42) to compensate for fluorochrome spectral overlap. We used pulse processing to exclude cell aggregates. All data was analyzed using FlowJo v10 software (Trestar, Oregon).

Immunohistochemistry and histology
For immunohistochemistry, freshly harvested tissues were fixed immediately in 10% buffered formalin solution overnight and embedded in paraffin and processed as previously reported 9,58. 
Oil Red O (ORO) staining was performed in O.C.T. frozen sections. After fixing them with 10% buffered formalin solution for 10 min, sections were incubated in 60% isopropanol for 15 min and then in ORO working solution (60% 0.3% ORO concentrated solution in isopropanol, 40% distilled water) for 15 min. Slides were washed, counterstained with hematoxylin, and mounted with aqueous mounting medium.
Sirius Red staining was performed in paraffin-embedded sections. Tissue sections of 3 m were deparafinized, rehydrated and fixed with pre-warmed Bouins’ solution at 55 ºC for 1 h. After section washing with water, slides were incubated with 0.1% Fast green solution for 10 min, followed by 2 min incubation with 1% acetic acid. Then, sections were washed and stained with 0.1% Sirius red solution for 30 min. Slides were washed and dehydrated.

Cell culture
3T3-L1 cells (ATCC® CL-173™) were kindly provided by José M. Fernández-Real (Idibgi, Girona, Spain). Cells were maintained in DMEM supplemented with 10% faetal calf serum (FCS), 100 unit/ml penicillin and 0.1 mg/ml streptomycin (1 x P/S). Subculture samples were frozen in a mixture of DMEM with 20% FCS and 10% DMSO. Differentiation was induced by culturing the cells with DMEM containing 10% faetal bovine serum (FBS) and 1 x P/S (differentiation media), supplemented with 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), lM dexamethasone, 850 nM insulin, and 2M rosiglitazone. After 48 h, the medium was changed to differentiation media containing 850 nM insulin and 2M rosiglitazone for 48 h. On day 5, the medium was changed to differentiation media containing 850 nM insulin and refreshed every two days until cells were fully differentiated. Prior to treatment with different compounds, cells were rinsed three times with PBS and starved of FBS for 16 h. Then cells were stimulated with rIL-17A (GenScript, Z03031-50) at different concentrations and/or with 5 M roscovitine (Abmole, M1974). After stimulation, cells were harvested for qRT-PCR or WB analysis.

Immunoblotting 
For immunoblotting, frozen tissues were lysed using RIPA lysis buffer containing: 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl, 2 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40, 10% glycerol, and supplemented with 10 g/ml protease inhibitor aprotinin and 0.5 mM PMSF. Lysates and immunoblots were processed as previously described 9.

Genotyping and qRT-PCR
Genotyping and qRT-PCR were performed as previously reported 58,59. Primers used for genotyping and qRT-PCR are listed in Supplementary Tables 6 and 7, respectively.

Human data
Visceral white adipose tissue samples and data from patients with morbid obesity included in this study were provided by the Aragon Health Sciences Institute in the framework of the Biobank of Aragon and were processed following standard operation procedures with the approval of the Ethical and Scientific Review Boards. Informed consent was obtained from all subjects.
For the epidemiological study, Dr. Roberto Elosua from Hospital del Mar Medical Research Institute in Barcelona collected and used data from a population-based cross-sectional survey conducted in Girona (Spain) with the objective of studying the burden and the determinants of cardiovascular disease at population level (Registre Gironí del Cor, REGICOR Study). The characteristics of the study have been detailed elsewhere 61. In this study, the data of 6,352 individuals aged from 35 to 79 years recruited in 2005 was included. Participants were randomly selected from the census and invited to participate. Participation rate was >70%. The study protocol was approved by the Parc Salut MAR (PSMAR) Ethics Committee and each participant signed an informed consent at enrolment.
All the participants reported the drugs that they were taking, including digoxin. Moreover, a group of trained nurses followed a standardized physical examination protocol to obtain the weight and height of all the participants. Body mass index (BMI) was calculated as weight (in kilograms) / height2 (in meters). Participants were asked to fast for at least 10 h before their appointment at the health examination site. Fasting blood samples were taken and total cholesterol was determined. Low density lipoprotein cholesterol (LDL-C) levels were estimated using the Friedewald equation when triglycerides were <300 mg/dL. The association between the use of digoxin and anthropometric and laboratory measurements was assessed by multivariate linear regression analyses.

Statistics and reproducibility
Image analysis and quantifications
To analyse stainings, 10 images per slide from each mouse were taken at 20 X magnifications and quantified using color de-convolution, available in ImageJ 1.7v software.
To quantify ORO, SR, UCP-1, and F4/80 stainings, percentage of area stained was measured.
To quantify B220, CD3, and MPO stainings, positive cells per area were counted.
To quantify adipocyte size, images of H&E stainings were taken at 20X magnification and adjusted to gray-scale using ImageJ 1.7v software. The area of each adipocytes was measured manually using the ROI manager tool available in Image J 1.7v software. 
To quantify temperature in infrared images, pictures were processed with in ImageJ 1.7v software. RGB pictures were converted to a 3-sliced stack. Plot profile of each channel was quantified and temperature was determined with the scale of temperature. Software for data analysis is described in Supplementary Table 8.

Statistical analysis
Statistical analyses were performed using GraphPad Prism V5.0 software. Statistical analyses for total EE was performed using R Statistical Computing Programme (R Statistical Package V4.0.3 and RStudio) (Supplementary Table 1). Statistical analysis in the population-based epidemiological study was performed using R Statistical Computing Programme (R Statistical Package V2.0). Software for statistical analysis is described in Supplementary Table 8. Statistical significance (p value) (p ≤ 0.05 = *, p ≤ 0.01 = ** and p ≤ 0.001 = ***) between the means of two groups was determined using unpaired two-tailed Student’s t test, two-way ANOVA, or linear regression analysis. Results are expressed as the mean value ± Standard Error of the Mean (SEM). For the statistical analysis of total EE, we have first checked whether the groups show different association between EE and mass (slope of EE on mass is significantly different) by applying the two-way ANOVA with interaction test (also called ANCOVA for non-parallel slopes test). When the different groups have different association between EE and mass (slope of EE on mass was significantly different) then, the ANOVA with interaction test is directly followed by two-tailed Tukey’s post-hoc comparison. If the ANOVA with interaction test demonstrates no significant group by mass interaction (slope of EE on mass is the same for each group and no significant differences are detected), then the “conventional” two-way ANCOVA test, using body weight as covariate, is applicable, followed by two-tailed Tukey’s post-hoc comparison (Supplementary Table 1). "n" represents number of mice used in each experiment, as indicated in figure legends, or number of independent experiments performed with cells, as indicated in figure legends. All results including WB are representative of at least three independent experiments.  

Data availability
All data in this study are available in the Article and its Supplementary Information files, which include Extended Data Figs. 1–8 and Source Data Figs. 1 and 2. The data that support the findings of this study are available from the corresponding author upon request.
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